DOI: 10.5061/dryad.w6m905qm6
title: Inter- and intraspecific variation in juvenile metabolism and water loss among five biphasic amphibian species

description: Population persistence is informed by the ability of individuals to cope
 with local abiotic conditions, which is commonly mediated by physiological
 traits. Among biphasic amphibians, juveniles—which are infrequently
 studied but play a key role in amphibian population dynamics—are the first
 life stage to experience terrestrial conditions following the aquatic
 larval stage. To illuminate phenotypic variation that may allow juveniles
 to survive the physiological challenges presented by this transition, we
 examined respiratory surface area water loss (RSAWL) and standard
 metabolic rates (SMR) among juveniles reared under common larval
 conditions for five salamander species (Ambystoma annulatum, A. maculatum,
 A. opacum, A. talpoideum, and A. texanum) collected across ~200 km of
 latitude in Missouri, USA. We found that SMR described 34% of variation in
 RSAWL, suggesting that physiological water conservation may be limited by
 energetic regulation among these species, and vice versa. On average,
 species differed in juvenile SMR and residual values of RSAWL (corrected
 for body size/shape) by 0.04 mL CO2  and 0.16, respectively,
 possibly because of distinct species ecologies. For example, A. annulatum
 had higher SMR and RSAWL compared to broadly distributed study species,
 potentially associated with a relatively narrow range of environmental
 conditions experienced across the small geographic distribution of A.
 annulatum. Latitude correlated negatively with temperature and
 precipitation, and positively with RSAWL, suggesting that variation in
 RSAWL may be adaptive to local conditions. We provide evidence that
 species differences likely have a genetic basis, reflecting selection
 favoring species divergence to effectively use distinct microhabitats.
description: Using a flow through Field Metabolic System (FMS; Sable Systems
 International, Inc.), we collected rates of water loss and
 CO<sub>2</sub> production from each juvenile salamander 24
 July–12 August 2017 (see Table 1 for population-specific sample sizes). A
 120 VAC/60 Hz pump pushed ambient air through a drying column to an
 eight-line external manifold and flow control bar (FB-8; Sable Systems
 International, Inc.). The FB-8 released the dry air at 60 mL/min to one of
 eight 5 cm <i>×</i> 16 cm (volume ~ 314 mL) capped cylindrical
 glass animal chambers (RC-2; Sable Systems International, Inc.) housed
 within an incubator set to 27°C. Each air stream flowed from one of the
 chambers, out of the incubator, and into an eight-channel multiplexer
 (RM-8; Sable Systems International, Inc.). The multiplexer directed the
 airstream from each chamber in sequence to the FMS, which recorded water
 vapor pressure (<i>WVP</i>;<i> </i>kPa) and
 percent CO<sub>2</sub> content of the air. The air stream was
 scrubbed of water vapor before CO<sub>2</sub> analysis. The
 multiplexer cycled through the chambers five times, with six-minute
 intervals of data collection per chamber. We recorded temperature inside
 the incubator each second using a stationary thermistor probe (SA2; Sable
 Systems International, Inc.). We selected juvenile
 salamanders for respirometry trials haphazardly across species, mesocosms,
 and date of metamorphosis until all <i>n </i>= 357 individuals
 had been measured. Due to the moss substrate, we could not assure the
 absence of food for all salamanders prior to trials, but no salamanders
 were fed within 48 hours of respirometry. We recorded snout-to-vent length
 (SVL), total length (TL), and the diameter of the midpoint between the
 forelimbs and hind limbs (DM) of each juvenile immediately prior to data
 collection using digital calipers (± 0.01 mm). Before the assigned trial,
 we soaked each juvenile in spring water for ≥20 minutes to ensure
 hydration across individuals. We then allowed each individual to walk
 across a paper towel to remove excess water and collected the mass of each
 salamander (± 0.0001 g). To limit movement within the animal chamber, we
 secured each salamander in a 3 cm <i>×</i> 11 cm 0.13-cm mesh
 plastic envelope closed with binder clips. We subsequently loaded a single
 juvenile into each animal chamber, with the first chamber remaining empty
 in all trials as a baseline, and up to seven juveniles being measured per
 trial. Following the trial period, we again collected the mass of each
 individual and allowed the salamanders to soak in spring water for ≥20
 minutes to facilitate rehydration. Each juvenile was subsequently replaced
 into its individual container. No salamanders perished as a result of
 these procedures. Data from animals that we observed to have defecated
 during the trial period (<i>n</i> = 4) were excluded from
 further analyses.  We used batch file processing in
 ExpeData PRO software v. 1.9.10 (Sable Systems International, Inc.) to
 transform <i>WVP </i>data to respiratory surface area water
 loss (<i>RSAWL)</i> for each juvenile by first calculating
 water vapor density (<i>ρ<sub>v</sub></i>; g
 <i> ∙ </i>m<sup>-3</sup>; Equation S1 in
 Supporting Information) and then evaporative water loss
 (<i>EWL</i>; mg <i>∙ </i>
 hr<sup>-1</sup>; Equation S2 in Supporting Information) as in
 Riddell and Sears (2015) (details available in Physiological Calculations
 subsection of Supporting Information).