README file - Thermal tolerance and physiology of Eleutherodactylus coqui along an elevational gradient in HawaiÔi.

Data file Title: Coqui_ThermalTolerance_data.csv

Name and contact information of co-PI: 
Name: Susannah French
Position: Professor
Institution: Utah State University
Address: 5305 Old Main Hill, Logan, UT, 84322-5230, USA
Email: susannah.french@usu.edu

Name and contact information of co-PI: 
Name: Karen Beard
Position: Professor
Institution: Utah State University
Address: 5230 Old Main Hill, Logan, UT, 84322-5230, USA
Email: karen.beard@usu.edu

Name and contact information of primary author: 
Name: Jack Marchetti
Position: MS student
Institution: Utah State University
Address: 5305 Old Main Hill, Logan, UT, 84322-5230, USA
Email: jack.marchetti@usu.edu

Funding Sources: USDA-APHIS National Wildlife Research Center, Utah State University Department of Biology, and Utah State University Ecology Center.  

Description of data collection: 
	The purpose of this study was to determine if the thermal tolerance and physiology of the coqui frog changed along HawaiÔiÕs elevation gradient. A short acclimation experiment was used to test the baseline thermal tolerance and physiology, and an extended acclimation experiment to test the acclimation ability of the thermal tolerance and physiology metrics of the coqui frogs collected from low (<150 m), medium (350-550 m) and high elevations (>750 m). 
	For the short acclimation experiment, 30 frogs were collected from 12 field sites (four at low, four at medium, and four at high elevation) and kept them in uniform conditions for three days. Critical thermal minimum (CTmin), blood glucose, oxidative stress (dROMs) and corticosterone of coqui were measured following the three-day acclimation. For the extended acclimation experiment, 30 frogs from the same 12 field sites were collected and randomly assigned them to a cold (16 degree C) or warm (23 degree C) temperature treatment. Frogs were kept in these temperature treatments for three weeks before CTmin, glucose, dROMs, and corticosterone were measured in the same way as the short acclimation study. 306 individuals (30 females, 276 males) were collected for the short acclimation experiment, and 317 individuals (43 females, 274 males) were collected for the extended acclimation experiment. 
	CTmin was measured by using a temperature regulated chamber device (TReCS) device in which five aluminum cradles sit on top of an aluminum heatsink. Each cradle holds a plastic tube. The cradle temperature is controlled by two peltier devices beneath each cradle. CTmin testing began at 2000h after the three day and three-week acclimation periods for the short and extended acclimation frogs, respectfully. Each trial consisted of testing the CTmin of five frogs in the TReCS. At the beginning of each trial, we placed frogs in the plastic tubes and the TReCS began cooling the cradles starting at 20-23¡C. Once a cradle temperature reached 15 ¡C, we periodically spun each tube on the cradles to flip the frog on its back and elicit a righting response. If the frog did not right itself after 30 seconds, we recorded the TReCS cradle temperature (righting loss temperature; hereafter RL-temp). RL-temp served as a close approximation of CTmin, but not an exact measurement. We also recorded the elapsed time from the start of the trial to the time of righting loss (righting-loss time; hereafter RL-time). This was also important for approximating the coqui frogÕs CTmin. 
	We measured glucose using an Accu-Chek Active blood glucose meter. dROMs were measured using an assay kit (MC435, Diacron International, Italy), ?which detects levels of hydroperoxides that oxidize an alkyl-substituted aromatic amine (A-NH2) to assess chronic oxidative stress. Corticosterone was measured using an ?enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Sciences, ?Farmingdale, NY) following a validation protocol described by Hudson et al. (2020) for blood plasma (8 µl). 

Description of data file columns:
Study: Short acclimation (SA) or extended acclimation (EA).
Site: Field site frogs were collected from.
Frog: Coqui frog ID.
Elev.Cat: Elevation category (low, med, high).
Elev: Elevation in meters.
Date.collected: Date frogs were collected from field site.
Date.tested: Date the CTmin was measured and blood samples were collected from frogs.
Block.num: TReCS block number the frog was in during CTmin testing (1-5).
Trial: Sequential order of frog groups tested in the TReCS for each study. 
Acclim.temp: Acclimation temperature of frogs in extended acclimation experiment (16 or 23). 
Ambient: Air temperature of the room at the start of a CTmin trial.
S.block: Start block temperature. TReCS block temperature at the start of the trial.
Cloacal: cloacal temperature of coqui frog after loss of righting response. 
F.block: Final block temperature. TReCS block temperature at loss of righting response.
RL.time: Righting-loss time. Duration of time from trial start to loss of righting response. 
Cooling.rate: Rate of cooling of TReCS cradle.
SVL: Snout-vent length
Sex: Sex of coqui frog (male, female).
Glucose: Blood glucose concentration.
Plasma: Plasma volume separated from blood sample.
TAS: Time at stop. Time of day when frogs lost their righting response. 
TOB: Time of bleed. Time of day when blood samples were collected from frogs. 
Waiting.time: Amount of time between when frogs lost their righting response and when blood samples were collected. 
Notes: Notes taken during CTmin process. 
Cort: Corticosterone concentration.
Cort.SD: Corticosterone standard deviation.
drom: dROM concentration.
rlt.pca. Righting loss time/temp Principal Component Analysis. PCA between F.block and RL.time to serve as a CTmin index. 
HSP: Heat shock protein.
HSP.SD: Heat Shock protein standard deviation.
True.HSP: True heat shock protein. HSP concentration when dilution is accounted for.