DOI: 10.5061/dryad.k0p2ngf4b
title: Data from: Rain shadow effects predict population differences in thermal tolerance of leaf-cutting ant workers (Atta cephalotes)

description: Tests of hypotheses for the evolution of thermal physiology often rely on
 mean temperatures, but mounting evidence suggests geographic variation in
 temperature extremes is also an important predictor of species’ thermal
 tolerances. Although the tropics are less thermally variable than higher
 latitude regions, rain shadows on the leeward sides of mountains can
 experience greater diel and seasonal variation in temperature than
 windward sites. Rain shadows provide opportunities to test predictions
 about the relationships of extreme temperatures with thermal physiology
 while controlling for latitude. We tested the hypothesis that populations
 of leaf-cutting ants (Atta cephalotes) in leeward, montane, and windward
 sites in Costa Rica would differ in upper thermal tolerances (CTmax) of
 workers. As predicted from rain shadow effects via extreme high
 temperatures, the leeward rain-shadow site yielded the highest mean CTmax
 (rain shadow site 42.1±0.3 °C, Montane site 38.2±0.5 °C, windward site
 38.2±0.3 °C). This suggests that high-temperature extremes in tropical
 rain shadow forests can select for higher thermal tolerances. CTmax
 increased with worker body size within sites, but CTmax increased with
 body size more gradually at the two lowland sites, as predicted if local
 high temperatures selected more strongly on the most thermally vulnerable
 society members (small workers). This suggests that warmer lowland
 climates selected for colonies with less variation in heat tolerance than
 cooler high elevation climates.
description: Ant
 collections We studied thermal tolerance of
 the leaf cutting ant <i>Atta cephalotes</i> across its
 elevational range extremes in Costa Rica, sampling high elevations near
 the continental divide and lowlands on both the Pacific rain shadow
 (leeward) and Caribbean less variable (windward) slopes of the Tilarán
 mountain range. All field work was performed during the rainy seasons of
 2014 and 2016 in premontane wet forests of Monteverde (N10.31°, W84.31°
 1296-1311 m asl), lowland rainforests of La Selva Biological Station
 (N10.42°, W84.02° 50-58 m asl), and seasonal dry forests of Santa Sosa
 National Park (N10.85°, W85.64° 297-300 m asl). Although we searched the
 continental divide near Monteverde up to over 1800 m above sea level
 (masl), <i>A. cephalotes </i>was only common at and below
 circa 1300 masl, suggesting that our sampling was representative of the
 elevational extremes for this species in the region. Within each site,
 active and mature nests of <i>Atta cephalotes </i>at least 500
 m linear distance apart<i> </i>from one another were selected.
 Twenty worker ants across the range of body sizes we encountered were
 collected from the tops of the nest mounds and transported (within 2 hours
 of collection) to the lab to use as subjects in thermal tolerance assays.
 Three colonies were sampled in Santa Rosa and La Selva, and two colonies
 were sampled in Monteverde. Voucher specimens from each colony were
 deposited in the entomology collection of the Academy of Natural Sciences
 of Drexel University, Philadelphia, PA, U.S.A.
   Ambient temperature measurements
 Daily maximum, minimum, and mean temperatures were calculated
 from temperatures recorded round-the-clock in each site using iButton
 thermochron data loggers (Maxim Integrated, San Jose, CA). Temperature
 sampling was concurrent with CT<sub>max</sub> sampling of the
 ants (rainy seasons of 2014 and 2016). Each temperature logger was placed
 in a plastic housing and beneath a plastic sheet (to shield the probe from
 direct insolation) and inserted beneath the leaf litter on the forest
 floor. Loggers recorded surface soil temperature once every 5 minutes for
 three continuous days. Loggers were arranged in transects of three, spaced
 5 m apart from one another and 3 m into the forest alongside dirt trails
 at each site. A total of 8 such transects were deployed in Santa Rosa, 9
 in Monteverde, and 2 in La Selva. Both raw ibutton temperature data files
 and summaries of these temperature data showing daily temperature range,
 means and standard devition have been uploaded to this digital
 repository. We also obtained bioclim extrapolations of local temperatures
 at each site. These extrapolated climate data are reported in the
 master file alonside CT<sub>max</sub> and head
 width.    Thermal tolerance
 assays We ran standard dynamic heat tolerance
 assays using digital dry heat blocks (USA Scientific). Ants were placed in
 1.5 ml microcentrifuge tubes stoppered with cotton at the top to prevent
 access to thermal refuge in the tubes during the assay. Tubes were placed
 in the dry heat block and started at 30ºC, increasing in temperature at a
 rate of 1ºC every 10 minutes. Every 10 minutes we checked for loss of
 mobility response to light tapping on the tube. For each ant lack of motor
 response to tapping was interpreted as having surpassed the critical
 thermal maximum (CT<sub>max</sub>). For each colony, five ants
 across the range of caste sizes were kept in tubes but not put into heat
 blocks for the duration of the assays, to ensure that factors other than
 increasing temperature did not account for loss of mobility. None of the
 control ants in this study lost mobility, suggesting that the cause of
 loss of mobility was due to temperatures experienced within the heat
 blocks rather than handling and confinement.
   Body size After
 performing CT<sub>max</sub> assays, we collected all ant
 workers and measured body sizes from head capsules. Each ant was
 photographed at 15X to 40X magnification (depending on size of ant) using
 a scope-mounted digital camera. ImageJ software was used to measure the
 width of heads at eye-height from photographs. A micrometer accurate to
 0.1 mm was used to convert head width measurements from pixels to
 millimeters. Head width was used as a proxy for body size across all
 analyses. Both CT<sub>max</sub> and head width data are
 reported for each assayed ant in these uploaded files.
description: Most headers have been named intuitively, with abbreviations such
 as "Avg." meaning "average" and "SD"
 indicating "standard deviation".  All temperatures and
 CT<sub>max</sub> values are reported in degrees celcius. Below
 is a list of less intuitive headers within these files and their exact
 meaning.   <strong>Downloaded
 bioclim data based on nest coordinates:</strong>
 BC_MAT - mean annual temperature
 (BIO<sub>1</sub>) BC_MDRngT - Mean Diurnal
 Range (Mean of monthly (max temp - min temp))
 (BIO<sub>2</sub>) BC_TARng - temperature
 annual range (BIO<sub>7</sub>) BC_Aprecip -
 Annual precipitation (BIO<sub>12</sub>)
 BC_PrecipSnlty - Precipitation seasonality (Coefficient of
 Variation) (BIO<sub>15</sub>) BC_MaxTWM -
 Maximum temperature of warmest month
 (BIO<sub>5</sub>) BC_MinTCM - Minimum
 temperature of coldest month (BIO<sub>6</sub>)
   <strong>Ibutton-based data in master
 CTmax file:</strong> ibutton_Min - average
 minimum daily temperature for this site ibutton_Max -
 average maximum daily temperature for this site
 ibutton_Avg - average daily temperature for this site
   <strong>Warming tolerances (discussed
 only in supplementary material):</strong>
 Night_Warming_Tolerance - individual ant CTmax minus average
 daily minimum temperature (ibutton data)
 Day_Warming_Tolerance - individual ant CTmax minus average daily
 maximum temperature (ibutton data)
 Mean_Warming_Tolerance - individual ant CTmax minus average mean
 daily temperature (ibutton data)