title: Meeting the challenge of DNA barcoding Neotropical amphibians: polymerase chain reaction optimization and new COI primers.
abstract: Amphibians are one of the most threatened vertebrate classes, yet at the same time new species are being described every year, demonstrating that the number of existing species is grossly underestimated. In groups such as amphibians, with high extinction rates and poorly known species boundaries, DNA barcoding is a tool that can rapidly assess genetic diversity and estimate species richness for prioritizing conservation decisions. However, reliable recovery of the 5' region of the cytochrome c oxidase subunit 1 (COI) gene is critical for the ongoing effort to gather DNA barcodes for all amphibian species. Here, we provide new PCR conditions and tested new primers that increase the efficiency of barcode recovery in amphibians. We found that a low extension temperature for PCR cycles significantly improves the efficiency of amplification for all combinations of primers. Combining low PCR extension temperature and primers AnF1 + AnR1, we were able to recover COI sequences for 100% of the species analysed (N = 161), encompassing ~15% of the species known from Brazil (representing 77 genera and 23 families), which is an important improvement over previous studies. The preliminary assessment of species diversity suggested that number of species might be underestimated by about 25%. We conclude that DNA barcoding is an efficient, simple, and standardized protocol for identifying cryptic diversity in amphibians and advocate for its use in biodiversity inventories and across widespread populations within known species.
authors.affiliation: Departamento de Zoologia, Instituto de Biociências, UNESP - Univ Estadual Paulista, Campus Rio Claro, Av. 24 A, No. 1515, Bela Vista, CEP 13506-970, Rio Claro, SP, Brazil.
authors.forename: Mariana L
authors.identifier: http://orcid.org/0000-0002-7863-4965
authors.initials: ML
authors.lastname: Lyra
authors.affiliation: Departamento de Zoologia, Instituto de Biociências, UNESP - Univ Estadual Paulista, Campus Rio Claro, Av. 24 A, No. 1515, Bela Vista, CEP 13506-970, Rio Claro, SP, Brazil.
authors.forename: Célio F B
authors.initials: CFB
authors.lastname: Haddad
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética and Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Rua Cândido Rondon No. 400, CEP 13083-875, Campinas, São Paulo, Brazil.
authors.forename: Ana Maria L
authors.initials: AML
authors.lastname: de Azeredo-Espin
mesh_terms: D000663:Amphibians / Q000145:classification* / Q000235:genetics*; D000818:Animals; D001938:Brazil; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D017931:DNA Primers; D003576:Electron Transport Complex IV / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D003576:Electron Transport Complex IV
keywords: 16S; Anura; COI; DNA barcode; low PCR extension temperature; species diversity