UNIVERSITY OF NIGERIA, NSUKKA

FACULTY OF BIOLOGICAL SCIENCES

DEPARTMENT OF PLANT SCIENCE AND
BIOTECHNOLOGY

A SEMINAR REPORT PRESENTED IN PARTIAL

FULFILLMENT OF THE REQUIREMENT FOR THE
COURSE: PSB 481 (SEMINAR IN PLANT SCIENCE
AND BIOTECHNOLOGY)

TOPIC
DNA BARCODING: ITS APPLICATION AND
IMPLICATIONS

BY
ONODI, IJEOMA MARY
2016/239490

SUPERVISOR
MR. UDOH O.

MARCH, 2020
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TITLE PAGE
DNA BARCODING: ITS APPLICATION AND IMPLICATIONS
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APPROVAL PAGE

This is to certify that this write-up has been presented orally, written in the present form and presented to
the Department of Plant Science and Biotechnology, Faculty of Biological Sciences in partial fulfillment
for the award of Bachelor of Science (B.Sc.) degree in Plant Science and Biotechnology.

PREPARED BY
ONODI, IJEOMA MARY

_______________________________
Signature & Date

___________________ ___________________
MR. UDOH O. DR. AJUZIOGU N.
Seminar Supervisor Seminar Coordinator
Date: _____________ Date: _____________

___________________
DR. ABU N.
Head of Department
Date: ______________
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DEDICATION
This work is dedicated to God Almighty, the author and finisher of all good things.
 iv

ACKNOWLEDGEMENT

My gratitude goes to God for his mercies and grace. Mostly, I thank the head of my department, Dr.
Ngozi Abu, for her motherly care towards her students. I thank my supervisor, Mr. Udoh Obiora, for his
patience and tutelage in the process of compiling this work. I also thank lecturers and staff of the
department. Lastly, I would like to acknowledge my parents, Mr and Mrs Onodi, for their support in my
academic pursuit.
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SUMMARY

DNA barcoding is a taxonomic technique for fast and accurate identification of species using a short
section of DNA obtained from a specific gene or genes, instead of the whole genome. The basics of DNA
barcoding is the comparison of DNA sections reference library, in which an individual sequence can be
used to identify unknown species, parts of a species and its taxonomy. The short DNA sequence generated
from the standard region of the genome is known as a marker. This marker is different for various species
like cytochrome oxidase 1 (CO1) for animals, matK and RuBisCO for plants, and Internal Transcribed
Spacer (ITS) for fungus. The applications of DNA barcoding are numerous. It can be applied in the
identification of new plant species, plant leaves without members, pollen, medicinal plants, endangered
and threatened species. DNA barcoding allows non-ecologists to identify vector species that can cause
serious infectious diseases to plants, animals and humans, thereby understanding these diseases and how
to cure them. It can be used to assess the presence of endangered species for conservation efforts, or the
presence of indicator species reflective to specific ecological conditions. Identification of species listed in
the Convention of the International Trade of Endangered Species (CITES) appendixes is used in
monitoring of illegal trade of species. When barcoding is used to identify sample containing DNA from
more than one organism, the term DNA metabarcoding is used. Barcoding and metabarcoding is used to
screen ecosystems for invasive species and to distinguish between invasive, native, morphologically
similar species. DNA barcoding is faster than traditional morphological methods since it takes less time to
gain expertise and the workflow is generally quicker. Despite the advantages offered by DNA barcoding,
it has its limitations. The major limitation of the barcoding method is that it relies on barcode reference
libraries for the taxonomic identification of the sequences. The taxonomic identification is accurate only if
a reliable reference is available. However, most databases are still incomplete, misidentified with spelling
mistakes and other errors. DNA barcoding also carries methodological bias, from sampling to
bioinformatics data analysis. Even as DNA barcoding is more widely used and applied, there is no
agreement concerning the methods for DNA preservation or extraction, the choices of DNA markers and
primers set, or PCR protocols. The parameters of bioinformatics pipelines are at the origin of much debate
among DNA barcoding users. DNA sequence databases like GenBank contain many sequences that are
not tied to vouchered specimens. This is problematic in the face of taxonomic issues such as whether
several species should be split or combined, or whether past identifications were sound. Therefore, best
practice for DNA barcoding is to sequence vouchered specimens.
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TABLE OF CONTENTS

Title Page - - - - - - - - - - i
Approval Page- - - - - - - - - - ii
Dedication - - - - - - - - - - iii
Acknowledgement - - - - - - - - - iv
Summary - - - - - - - - - - v
Table of Contents - - - - - - - - - vi
Table of Figures - - - - - - - - - vii

CHAPTER ONE: INTRODUCTION

1.1 Background of Study - - - - - - - - 1-2
1.2 DNA Barcoding Markers - - - - - - - - 3-4
1.3 DNA Reference Library - - - - - - - - 4
1.4 DNA Barcoding History and Gene Region Criteria - - - - 5

CHAPTER TWO: PROCESSING, METHODS AND APPLICATION OF DNA BARCODES

2.1 Prerequisite before Processing - - - - - - - 6-7
2.2 DNA Barcoding Methods in Brief - - - - - - 7
2.3 Current Applications of DNA Barcoding - - - - - - 8-12
2.4 Futuristic Applications - - - - - - - - 12-13

CHAPTER THREE: BENEFITS AND LIMITATIONS OF DNA BARCODING

3.1 Benefits OF DNA Barcoding - - - - - - - 14
3.2 Limitations of DNA Barcoding - - - - - - - 14-18

CHAPTER FOUR
4.1 Successful Uses of DNA Barcoding in Medicinal Plants - - - - 19

Conclusion - - - - - - - - - - 19
References - - - - - - - - - - 20-22
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TABLE OF FIGURES

Figure 1. Barcode regions of plants

Figure 2. Organism groups and marker
Figure 3. Applications of DNA barcoding
Figure 4. How does DNA barcoding work
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CHAPTER ONE
INTRODUCTION

1.1 BACKGROUND OF STUDY

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 (Hebert et al., 2003). 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. By harnessing advances in molecular genetics, sequencing
technology, and bioinformatics (Savolainen et al., 2005). 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 Earth ’s immense and
changing biodiversity.

DNA barcoding is a system for fast and accurate species identification. It’s creates ecological system more
accessible by using short DNA sequence instead of whole genome and is used for eukaryotes and
prokaryotes. The short DNA sequence is generated from standard region of genome known as marker.
This marker is different for various species like CO1 cytochrome c oxidase 1 for animals, matK and rbcL
for plants and Internal Transcribed Spacer (ITS) for fungus. A DNA barcode, in its simplest definition, is
one or shorter gene sequences taken from a standardized portion of the genome used to identify species
(Hebert et al., 2003). The short DNA sequence is taken from standard region of genome to generate DNA
barcode. DNA barcode is short DNA sequence made of four nucleotide bases A (Adenine), T (Thymine),
C (Cytosine) and G (Guanine). Each base is represented by a unique color in DNA barcode. Even non-
experts can identify species from small, damaged or industrially processed material. The standard region
used to generate DNA barcode is known as marker. The use of such short DNA sequences for biological
identifications was first proposed by Paul Hebert and colleagues 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. However, a standard DNA barcode
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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, three
plastids (rbcL, matK, and trnH-psbA) and one nuclear (ITS) gene regions (Chen et al., 2010) have become
the standard barcode of choice in most applications for plants and fungi.

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 (Chen et al., 2010). As a biodiversity discovery tool, barcoding helps to flag species
that are potentially new to science, especially undescribed and cryptic species. DNA barcodes are now
also being used to address fundamental ecological and evolutionary questions, such as how species in
plant communities are assembled and the degree of specialization in tropical versus temperate zone
herbivores (Hebert et al., 2003). It was not a coincidence that DNA barcoding developed in concert with
genomics-based investigations in the first decade of the twenty-first century. 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.
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1.2 DNA BARCODING MARKERS

A marker is a fragment of DNA that is associated with a certain location within the genome. It is a
segment of DNA used for identification especially when closely linked to a trait or genetic material that is
difficult to identify. Markers used for DNA barcoding are called barcodes. One gene sequence containing
a specific marker should be used for all taxonomic groups, from viruses to plants and animals. However,
no such gene region/sequence has been found yet, therefore, different barcodes are used for different
groups of organisms. For animals, the most widely used marker/barcode is mitochondrial cytochrome C
oxidase I (COI) locus. Other mitochondrial genes, such as Cytb, 12S or 18S are also used. Mitochondrial
genes are preferred over nuclear genes because of their lack of introns, their haploid mode of inheritance
and their limited recombination (Song et al., 2008; Hebert et al., 2004). Cytochrome c oxidase I (COI) is
used for species discrimination though it cannot be used for plants due to its limited divergence and also
the generally low rate of nucleotide substitution in plant mitochondrial genomes which precludes the use
of CO1 as a universal plant barcode sequencing errors. For bacteria, the small subunit of ribosomal RNA
(16S) gene can be used for different taxa, as it is highly conserved. Barcoding fungi are more challenging,
and more than one primer combination might be required (Manoylov, 2014; Hall et al., 2010, Buchheim
et al., 2011). The COI marker performs well in certain fungi groups, but not equally well in others.
Therefore, additional markers are being used, such as ITS rDNA and the large subunit of nuclear
ribosomal RNA (LSU) (Chen et al., 2010). Efforts are going on to find suitable universal barcode loci for
plants.

1.2.1 DNA BARCODING MARKERS IN PLANTS

Since the standard DNA barcode for most animals, a fragment of the mitochondrial CO1 gene, was
inapplicable for plants because of the low and uneven mutation rate of plant mitochondrial DNA, it was
decided to choose a barcoding region among the chloroplast regions of the genome. Few genes/marker
have been found in the chloroplast genome, the most promising being maturase K gene (matK) by itself or
in association with other genes. The best discrimination between plant species has been achieved when
using two or more chloroplast barcodes (like matK and rbcL). Plant marker regions used as DNA barcode
on plants include matK, rbcL, trnH-psbA, ITS, trnL-F, 5S-rRNA, and 18S-rRNA (Newmaster et al.,
2008). No single region is able to serve as a barcode for all plants; for this purpose, combinations of DNA
regions are used. For the identification of plant species, two defined regions of the chloroplast DNA
(maturase K) matK and (ribulose-1,5-biphosphate carboxylase oxygenase large subunit) rbcL have been
widely used for standard barcodes as endorsed by the Plant Working Group of the Consortium for the
Barcode of Life (CBOL) (Pawlowski et al., 2012).
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matK is the nearest plant analogue to COI, the animal DNA barcode. It typically provides high resolution,
leading to good species identification as a result of its speedily evolving coding fragment among the
plastid genome. Unfortunately, matK can be difficult to amplify using existing primer sets, particularly in
non-angiosperms. As compared to matK, the barcode marker rbcL (ribulose-1,5-bisphosphate
carboxylase/oxygenase large subunit) is easy to amplify and sequence. It is an important candidate for
plant DNA barcoding even though its discriminatory power is not as good as matK. matK and rbcL have
been suggested to be the core DNA barcodes for plants. Other than these, the plastid intergenic spacer
trnH-psbA is also used as a supplementary DNA barcode. It has higher species discrimination success and
variable intergenic spacers in plants (Chase et al., 2007).

1.3 DNA REFERENCE LIBRARY

DNA barcode serve fast and accurate identification of a plant species, and the sequences are available in
the sequence library such as GenBank and BOLD. The most common DNA sequence databases are the
Barcode of Life Database (BOLD) and the GENBANK database managed by the National Center for
Biotechnology Information (NCBI). They are the two main public databases of DNA barcode data for
animals, plants, and fungi. BOLD currently contains sequences of about 296,000 formally described
species (about 7 million specimens) (Ratnasingham et al., 2007). For a sequence to obtain a formal
barcode status in BOLD, several elements must be provided: species name, voucher data (storing
institution and catalog information), collection record, identifier of the specimen, sequence of >500 bp,
primer information, and the raw sequence data files. Once uploaded, BOLD administrators perform
quality checks of data prior to making it public (i.e., confirming that the sequence is not that of a
contaminant, is a true functional copy, and is of adequate quality). GenBank is much larger and contains
about 212 million sequences. GenBank also performs basic quality checks on all new submissions, such
as vector contamination, proper translation of coding regions, correct bibliographic citations and correct
taxonomy. However, unlike BOLD, GenBank does not store sequence chromatograms, collection
metadata or photographs. BOLD is a curation tool that also stores sequences, while GenBank is just a
sequence repository. Many sequences are duplicated between databases, as all BOLD sequences are
automatically submitted to GenBank (denoted by the key term BARCODE) and BOLD periodically
mines barcode sequences from GenBank (Ratnasingham et al., 2007). Ideally, all barcode sequences
contained in either database should have been derived from a vouchered specimen, which was initially
identified by a taxonomic expert. However, given the inherent nature of any public database, it is
inevitable that some erroneous data will be present.
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1.4 DNA BARCODING HISTORY AND GENE REGION CRITERIA

For a DNA barcode to be regarded as credible, a gene region must satisfy three criteria:
(1) It should contain significant species-level genetic variability and divergence,
(2) It must possess conserved flanking sites for developing universal PCR primers for the widest
taxonomic application, and
(3) It should 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. 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. The inability of CO1 to work as a barcode
in plants and fungi required that botanists find a more appropriate marker. A number of candidate gene
regions were immediately suggested as possible barcodes for plants, 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 in which the largest number of candidate
barcode markers with the largest set of data was 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 (Fi šer and
Buzan, 2014).

Figure 1. Barcode regions of plants Figure 2. Organism groups and marker

CHAPTER TWO: PROCESSING, METHODS AND APPLICATION OF
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DNA BARCODES

2.1 PREREQUISITE BEFORE PROCESSING

The DNA isolation and amplification methods used work for a variety of plants, fungi, and animals —and
many products derived from them. The collection of specimens may support a census of life in a specific
area or habitat, an evaluation of products purchased in restaurants or supermarkets, or may contribute to a
larger “campaign” to assess biodiversity across large areas. It may make sense to the use sampling
techniques from ecology. For example, a quadrat samples the plant and/or animal life in one square meter
(or ¼ square meter) of habitat, while a transect collects samples along a fixed path through a habitat. A
“Hula Hoop” can be used as an acceptable substitute for a quadrat. Use common sense when collecting
specimens. Respect private property; obtain permission to collect in any location. Respect the
environment; protect sensitive habitats, and collect only enough of a sample for barcoding. Do not collect
specimens that may be threatened or endangered. Be wary of poisonous or venomous plants and animals.
Consult an expert if in doubt about the safety or conservation status of a potential specimen. High quality
photographs of the organisms and a small sample for classical taxonomic analysis are needed to act as a
reference sample if one plans to submit one's data to GenBank. Do not take more sample than you need.
Only a small amount of tissue is needed for DNA extraction—a piece of plant leaf about ⅛- to ¼-inch
diameter or a piece of animal or fungal tissue the size of a grain of rice (Hollingsworth et al., 2011).

Minimize damage to living plants by collecting a single leaf or bud, or several needles. When possible,
use young, fresh leaves or buds. Flexible, non-waxy leaves work best. Tougher materials, such as pine
needles or holly leaves, can work if the sample is kept small and is ground well. Dormant leaf buds can
often be obtained from bushes and trees that have dropped leaves. Fresh, frozen leaves work well. Dried
leaves and herbarium samples are variable. Avoid twigs or bark. If woody material must be used, select
flexible twigs with soft pith inside. As a last resort, scrape a small sample of the softer, growing cambium
just beneath the bark. Roots and tubers are a poor choice, because high concentrations of storage starches
and other sugars can interfere with DNA extraction. For fungi, obtain fruit bodies (such as mushrooms)
when possible, as DNA is easier to obtain from fruiting bodies than mycelia. Only include multiple
fruiting bodies in the same sample when they are clearly growing together and appear similar, and avoid
contamination by other fungi. Fresh samples work well for DNA isolation, while dried samples give
variable results. Fungal fruiting is weather and climate dependent, so their abundance will vary. Small
invertebrate animals, such as insects, can be collected whole and euthanized in a kill jar by placing them
in a freezer for several hours (Virgilio et al., 2010). Samples of muscle tissue can be taken from animal
foods—such as fish, poultry, or red meat. Internal organs and bone marrow are also good sources of
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DNA. Fresh and frozen samples, and those preserved in ethanol, work well. However, bone, skin, leather,
feather, desiccated, and processed samples are challenging.

2.2 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
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. 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. 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.

2.3 CURRENT APPLICATIONS OF DNA BARCODING

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DNA barcoding has many applications in various fields like preserving natural resources, protecting
endangered species, controlling agriculture pests, identifying disease vectors, monitoring water quality,
authentication of natural health products and identification of medicinal plants. The use of plant DNA
barcodes has skyrocketed with several reviews of these applications already published. Categories of use
include species level taxonomy, biodiversity inventories, phylogenetic evaluation, biosecurity and public
health, conservation assessment and environmental preservation, specie interactions and ecological
networks, cryptic diversity information, DNA barcoding metadata, ecological forensics, community
assembly, traffic in endangered species, and monitoring of commercial products. In some cases, the
methodologies are now advanced, while others remain in their infancy.

2.3.1 CONTROLLING AGRICULTURAL PEST, IDENTIFYING DISEASE VECTORS AND

MONITORING WATER QUALITY
DNA barcoding can help in identifying pests in any stage of life making easier to control them saving
farmers from cost of billion dollars from pest damage. The global tephritid barcoding initiative
contributes to management of fruit flies by providing tools to identify and stop fruit flies at border. DNA
barcoding allows non ecologists to identify the vector species that can cause serious infectious diseases to
animals and humans, to understand these diseases and cure them (Hebert and Gregory, 2005). A global
mosquito barcoding initiative in building a reference barcode library that can help public health officials
to control these diseases causing vector species more effectively and with very less use of insecticides.
Drinking water is a process resource for living being. By studying organism living in lakes, rivers and
streams, their health can be measured or determined. DNA barcoding is used to create a library of these
species that can be difficult to identify. Barcoding can be used by environmental agencies to improve
determination of quality and to create better policies which can ensure safe supply of drinking water
(Hebert et al., 2003).

2.3.2 COMMUNITY PHYLOGENY AND SPECIES ASSEMBLY

DNA barcodes, as a tool, has greatly expanded the collaboration between systematists, who focus on
species identification and evolutionary relationships, and ecologists, who investigate species interactions
and patterns of associations (Chase et al., 2005). Plant DNA barcoding has been a boon to community
ecologists seeking to understand the factors, such as species diversity pools and functional traits, which
control the assembly of species into ecological communities. Estimating the third component controlling
species assembly, namely evolutionary history, has always been hampered by the lack of well-resolved
phylogenetic hypotheses on species relationships in communities. Phylomatic, a tool for estimating
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phylogenetic trees for plant communities, was a giant step forward for ecologists. However, the
publication of the first community phylogeny based on DNA barcode sequence data for the trees in the
forest dynamics plot on Barro Colorado Island in Panama set off a storm of new investigations that were
able to add a well-supported evolutionary component to understanding species diversity and assembly
(Cowan et al., 2006). Determining if species in a community are more closely related than by chance
(phylogenetic clustering), more distantly related than by chance (phylogenetic overdispersion), or
randomly distributed across the plant tree of life can now be ascertained by building a DNA barcode
library of these species assemblages and generating a phylogenetic tree based on the sequence data. The
assumption follows that species in a community that are phylogenetically clustered are more likely to
have similar ecological niches (i.e., phylogenetic niche conservation) and have been assembled via abiotic
filtering. The generation of community phylogenies using DNA barcode data across multiple plots in
varied habitats and environments has great promise for further testing the basic assumptions and rules
governing species assemblies in plant communities. And it is clear that this approach has yet to reach its
full potential (Cowan et al., 2006).

2.3.3 FUNCTIONAL TRAITS AND SPECIES ASSEMBLY

Ecologists have long been interested in quantifying critical plant traits that allow species to function in
specific environments, and hence assemble into communities. Measuring the degree of similarity of traits
in an assemblage provides insights into those features that allow these species to coexist or not.
Quantitative information on functional traits together with well-resolved evolutionary histories gives
ecologists a powerful tool for understanding the processes of community assembly. DNA barcodes alone
do not provide specific new insights into the role of functional traits in determining plant species
assemblages. However, the DNA sequence data provide sufficient signal to derive phylogenetic
hypotheses on the role of evolutionary signal in assembling species. It was hoped that the relationship of
traits and phylogeny would allow the latter to be a strong predictor in measuring trait similarity across
species. Unfortunately the relationship between phylogeny and functional traits is not always a direct
correlation thereby preventing phylogenetic signal from being a proxy for ecological similarity.
Nonetheless, since the publication of the first DNA barcode based community phylogeny of tree species,
a host of investigations have combined data from functional traits with community phylogenies that
together have allowed ecologists to explore the processes determining community assembly in temperate,
subtropical, and tropical forests.

2.3.4 SPECIES INTERACTIONS: IDENTIFYING UNKNOWN PARTNERS

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In order to fully understand the ecology and evolution of interactions among species in natural and
human-altered environments, accurate and repeatable identifications of the interacting partners are
imperative. Generalized interactions can be studied to some degree without clear identifications at the
species-level of the organisms involved, i.e., only identifying to genus or family (Christenhusz and Byng,
2016). Specialized interactions, including mutualisms and antagonisms, require unambiguous species
identifications. The development of DNA barcodes as species-level markers has already begun to
revolutionize our understanding of species interactions and the community networks they form, especially
in tropical habitats where the most complex interactions have evolved. One of the earliest applications of
plant DNA barcodes to investigate species interactions was employed almost simultaneously in both
temperate and tropical ecosystems. The below ground interactions of plants in a community with each
other and with micro communities in soils has been exceptionally problematic to investigate because of
difficulty in the identification of plant roots based on morphology alone. However, once a DNA barcode
library is developed for a community based on the presence of aboveground representatives, species-
specific genetic identification of the belowground roots is facilitated. In general, species interactions and
spatial overlap is greater belowground than expected based on above ground stem densities. Food web
interactions have been greatly clarified with the application of DNA barcodes. Using the CO1 DNA
barcode marker, the food web structure of the spruce budworm and its numerous parasitoids are
investigated to understand the population dynamics of this major pest of trees in boreal forests. With
regards to plant-herbivore interactions, several teams of ecologists have been able to demonstrate the
utility of DNA barcodes to identify the diversity of host plants for herbivorous beetles in both neotropical.
However, these studies used a limited number of molecular markers and were only able to identify the
hosts at the generic or familial level.

2.3.5 SPECIES BOUNDARIES AND BIODIVERSITY DISCOVERY

Taxonomists have been using morphological features for the identification of both plants and animals
since before the time of Carl von Linnaeus. Yet, even after hundreds of years of work by taxonomists
perhaps only 20% of the species on earth have been formally recognized and named. Much work remains
to be done. DNA barcoding provides a relatively new and significant tool to aid in the determination of
species boundaries and discovery of new taxa (Vijayan and Tsou, 2010). Botanists have also applied
DNA barcodes to species inventories even though the discriminatory power of the barcode markers for
plants is less than the barcode markers for insects. Early studies mostly focused on trees in tropical forest
monitoring plots and demonstrated the difficulties, especially the low identification rates (e.g., 70%), of
using DNA barcodes. The same studies also pointed out the significant gains in being able to more
accurately identify sterile and juvenile specimens lacking traditional morphological features required for
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identification. The critical role in species identification and discovery played by herbarium voucher
specimens, even if lacking flowers or fruits, and the field data associated with these collections cannot be
overemphasized (Kress et al., 2005). Forest inventory plots in which trees are tagged for long-term
monitoring allow taxonomists to resample and collect additional data from these individuals in the future
if necessary. Standardizing the DNA barcode markers and bioinformatics tools being used in different
forest inventory projects will facilitate species discovery and taxonomic consistency across broad-scale
geographic zones. So far, such standardizations have not been fully adopted. A recent example of how
DNA barcodes could play a decisive role in assisting taxonomic clarity is in the tree flora of the Amazon
Basin of South America (Christenhusz and Byng, 2016).

2.3.6 DNA BARCODE FORENSICS OF COMMERCIAL PRODUCTS, ENDANGERED

SPECIES, HERBAL SUPPLEMENTS, AND ETHNOBOTANY
The correct identification of plants and animals is equally important in the non-scientific, commercial
world as it is to ecologists and taxonomists. Broadly termed “DNA barcode forensics,” genetic markers
are being employed to insure commercial product identity and purity, to protect endangered species in
illegal trading, and to document the use of forest plants by local people. For example, the use of DNA
barcodes in determining species responsible for bird-strikes of commercial aircraft is now routine. More
widespread is the utilization of these markers in the authentication of animal and other wild-collected
commercial products sold in markets around the world. The desire for an accurate, reliable, and
inexpensive tool for the identification of illegal timber products has been one of the driving forces in
recent applications of DNA barcode technologies in several diverse regions of the world (Mishra et al.,
2016). Using DNA barcoding, natural resource managers can monitor illegal trade of products made of
natural resources like hardwood trees. Fishbol is reference barcode library for hardwood trees to improve
management and conservation of natural resources. An analysis carried out tested a number of possible
DNA barcode markers for the identification of species of trees in the commercially important mahogany
family (Meliaceae). Although most markers fell short of expectations for discriminating species, ITS was
able to identify some species of this family that are listed in the Convention on International Trade of
Endangered Species (CITES). A higher level of discrimination was demonstrated among commercially
important, but threatened species of trees of the tropical dry forests of India. Most recently, DNA
barcoding was employed to monitor illegal timber trade in the biodiversity hotspot of Madagascar, where
species of Dalbergia (Fabaceae), the rosewoods, are under threat. The limitations of the standard genetic
markers in identifying closely related species was discouraging in this genus although some success was
achieved. Nonetheless regulators are in general optimistic that DNA barcode tools will be of assistance in
 12

recognizing species currently protected by government legislation, but under threat from illegal timber
operations. In addition to timber trees, DNA barcode libraries have been developed for other taxonomic
groups of threatened and endangered taxa listed in CITES, e.g., orchids and it is expected that this
technology will eventually become standard in the monitoring of illegal trade. Timber is not the only
commercial plant product in need of accurate species identifications by regulators and quality control
specialists. Traditional medicines, teas, and herbal supplements together are an important and large
component of the commercial market in biodiversity, locally, nationally, and internationally. From the
early development of plant DNA barcodes, applications to monitor this market have been in development.
One arena that is only now receiving sufficient attention is the use of plant DNA barcodes in the
documentation of traditional ethnobotanical knowledge of indigenous people.

2.3.7 SPECIES AND HABITAT CONSERVATION

One of the major challenges facing biologists today is conserving biodiversity under severe threat due to
major habitat degradation and environmental change caused by humans. DNA barcoding, as a tool
primarily for species identification, can be used in two specific ways to address biodiversity conservation:
1) as a means of more accurate an eventually more rapid biodiversity monitoring both before and after
conservation actions, and 2) by providing data that will assist in estimations of phylogenetic diversity for
setting conservation priorities (Erickson et al., 2008). Making accurate taxonomic determinations for
conservation monitoring can be greatly aided with plant DNA barcodes, especially in tropical biomes
where biodiversity is poorly known and many species lack verified scientific names. As pointed out above
with respect to herbal supplements and medicines, the deficiency of uniform taxonomy is a significant
problem in assessing species diversity and identification in local market products. The same applies to
poorly known tropical forests requiring conservation in which species identification is extremely difficult,
especially when using non-fertile specimens often only labeled as “morphospecies”. In such cases DNA
barcoding offers a solution for more uniform identifications, although some logistical hurdles may still
impede the widespread use of DNA barcodes in this fashion. With regard to determining conservation
priorities, it has been demonstrated that plant DNA barcodes can play a key role in estimating species
richness in the relatively poorly known northern tropical forests of Queensland, Australia.

2.4 FUTURISTIC APPLICATIONS

Since the time of their introduction into the botanical community over a decade ago DNA barcodes have
been applied to a variety of investigations in both basic and applied research in plants. One of the main
reasons that plant systematists have not yet universally accepted DNA barcoding as a core tool in their
arsenal for identifying species is that no single marker is able to completely discriminate among species in
 13

most taxonomic groups. In contrast ecologists have been more willing to find new and unique applications
of DNA barcodes to address some of their basic research questions because in general they work in
systems made up of multiple lineages of plants that can be uniquely identified by a combination of DNA
barcode loci. Looking to the future, plant DNA barcoding will advance in two key ways to serve the
botanical community by building a more comprehensive global plant DNA barcode library for universal
use, and developing new markers and adopting new sequencing technologies. Besides the core DNA
barcode rbcL and matK, plant barcoding needs some supplementary markers such as trnH-psbA and ITS.
Moreover, in closely related and cryptic taxa DNA barcoding is always ambiguous and demands more
group specific markers. However, DNA barcoding has significant impact on molecular phylogeny,
population genetics, evolution and ecology, biosecurity and food product regulation. Recently developed
tools such as metabarcoding coupled with high-throughput sequencing (HTS) are rapid, accurate, and
cost-effective alternative to resolve cryptic taxa (Vijayan and Tsou, 2010). Moreover, environmental
DNA (eDNA) metabarcoding, which includes universal DNA barcodes and HTS to characterize
biological communities from terrestrial and aquatic environmental samples can be effectively used.

Figure 3. Applications of DNA barcoding

CHAPTER THREE
 14

BENEFITS AND LIMITATIONS OF DNA BARCODING

3.1 BENEFITS OF DNA BARCODING

Information gathered from DNA barcodes can be used beyond taxonomic studies and will have far-
reaching implications across many fields of biology, including ecology (rapid biodiversity assessment and
food chain analysis), conservation biology (monitoring of protected species), biosecurity (early
identification of invasive pest species), medicine (identification of medically important pathogens and
their vectors) and pharmacology (identification of active compounds) (Mohammed et al., 2017). DNA
barcodes are likely to play a major role in the future of taxonomy. It relieves the enormous burden of
identifications from taxonomists, so they can focus on more pertinent duties such as delimiting taxa,
resolving their relationships and discovering and describing new species. It will also help in pairing up
various life stages of the same species (e.g. seedlings, larvae).

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. A rapid and accurate method is now
being developed for the quick identification of plant species based on extracting DNA from a tiny tissue
sample of a leaf, flower, or fruit. The direct benefits of DNA barcoding is to make the outputs of
systematics available to a large number of end-users by providing standardized and high-tech
identification tools, e.g. for biomedicine (parasites and vectors), agriculture (pests), environmental assays
and customs (trade in endangered species). It will provide a bio-literacy tool for the general public. DNA
based species identification will help opens the treasury of biological knowledge, which is currently
underused partly because taxonomic expertise for species identification is relatively inaccessible. The
most important aspect of DNA barcoding is that it will facilitate basic biodiversity inventories. DNA
barcoding can be likened to aerial photography, in that it provides an efficient method for mapping the
extent of species, though in sample space rather than physical space.

3.2 LIMITATIONS OF DNA BARCODING

DNA barcoding is faster than traditional morphological methods all the way from training through to
taxonomic assignment. It takes less time to gain expertise in DNA methods than becoming an expert in
taxonomy. The DNA barcoding workflow (i.e. from sample to result) is generally quicker than traditional
morphological workflow and allows the processing of more samples. Despite the advantages offered by
DNA barcoding, it has also been suggested that DNA barcoding is best used as a complement to
traditional morphological methods. This recommendation is based on multiple perceived challenges
 15

(Collins et al., 2013). The major limitation of the barcoding method is that it relies on barcode reference
libraries for the taxonomic identification of the sequences. The taxonomic identification is accurate only if
a reliable reference is available. However, most databases are still incomplete, especially for smaller
organisms e.g. fungi, phytoplankton, nematoda etc.

3.2.1 ABSENCE OF UNIVERSAL BARCODE AND SELECTION OF APPROPRIATE

BARCODE REGION
In DNA barcoding, the universality of the barcode is still a big problem. It is difficult to attain the
universality of barcode due to the insufficient information of genetic variation in the less studied
taxonomic group. This problem is majorly found in plants as compared to animals. The differentiation and
identification of species relying on interspecific variation among DNA sequences are due to the resolution
capability of a barcode. Thus, there is a challenge in defining a good quality barcode consisting of a small
and variable DNA sequence flanked by conserved regions. The most important task of DNA barcoding is
the identification of universal primers amplifying fragments with high resolution. However, it has been
argued that a single short fragment will be sufficient to discriminate the organism at species level
identification The single-locus DNA barcodes lack adequate variation in the closely connected taxonomic
group, so for the identification of plants, no loci are available.

3.2.2 ERROR FOUND IN DNA BARCODING WHEN MITOCHONDRIAL SEQUENCES ARE

USED
DNA barcoding faced limitation due to the presence of the same copy of a gene of interest in the
mitochondrial genome because of heteroplasmy in mtDNA, bacterial infection biasing, nuclear
integration, and introgression in mtDNA (Song et al., 2008). The duplication of a gene, i.e., if a portion of
cytochrome oxidase I (COI) are duplicated in a given species, typical PCR may amplify these fragments.
Thus, it will not be clear whether the paralogous copy had diverged from duplication of COI. The
heteroplasmy is the combination of more than one type of mitochondrial genome in a species. The
overestimation of the quantity of distinctive species in barcoding results due to occurrence of co-
amplification in divergent heteroplasmic copies of mtDNA. The bacterial infection found in mtDNA due
to the maternally inherited symbionts can cause linkage disequilibrium, and each individual becomes
infected with such symbionts. These symbionts among closely connected species break the species barrier
by conjugation followed by selective sweep leading to the identical mtDNA sequences in different
species. The nuclear integration of mtDNA creates error for barcoding. Nuclear mitochondrial
pseudogenes (numts) are a nonfunctional duplication of mtDNA in the nucleus and occur in the major
clades of eukaryotes. The presence of numts in the nuclear region creates a problem in DNA barcode data
 16

library construction and species identification. There are limitations of using mtDNA in infer species
boundaries with the retention of ancestral polymorphism, male-biased gene flow, and selection on any
mtDNA nucleotide (the whole genome is one linkage group). The introgression along with hybridization
and paralogy results in the transfer of mtDNA gene copies to the nucleus. These factors in mtDNA create
a problem for both animal and plant DNA barcoding (Rubinoff et al., 2006).

3.2.3 LACK OF COMPREHENSIVE REFERENCE DATABASE

DNA barcoding is affected due to incomplete a priori identification of specimen in the reference database.
The conflictions are created in data assessment; different laboratories work on the same taxa and explain
different nomenclatures of the same species through morphological identification. If the reference
database is not comprehensive, it will create misidentification of the taxa (Vijayan and Tsou, 2010). DNA
barcoding faces limitations when the selected individual represents to every taxon within the reference
database. The unknown specimen had taken from undescribed biodiversity causes problems in the
identification. The reference sequences from taxonomically verified specimen lead to the validity of DNA
barcoding. In the absence of the reference data, DNA barcoding will face limitations and challenges.
DNA barcoding will also face difficulty when the query sequence lacks its target in the reference
database. Therefore, the barcoding-based identification of the query at the species level fails. The
reference sequences are verified from voucher specimen that is documented by experienced taxonomists
(Erickson et al., 2008). Due to lack of reference database, there will be no authentic library for recently
identified query sequences. As a result, there will be a large quantity of legacy data in the form of
sequences that are available in GenBank. These will not be used as a barcode. Thus, DNA barcoding does
not improve the speed of cataloging the life on earth.

3.2.4 LACK OF STATISTICAL SOLUTION

DNA barcoding is a useful tool for the identification of unknown species. For this methodology, the
threshold values providing a distinction between intraspecific and interspecific variation values are
required. If the unknown sequence differs from the nearest reference sequence by a variation above the
threshold, the organism containing the sequences will belong to a specific species, suggesting its
classification needs additional investigation. The wide range of overlap between intra- and interspecific
divergence values creates major problems. These overlaps seem comparatively restricted and far from the
respective average values. Thus, only the mean values for intra- and interspecific comparisons of closely
connected sibling species are required. The use of a different threshold considering the tenfold rule (gap
corresponds to a generic ten times the value of intraspecific divergence) has been proposed. This law has
been extensively criticized. To overcome this difficulty, currently it is predicted that the interspecific
 17

sequence divergence should increase to the threshold of 2 or 3% dissimilarity. This threshold has been set
on the basis of experimental proof observation of sequence variations among congeneric species. This
approach might be simple to neglect the inconclusive or inaccurate results. Thus, there is a requirement of
for statistical strategies when a sampled query sequence is the same as the specific database sequence to
proof a species assignment of the query. The strong assumptions based on the population genetics of the
analyzed species revealed the statistical uncertainty in DNA barcoding. The unrealistic assumption of
excellent sequence identity at intraspecies level is abandoned. Thus, with not creating population genetic
assumptions, the DNA barcoding is not possible. It has been observed that with robust population
subdivision within species, the species assignment might fail due to the underlying demographics that
have not been modeled capably. Another case is sequence sampled from a sub-population with no gene
flow with any of the population listed in the database. The DNA barcoding statistical methods which are
used here do not categorize the query sequence as a member of the parental species, even though
taxonomists would identify it as belonging to it. So, DNA barcoding might fail as a result of the
recognition of taxonomical units corresponding to a population that is reproductively isolated and
additionally if centered on a range of nucleotide changes (K) as a statistics within the hypothesis-based
mostly approach. DNA barcoding faces the problem to check the clear hypothesis meaning alternative of
inappropriate or suboptimal analytical technique because of confusion on the objectives of the study
(Nielsen and Matz, 2006).

3.2.5 LIMITATION OF DISTANCE-BASED AND TREE-BUILDING METHOD USED IN DNA

BARCODING
It is noted that DNA barcoding fails in the form of taxonomic approach because it does not recover
correct species tree. Some criticism has arisen due to distance-based and character-based methods. Some
reports have mentioned that the distance-based method should not be used for DNA barcoding, as it is a
phenetic measure and is not appropriate for species identification. In the distance-based method, NJ tree
acts as a standard part of the procedure for DNA barcoding. But, there is a good documentation about the
poor performance of NJ trees on the basis of trial and error and theoretical (Chase et al., 2007). The
inappropriate use of NJ trees for identification can decrease the effectiveness of DNA barcoding. This will
ensue either mtDNA paraphyly or misidentification of species independently. The NJ trees do not seem to
be resolved by exploitation the other tree inference ways. The character-based methodology has failed to
break into the most stream of DNA barcoding. However, currently, DNA barcoding via tree-based
approach did not stop at distance vs. character based approach. Avoiding any tree-building analysis due to
its impression of inferring phylogenies and relationships with single-gene tree is well known as a problem
of phylogenetics. Generally, the phylogenetic technique has been proposed as a data analysis in order to
 18

overcome the limitations of the threshold-based methodology in DNA barcoding. However, the
application of these threshold-based approaches leads to some problem in a study on the relationship
between DNA barcoding and molecular phylogeny. DNA barcoding is not a phylogenetic reconstruction.
Still, these methods are being used along with the debate in phylogeny and identification in the area of
DNA barcoding. The bootstrap resampling can further decrease the already low identification success
rates associated with NJ trees. The use of bootstrap resampling in DNA barcoding studies creates
confusion between species discovery and specimen identification.

3.2.6 LIMITATION IN AVAILABLE BIOINFORMATICS TOOLS AND ALGORITHM

The biases occurred in methods used for the original cohort of DNA barcoding are being replicated by
various studies and assisted by the analytical tools obtainable from the BOLD. A character-based tool,
i.e., BLOG, has been made along with BOLD. But, presently it is available only on the Barcode of Life
Data Portal (BDP) instead of various BOLD websites (Ratnasingham et al., 2007). The current popular
methods could be a product of routine instead of wise selection. This means a systematic appraisal of taxa
has not been capitalized by the barcoding movement (Gustav and Meyer, 2005). For DNA barcoding,
easy-to-handle tools are required for species discrimination and identification. These tools use pairwise
global alignment or alignment-free and automated selection of data partitions of an alignable group of
samples. Micro inversions are common in noncoding regions leading to multiple groupings of samples.

Figure 4. How does DNA barcoding work

CHAPTER FOUR
 19

4.1 SUCCESSFUL USES OF DNA BARCODING IN MEDICINAL PLANTS

In many studies from 2003 to 2016, the results of DNA barcoding can provide accurate identification of
many medicinal plant materials that are not morphologically distinguishable. DNA barcoding has found
its applications in several areas like forensic science, biosecurity, tracing of illegal trading of organisms
and pharmaceutical and herbal industries, among others. When there is an insufficient morphological or
anatomical data for the identification of a sample, a stretch of DNA sequence might be helpful in
identifying a species. Samples with multiple fragments can now provide multiple species identification,
giving a clear picture of habitat that offers a critical clue to the investigators. DNA barcoding works with
different identification fields and gives more accurate results of medicinal plant identification, i.e., DNA
barcode identification with chemical analysis and next-generation sequencing. The plant materials are
frequently encountered in criminal investigations but often overlooked as potential evidence. A forensic
investigation that seeks to match evidence to a particular plant would require an updated database of
samples. This requires the collection and genotyping of many samples from or near the crime scene. The
law enforcement officers and attorneys are not very much familiar with the science of botany
(Mohammed et al., 2017). So, the important plant-based evidence is often overlooked. Development of a
robust DNA barcode database with highly authenticated sequence information will greatly contribute to
the future of forensic botany. Hallucinogenic compounds are pharmacological agents banned in most of
the states or countries. They cause changes in perception, thought, emotion, and consciousness. Such kind
of plants producing hallucinogens has been detected using DNA barcoding technique in some of the
forensic studies (Mishra et al., 2016).

CONCLUSION
DNA barcoding is a system for fast and accurate species identification which will make ecological system
more accessible. It has many applications in various fields like controlling agricultural pests, sustaining
natural resources, protecting endangered species, monitoring water quality, preserving natural resources,
protecting endangered species and identification of medicinal plants. Since the last decade, DNA
barcoding has been attracting a lot of interest all over the world. Researchers working in this field are
busy in finding a more superior and desirable universal DNA barcode for an efficient conservation of the
biodiversity. Since a major problem of barcoding lies in the case of plants, the research carried out so far
in this area has been reviewed including the futuristic approaches.

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