Multilocus DNA barcoding – Species Identification with Multilocus Data

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.

High-Quality Genome Assembly and Annotation of the Big-Eye Mandarin Fish (Siniperca knerii)

“…The default settings of PSMC were adopted. A generation time of 2.5 years for Siniperca and a substitution rate of 2.22 • 10-9 per site per year were set for the PSMC run (Liu et al 2017).…”

A new forest dwelling button spider from South Africa (Araneae, Theridiidae, Latrodectus)

“…as a new species. We suggest that in addition to natural history and morphology, multiple gene loci, including nuclear loci, should be considered over single locus approaches for automated species identification (Will et al 2005;Dupois et al 2012;Dowton et al 2014;Liu et al 2017;Yang & Rannala 2017). Coloration is clearly diagnostic for this new species, as it is in some other Latrodectus species.…”

A method to generate multilocus barcodes of pinned insect specimens using MiSeq

“…To this end, it is preferable to focus the sequencing efforts on a selection of genes that have been verified for use in insect barcoding and phylogenetic analysis (Cruaud et al, 2017). The hybrid capture method can be used for multilocus barcoding of type specimens because it is capable of targeting specific loci (Burrell et al, 2015;Liu et al, 2017), has been optimized to use low DNA quantities from highly degraded DNA samples (Sproul & Maddison, 2017) and allows multiplexing several samples (Burrell et al, 2015;McCormack, Hird, Zellmer, Carstens, & Brumfield, 2013). However, it is technically demanding and has high upfront costs (Burrell et al, 2015), which make it less accessible to typical laboratories.…”

“…Mulitilocus barcoding can enhance accurate species identification in taxa where the CO1 marker (658 bp of the 5′ end of CO1) is found to be problematic (Cruaud, Rasplus, Rodriguez, & Cruaud, 2017;Klopfstein, Kropf, & Baur, 2016;Liu et al, 2017;Paknia, Bergmann, & Hadrys, 2015;Rach et al, 2017). Examples include the lack of a sufficient barcoding gap in the standard CO1 barcode sequence of some taxa in the orders Diptera and Odonata (Koroiva & Kvist, 2018;Meier, Shiyang, Vaidya, & Ng, 2006), and the presence of nuclear-mitochondrial DNA pseudogenes (NUMTS) in the CO1 sequences of several taxa in the order Orthoptera (Moulton, Song, & Whiting, 2010;Song, Buhay, Whiting, & Crandall, 2008), blue banded bees (Amegilla; Leijs, Batley, & Hogendoorn, 2017) and some species of the genus Sitobion (Hemiptera; Sunnucks & Hales, 1996).…”