DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples

Authors

  • PAULO B. CHAVES,

    1. Programa de Pós-Graduação em Zoologia, Pontifícia Universidade Católica do Rio Grande do Sul, Faculdade de Biociências, Av. Ipiranga, 6681, 90619-900 Porto Alegre, RS, Brazil
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  • VANESSA G. GRAEFF,

    1. Programa de Pós-Graduação em Zoologia, Pontifícia Universidade Católica do Rio Grande do Sul, Faculdade de Biociências, Av. Ipiranga, 6681, 90619-900 Porto Alegre, RS, Brazil
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  • MARÍLIA B. LION,

    1. Programa de Pós-Graduação em Biologia Animal, Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Ecologia, Asa Norte, 70910-900 Brasília, DF, Brazil
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  • LARISSA R. OLIVEIRA,

    1. Laboratório de Ecologia de Mamíferos, Universidade do Vale do Rio dos Sinos, Av. Unisinos, 950, 93022-000 São Leopoldo, RS, Brazil
    2. Grupo de Estudos de Mamíferos Aquáticos do Rio Grande do Sul, Av. Tramandaí, 976, 95625-000 Imbé, RS, Brazil
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  • EDUARDO EIZIRIK

    1. Laboratório de Biologia Genômica e Molecular, Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga, 6681, 90619-900 Porto Alegre, RS, Brazil
    2. Instituto Pró-Carnívoros, Atibaia, SP, Brazil
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Eduardo Eizirik, Fax: (5551) 3320 3568; E-mail: eduardo.eizirik@pucrs.br

Abstract

Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA and allowed an assessment of prey DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora.

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