Coupling molecular evolution with macroevolutionary transitions in a pheromone system including the three amphibian orders

Chemical communication is a key mechanism for modulating social and reproductive behaviours. Among the most studied chemical signals are the sodefrin precursor-like factors (SPFs), a group of pheromones that enhances reproduction receptivity in salamanders and frogs. The origin of these pheromones is inferred to date back to the last common ancestor of amphibians. Despite being extensively investigated, the diversity and evolutionary history of the SPF gene family remain incomplete, as the third extant amphibian order, caecilians (order Gymnophiona), has been overlooked. Here, I revisited the molecular evolution of SPF pheromones in amphibians by including candidate SPF sequences from caecilian transcriptome data. To uncover gene duplication events, I inferred the orthology relationships of amphibian SPFs from 35 salamanders, five frogs, and eight caecilians. I performed phylogenetic comparative analyses to describe the evolution of pheromone delivery, linking transmission environment with SPF gene duplication events. SPFs comprised several orthologous groups, potentially revealing multiple ancestral gene duplications during the evolution of this gene family. Several candidate caecilian SPFs were clustered with two types of well-known SPFs (Alpha and Beta SPFs). Importantly, one of these types (Alpha SPFs) has previously been documented only in salamanders. The last common ancestor of amphibians could have delivered their pheromones in terrestrial environments, with derived aquatic courtships associated with SPF gene expansions in salamanders, but not in caecilians. These findings highlighted the importance of including the neglected order of caecilians to study amphibian biology and the evolution of pheromone systems.