DOI: 10.5061/dryad.83bk3j9nr
title: Data from: Volatile fatty acid and aldehyde abundances evolve with behavior and habitat temperature in Sceloporus lizards

description: Animal signals evolve by striking a balance between the need to convey
 information through particular habitats and the limitations of what types
 of signals can most easily be produced and perceived. Here, we present new
 results from field measures of undisturbed behavior and biochemical
 analyses of scent marks from 12 species of Sceloporus lizards to explore
 whether evolutionary changes in chemical composition are better predicted
 by measures of species behavior , particularly those associated with
 visual displays, chemoreception, and locomotion, or by measures of habitat
 climate (precipitation and temperature). We found that more active lizard
 species used fewer compounds in their volatile scent marks, perhaps
 conveying less specific information about individual, sex and species
 identity. Scent marks from more active lizard species also had higher
 proportions of saturated fatty acids, and the evolution of these compounds
 has been tracking the phylogeny closely as we would expect for a metabolic
 by-product. In contrast, the proportions of unsaturated fatty acids were
 better explained by evolutionary shifts in habitat temperature (not
 precipitation), with species in warmer climates using almost no volatile
 unsaturated fatty acids. The proportion of aldehydes was explained by both
 behavior and environment, decreasing with behavioral activity and
 increasing with habitat temperature. Our results highlight the
 evolutionary flexibility of complex chemical signals, with different
 chemical compounds responding to different elements of the selective
 landscape over evolutionary time.
description: <em>Collection of chemical and behavioral
 data</em> We collected femoral pore secretions
 and recorded behavior of adult males in the <em>Sceloporus
 </em>lizard genus between May 2012 and August 2016 at sites and
 using methods described in Campos et al., 2020 as part of a larger study
 of the evolution of multimodal signals in <i>Sceloporus</i>
 lizards (Hews and Martins, 2013; Martins et al., 2015; Pruett et al.,
 2016; Romero-Diaz et al., 2019). For each species, we collected behavioral
 and chemical data from the same population, collecting data at comparable
 points in species breeding seasons, such that the intensity of male
 territorial aggression was similar. Since reproductive timing can vary
 annually with environmental factors (Smith et al., 1995), we also
 confirmed mating activity through observed courtship and aggressive
 territorial displays by males. We first filmed a lizard and subsequently
 caught the lizard to collect its secretions, when
 possible. This data set includes chemical and behavioral data
 from <em>Sceloporus virgatus, Sceloporus jarrovii,
 Sceloporus megalepidurus, Sceloporus grammicus, Sceloporus undulatus,
 Sceloporus parvus, Sceloporus variabilis, Sceloporus cozumelae, Sceloporus
 siniferus, Sceloporus merriami, Sceloporus graciosus </em>and
 <em>Sceloporus occidentalis. </em>
 We recorded the undisturbed behavior of 20-30 adult
 males per species during their respective breeding season, approaching
 individuals to an average distance of 4-5 m, and filming for up to 10 min
 during their daily activity period. We scored videos for chemical and
 visual displays and locomotion, then calculated species’ averages for
 rates of behavior. <b>Chemical behavior</b>, involved in
 olfaction and vomerolfaction, included licks (tongue touch to substrate),
 tongue flicks (tongue in air), lip smacks (rapid opening and closing of
 the mouth), and chin wipes (jaw rub across substrate). For contrast, we
 also counted the number of <b>Headbob Displays</b>,
 stereotyped and species-typical series of up-and-down movements of the
 head and torso used in social interactions. Many forms of chemical
 behavior occur as lizards move around their environments, so we also
 scored general activity levels in two ways. First, we counted the number
 of short <b>Locomotion Bouts</b>, or episodes in which the
 subject moved distances &lt;10 cm, which were often associated with
 chemical sensing or visual display behavior in this study. Second, we
 calculated the number of all behavioral acts as a measure of total
 <b>Activity</b>, summing the total number of<i> short
 distance locomotory movements (&lt; 10 cm)</i>, <i>long
 distance movements (&gt; 10 cm),</i> <i>chemical behavior
 (tongue flicks plus jaw rubs, counted individually),</i>
 <i>visual display behavior</i> <i>(headbob displays plus
 shudder displays, counted as individual bouts),</i>
 <i>adjustments</i> <i>(small motions of the head or
 torso that do not move the lizard from one place to another),</i>
 <i>attacks </i>and <i>tail
 wags</i>. For each behavioral measure, we estimated the
 count of behavior per hour and used this frequency in statistical
 analyses. <i>Chemical analysis of
 secretions</i> We extracted secretions from
 lizard pores using alcohol-cleaned forceps and wearing nitrile gloves,
 then stored secretions in 2 mL glass vials with Teflon®-lined screw caps
 at -20 ºC. At Indiana University’s Institute for Pheromone Research, we
 used stir bar (Gerstel Twister® PDMS-coated magnetic stir bars) sorptive
 extraction to chemically analyze secretions (Pruett et al., 2016; Soini et
 al., 2005). For <i>S.</i> <i>graciosus,</i>
 <i>S.</i> <i>variabilis, S.</i>
 <i>occidentalis</i>, and <i>S. grammicus</i>, we
 analyzed samples from six individuals per species. Samples weighed 1.1–2.2
 mg, 1.8–2.4 mg, 1.5–1.8 mg, and 2–2.7 mg for each species, respectively.
 For other species, we pooled samples from multiple individuals to obtain
 sufficient quantities for analysis. We combined secretions from 17
 <i>S. virgatus</i>, 7 <i>S. jarrovii</i>, and 13
 <i>S. megalepidurus</i> males to create six pooled samples per
 species weighing 1.6–2.4 mg, 1.8–2.5 mg, and 1.8–2.4 mg, respectively. We
 pooled secretions from 14<i> S. undulatus</i> individuals into
 three samples, weighing 0.7–2.9 mg. For <i>S. cozumelae</i>,
 <i>S.</i> <i>parvus</i>, <i>S.</i>
 <i>siniferus</i>, and <i>S. merriami</i> we used
 samples and measures previously described in Pruett et al. (2016), which
 are two samples per species, each pooling secretions from 7–10 individuals
 and weighing 2 mg. We also added secretions from 13 <i>S.
 merriami</i>, pooled into four samples (2–4 males per sample, each
 weighing 1.5–2.0 mg), for a total of six pooled samples for this
 species. We chemically analyzed samples using
 gas-chromatography/mass-spectrometry. We also processed reagent blanks
 [<i>2 mL OmniSolv™ water</i> (EMD Millipore Corporation,
 Billerica, MA) <i>with 50 mg ammonium sulfate</i>
 (Sigma-Aldrich, St.Louis, MO)] as a control. We added an internal standard
 (IS) of <i>8 ng of synthetic 7-tridecanone</i> (Sigma-Aldrich,
 Saint Louis, MO) <i>dissolved in 5 μL methanol</i> (Baker
 Analyzed, Mallinckrodt Baker Inc., Phillipsburg, NJ) to every sample. We
 analyzed volatile-embedded stir bars in a Thermal Desorption Autosampler
 with a Cooled Injection System (TDSA-CIS 4 from Gerstel GmbH) connected to
 an Agilent 6890 N gas chromatograph – 5973iMSD mass spectrometer (Agilent
 Technologies, Inc., Wilmington, DE). Other equipment specifications,
 including oven program and temperature protocol details, are provided in
 Pruett et al. (2016). We identified volatile compounds
 by comparing mass spectra and retention times against references,
 reference standard compounds when available, and the National Institute of
 Standards and Technology (NIST) database. We used post-run selected ion
 currents (SIC) and measured the peak area of each compound. To estimate
 the relative abundance of a compound, we divided the compound peak area by
 the IS peak area and sample mass <i>[compound peak area / IS peak
 area / sample mass]</i>. We calculated species’ averages for
 <b>Chemical Richness</b>, or the total number of different
 volatiles, as a proxy for signal complexity (Hebets and Papaj,
 2005). <i>Climate measures</i>
 We used climate data deposited in the Dryad Digital Repository to
 support Lawing et al. (2016). Lawing et al. (2016) estimated mean climate
 profiles for each <i>Sceloporus</i> species by averaging
 bioclimate data (from WorldClim database) for each point at which that
 species has been reported (in the Global Biodiversity Information
 Facility). They found that BIO4 (temperature seasonality = standard
 deviation of temperature*100), BIO6 (lowest minimum temperature of the
 coldest month), BIO12 (mean annual precipitation), and BIO15
 (precipitation seasonality = coefficient of variation) were especially
 important in describing changes in suitable <i>Sceloporus</i>
 habitat over evolutionary time. To these measures, we added BIO1 (mean
 annual temperature) in our analysis as a point of comparison to other
 studies. As the best available estimate of the climate experienced by the
 species as a whole, we used mean climate measures averaged across all
 recorded locations for each species. Although these measures may not
 accurately reflect the specific climates experienced by the lizards we
 sampled in the field, they are more appropriate for our phylogenetic
 comparative analyses of long-term evolutionary history. Furthermore,
 Parker and Mason (2009) demonstrated in snakes that annual periods of low
 temperature dormancy (as occurs in many seasonal breeders) significantly
 boosts the synthesis and concentration of sex pheromones, suggesting that
 habitat temperatures during periods of behavioral inactivity can still be
 important in shaping signal composition.
description: <em>Species</em> These data are
 from 12 lizard species in the Sceloporus genus,
 including <em>Sceloporus virgatus, Sceloporus jarrovii,
 Sceloporus megalepidurus, Sceloporus grammicus, Sceloporus undulatus,
 Sceloporus parvus, Sceloporus variabilis, Sceloporus cozumelae, Sceloporus
 siniferus, Sceloporus merriami, Sceloporus graciosus </em>and
 <em>Sceloporus occidentalis. </em>The genus name has been
 ommitted from the "Species" column in our data
 set. <em>Compound
 abundances</em> The relative abundances reported
 for individual compounds in this data set (c1-c47) were calculated
 as <i>compound peak area / IS peak area / sample mass. </i>In
 order to calculate the actual abundance of a compound (in ng) in lizard
 femoral gland secretions (in mg), the numbers we report must be multiplied
 by 8 ng (i.e., the abundance of Internal Standard added to each sample) to
 give <em>compound ng / secretion mg.</em>
 <i>Sample masses</i> We report the
 mass of each sample to the nearest tenth of a mg. Some samples contained
 the femoral pore secretions of one to several adult males in order to
 obtain sufficient quanitites for GC-MS detection. 
 <em>Total Abundances</em> We
 summed the abundances of compounds 1-47 for each sample and report
 this total abundance in the "abundance" column. 
 <em>Richness</em> The
 "Richness" column is the total number of compounds detected in a
 given sample of those compounds measured in this study.
 <em>Bioclimate variables</em>
 Using bioclimate data reported in Lawing et al. (2016), we
 calculated and report the average for each variable across the entire
 geographic distribution of each species used in this study. 
 <em>Behavior</em> Behavioral
 activity measures are reported as frequencies, or the count of behavior
 per hour. These measures were calculated from baseline (undisturbed)
 behavior of adult males, and includes frequencies of headbob behavior
 (BaseHB), chemical display behavior (BaseChem), locomotor behavior
 (BaseMv10), and behavioral activity (BaseAct).