Meeting the challenge of DNA barcoding Neotropical amphibians: polymerase chain reaction optimization and new COI primers

Author(s)

Lyra, Mariana L. ^[1] ; Haddad, Celio F. B. ^[1] ; de Azeredo-Espin, Ana Maria L. ^{[2, 3]}

Affiliation

^[1] Univ Estadual Paulista, UNESP, Dept Zool, Inst Biociencias, Campus Rio Claro, Av 24 A, 1515, BR-13506970 Rio Claro, SP - Brazil

^[2] Univ Estadual Campinas, Inst Biol, Ctr Biol Mol & Engn Genet, Rua Candido Rondon 400, BR-13083875 Campinas, SP - Brazil

^[3] Univ Estadual Campinas, Inst Biol, Dept Genet Evolucao & Bioagentes, Rua Candido Rondon 400, BR-13083875 Campinas, SP - Brazil

• Document type: Journal article
• Source: MOLECULAR ECOLOGY RESOURCES; v. 17, n. 5, p. 966-980, SEP 2017.
• Web of Science Citations: 31

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 50 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 similar to 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. (AU)

• FAPESP's process: 13/50741-7 - Diversity and conservation of Brazilian amphibians
• Grantee: Célio Fernando Baptista Haddad
• Support Opportunities: BIOTA-FAPESP Program - Thematic Grants
• FAPESP's process: 08/50928-1 - Speciation of frogs in high-altitude environments
• Grantee: Célio Fernando Baptista Haddad
• Support Opportunities: Research Projects - Thematic Grants