Integrative taxonomy of osteocephalus planiceps (Anura : Hylidae) : new insights into its phenotypic and genetic variation and their implications for systematics

Abstract

The western Amazon Basin harbors the largest number of amphibian species by area in the world. Despite the increase in phylogenetic studies and descriptions of new species, there are still large gaps in knowledge of the described species. This creates taxonomic uncertainty and difficult progress of biodiversity knowledge and conservation. These gaps are frequently associated with species described in the 19th century based on one or a few specimens, often poorly preserved, and with ambiguity in material designation and/or type locality. This is the case of the treefrog Osteocephalus planiceps (Hylidae), briefly described from a single specimen in 1874 by Cope. What we currently known about O. planiceps derives from a 171 years old cursory characterization of its external morphology and additional indirect information from studies focusing on the description of new species, with no reports of topotypical specimens since the original description. Considering this context, we performed expeditions to three localities in Loreto, Peru: Estación Biológica José Álvarez Alonso (EBJAA), Centro de Investigación Jenaro Hererra – José Lopez Parodi (CIJH), and Comunidad Frontera. We studied genotypic and phenotypic variation, including external morphology, morphometry, osteology and DNA sequences of O. planiceps. We collected 50 specimens (19 males, 26 females and 5 juveniles) in addition, we studied 53 specimens of Osteocephalus from the collections of the Museu de Ciências e Tecnologia da PUCRS and Instituto Nacional de Pesquisas da Amazônia. We measured 16 morphometric variables from 49 individuals. We extracted DNA and sequenced two fragments of mitochondrial genes 16S (~590 bp) and COI (~610 bp) from 29 individuals from different locations. We studied the osteology of 9 specimens using microCT-scan and 3D models. Because O. planiceps and O. vilarsi are the most similar congeneric species, we included detailed comparisons of the two species for all sets of characters. Our results reveal: (i) previously unknown genetic variation and hierarchical structure within both O. planiceps and O. vilarsi; (ii) sister phylogenetic relationship between the aforementioned species; (iii) first detailed and complete osteological description for both species; (iv) previously unknown phenotypic variation for both species, including the discovery intraspecific polymorphism of bone color in O. planiceps, and overlap of all previously suggested diagnostic characters; (v) new data on the distribution and natural history of O. planiceps, including reidentification of populations wrongly assigned to O. planiceps and its first confirmed record for Brazil; (vi) the proposal of four new diagnostic osteological characters between O. planiceps and O. vilarsi; and (vii) new insights into the species diversity and distribution of the O. leprieurii species group derived from phylogenetic analysis of DNA sequences. In light of our results, we discuss the taxonomic status of O. vilarsi in relation with O. planiceps, the relevance of some phenotypic characters (i.e., bone coloration, frontoparietal ridges, and a newly discovered process of the coracoid) in Osteocephalus systematics, and the species diversity within O. leprieurii species group.