# Genetic Diversity of Sodefrin-Variant Pheromones and Pheromone Responsiveness in Subspecies of the Japanese Sword-Tail Newt Cynops ensicauda

## TL;DR

This study explores the genetic diversity of pheromones in two subspecies of the Japanese sword-tail newt and how these pheromones influence mating behavior.

## Contribution

The study identifies a novel pheromone, caudarin, and reveals genetic and behavioral differences in pheromone use between newt subspecies.

## Key findings

- The [Gln8]sodefrin precursor mRNA is widely expressed in the abdominal glands of both C. e. ensicauda and C. e. popei.
- Caudarin attracts conspecific females, and silefrin is additionally expressed in C. e. popei males.
- Imorin attracts C. e. ensicauda males but not C. e. popei males, indicating subspecies-specific pheromone responsiveness.

## Abstract

Cynops newts inhabiting the Japanese archipelago have been classified into two species, namely, Cynops pyrrhogaster and C. ensicauda, with the latter being divided into two subspecies, namely, C. ensicauda ensicauda and C. e. popei. Experiments were conducted to determine whether mRNA expression of a sodefrin-like female attractant pheromone precursor is differently expressed in the abdominal glands of these subspecies. The results indicate that the precursor mRNA encoding [Gln8]sodefrin is widely detected in the abdominal glands of both subspecies. Preference tests demonstrated that [Gln8]sodefrin attracts conspecific females. This novel pheromone was named caudarin. Modification of the pheromone molecules was discussed in consideration of the geographical and chronological location of the newts’ habitat.

Experiments were conducted to examine whether the abdominal glands of Cynops ensicauda subspecies (C. e. ensicauda and C. e. popei) express different mRNAs for sodefrin-like female-attracting pheromone precursors. A precursor mRNA encoding a novel sex pheromone candidate, [Gln8]sodefrin, was widely detected in the abdominal glands of both of C. ensicauda subspecies. Female partners exhibited a considerable preference for synthetic [Gln8]sodefrin. Thus, [Gln8]sodefrin was designated caudarin. In C. e. ensicauda, the abdominal glands of males expressed sodefrin precursor mRNA as well, and sodefrin was also effective in attracting female partners. In addition to caudarin precursor mRNA, the abdominal glands of C. e. popei males expressed silefrin ([Leu3, Gln8]sodefrin) precursor mRNA, as reported previously. Moreover, imorin, a male-attracting pheromone previously identified in C. pyrrhogaster females, was effective in attracting C. e. ensicauda males but not males of C. e. popei. Molecular phylogenetic analysis indicated that trans-specific possession of sodefrin genes was likely because of gene occurrence preceding speciation of C. ensicauda and C. pyrrhogaster, and that the additional occurrence of silefrin in C. e. popei possibly resulted from duplication of the caudarin gene. Differences in pheromone components among Cynops species and subspecies reflect the speciation route estimated by previous molecular genetic analyses.