Fifty years after: A taxonomic revision of the amphibian species from the Ecuadorian biodiversity hotspot Abra de Zamora, with description of two new Pristimantis species

Remarks. Despite intensive searches carried out since 2016, we were not able to find this species. We managed to survey an area of almost 100 km2 around the type locality, but unfor- tunately, we were not able to find any G. psychrophila individuals. The results are even more worrisome as our intensive searches in the type locality failed to find any traces of marsupial frog presence (no tadpoles or calls), although if the ecosystem remained practically unchanged since the species was found in 1968 (Fig 2A). Two other Gastrotheca species were encountered, relatively close to the type locality: G. elicioi (on the western slope) and G. testudinea (on the eastern slope), but both well below G. psychrophila’s altitudinal range of 2700–2800 m. The identity of the encountered Gastrotheca species was confirmed through non-destructive DNA tissue sampling (using buccal swabs). In the light of these current findings, we are considering G. psychrophila probably extinct. Superfamily Brachycephaloidea Gu¨nther, 1858 Family Strabomantidae Hedges, Duellman, and Heinicke, 2008 Subfamily Pristimantinae Pyron and Wiens, 2011 Genus Lynchius Hedges, Duellman, and Heinicke, 2008 Phylogeny For Lynchius, PartitionFinder under AICc (for ML) identified five partition schemes as the best strategy (best model in parentheses): 12S (GTR+I+G), 16S (GTR+I+G), RAG-1 1st posi- tion (TRNEF+G), RAG-1 2nd position (K81UF+G) and RAG-1 3rd position (GTR+G). Under BIC (for BI) PartitionFinder identified three partitions: 12S and 16S (GTR+I+G), RAG-1 1st

position (K80+G) and RAG-1 2nd and 3rd positions (HKY+G). The phylogenetic trees con- structed by Bayesian inference and Maximum likelihood showed the same topology, with the only difference being the position of L. nebulanastes, which remained non-resolved in both trees (Fig 3). The phylogram of our analysis showed exactly the same topology with the one constructed by Sanchez-Nivicela et al. [28]. The L. flavomaculatus sequence (KU 218210) used in all published studies (e.g. [27, 28, 41, 42]), was obtained from a specimen collected in 1990, from 19.4 km south of Yangana, Loja province, which is located more than 60 km south from the type locality, Abra de Zamora. In June 2018 we encountered an adult female (MUTPL 342) in Parque Nacional Podocarpus (Cajanuma entrance), from about 13 km south of the type locality, and in the same type of eco- system as of the type locality (shrub pa´ramo). The uncorrected p-genetic distance for the gene 16S between the two samples is 3.1% (S3 Table). Based on the conservative 3% threshold of the genetic distances in the 16S gene for species delineation [43], together with observed morpho- logical differences between several similar species from the nearby areas (pers. obs.) we con- sider the specimen from Yangana as belonging to a different, currently undescribed species. Thus, it seems that the previously known L. flavomaculatus is in fact a complex of cryptic spe- cies, composed of at least four different species in Ecuador: one species from Yangana, L. flavo-

maculatus sensu stricto from the type locality (Abra de Zamora), one species from San Lucas and Saraguro (we are preparing its description) and one species nearby Plan de Milagro, Mor- ona Santiago province. In addition, very likely the specimen collected in Peru [25] is a differ- ent, currently undescribed species. Lynchius flavomaculatus (Parker, 1938) (Fig 4) Eleutherodactylus flavomaculatus Parker, 1938 Niceforonia flavomaculata: Lynch, 1969 Phrynopus flavomaculatus: Lynch, 1975 Eleutherodactylus flavomaculatus: Lehr, 2006 PLOS ONE Taxonomic revision of the amphibian species from Abra de Zamora PLOS ONE | https://doi.org/10.1371/journal.pone.0238306 September 10, 2020 9 / 59