DOI: 10.5061/dryad.3xsj3txq5
title: Data from: Levels of sex steroids in plethodontid salamanders: A comparative study within the genus Aneides

description: Derived monomorphism is a condition in which males and females are
 phenotypically similar, but the similarity is derived.  Derived
 monomorphism typically evolves from sexual dimorphism or from a different
 monomorphic state.  We examined the hormonal basis of derived
 monomorphism in the salamander genus Aneides (Plethodontidae).
  We reject our hypothesis that circulating levels of androgens
 explain the derived traits, such as enlarged jaw musculature, in females
 (some would call them “male-like traits”).  There was no clear
 pattern of differences in androgen levels or degree of dimorphism in
 androgen levels, between the sexually dimorphic Aneides hardii and the
 other, derived monomorphic, species studied.  Concentrations of
 testosterone and dihydrotestosterone were higher in males than in females
 in all species examined. The degree of sexual dimorphism in androgen level
 was also consistent among the species studied. Levels of androgens in
 female plethodontids have not been previously reported.
description: <strong>Sample collection</strong>
 Individuals of <em>A. lugubris</em>, <em>A.
 ferreus</em>, <em>A. flavipunctatus</em>, <em>A.
 hardii</em>, and <em>P. elongatus</em> were hand
 collected in the field (see Table 1 for sample sizes and Appendix for
 localities and dates), sacrificed by decapitation within 3 minutes of
 capture, and blood samples collected in heparinized capillary tubes and
 stored on ice.  Within three hours of collection, blood samples were
 centrifuged and the plasma frozen.  Samples were kept at –80°C until
 radioimmunoassays (RIAs) were performed.  Animals were fixed in 10%
 neutral buffered formalin, rinsed in water, and stored in 70% ethanol. 
 Sex was determined via gonad inspection.  One testis was removed from
 males for histological analysis.  Authorization for this work included:
 Oregon Dept of Fish and Wildlife permits 437-93, 051-94, 038-95, 045-96;
 New Mexico Dept of Game and Fish permit 1702 (1993-96)<em>;
 </em>California Department of Fish and Game permits 4023 1993-95,
 4755 1995-1997.   Blood samples were collected across
 the animal’s active season: <em>A. lugubris</em>
 (October-May), <em>A. ferreus</em> (September – May), and
 <em>A. hardii</em> (May-August).  <em>A.
 flavipunctatus</em> samples were collected in March only.  For
 <em>P. elongatus</em>, samples were collected over several
 months (October, November, January, March), but sample sizes were too low
 to analyze seasonally.
 <strong>Radioimmunoassays</strong>
 Plasma Estradiol (E), testosterone (T), and dihydro-testosterone
 (DHT), were separated using column chromatography and measured by
 radioimmunoassay (RIA).  Extraction, Celite microcolumn chromatography,
 and radioimmunoassay techniques followed the protocol in Wingfield et al.
 (1982).  Basically, plasma samples were equilibrated overnight with
 800-1000 cpm of each steroid hormone to be measured to determine recovery
 efficiency.   Plasma was extracted with diethyl ether, dried under
 nitrogen gas, and resuspended in 10% ethyl acetate in iso-octane.  Samples
 were chromatographed on microcolumns of Celite:propylene/ethylene glycol. 
 The eluate solvents used were 10% ethyl acetate in iso-octane for DHT, 20%
 ethyl acetate in iso-octane for T, and 40% ethyl acetate in iso-octane for
 E.  Column aliquots were dried under nitrogen and resuspended in phosphate
 buffer.  Two aliquots were taken (1, 1:20 for DHT, and T, and duplicates
 for E).  Aliquots were incubated overnight at 4C with the tritiated
 steroid to be measured and its respective antibody (E antibody, 17-94 from
 Endocrine Sciences, Tarzana, CA; androgen antibody, T3003).<strong>
 </strong>Following the incubation period, the assay tubes received a
 5% charcoal: 0.5% dextran mixture to remove unbound steroid.  Tubes were
 vortexed, incubated for 15 min at 4°C, and centrifuged for 10 min at 3,000
 rpm.  The supernatant was collected from each sample, equilibrated
 overnight, and counted in the Beckman scintillation counter the following
 day (see Navara et al. 2006, Wingfield et al. 1982).
 Sensitivities were 15 pg/ml for T, DHT, and E. Inter-assay
 coefficients of variation were E: 13% (n=11), T: 10% (n=11), DHT: 8%
 (n=10).  Values for intra-assay variation were E: 8% (n=138), T: 7%
 (n=150), DHT: 8% (n=131).   
 <strong>Histology</strong> Using
 standard histological procedures, testes were embedded in paraffin,
 sectioned at 12 um, mounted on slides, and stained with hematoxylin and
 eosin (Presnell and Schreibman 1997). 
 <strong>Statistical Analysis</strong>
 To examine our hypothesis, we modeled sex steroid concentration
 as a function of sex and species for <em>A. hardii, A. ferreus, A.
 flavipunctatus, A. lugubris</em>, and <em>P.
 elongatus</em> (i.e. five-species model). Because little has been
 published on sex steroid variation in these species, we first modeled E,
 T, and DHT concentration as a function of month and sex, to determine
 which months to include in the five-species model. We first fit
 interaction models of month by sex for adult <em>A. hardii, A.
 ferreus</em> and <em>A. lugubris</em>. If interactive
 effects were not significant, we dropped the interaction term, refit and
 interpreted main effects models of month and sex. Our <em>P.
 elongatus</em> and <em>A. flavipunctatus</em> sample
 sizes were too small to fit sex by month models.
 Following our interpretation of the sex by month models (see
 Results, Seasonal Variation), our five-species model included June, July,
 and August observations for<em> A. hardii, </em>January,
 February, March, and November observations for<em> A. lugubris,
 </em>and January, February, March, May, August, October, November,
 and December observations for <em>A. ferreus, </em>March
 observations for<em> A. flavipunctatus</em>, and January,
 March, and November observations for <em>P. elongatus</em>.
 Our hypothesis predicted we would observe higher levels of T and DHT in
 males than in females of the sexually dimorphic<em> A. hardii,
 </em>but higher levels of T and DHT in female <em>A. ferreus,
 A. flavipunctatus, </em>and<em> A. lugubris,
 </em>compared to female<em> A. hardii, </em>due to
 derived monomorphism. We therefore expected to observe a large sex by
 species interaction in the five-species model, with pairwise comparisons
 of sex within species revealing different levels of T and DHT in
 <em>A. hardii, </em>and less of a difference in<em> A.
 ferreus, A. flavipunctatus,</em> and <em>A.
 lugubris</em>. Because little is known about sex
 steroid variation in these species, we also report results of modeling E,
 T, and DHT as a function of sex and species in immature <em>A.
 hardii</em> and <em>A. ferreus</em>. Sample sizes for
 <em>A. lugubris</em> immatures were too low to be included in
 the modeling analyses. For all models, we included data
 with and without potential outliers, as suggested by Pollet and van der
 Meij (2017). We identified potential outlier observations as those more
 than 1.5 interquartile range units away from the median, within sex by
 month groups. Modeling results were equivalent with and without outliers,
 so we retained all observations for modeling. We log-transformed our
 hormone observations to stabilize variance and analyzed residuals to
 evaluate model fit.  We made post-hoc comparisons using the Tukey HSD
 test. We also provide summary statistics for individual T/DHT ratios by
 sex and species, and mean male/female T and DHT ratios by species. All
 analysis was done using R (R Core Team 2023).
description: **Hormone levels in Aneides and Plethodon** R code reads the file
 "Aneides_Plethodon_hormones_eggs.csv"  which is the complete
 data set. The complete data set includes some egg data (see below for
 abbreviations) and other miscellaneous information. Eleven "
 p_val.csv" files are included for printing p-value brackets on
 figures. Just the hormone levels are in
 “Steroid_levels_salamander_Aneides_Plethodon.xlsx” organized by species in
 5 different
 sheets/tabs: hardii, ferreus, flavipunctatus, lugubris, P.elongatus All
 hormone measurements are in ng/ml For blank data cells, treat them as
 blank rather than zero. abbreviations for
 "Aneides_Plethodon_hormones_eggs.csv" species: HAR = Aneides
 hardii, FER = Aneides ferreus, FLA = Aneides flavipunctatus, LUG = Aneides
 lugubris, ELO = Plethodon elongatus ID = specimen identification number on
 tag on animal itself. These are NLS (Nancy Lynn Staub) numbers and the
 animals will be accessioned into the Museum of Vertebrate Zoology, UC
 Berkeley’s collection in 2025. Collection date refers to month and year
 collected: e.g., 395 = March 1995 Month refers to month collected Fix date
 refers to month and year the animal was bled and fixed. If blank, is equal
 to collection date. Bngml = cortisol E = estradiol T = testosterone DHT =
 dihydotestosterone Sex m = male, f = female Maturity. m = mature, im =
 immature eggsunits.3 = microscope micrometer measurement for 3 ova,
 diameter in ocular micrometer units eggsmm.3 = measurement total for 3
 ova, diameter in mm avg.1egg = average of egg measurements (= eggsmm.3/3)