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Syst. Biol
Vol.58, No.
4, 2009; Pages: 445–451

Barcoding Bamboozled by Bacteria: Convergence to Metazoan Mitochondrial
Primer Targets by Marine Microbes

MARK E. SIDDALL1*, FRANK M. FONTANELLA2, SARA C. WATSON1, SEBASTIAN KVIST3, AND CHRISTER ERS´EUS4

Sackler Institute for Comparative Genomics, American Museum of Natural History, New York, New York, USA.

Abstract

DNA barcoding has become a legitimate tool for assessment of global biodiversity patterns in a manner intended to allow diagnoses of known species to nontaxonomists. Relying on a standardized region (∼650 nucleotides) of the cytochrome c oxidase subunit I mitochondrial gene (COI), a variety of taxon-based initiatives (www.barcoding.si.edu/major projects.html), as well as large-scale locality-based barcoding projects (e.g., bscit.berkeley.edu/biocode/), are well underway. If DNA barcoding is to achieve its promise, these projects are essential as they will provide much-needed comparative baseline data for taxonomic groups, or for regional assemblages, upon which future identification efforts will rely. Thorough coverage of clades and guilds is essential to the success of DNA barcoding, but so too is highly accurate taxonomic identification of the source organisms. Although surreptitious amplification of nuclear mitochondrial pseudogenes (numts) may lead the unwary to overestimation of species diversity (Song et al. 2008), it may not necessarily lead to incorrect taxonomic determinations so long as the source organism was accurately identified. Erroneous taxonomic identification of a reference organism is a more serious problem (Vilgalys 2003; Traub et al. 2007), yet one that can be mostly mitigated by expert identification and appropriate deposition of reference specimens in natural history collections (Smith et al. 2008). Even when great care is taken to avoid contamination, inadvertent amplification of surface-adhering or tissue-infecting organisms may be impossible to avoid (e.g., Zhang et al. 1997; Nilsson et al. 2006). As such, underlying broad barcoding efforts is a presumption that the primers used are more likely to amplify the animal of interest than they are likely to amplify (e.g.) an unobserved bacterial or fungal contaminant. The LCO1490 and HCO2198 primers that provided the basis for the DNA barcoding standard were described by Folmer et al. (1994) as being “universal” for 11 invertebrate phyla. Their universality, however, is belied by several groups of animals not amplifying at the predicted optimal annealing temperature for these primers (e.g., Halanych and Janosik 2006). This difficulty has led to the use of modified or degenerate primers (Bely andWray 2004; Hebert et al. 2004; Ivanova
et al. 2007) and to greatly reduced primer annealing temperatures (de Waard et al. 2008). In contrast to universality, little attention has been given to the exclusivity with which barcoding primers will amplify target versus nontarget taxa. Here we demonstrate that primers widely used for DNA barcoding may prove problematic in light of their predicted, and repeatedly demonstrated, ability to amplify nontarget cold-adapted marine gammaproteobacteria.

Keywords: Marine Microbes,mitochondrial gene,Xylotrya setacea,Penitella penita,Paranais botniensis,marine biodiversity.


Corresponding author:

E-mail: siddall@amnh.org

 

 
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