DOI: 10.5061/dryad.w9ghx3fvd
title: How new communication behaviors evolve: Androgens as modifiers of neuromotor structure and function in foot-flagging frogs

description: How diverse animal communication signals have arisen is a question that
 has fascinated many. Xenopus frogs have been a model system used for three
 decades to reveal insights into the neuroendocrine mechanisms and
 evolution of vocal diversity. Due to the ease of studying central nervous
 system control of the laryngeal muscles in vitro, Xenopus has helped us
 understand how variation in communication signals between sexes and
 between species is produced at the molecular, cellular, and systems
 levels. Yet, it is becoming easier to make similar advances in non-model
 organisms. Here, we summarize our research on a group of frog species that
 have evolved a novel hind limb signal known as ‘foot flagging.’ We have
 shown that the evolution of foot flagging in multiple species is
 accompanied by the evolution of higher androgen hormone sensitivity in the
 leg muscles and an increased density of spinal interneurons in the
 neuromotor system that controls the hind limb. Comparing this work to
 prior work in Xenopus, we highlight which patterns of hormone sensitivity
 and neural circuit properties are shared between Xenopus and foot-flagging
 frogs and which appear to be species-specific. Overall, we aim to
 illustrate the power of drawing inspiration from experiments in model
 organisms, in which the mechanistic details have been worked out, and then
 apply these ideas to a non-traditional model species to reveal new
 details, further complexities, and fresh hypotheses.
description: Data on androgen receptor (AR) expression was generated using
 radioactive in situ hybridization. See manuscript supplemental files for
 complete methods. Data were collected from photomicrographs. Tissue slides
 were developed in photographic emulsion and silver grains were developed
 over cells that contained androgen receptor mRNA. AR expression data were
 collected from photomicrographs taken on a light microscope. Tissue
 sections were counterstained with Nissl stain so we could also see the
 cells. We quantified the number and area of developed silver grains in
 each image, then subtracted the number of background silver grains outside
 for the target area, and divided by the total number of identified neurons
 in the image/field of view. Thus, AR expression data can be expressed as
 the total area covered by AR grains, percent area covered by AR grains,
 number of grains above the background, and number of grains above the
 background per cell. Additionally, we can average the values for each frog
 and compute a ratio of AR grains for the lumbar vs. brachial spinal cord
 segments. Data on neuron cell numbers (motoneurons, interneurons) was
 generated using ImageJ. A trained observer manually classified and counted
 all cells in the photomicrographs.
description: # How new communication behaviors evolve: Androgens as modifiers of
 neuromotor structure and function in foot-flagging frogs --- ##
 Description of the data and file structure ### Filename: ARData Each row
 in the data file represents one observation. There were multiple
 measurements of AR and cells from a single frog (grouped by SubjectID).
 Columns represent each of the independent variables (species, spinal cord
 segment) and dependent variables (AR grains above background, cell
 counts).  Column names: * "MotorNuc" = spinal cord segment that
 data were taken from, L= lumbar, B=brachial * "Sample" = denotes
 how many image samples were taken from each subject, with 1=most anterior
 location in the spinal cord * "MNCount" = number of motoneurons
 counted in each photomicrograph; "INCount" = number of
 interneurons counted in each photomicrograph * "TotalCells" =
 MNCount+INCount * "TotAreaGrains" = area covered by silver
 grains in square pixels * "PercAreaGrains" = percent area of the
 image that is covered by silver grains, as measured in pixels *
 "GrainsAbBck" = the number of grains above the background that
 are counted in each photomicrograph, calculated by total no. grains - no.
 grains counted in a photomicrograph taken of an area of the slide with no
 tissue (background) * "GrAbBackPerCell" =
 GrainsAbBck/TotalCells. Data are from three frog species: *Staurois
 parvus* (=Sp), *Rana (Lithobates) pipiens* (=Rp), and *Xenopus laevis*
 (=XL). ### Filename: ARRatio Each row in the data file represents a ratio
 computed from the average of all observations for one individual frog of
 each species. To compute the ratio, we averaged measurement values across
 all lumbar spinal cord samples from an individual, then divided it by the
 average value in that individual's brachial spinal cord samples.
 Ratio values around 1 reflect equal proportioning between the lumbar and
 brachial spinal cord segments in an individual, whereas ratio values &gt;1
 mean greater proportioning to the lumbar spinal cord. Column Names: *
 "Species" = species of frog, either *Staurois parvus* (=Sp),
 *Rana (Lithobates) pipiens* (=Rp), and *Xenopus laevis* (=Xl) *
 "SubjectID" = letter/number code representing individual frogs *
 "TotCellsRatio"= average of total no. cells counted in
 photomicrographs of the lumbar/brachial spinal cord * "MNRatio"
 =  average of no. motoneurons counted in photomicrographs of the
 lumbar/brachial spinal cord * "INRatio" = average of no.
 interneurons counted in photomicrographs of the lumbar/brachial spinal
 cord * "ARRatio"=average of AR expression (measured as no. of
 silver grains above the background per cell) in photomicrographs of the
 lumbar/brachial spinal cord. ## Sharing/Access information No other data
 sources. ## Code/Software The code was written in R Studio Version
 1.2.5001.