Chapter 1

DNA Barcodes: Methods and Protocols

W. John Kress and David L. Erickson
Abstract
DNA barcoding, a new method for the quick identification of any species based on extracting a DNA
sequence from a tiny tissue sample of any organism, is now being applied to taxa across the tree of life. As
a research tool for taxonomists, DNA barcoding assists in identification by expanding the ability to diagnose
species by including all life history stages of an organism. As a biodiversity discovery tool, DNA barcoding
helps to flag species that are potentially new to science. As a biological tool, DNA barcoding is being used
to address fundamental ecological and evolutionary questions, such as how species in plant communities
are assembled. The process of DNA barcoding entails two basic steps: (1) building the DNA barcode
library of known species and (2) matching the barcode sequence of the unknown sample against the
barcode library for identification. Although DNA barcoding as a methodology has been in use for less than
a decade, it has grown exponentially in terms of the number of sequences generated as barcodes as well as
its applications. This volume provides the latest information on generating, applying, and analyzing DNA
barcodes across the Tree of Life from animals and fungi to protists, algae, and plants.
Key words: DNA barcode, Identification, Taxonomy, Discovery, Ecology, Evolution

1. What Is DNA
Barcoding?
The taxonomic impediment that exists today for many systematists,
field ecologists, and evolutionary biologists, i.e., determining the
correct identification for any plant or animal sample in a rapid,
repeatable, and reliable fashion, is a reality we all must accept (1).
This taxonomic problem was a major reason for the development of
a new method for the quick identification of any species based on
extracting a DNA sequence from a tiny tissue sample of any organism. Appropriately called DNA barcoding, referring to the
UPC labels one finds on commercial products, DNA barcodes
consist of a standardized short sequence of DNA between 400
and 800 bp long that, in theory, can be easily isolated and characterized for all species on the planet (2, 3). By harnessing advances
W. John Kress and David L. Erickson (eds.), DNA Barcodes: Methods and Protocols, Methods in Molecular Biology, vol. 858,
DOI 10.1007/978-1-61779-591-6_1, Springer Science+Business Media, LLC 2012

W.J. Kress and D.L. Erickson

in molecular genetics, sequencing technology, and bioinformatics,

DNA barcoding is allowing users to quickly and accurately recognize known species and retrieve information about them. It also has
the potential to speed the discovery of the thousands of species yet
to be named. DNA barcoding has become a vital new tool for taxonomists who are charged with the inventory and management of
the Earths immense and changing biodiversity (4).
A DNA barcode, in its simplest definition, is one or more short
gene sequences taken from a standardized portion of the genome
used to identify species. The use of such short DNA sequences for
biological identifications was first proposed by Paul Hebert and
colleagues (2, 5) with the ultimate goal of quick and reliable
species-level identifications across all forms of life, including
animals, plants, and microorganisms. The concept of a universally
recoverable segment of DNA that can be applied as an identification marker across species was initially applied to animals (5).
However, a standard DNA barcode locus for plants was not
accepted by the botanical community until 6 years after Hebert
published his first paper on barcoding animals. After several broad
screenings of gene regions in the plant genome (e.g., refs. 610),
three plastid (rbcL, matK, and trnH-psbA) and one nuclear (ITS)
gene regions have become the standard barcode of choice in most
applications for plants and fungi (1113).

2. The Uses of DNA

Barcoding
From its inception the primary use of DNA barcodes has been for
species identification. As a research tool for taxonomists, barcoding
assists in identification by expanding the ability to diagnose species
by including all life history stages of an organism (e.g., seeds, seedlings, eggs, larvae, mature individuals both fertile and sterile), unisexual species, damaged specimens, gut contents, scats, and fecal
samples. In addition systematists have the potential to quantify the
consistency of their species definitions with a universal measure of
genetic variability based on the barcode sequence data. For the
applied users of taxonomy, barcoding is a tool to identify regulated
species, including invasive and endangered species, as well as to test
the identity and purity of biological products, such as seafood,
herbal medicines, and dietary supplements. As a biodiversity discovery tool, barcoding helps to flag species that are potentially new
to science, especially undescribed and cryptic species (see ref. 14).
DNA barcodes are now also being used to address fundamental
ecological and evolutionary questions, such as how species in plant
communities are assembled (12, 15) and the degree of specialization in tropical versus temperate zone herbivores (e.g., ref. 16;
see below).

DNA Barcodes: Methods and Protocols

It was not a coincidence that DNA barcoding developed in

concert with genomics-based investigations in the first decade of
the twenty-first century (17). DNA barcoding (a rapid tool for
species identification based on DNA sequences) and genomics
(a broad-based comparative approach to entire genome structure
and expression) share an emphasis on large scale genetic data acquisition that offers new answers to questions previously beyond the
reach of traditional disciplines. DNA barcodes, which in principle
will eventually be generated and characterized for all species on the
planet, are intended to be stored in an online digital library of
sequences for matching and recognizing unidentified biological
samples. Genomics has accelerated the process of recognizing
novel genes and gene function through the comparisons of vast
amounts of sequence data of the entire genomes of a limited number of taxa. In other words, the aim of DNA barcoding is to utilize
the information of ONE OR A FEW gene regions to identify ALL
species of life whereas genomics, the inverse of barcoding, describes
in ONE OR A FEW (but eventually many) selected species the
function and interactions across ALL genes. All other types of
DNA sequence-based investigations of organisms, including population genetics and phylogenetics, fall between these two ends of
the DNA spectrum.

3. DNA Barcoding
Methods in Brief
The process of DNA barcoding entails two basic steps: (1) building
the barcode library of known species and (2) matching, or assigning
the barcode sequence of the unknown sample against the barcode
library for identification. The first step requires taxonomic expertise in selecting one or preferably several individuals per species to
serve as reference samples in the barcode library. All taxonomists
should generate DNA barcodes for the taxa in their monographs
or at the least they should deposit verified DNA samples with their
associated voucher specimens in core DNA barcode institutions.
Tissue samples that yield high-quality DNA extractions in some
cases can be obtained from specimens already housed in museum
collections and herbaria. However, in most cases new tissues will
be taken directly from live specimens in the field before they are
prepared, labeled, and stored as voucher specimens in museum collections. These vouchers then serve as the permanent record that
connects the DNA barcode to a particular species of plant, fungus,
or animal.
Once the reference barcode library is complete for the organisms under study, whether they comprise a geographic region, a
taxonomic group, or a target assemblage (e.g., medicinal plants,
timber trees, etc.), then the DNA barcodes generated for the

W.J. Kress and D.L. Erickson

unidentified samples are compared to the known barcodes using

some type of matching algorithm. Most practical algorithms for
species assignment start by comparing two DNA sequences to produce a distance measure between the sequences. In DNA barcoding,
a sequence alignment algorithm is usually employed to assign an
unknown sample to a known species by finding the closest database
sequence to the sample sequence (18). Basic local alignment search
tool (BLAST) is a matching tool that is provided through GenBank
to search for correspondence between a query sequence and a
sequence library. Two additional commonly used distance measures are the Kimura-2-Parameter Distance and the Smith
Waterman Algorithm (similar to BLAST) for Local Alignment
Similarity (see ref. 19).
For many users of DNA barcodes, the process ends after the
unknown sample is correctly identified. However, barcodes can
also be applied as tools for answering fundamental biological questions, such as how species are assembled into communities. This
aspect of DNA barcoding has only recently been considered, but
offers some of the most exciting prospects for using this new taxonomic tool.

4. A Short History
of DNA Barcoding
To be practical as a DNA barcode, a gene region must satisfy three
criteria: (1) contain significant species-level genetic variability and
divergence, (2) possess conserved flanking sites for developing
universal PCR primers for the widest taxonomic application, and
(3) be of appropriate sequence length so as to facilitate current
capabilities of DNA extraction and sequencing. A fourth criterion for a successful DNA barcode relates to sequence quality and
has been proposed by some as an important consideration (see
CBOL Plant Working Group 2009). A short DNA sequence of
600 bp in the mitochondrial gene for cytochrome c oxidase subunit 1 (CO1; 2) generally fits these criteria and was accepted early
on as a practical, standardized species-level barcode for many
animals (see http://www.barcoding.si.edu/). The inability of CO1
to work as a barcode in plants and fungi (6, 8) required that botanists find a more appropriate marker. A number of candidate gene
regions were immediately suggested as possible barcodes for plants
(e.g. refs. 6, 7, 9, 10, 20, 21), but until 2009 none were universally
accepted by the plant taxonomic community. This lack of consensus was in most part due to the limitations inherent in a plastid
marker (i.e., low sequence variability) relative to CO1. In 2008,
The Consortium for the Barcode of Life Plant Working Group
convened a lengthy discussion on selecting an appropriate plant
barcode and eventually published a paper (11) in which the largest

DNA Barcodes: Methods and Protocols

number of candidate barcode markers with the largest set of data

were evaluated. Their results identified three markers that have
become the most widely used barcode loci today: rbcL, matK, and
trnH-psbA. Two, rbcL and matK, were identified as the core barcode loci and the third, trnH-psbA, was designated as an important
supplementary marker to be further tested and used in appropriate
cases. Some research groups continue to advocate additional markers for plants, such as ITS, for specific purposes or specific taxa.

5. What This Book

Is About
DNA Barcodes: Methods and Protocols provides the latest information on generating, applying, and analyzing DNA barcodes across
the Tree of Life from animals and fungi to protists, algae, and
plants. Background material is provided on the rationale for the
use of DNA barcodes as well as detailed protocols and methodologies for barcoding various types of organisms. Topics include sample acquisition and archiving, laboratory tracking of tissues and
sequences, sequencing protocols, data analyses, and informatics as
well as case studies of particular taxonomic groups and DNA
barcoding campaigns. In addition to these chapters on specific
laboratory methodologies, information on the applications of
DNA barcodes in the fields of systematics, phylogeny, and community ecology are provided for those who want to go beyond
generating sequences for particular taxonomic groups. DNA
barcoding is a new and powerful basic research tool with exceptional potential for the incorporation of new technologies and for
future applications. This book should be of benefit and interest to
all biologists and technicians interested in the relevance and application of molecular biology and DNA sequencing to identification,
taxonomy, evolution, and ecology.
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