Historical DNA Places an Apparently Extinct Gladiator Frog in the Phylogeny of the Boana pulchella Group (Anura: Hylidae)

Abstract

Boana cymbalum has not been collected for the last six decades and is one of the two species of Brazilian frogs considered to be extinct. It is the only species of the B. pulchella group that has never been included in molecular phylogenies to date, because no fresh tissue is available. Using specialized historical DNA (hDNA) extraction protocols and high-throughput sequencing, we obtained mitochondrial and nuclear sequences from the 62-year-old holotype of B. cymbalum. Our results place B. cymbalum as the sister taxon of the clade formed by B. prasina, B. cordobae, and B. pulchella, supporting its inclusion in the B. prasina clade. The scars on the dorsum of adult males suggest male–male combat. Micro-computed tomography revealed the presence of a large post-articular process and a distal prepollex directed laterad to pass ventral to metacarpal II, two known synapomorphies of the B. pulchella group. On the basis of recordings from 1963, we provide an expanded description of the vocalization of B. cymbalum, consisting of a short, tonal note (Note A) and a longer, multipulsed note (Note B). Finally, we discuss the conservation status of B. cymbalum and the potential application of the newly generated hDNA sequences to screen water bodies near the type locality using environmental DNA in an effort to rediscover this species.