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.

Trimitomics: An efficient pipeline for mitochondrial assembly from transcriptomic reads in nonmodel species

“…Despite awareness that RNA-seq data contain high numbers of mt genome reads (Smith, 2013), the majority of available methods for mt genome assembly are customized for recovering these from genome sequencing projects (either whole genome sequencing or genome skimming). Nevertheless, the mining of mt genomes from RNA-seq data has been successfully performed in a handful of eukaryotes, including algae (Tian & Smith, 2016), reptiles (Lyra, Haddad, & Azeredo-Espin, 2017), sea urchins (Dilly, Gaitán-Espitia, & Hofmann, 2015) and bony fishes (Moreira, Furtado, & Parente, 2015), yet using disparate software approaches, including Bowtie2/ Trinity, Trinity assembly, MIRA version 4.0/MITObim version 1.8 (Lyra et al, 2017), and blasting against the mt genomes of closely related species (Dilly et al, 2015). In the case of incomplete mt genome recovery from transcriptomic data, gaps were filled by combining with the genomic data (Nan Song, An, & Yin, x., Cai, w., Li, H., 2016;Fabre, Jønsson, & Douzery, 2013).…”

More sex chromosomes than autosomes in the Amazonian frog Leptodactylus pentadactylus

“…We amplified and sequenced two mitochondrial (16S ribosomal RNA gene (16S), cytochrome oxidase c subunit I (COI)) and two nuclear (brain-derived neurotrophic factor gene (BDNF), cellular myelocytomatosis gene (C-myc)) gene fragments for all analyzed specimens. DNA fragments were amplified by standard PCR technique using the primers listed in Table S2 -Electronic supplementary material (Crawford 2003;Lyra et al 2016;Palumbi et al 1991;Van der Meijden et al 2007;Wiens et al 2005). PCR products were purified via enzymatic reaction and sequenced with the BigDye™ terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, CA, USA) in an automatic Sequencer 3730XL by Macrogen Inc., Seoul, South Korea.…”

A New Amazonian Species of Adenomera (Anura: Leptodactylidae) from the Brazilian State of Pará: A Tody-Tyrant Voice in a Frog

“…For newly generated sequences, genomic DNA was extracted from ethanol-preserved mus¬ cle tissue using standard ammonium acetate extraction protocol adapted from Maniatis et al (1982). Primers employed for amplification (COI gene) and PCR enzymatic reaction conditions follow those of Lyra et al (2017), and the resulting amplified fragments were sequenced by Macrogen Inc. (Seoul, South Korea). Chromatograms were checked manually, assembled, and edited using Geneious v. 7 (Biomatters Ltd.).…”