DOI: 10.5061/dryad.t1g1jwt8f
title: Species and sex specific chemical composition in a concealed gland from an internal gland-like tissue of an African frog family

description: Intraspecific chemical communication in frogs is understudied and the few
 published cases are limited to externally visible and male-specific
 breeding glands. Frogs of the family Odontobatrachidae, a West African
 endemic complex of five morphologically cryptic species, have large, fatty
 gland-like strands along their lower mandible. We investigated the general
 anatomy of this strand and analyzed its chemical composition. We found the
 strand to be present in males and females of all species. The strand
 varies in markedness, with well-developed strands usually found in
 reproductively active individuals. Although the strands are situated under
 the skin, they are connected to particular skin sections, the vocal sac in
 male frogs and a respective area in females. Gas-chromatography/mass
 spectrometry and multivariate analysis revealed, that the strands contain
 sex- and species-specific chemical profiles, which are consistent across
 geographically distant populations. The profiles varied between
 reproductive and non-reproductive individuals. Our results indicate that
 the gland-like strand in the Odontobatrachidae comprise a so far
 overlooked type of breeding related organ, potentially a gland, that most
 likely plays a role in the mating and/or breeding behavior of the five
 Odontobatrachus species. Our results highlight the relevance of multimodal
 signaling in anurans, and indicate that chemical signaling may not be
 restricted to sexually dimorphic, apparent skin glands.
description: We collected specimens of all
 five <em>Odontobatrachus</em> species, <em>O.
 arndti</em>, <em>O. fouta</em>, <em>O.
 natator</em>, <em>O. smithi</em> and <em>O.
 ziama</em>. We euthanized, killed and dissected the animals to
 obtain tissue samples for chemical analysis. Tissue samples where then
 extracted in dichloromethane and analyzed by gas chromatography/mass
 spectrometry (GC/MS). To search for sex and species-specific differences
 in gland composition, we sampled both sexes of each species from at least
 two locations per species. We also sampled <em>O.</em>
 <em>arndti</em> under wet and dry season conditions on the
 same river, to screen for seasonal effects. To check for the effect of
 geographical distance between populations, we sampled
 <em>O.</em> <em>arndti</em> males and
 <em>O.</em> <em>smithi </em>females over a large
 part of their distribution range. To investigate ontogenetic effects on
 strand composition, we sampled metamorphic and sub-adult <em>O.
 ziama</em> frogs and compared them to sexually mature animals.
 For histological and anatomical analyses, we
 used voucher specimens from the collection of the Museum für Naturkunde
 Berlin (ZMB). 
description: # Species and sex specific chemical composition in a concealed gland in an
 internal tissue of an African frog family - Electronic Supplement
 Contains 6 files:
 includingone csv file with original GCMS data (1), one csv file with
 geospatial data (2), one excel file with additional peak information for
 the GCMS data file (3), and two high-resolution histological crosssection
 scans of the mandibular strand in a male and female *Odontobatrachus
 arndti (4), as well as the annotated R Code. Scans and Code are stored in
 Zenodo as Supplementary and Software respectively. Description of the data
 and file structure 1. GC_data.csv (the original GCMS data): depicts
 metadata and GC count data for the 161 strand samples (ZFG_XXX) from 1178
 peaks and contains the following relevant columns: "ID"= sample
 identification, "species" = species identification
 "date"= day of collection, "season"= corresponding wet
 or dry season, "river"=name of river where sample was collected,
 "size"= bodysize (SVL) of speciemen in mm, "weight"=
 weight of speciemen in g, "sex" = sex of the collected frog
 specimen, "Detail" = either the microhabitat (slope, cascade,
 plain; see manuscript for details) or apparent developmental status of the
 gland (good, bad, and moderate) of collected specimen.
 "Peaks.1-1178" = relative abundance (area under the curve of
 respective peaks, normalized over the total ioncounts of entire sample)
 for the 161 individual samples (ZFG_XXX). 2. geodistance.csv (Geospatial
 data): depicts GPS coordinates for each of the 161 strand samples
 (ZFG_XXX) and contains the following columns: "ID"= sample
 identification, "latitude" = easting, "longitude" =
 northing 3. GC_RT_alligned.xlsx: depicts the retention times of the 1178
 peaks and contains the following columns: "mean rt" = mean
 retention time for peaks 1-1178 derived from, "sd" = the
 respective standard deviation for the before-mentioned values, "min
 rt" = minimal retention time found for the respective for peaks,
 "max rt" = maximal retention time found for the respective for
 peaks. "samples with peaks"= the number of samples that
 contained the respective peak. File also constitutes a presence absence
 matrix for each peak (1-1178) in each of the 161 samples (ZFG_XXX). In
 this matrix 1 refers to presence (irrespective of the intensity of the
 signal) empty cells refer to the absence. 4. High resolution histological
 sections are based on individual scans at 20 fold magnification on a Zeiss
 Axioscop stereomicrope, merged manually using Microsoft Photoshop and
 Powerpoint. ## Sharing/Access information Original strand extracts and
 unprocessed GCMS data stored at University of Würzburg, Department of
 Animal Ecology and Tropical Ecology. Histological material based on museum
 specimen from the collection of the Museum für Naturkunde Berlin (ZMB):
 Voucher numers ZMB 78360 and ZMB 78361. For further details on geolocation
 of specimen as well as used museum specimen, see manuscript. ##
 Code/Software Annotaded R- Code for statistical and graphical analysis:
 GCMS data were ordinated by an NMDS using Bray-Curtis dissimilarity
 indices, respective group homogeneity was tested with permutational
 analyses of multivariate dispersions (PERMDISP) and permutational
 multivariate analysis of variance (PERMANOVA) were computed for
 differences in group means. For spatial analysis, we additionally
 performed Mantel tests to control for correlation between the
 dissimilarity matrix and a distance matrix computed from the localities of
 the respective samples. To identify important decisive peaks in the
 chemical profiles, we trained random forests on a labelled dataset and
 used these models (one for sex and one for species) to predict the
 assignment of sex and species of an unlabelled dataset. All data handling
 and analysis was computed with R software 4.2.1, using the following
 packages: caret, dplyr, geodist, gplot2, randomForest, recipes, vegan