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<p>Neotropical biomes are characterized by both high biodiversity and significant threats. To address the urgent need for biodiversity assessment in these threatened and largely understudied ecosystems, like the ones in Brazil, modern techniques like metabarcoding have been employed to generate detailed taxonomic inventories. However, selecting appropriate genetic markers remains challenging, as different markers and primer pairs may introduce biases by preferentially amplifying certain taxonomic groups, depending on their nucleotide sequences and the reference database used. This study advances biodiversity research by developing a customized reference database and optimizing primers for metabarcoding Brazilian mammals. Our database, covering 72% of native species, including endangered ones, enhances taxonomic identification accuracy across all Brazilian biomes. Both <i>in-silico</i> and <i>in-vitro</i> tests were conducted to evaluate and optimize the most suitable genetic markers and primer pairs, comparing their taxonomic coverage and resolution. Customized modifications made to COI primers reduced mismatches against Brazilian mammal sequences, significantly enhancing taxonomic coverage and outperforming commonly used primers in literature. We demonstrate that combining COI with RNA12S and RNA16S provides important and complementary information, improving the robustness of taxonomic assignments and addressing challenges posed by the Neotropical mammalian diversity. This multi-marker approach offers a reliable strategy for enhancing metabarcoding identification in the Neotropical region, supporting more effective conservation efforts.</p>

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Metabarcoding markers and a reference database for neotropical mammals

  • 期刊论文

  • 发表日期：

    2025年9月27日

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  • Barbara R. N. Chaves,
  • Jose Eustaquio Santos-Junior,
  • Fabricio R. Santos

摘要

Neotropical biomes are characterized by both high biodiversity and significant threats. To address the urgent need for biodiversity assessment in these threatened and largely understudied ecosystems, like the ones in Brazil, modern techniques like metabarcoding have been employed to generate detailed taxonomic inventories. However, selecting appropriate genetic markers remains challenging, as different markers and primer pairs may introduce biases by preferentially amplifying certain taxonomic groups, depending on their nucleotide sequences and the reference database used. This study advances biodiversity research by developing a customized reference database and optimizing primers for metabarcoding Brazilian mammals. Our database, covering 72% of native species, including endangered ones, enhances taxonomic identification accuracy across all Brazilian biomes. Both in-silico and in-vitro tests were conducted to evaluate and optimize the most suitable genetic markers and primer pairs, comparing their taxonomic coverage and resolution. Customized modifications made to COI primers reduced mismatches against Brazilian mammal sequences, significantly enhancing taxonomic coverage and outperforming commonly used primers in literature. We demonstrate that combining COI with RNA12S and RNA16S provides important and complementary information, improving the robustness of taxonomic assignments and addressing challenges posed by the Neotropical mammalian diversity. This multi-marker approach offers a reliable strategy for enhancing metabarcoding identification in the Neotropical region, supporting more effective conservation efforts.