DOI: 10.5061/dryad.vq83bk45g
title: Histological and gene expression analyses of the arm and finger macroglands of two <em>Hyloxalus</em> frogs (Dendrobatidae)

description: In some Neotropical poison frogs, males exhibit specialized mucous glands
 (SMGs) in the hand integument that express high levels of sodefrin
 precursor-like factors (SPFs), a well-known amphibian sex pheromone. Some
 species also show integumentary swellings at the distal upper arm, known
 as the black arm gland (BAG), of unclear function. Here, we used histology
 and RNA sequencing to analyze the arm and finger integument of Hyloxalus
 nexipus and H. azureiventris to examine glandular composition, gene
 expression patterns, and potential pheromone production. We
 confirmed the co-occurrence of two sexually dimorphic macroglands—swollen
 fingers and BAG—in H. nexipus, a rare trait in dendrobatids. 
 Both structures exhibited differential SPF expression, a well-known
 pheromone in salamanders. Notably, SPF expression in the BAG of H. nexipus
 indicates that SSGs, previously not linked to this function, can also
 produce proteinaceous pheromones. In H. azureiventris, no
 differential SPF expression was found in the arm, making its reproductive
 role uncertain.  Conversely, both species expressed SPF in their
 fingers, suggesting that H. azureiventris also possesses specialized
 glands despite lacking visible swelling. Overall, our findings
 reveal novel pheromone-producing gland types and emphasize the complexity
 of chemical communication during reproduction in dendrobatid frogs.
description: # Histological and gene expression analyses of the arm and finger
 macroglands of two *Hyloxalus* frogs (Dendrobatidae) #### Description of
 the data and file structure This repository is associated with Abondano
 Almeida, D., Anganoy-Criollo, M., Grant, T., Klimovych, S., & Schulte,
 LM (2025). Histological and gene expression analyses of the arm and finger
 macroglands of two Hyloxalus frogs (Dendrobatidae). Molecular Ecology,
 34(22), e70162.
 [https://doi.org/10.1111/mec.70162](https://doi.org/10.1111/mec.70162) We
 studied the integument of the arms and fingers of the Hyloxalus nexipus
 and H. azureiventris males, focusing on glandular composition, gene
 expression patterns, and potential pheromone production. For this, we used
 a whole-transcriptome sequencing (RNAseq) approach and histology analysis.
 For RNA sequencing, we followed the methods and pipeline of
 Abondano-Almeida et al. (2024). Our RNA-seq results provide evidence of
 the co-occurrence of two sexually dimorphic macroglands—swollen fingers
 and BAG—in H. nexipus, which is a rare trait in dendrobatids. Both
 structures differentially expressed SPF, suggesting complementary roles in
 courtship. In H. azureiventris, however, no differential SPF expression
 was found in the arm, leaving its reproductive role uncertain. Both
 species expressed SPF in their fingers. Thus, based on other dendrobatids
 where SPF is upregulated in male fingers with SMGs linked to pheromone
 production, we hypothesize that H. azureiventris may have specialized
 glands despite lacking visible swelling. Our findings reveal a novel
 pheromone-producing gland and underscore the complexity of chemical
 communication in dendrobatid reproduction.  This repository contains the
 assembliesthath are necessary for performing annotation and the
 differential expression (DE) analyses. In turn, the DE analysis was the
 input for the volcano plots, graphs that show the gene expression in the
 two conditions (glandular tissue vs. non-glandular tissue). The DE
 analysis was conducted using the R package DESeq2 (version 4.2). The raw
 sequencing reads necessary for all gene expression analyses are available
 from the European Nucleotide Archive under project accession number
 PRJEB102072. #### Files: • Hyloxalus.azureiventris_arm-leg_assembly.tr •
 Hyloxalus.azureiventris_finger-toe_assembly.tr •
 Hyloxalus.nexipus_arm-leg_assembly.tr •
 Hyloxalus.nexipus_fingers-toe_assembly.tr Description: The assemblies are
 presented in fasta files (.tr). Those files contain the final assembly per
 species from all males per tissue type (glandular tissue or non-glandular
 tissue). For each individual, we used kmer sizes of 25, 53, and 75 in
 SPAdes v.3.12 (Bankevich et al. 2012), incorporating both sets of reads
 (arm and leg skin; fingers and toes). With four individuals sequenced per
 species, this resulted in a total of 12 assemblies for each tissue type
 per species. For H. nexipus, only 11 assemblies were produced for finger
 and toe samples due to computational limitations preventing the use of
 kmer 25. TABLE_S11.xlsx Description: The list of annotated genes and
 differential expression analysis from both species, H. nexipus and H.
 azureiventris, per tissue type (arm-leg or finger-toes) is presented in an
 Excel table. For each species and tissue type (in different tabs in the
 Excel file), we annotated the final merged (evigene) assembly (using
 Diamond v.0.9.22.123; Buchfink et al., 2015), using Blastx mode to search
 assembly transcripts against the Identical Protein Groups database from
 NCBI (NCBI, 2022), which was filtered to include only vertebrate
 sequences. Diamond was run with max-target-seqs 1 and max-hsps 1 in order
 to retain a single, best hit for each transcript. The differential
 expression (DE) analysis was performed with the R package “DESeq2”
 (version 4.2). The Benjamini-Hochberg correction was applied to p-values
 to account for multiple hypothesis testing (Love et al., 2014). Given that
 we employed a paired-sample design (gland and control tissues taken from
 each individual), the final model included tissue type as a fixed effect
 and individual as a random effect to account for both treatment and
 individual variation. The dataset has a header, and this file can be
 opened with any text editing software. 3.1 Tabs: H.azureiventris.arm-leg
 H.azureiventris.finger-toe H.nexipus.arm-leg H.nexipus.finger-toe 3.2
 Variables-Header • id_transcript (Column A): Refers to the NCBI code for a
 coding protein (annotation) given to the transcript. Thus, this value is
 not unique and can be present several times in the column\ • transcript
 (Column B): a unique value for every transcript in the final assembly •
 annotation (Column C): NCBI code and name for the protein coding gene •
 baseMean (Column D): metrics from the DE analysis • log2FoldChange (Column
 E): metrics from the DE analysis. Quantifies the magnitude and direction
 of gene expression change between experimental conditions • lfcSE (Column
 F): metrics from the DE analysis • stat (Column G): metrics from the DE
 analysis • pvalue (Column H): metrics from the DE analysis • padj (Column
 I): metrics from the DE analysis. The adjusted p-value, is the crucial
 output for identifying significant differentially expressed genes after
 correcting for the massive number of tests performed (multiple testing
 problem) using the Benjamini-Hochberg (BH) method to control the False
 Discovery Rate (FDR) • TPM_1arm (Columns J-Q): Transcript per Million
 (TPM) from the individual one (1) from the arm. In each consecutive column
 (e.g., column K= TPM_2arm), the TPM is followed by the individual number
 and the tissue type. REFERENCES Abondano Almeida, D., Twomey, E.,
 Vargas-Salinas, F., Meyer, C. & Schulte, L.M. (2024) Sexy fingers:
 Pheromones in the glands of male dendrobatid frogs. Molecular Ecology, 33:
 e17476.
 [https://doi.org/10.1111/mec.17476](https://doi.org/10.1111/mec.17476)