Putative pheromones in Hyloxalus macroglands

Chemical communication during courtship is well documented in salamanders and newts, but its role in frogs is less understood. 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), an amphibian pheromone. Some species also show integumentary swellings at the distal upper arm—known as the black arm gland (BAG)—of unclear function. 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, 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 differentially expressed SPF, suggesting complementary roles in courtship. We also provide the first histological characterization of the BAG in H. nexipus and the homologous region in H. azureiventris, revealing that both are composed of specialized serous glands (SSGs). 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. Both species expressed SPF in their fingers, thus, we hypothesize that H. azureiventris may have specialized glands despite lacking visible swelling, based on other dendrobatids where SPF is upregulated in male fingers with SMGs linked to pheromone production. Our findings reveal novel pheromone-producing gland and emphasize the complexity of chemical communication in dendrobatid reproduction.