Multilocus DNA Barcoding-Species Identification With Multilocus Data

DNA barcoding is a species identification method with a short DNA sequence.DNA barcoding has been widely used in many applied fields because it is a rapid and successful method,ranging from routine species identification to the discovery of cryptic species,tracking of invasive species,conservation,and community ecology.The mitochondrial cytochrome c oxidase subunit I gene(COI)has a good amount of variation and it is easy to amplify using PCR based approaches in most animal groups.It has become the most commonly used marker for animal DNA barcoding since it was first proposed more than a decade ago.In most cases,single-locus(COI)DNA barcoding results in high successfully rate in species identification.However,the success rate of species identification was low for species complexes with gene flow or where species had only recently diverged.Currently,barcoding methods are usually based on a single mitochondrial locus,such as cytochrome c oxidase subunit I(COI).This type of barcoding is not always effective when applied to species separated by short divergence times or that contain introgressed genes from closely related species.Although haplotypes at a single locus,such as COI can be shared between two species,it is unlikely that individuals of two species share alleles across multiple independent genes.Accordingly,multilocus data should perform better for species identification than any single locus could.Dowton et al.proposed “next-generation” DNA barcoding based on multilocus data in which they incorporated multispecies coalescent species delimitation in 2014.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 which only with COI gene,but their efforts were counterargued due to its limited number of loci and insufficient taxa selected.Herein we introduce a more effective multi-locus barcoding framework that is based on gene capture and “next-generation” sequencing and provide both empirical and simulation tests of its efficacy.Results revealed that distinctness between species increased as more independent loci were added.We also investigated effect of increasing length of loci on species discrimination and identification using simulated data and found that the number of loci other than the sheer length of locus determines the discrimination power of the data.We decided to pick 500 loci to be markers according our results for multilocus DNA barcoding to do species identification,which were developed for all ray-finned fishes.We devised a pipeline with three steps for multilocus DNA barcoding.Firstly,the p-distance between the unknown query and the other individuals in the database was calculated.Secondly,based on the sorted list of p-distances,we selected five closely related taxa,including the query,and use these individuals to reconstruct a species tree.Thirdly,we ran a BFD* test to delimitate the unknown query.Under limited gene flow and enough separation time,we can correctly identify species using multiple loci when single locus DNA barcoding fail.We anticipate that this multilocus barcoding approach will complement existing DNA barcoding methods based on a signle locus to better understand the current diversity of the living world.