Abstract
Species identification using DNA sequences, known as DNA barcoding has been widely used in many applied fields. Current barcoding methods are usually based on a single mitochondrial locus, such as cytochrome c oxidase subunit I (COI). This type of barcoding method does not always work when applied to species separated by short divergence times or that contain introgressed genes from closely related species. Herein we introduce a more effective multi-locus barcoding framework that is based on gene capture and “next-generation” sequencing. We selected 500 independent nuclear markers for ray-finned fishes and designed a three-step pipeline for multilocus DNA barcoding. We applied our method on two exemplar datasets each containing a pair of sister fish species: Siniperca chuatsi vs. Sini. kneri and Sicydium altum vs. Sicy. adelum, where the COI barcoding approach failed. Both of our empirical and simulated results demonstrated that under limited gene flow and enough separation time, we could correctly identify species using multilocus barcoding method. We anticipate that, as the cost of DNA sequencing continues to fall that our multilocus barcoding approach will eclipse existing single-locus DNA barcoding methods as a means to better understand the diversity of the living world.
Highlights
DNA barcoding has been very successfully employed in many applied fields, ranging from routine species identification[1,2,3], to discovery of cryptic species[4,5], tracking of invasive species[6,7,8], conservation, and community ecology[9,10,11,12]
Haplotypes at a single locus, such as c oxidase subunit I (COI) can be shared between two species, it is unlikely that individuals of two species share alleles across multiple independent genes
Tools for finding thousands of nuclear gene markers[32,33,34] and collecting their sequences through cross-species gene capture and next-generation sequencing are available[35], providing an opportunity to rigorously test the power of multilocus DNA barcoding
Summary
DNA barcoding has been very successfully employed in many applied fields, ranging from routine species identification[1,2,3], to discovery of cryptic species[4,5], tracking of invasive species[6,7,8], conservation, and community ecology[9,10,11,12]. The mitochondrial cytochrome c oxidase subunit I gene (COI) has a good amount of variation and is easy to amplify using PCR based approaches in most animal groups[13,14,15,16] It has become the most commonly used marker for animal DNA barcoding since it was first proposed more than a decade ago[13]. Dowton et al.[26] proposed “next-generation” DNA barcoding based on multilocus data in which they incorporated multispecies coalescent species delimitation They analyzed Sarcophaga flesh flies with two loci, mitochondrial COI and nuclear carbomoylphosphate synthase (CAD), and found out that their coalescent-based *BEAST/BPP approach was more successful than standard barcoding method[26]. Our goal is to develop a multilocus barcoding approach for identifying species that are indistinguishable based on the current DNA barcoding method
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