DNA (meta)barcoding of biological invasions: a powerful tool to elucidate invasion processes and help managing aliens

Abstract

Biological invasions are a major threat to the world’s biodiversity with consequences on ecosystem structure and functioning, species evolution, and human well-being (through ecosystem services). Conservation of biological diversity and management of biological resources require multi-level management strategies on non-native species, in order to (1) prevent biological introductions, (2) detect non-native species at an early stage of the introduction, and (3) eradicate or maintain at a low level of population density non-native species that were successfully introduced. A pre-requisite to any control measures on non-native species is the ability to rapidly and accurately identify the putative threatening alien species. DNA barcoding, and its recent extension, DNA metabarcoding are complementary tools that have proved their value in the identification of living beings. Here we review their use in the identification of non-native species at several steps of the introduction processes, and how they can be applied in the control and management of biological introductions. Through examples covering various taxa and ecosystems (terrestrial, freshwater, marine), we highlight the strengths and weaknesses of approaches that we foresee as crucial in the implementation of early warning strategies.

Full text

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Biological Invasions
ISSN 1387-3547
Volume 17
Number 3
Biol Invasions (2015) 17:905-922
DOI 10.1007/s10530-015-0854-y
DNA (meta)barcoding of biological
invasions: a powerful tool to elucidate
invasion processes and help managing
aliens
Thierry Comtet, Anna Sandionigi,
Frédérique Viard & Maurizio Casiraghi

1 23
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MOLECULAR TOOLS
DNA (meta)barcoding of biological invasions: a powerful
tool to elucidate invasion processes and help
managing aliens
Thierry Comtet •Anna Sandionigi •
Fre
´de
´rique Viard •Maurizio Casiraghi
Received: 18 May 2014 / Accepted: 4 February 2015 / Published online: 10 February 2015
ÓSpringer International Publishing Switzerland 2015
Abstract Biological invasions are a major threat to
the world’s biodiversity with consequences on ecosys-
tem structure and functioning, species evolution, and
human well-being (through ecosystem services).
Conservation of biological diversity and management
of biological resources require multi-level manage-
ment strategies on non-native species, in order to (1)
prevent biological introductions, (2) detect non-native
species at an early stage of the introduction, and (3)
eradicate or maintain at a low level of population
density non-native species that were successfully
introduced. A pre-requisite to any control measures
on non-native species is the ability to rapidly and
accurately identify the putative threatening alien
species. DNA barcoding, and its recent extension,
DNA metabarcoding are complementary tools that
have proved their value in the identification of living
beings. Here we review their use in the identification
of non-native species at several steps of the introduc-
tion processes, and how they can be applied in the
control and management of biological introductions.
Through examples covering various taxa and ecosys-
tems (terrestrial, freshwater, marine), we highlight the
strengths and weaknesses of approaches that we
foresee as crucial in the implementation of early
warning strategies.
Keywords DNA barcoding DNA metabarcoding 
Alien species Early warning Environmental DNA 
Next generation sequencing High throughput
sequencing
Introduction
For many centuries, human-related actions have
brought together, both accidentally or deliberately,
species that have typically diverged millions of years
ago. Biological introductions of non-native species in
distant regions have thoroughly changed our percep-
tion of the world’s biodiversity, in some cases with
relevant consequences (e.g. Chapin et al. 2000; Sakai
et al. 2001). Where established introduced species
became proliferating (both widespread and locally
dominant, i.e. invasive, according to the definitions of
Colautti and MacIsaac 2004), they may pose severe
problems both environmentally and economically.
Biological invasions are thus one of the major open
challenges in the latter-day societies (Simberloff et al.
2005,2013). To cope with this huge problem,
management measures have to be implemented which
T. Comtet F. Viard
Sorbonne Universite
´s, CNRS, UMR 7144, Station
Biologique de Roscoff, UPMC Univ Paris 06, Place
Georges Teissier, 29688 Roscoff Cedex, France
A. Sandionigi M. Casiraghi (&)
ZooPlantLab, Department of Biotechnology and
Biosciences, University of Milan-Bicocca, Piazza della
Scienza, 2, 20126 Milan, Italy
e-mail: maurizio.casiraghi@unimib.it
123
Biol Invasions (2015) 17:905–922
DOI 10.1007/s10530-015-0854-y
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include preventing introductions, early detection of
introduced species, eradication or maintenance at a
low level of population density (Simberloff et al.
2005; Simberloff 2014). Regulations have been set up
in many countries to face these important issues. For
instance, in Europe, the Council Regulation No
708/2007 of 11 June 2007 was established to limit
the environmental risks related to the introduction of
non-native species in aquaculture. And even more
recently, the European Community adopted a new
regulation (CR No 1143/2014 of 22 October 2014)
about alien species to ‘‘prevent, minimize and mitigate
the adverse impact on biodiversity of the introduction
and spread within the Union, both intentional and
unintentional, of invasive alien species’’ (http://ec.
europa.eu/environment/nature/invasivealien/index_
en.htm). These regulations all recognize the priority of
the rapid identification and detection of non-native
species. To be effective these regulations and other
legal frameworks (e.g. International Convention for
the Control and Management of Ships’ Ballast Water
and Sediments; IMO 2004), however, required accu-
rate species identification, More specifically, powerful
tools are requested for: (1) identifying species at all
developmental stages; (2) detect biological introduc-
tions even at low concentration of the introduced or-
ganism; (3) develop efficient and affordable systems
for early warning.
To answer these questions, DNA-based molecular
tools have been widely applied (Darling and Blum
2007; Le Roux and Wieczorek 2009). DNA-based
detection has numerous advantages over traditional
approaches (Darling and Blum 2007). In brief, these
methods (1) are standardized tool allowing direct
comparison among different users; (2) do not require
expertise on different taxonomic groups; (3) can be
applied to complex matrices (e.g. environmental
samples which comprise a mix of several species,
like a soil or a water sample) and (4) can be used in
early warning allowing detection of low concentra-
tions of potential invaders, or even imprints of a
potential invader (Deagle et al. 2003; Ficetola et al.
2008).
Among the plethora of molecular approaches
available for identification of living beings, DNA
barcoding has become very popular since the begin-
ning of the 2000 s’ when parallel initiatives to use
‘‘universal and standardized’’ molecular methods for
species identification were proposed (Floyd et al.
2002; Hebert et al. 2003a,b,2004a,b; Blaxter et al.
2005). These initiatives led to the International
Barcoding of Life, a global network of research
centres involved in molecular species identification.
Briefly, DNA barcoding is a diagnostic technique in
which short standard DNA sequence(s), called DNA
barcodes, can be used for species identification.
Generally speaking it is based on the principle that
the intraspecific sequence polymorphism is lower than
the interspecific divergence, the difference between
the two being known as the barcoding gap (Meyer and
Paulay 2005), even if some questions about the
existence of the gap have been raised (Wiemers and
Fiedler 2007; Ferri et al. 2009). The wider the
barcoding gap is, the more reliable species discrimina-
tion will be achieved. DNA barcoding mostly differs
from other molecular identification techniques by the
use of standard marker(s), i.e. that can be applied to a
wide range of taxa (such as coxI widely used in
metazoans; ITS in fungi; rbcL,matK, and ITS in
plants; see some of the standard barcodes at BoLD
System, http://www.boldsystems.org). It is now well
accepted that the benefits of DNA barcoding include:
(1) enabling species identification at any life stage or
fragment, sometimes even in degraded samples; (2)
facilitating species discoveries; (3) promoting devel-
opment of handheld DNA sequencing technology that
can be applied in the field for biodiversity inventories
and (4) providing insight into the diversity of life (e.g.
Savolainen et al. 2005; Valentini et al. 2009; Casiraghi
et al. 2010; Bucklin et al. 2011). As for most other
methods, there has been much debate on the use of
DNA barcoding, especially on the barcode(s) to be
used (e.g. Vences et al. 2005; Hollingsworth et al.
2009; Bhadury and Austen 2010; Hollingsworth et al.
2011) (Box 1). However, even with its known
limitations, DNA barcoding has proven to be a greatly
powerful tool for species discrimination, which ex-
plains its popularity in various research areas (Neigel
and Stake 2007; Valentini et al. 2009; Radulovici et al.
2010; Sweeney et al. 2011; Galimberti et al. 2013).
The success in using DNA barcoding as a powerful
identification tool relies primarily on the accuracy of a
reference database, composed of voucher specimens (a
kind of equivalent to type specimens in traditional
taxonomy), morphologically-identified based on exist-
ing criteria, and associated to the sequence of a given
barcode (e.g. BoLD, Ratnasingham and Hebert 2007).
Any unknown specimen might thus be identified by the
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comparison of its sequence with those from the most
comprehensive database: it requires that most of the
intraspecific molecular variability at this barcode is
recorded (extensive coverage of the geographic range)
and that interspecific divergence with closely related
species is documented (taxonomic coverage) (Meyer
and Paulay 2005;Schefferetal.2006). In case of
incomplete coverage of a given taxonomic group, DNA
barcoding includes the possibility to either identify
specimens at a higher taxonomic level or to delineate
species based on divergence threshold, allowing
identifying the so-called Molecular Operational Taxo-
nomic Units or MOTUs. However, a MOTU is ‘‘only’’ a
molecular variant whose specific status has to be
demonstrated (Galimberti et al. 2012). The generation
of the global BoLD database, which is the main goal of
the international DNA barcoding initiative (Ratnasing-
ham and Hebert 2007), will help building the most
comprehensive dataset possible. To achieve compre-
hensiveness, however, requires tradeoffs, and if the
BoLD database is composed of sequences associated to
voucher specimens, it also comprises sequences
Box 1 The right barcode region?
The basic principle of DNA barcoding is to identify species based on one or a few standard loci that can be sequenced routinely
and reliably in a wide range of taxa. These loci are thus required to be universal and to have a high discriminatory power. The
latter criterion relies on the assumption that the history of the target sequence reflects the evolution of the species, and that the
molecular divergence within species is lower than between species. However, this assumption is not always confirmed due to
evolutionary genetic processes that may bias the DNA barcoding approach (Galtier et al. 2009; Hollingsworth et al. 2011)
In metazoans, the 5’ end of the cox1 gene (mtDNA) is the standard DNA barcode (Hebert et al. 2003b). The presence of nuclear
pseudogenes of mitochondrial origin (numts) and heteroplasmy are concrete problems that may make the use of DNA barcoding
harder (Hebert et al. 2004a; Buhay 2009). For example, heteroplasmy may be linked to the inheritance mode of the target gene,
as illustrated in the well-known case of some bivalve mollusk species, especially marine and freshwater mussels (Fisher and
Skibinski 1990): mitochondria are transmitted through a Doubly Uniparental Inheritance process, which lead to two lineages of
the mitochondrial genome (one male, one female) in individuals, so that all male individuals are heteroplasmic (Breton et al.
2007). Inheritance of the plastid genome is also important in the use of the plastid DNA barcodes in plants (paternally-inherited
in conifers, maternally-inherited in angiosperms) (Hollingsworth et al. 2011)
Non-neutrality may also be a problem (Galtier et al. 2009). If selective sweeps would increase the DNA barcoding gap (then the
discriminatory power of barcoding), balancing selection (that maintains high intraspecific diversity) would reduce it, thus
limiting the performance of DNA barcoding (Galtier et al. 2009). Furthermore, hybrid introgression (which decreases the
interspecific diversity) might decrease the efficiency of DNA barcoding (Galtier et al. 2009; Hollingsworth et al. 2011). All these
processes might explain while DNA barcoding is not powerful in some metazoans (e.g. Anthozoa, Porifera, Hydrozoa, Diptera,
lycaenid Lepidoptera; Meier et al. 2006; Neigel and Stake 2007; Wiemers and Fiedler 2007; Huang et al. 2008; Shearer and
Coffroth 2008) and in some plants (e.g. Berberidaceae and other examples; Roy et al. 2010; Hollingsworth et al. 2011)
Heterogeneous evolutionary rates of the barcode among taxa might also be a problem, especially if DNA barcoding is used to help
delineating species within complex groups based on distance thresholds (Galtier et al. 2009). In such cases, DNA barcoding
alone would not give an accurate identification and should be combined with other methods (i.e. integrative taxonomy; Dayrat
2005; Galimberti et al. 2012; Schlick-Steiner et al. 2010)
Technical problems may also impede DNA barcoding approaches, especially dealing with the ‘‘universality of universal primers’’.
Cox1 is commonly amplified with the Folmer primers (Folmer et al. 1994), but they often fail or perform poorly (e.g. Hoareau
and Boissin 2010; Geller et al. 2013). This may not be a problem for some applications, where the unknown specimen can be
first identified at a higher taxonomic level for which taxon-specific primers are available, but may prevent some other uses (e.g.
in DNA metabarcoding approaches). Newly designed universal primers may help resolve such limitations (Geller et al. 2013)
Ways to overcome some of the above problems would be to use a combination of several markers, keeping in mind that they
should be used following standardized procedures. For example, the matK locus, one of the two core plant DNA barcodes, can
be difficult to amplify using existing primer sets, particularly in non-angiosperms (Hollingsworth et al. 2011), and is then used in
combination with the rbcL locus, which is easy to amplify, but has a lower discriminatory power (Hollingsworth et al. 2011).
Combining multiple markers will however increase the analytical costs. A way to reduce such costs has been proposed recently
by Shokralla et al. (2014). These authors used NGS technologies to barcode individual specimens, by tagging individual
specimens during PCR amplification using unique oligonucleotides attached to the DNA barcoding PCR primers
Finally, for some particular applications, shorter DNA barcodes may be used. For example, DNA barcodes as shorter as 100 bp
were used successfully to identify museum specimens characterized by degraded DNA (Hajibabaei et al. 2006). Similarly, the
current limitations of next-generation sequencing led Leray et al. (2013) to use 313 bp DNA barcodes
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imported from other databases (like Genbank), for
which vouchers are not necessarily available.
In the last years, next-generation sequencing tech-
nologies (NGS, now better defined as High Through-
put DNA Sequencing or HTS) led to a major
breakthrough in DNA-based species identification,
with the development of DNA metabarcoding. In
2012, Pierre Taberlet and colleagues introduced this
term to ‘‘designate high-throughput multispecies (or
higher-level taxon) identification using the total, and
typically degraded, DNA extracted from an environ-
mental sample (i.e. soil, water, faeces, etc.)’’ .
Although the general goal of DNA barcoding and
metabarcoding is similar, i.e. identifying species or
higher-level taxa, they differ in a number of ways, like
their biological targets (single specimens vs. commu-
nities or environmental samples), the sequencing
technologies involved, and the data processing (for
recent reviews see for instance Ji et al. 2013, Cristescu
2014). To avoid any misunderstanding, it is important
to note that the term ‘metabarcoding’ does not imply
any functional analysis at the genome level (i.e. the so-
called ‘metagenomics’).
Since the very beginning of DNA barcoding, its
potential role to answer questions related to invasive
species has been recognized (Armstrong and Ball
2005). DNA barcoding and metabarcoding may help
resolve processes involved at all steps of the invasion
history: before introduction (biosecurity and risk
assessment); at the early phase of introduction (early
detection and identification of dispersal vectors);
establishment and spread (e.g. by allowing identifica-
tion of early life stages in the study of demography,
population dynamics and dispersal). In the present
article we are underlining the vast potential for using
DNA barcoding and DNA metabarcoding in both
research and management issues on biological
invasions.
DNA barcoding as a global identification tool
in biological invasions
Taxonomy is central to all fields of biology as most of
the knowledge about an organism is directly linked to
its species name. Using erroneous taxonomy may then
lead to errors in the assessment of biodiversity, in
experimental biology (e.g. misidentification of a
model species), and in conservation biology, and
may also have socio-economic consequences (Borto-
lus 2008). Consequences of species misidentifications
are even more important dealing with several aspects
related to invasion biology (Pys
ˇek et al. 2013). The
three following examples illustrate different kinds of
bad taxonomy in this field.
(1) Misidentification of species deliberately intro-
duced may facilitate invasions. As part of a
restoration project of tidal salt marshes of
California, the dense-flowered cordgrass Sparti-
na densiflora, a non-native cordgrass acciden-
tally introduced from Chile to Humboldt Bay,
was introduced in the seventies into San Fran-
cisco Bay with negative effects on the tidal
marsh flora because it has been misidentified as
a growth form of the native cordgrass S. foliosa
(Faber 2000; Bortolus 2008).
(2) Predicting the spread of aliens based on previ-
ous occurrence records through ecological
niche modeling is particularly sensitive to
taxonomic uncertainty: the potential invasion
range of the plant Pilosella glomerata in North
America based on all occurrence records was
predicted to be substantially larger than consid-
ering only ‘‘taxonomically reliable’’ records
(Ensing et al. 2013).
(3) Predicting the spread of an alien based on the
knowledge of its biology requires its accurate
identification. In Humboldt Bay, California,
non-native calyptraeid gastropods putatively
identified as Crepidula fornicata were later
(20 years) identified through DNA barcoding as
C. convexa, a species with a drastically differ-
ent developmental mode and dispersal potential
(McGlashan et al. 2008), with consequences in
management strategies.
These examples illustrate that accurate and rapid
identification of non-native species is required at all
steps of the invasion process and the management of
invasive species, from prevention to eradication and
control (Smith et al. 2008; Pys
ˇek et al. 2013).
Molecular tools and especially DNA barcoding have
(and still will) greatly contributed to achieve this aim.
Identification of unknown specimens
Dealing with identification and detection of non-native
species, many molecular methods are available (Bott
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et al. 2010; Darling and Blum 2007), but most of them
are targeted to a single or a few already known species
of known origin which diagnostic needs to be con-
firmed. This is true for PCR–RFLP, in situ hybridiza-
tion, qPCR, or the new hybridization coupled with light
transmission spectroscopy method (Egan et al. 2013;
Mahon et al. 2013), which all involve specific primers
or probes. However, most of the time invasion
ecologists do not have a specific biogeographic focus
and require taxonomic information from much larger
areas (essentially the whole world) for many taxa
(Pys
ˇek et al. 2013), which is almost impossible.
Because DNA barcoding does not require any a priori
knowledge on the origin or identity of the species to be
identified, it is particularly well suited to the identifi-
cation of species out of their native range. Further, the
power of this approach relies on the amount and quality
of sequences composing the databases, as well as their
taxonomic coverage (Saunders 2009; Darling and
Blum 2007). The development of the international
database BOLD (Ratnasingham and Hebert 2007) now
makes possible identification of a large number of
species out of their native range. Of course, efforts to
supply additional data have to be continued, not only to
add new species, but also to better assess the intra-
specific (including geographic) diversity. For example,
considering only the list of 100 of the world’s worst
invasive alien species (Lowe et al. 2000), 11 % of them
have no barcode deposited in BOLD yet, and most have
fewer than 15 barcodes (Fig. 1). Thanks to the
development of such databases, large-scale surveys
may lead to serendipitous detection of introduced
species. This is the case of the alien macroalga
Gracilaria vermiculophylla introduced in British
Columbia (Saunders 2009). In a similar way,
Radulovici et al. (2009) documented the northeastern
expansion of the non-native amphipod Echinogam-
marus ischnus in the Saint Lawrence Estuary.
DNA barcoding is then very robust for groups with
well-known taxonomy. It is particularly useful in some
taxonomic groups, which are very difficult to identify,
because of the paucity of diagnostic morphological
criteria, especially in fungi (Migheli et al. 2009), algae
(Geoffroy et al. 2012), bryozoans (Mackie et al. 2012),
and ascidians (Callahan et al. 2010; Smale and Childs
2012; Bishop et al. 2013). In these taxa, many non-
native species may be overlooked because of poor
taxonomy at the species level and poor knowledge of
their biogeographic status (Bishop et al. 2013).
Identification of early life-history stages
Contrary to many other features (including morpho-
logical characteristics) DNA does not vary during
ontogeny and then can be used across all life-history
stages and genders for identification purposes (e.g.
Casiraghi et al. 2010). It can be used for the
identification of eggs, spores, seeds, larvae and
juveniles of many species, for which knowledge on
early life stages is lacking or which lack morpho-
logical diagnostic criteria at these stages, especially
dealing with non-native species (Frischer et al. 1997;
Evans et al. 1998; Johnson and Geller 2006; Bott et al.
2010; Kaplan et al. 2010; Darling and Mahon 2011).
Under such circumstances, DNA barcoding is very
useful in providing accurate and rapid identification of
non-native species at early stages (Ball and Armstrong
2006; Harvey et al. 2009; Ascunce et al. 2009;
Mitchell and Maddox 2010; Pieterse et al. 2010).
The identification of early life stages of non-native
species has several interests:
(1) Early life stages may be involved in the
introduction per se. This is the case of the larval life
stages of marine invertebrates that can be transported
in ships’ ballast waters (Carlton 1985; Carlton and
Geller 1993; Gollasch et al. 2002; Carlton 1985;
Carlton and Geller 1993), or the eggs of insects that are
deposited on hard surfaces such as containers or ship
superstructures (Armstrong et al. 2003; Armstrong and
Ball 2005; Armstrong et al. 2003). Identifying these
early life stages may allow identifying the vectors of
introduction and assessing the risks associated. In this
context, by using DNA barcoding Manghisi et al.
(2010) identified the introduced marine macroalga
Agardhiella subulata in Italy, and suggested that the
vector of introduction was likely oyster aquaculture,
based on the identification of an immature specimen
on the shell of a cultivated oyster (Crassostrea gigas)
from an imported stock. On the other hand, DNA
barcoding was used successfully to inventory inver-
tebrates living in ships’ ballast sediments, focusing on
their diapausing eggs (Rotifera, Crustacea), and to
study their potential as introduction vector (Briski
et al. 2010,2011a,b).
(2) Once a particular alien has been established,
DNA barcoding may serve in the study of its
population dynamics, by allowing identification of
their early life stages. DNA barcoding may allow
studying larval supply or the relative abundance and
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recruitment periods of several non-native and native
species. For example, by identifying with DNA
barcoding alien insect larvae on the sub-Antarctic
Marion Island, Chown et al. (2008) evidenced the
establishment of a population of Agrotis ipsilon,
whereas it was thought that this species was only a
transient alien.
(3) In cases where data on the life cycle are lacking,
DNA barcoding may help to elucidate parts of it by
determining the species identity of the various devel-
opmental stages, which allows reconstructing the
developmental sequence of this species. We do not
know examples for non-natives species but two
interesting examples are Thomas et al. (2005) and
Ko et al. (2013). Thomas et al. (2005) described the
larval stages of three species of amphibians, after their
identification with DNA barcoding, while Ko et al.
(2013) proved the efficacy of DNA barcoding in the
identification of unknown larvae of fishes.
(4) In some particular applications, like the control
of pests of cultured plants, detecting early infestation
to provide efficient control measurements often
requires the identification of early developmental
stages (eggs, larvae). This is the case of the invasive
citrus root weevil Diaprepes abbreviatus for which a
DNA barcoding approach was developed for the
diagnostic of infestation by eggs and larvae (Ascunce
et al. 2009).
Identification of ancient samples: a way to assess
invasion history
Historical knowledge is fundamental to better under-
stand biological invasions, for example by allowing to
determine the non-native status of a species (Carlton
2009) or to determine its invasion history (Civille et al.
2005; Chauvel et al. 2006; Hoos et al. 2010; Estoup
and Guillemaud 2010), and to help design strategies
for their management (Swetnam et al. 1999). Accurate
taxonomy in historical records is then crucial when
these are used for management purposes (Ensing et al.
2013). DNA barcoding may then be applied to
historical samples from museums and herbaria to
confirm historical diagnostics in the light of more
recent knowledge, and may help trace back the origin
of introduction events. With such an approach, Pringle
et al. (2009) studied samples from herbaria previously
recorded as the introduced mushroom Amanita phal-
loides, dating back to 1911. They confirmed the
identity of samples since 1938, earlier records being
Fig. 1 Standard DNA barcodes available for 100 of the world’s
worst invasive alien species (list from Lowe et al. 2000).
Number of species for which DNA barcodes are available in the
Barcoding of Life Database (http://www.boldsystems.org/)
(updated 27th Feb, 2014). Based on 99 species of this list (the
Banana bunchy top virus has been discarded). For each species,
DNA barcodes have been searched through the Taxonomy
search tool. When synonyms were available, the number of
DNA barcodes is the sum of DNA barcodes deposited under all
synonyms
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different species of Amanita, and then confirmed the
introduction of A. phalloides on the West coast of
North America to occur in 1938. In some cases, DNA
barcoding may drastically change our view of a
particular introduction. This is the case for the
invasion by the horse-chestnut leaf-mining moth
Cameraria ohridella, which has been controversial
since the insect was first described in 1986 in Europe.
By using minibarcodes on remains of larvae pressed
within leaves preserved in herbaria across Europe,
Lees et al. (2011) confirmed the introduction of
C. ohridellaas early as 1879, more than a century
earlier than previously thought.
Towards DNA metabarcoding: changing the scale
of invasion assessment
From DNA barcoding to DNA metabarcoding
DNA barcoding is a very powerful tool to identify
individual specimens at the species level and has thus
been employed to confirm morphological diagnostic
of target aliens and to discover previously unrecorded
introduced species. This regular approach of DNA
barcoding requires the sorting of individual specimens
and the preliminary identification of potential un-
knowns to be barcoded. For taxa with incomplete
taxonomy or which morphological criteria are lacking,
the number of specimens to be barcoded to distinguish
the native and non-native taxa may be huge and the
regular approach may be time-consuming and expen-
sive (deWaard et al. 2009; Porco et al. 2013).
High-throughput detection of non-native species
using DNA barcoding thus appeared as speculation
less than a decade ago (Darling and Blum 2007). It has
now been made possible through the development of
next generation DNA sequencing (NGS) or High
Throughput DNA Sequencing (HTS) technologies
(Shendure and Ji 2008; Metzker 2010; Glenn 2011).
Based on sequencing procedures different from the
classical Sanger technology, HTS constitutes a step-
change in sequencing capacities, providing billions of
sequence reads in a single run, quickly and at low cost.
HTS has revolutionized DNA-based research, includ-
ing biodiversity assessment from complex environ-
mental samples (i.e. comprising multiple species)
through DNA metabarcoding (Bik et al. 2012; Epp
et al. 2012; Shokralla et al. 2012; Taberlet et al. 2012;
Yoccoz 2012). Such a sequencing depth theoretically
allows recovering the whole diversity of the sample,
and offers the possibility to analyze simultaneously
several samples (through multiplexing) identified by
ligating unique short indexing oligonucleotide tags
(Parameswaran et al. 2007).
Although differing in the technology used to obtain
the barcodes of species to be identified, DNA
metabarcoding relies on the same general principle
as regular DNA barcoding for species identification, in
that unknown barcodes will be compared to barcodes
originating from well-identified voucher specimens
included in reference databases. In this regard, one
limitation of the use of metabarcoding in describing
diversity is the current limited availability of reference
databases (Cristescu 2014; see discussion on that point
below). The short read lengths generated have first
limited the use of NGS for DNA metabarcoding
purposes to the Roche 454 pyrosequencing technique
which allowed ca. 400 bp read length (for comparison,
the standard cox1 barcode is 650–700 bp). More
recent versions of this technology allow the sequenc-
ing of up to 600–800 bp reads (Loman et al. 2012;
Shokralla et al. 2012), but recent developments of
other technologies like the Illumina MiSeq have
brought some equivalent read length (2 9300 bp,
paired-end reads; http://www.illumina.com/systems/
miseq/performance_specifications.ilmn), with higher
throughput and lower costs (Loman et al. 2012).
Pending further improvements in read length, mini-
barcodes may constitute a successful alternative
(Leray et al. 2013).
Why have less when you can have more?
Given this potential power of DNA metabarcoding,
will it replace regular DNA barcoding in the future?
The discussion below aims to highlight how DNA
metabarcoding will complement DNA barcoding in
addressing different issues and processes, and to
discuss its main current limitations and how they
may be solved for a routine use.
Early detection
Early detection of non-native species implies our
ability to detect very low numbers of introduced
specimens lost within the native community. As an
identification tool, DNA barcoding would help
DNA (meta)barcoding of biological invasions 911
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identifying specimens that are suspected of being non-
native once they have been recovered, but does not
help finding them among thousands of native organ-
isms. Some examples however exist where DNA
barcoding was applied systematically to identify
multiple taxa (deWaard et al. 2009; Porco et al.
2013), a very time-consuming and expensive task. For
example, deWaard et al. (2009) barcoded more than
900 individual lepidopterans, thus identifying 190
species, including 31 non-natives, of which 4 were
new to the area. If DNA barcoding cannot help find the
needle in the haystack, DNA metabarcoding can,
thanks to its sequencing depth and its ability to detect
rare species.
Because metabarcoding provides millions of se-
quences from a bulk sample, it has the potential to
assess the whole diversity of a given community (e.g.
Cristescu 2014), with increased speed and lower cost
than traditional approaches. Integrated to regular
monitoring programs, it may then allow detecting
non-native species either through the comparison of
metabarcoding-based diversity assessment with spe-
cies lists available for a given area, or through long-
term metabarcoding surveys. DNA metabarcoding is
very sensitive and allows the detection of rare species,
provided that replication level is adequate to reduce
false negatives (Ficetola et al. 2014). Particular
attention must also to be paid when filtering sequences
to remove artifacts in Quality Control (QC) step
(Fig. 2), because overly stringent filtering level may
lead to a failure in the detection of rare species (Zhan
et al. 2014b). Recent studies focusing on the detection
of rare species in aquatic communities showed that
DNA metabarcoding through 454 pyrosequencing
allowed detecting as little as one larva of different
taxa (echinoderms and mollusks) in plankton and
sediment samples containing a wide array of eukary-
otes (Pochon et al. 2013; Zhan et al. 2013). However,
some counterexamples exist (e.g. Hajibabaei et al.
2011), but this ability to detect very low abundances
(down to 2.3 910
-5
% of sample biomass in the
example of Zhan et al. 2013) makes DNA metabar-
coding a very promising method to allow early
detection of new (i.e. not documented yet) non-native
species. Furthermore, applying DNA metabarcoding
to the analysis of environmental DNA (Box 2;
Bohmann et al. 2014) would allow detecting DNA
traces originating from putative non-natives (Ficetola
et al. 2014), even if in some cases the approach may
fail to detect introduced species (Andersen et al.
2012).
Relative abundance of non-natives
within the community
Species richness can be assessed with DNA metabar-
coding, including rare species (see above). However,
estimating abundances remains a challenge, and some
authors even suggested using metabarcoding data only
as presence/absence data (Yu et al. 2012). If absolute
abundances (number of individuals) cannot be
assessed, the estimation of relative abundances within
a sampled community is however crucial to describe
its biological diversity and its variations. This is also
true when dealing with biological invasions, relative
abundances of native and non-native species being
essential to assess the status of the introduced targets
(early stage, established, invasive) or the potential
interactions of the aliens and the native species, and
how they vary on a temporal scale. Evaluating the
potential for accurate estimation of relative abun-
dances requires the use of artificial communities
whose composition is controlled (i.e. ‘‘mock commu-
nities’’), or natural communities spiked with known
number of target specimens (Zhan et al. 2013,2014b),
or may be based on the comparison between metabar-
coding and more classical approaches based on
morphological identification (Lindeque et al. 2013).
Although some early studies failed in finding a
correlation between the relative number of sequences
assigned to a taxon and the relative abundance of this
taxon in a given sample (Porazinska et al. 2009),
others found such a correlation and suggest that
relative abundance of sequences may be used as a
proxy for relative abundance of taxa within sampled
communities (Porazinska et al. 2010; Hajibabaei et al.
2011), Similarly, a correlation between relative abun-
dance of sequences and relative biomass or relative
abundance of individual species has been shown from
eDNA samples (Thomsen et al. 2012; Andersen et al.
2012). Further improvements in approaches standard-
ization, optimised sampling strategy and development
of analytical methods (McMurdie and Holmes 2014)
would led to better estimations of relative abundances.
For example, an alternative solution to improve the
estimation of relative abundances has recently been
proposed by Zhou et al. (2013), avoiding the use of
PCR amplifications in the HTS pipeline. Based on the
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recovery of mitochondrial gene sequences, it consists
in a mitochondrial enrichment directly followed by
Illumina shotgun sequencing, at an ultra-high se-
quence volume. Using this technique, Zhou et al.
(2013) found a relatively strong correlation between
the sequencing volume (number of nucleotides) and
the total biomass for a given species from a bulk
sample of terrestrial insects.
Taxonomic assignment
In DNA metabarcoding, even more than for traditional
DNA barcoding, the availability of curated reference
databases is a major limitation to the assignment of
sequences to species, primarily because with such a
massive sequencing many species have the chance to
be absent from global databases (Cristescu 2014)
(local databases might be an alternative; Casiraghi
et al. 2010). However, as in standard DNA barcoding,
barcodes might be grouped as unassigned (to the
species level) MOTUs. Although they are difficult to
use for a management point of view, MOTUs might
still be of value (Cristescu 2014). MOTUs can first be
assigned to supra-species levels, like genus or family,
which could be recognized as non-native. Second, in
cases where metabarcoding is used in long term
surveys, the discovery of new unassigned MOTUs can
suggest the introduction of a non-native species or the
proliferation of a native that was quite rare before.
Although the exact reason for such an appearance
might not be known, the observation would draw
attention and advocate for further investigations. In all
A
B
Fig. 2 Schematic workflow of the DNA metabarcoding pipe-
line when reference databases are or are not available. The first
part of the scheme is relative to the standard procedures in HTS
environments, and then two scenarios are possible: awhen a
comprehensive reference database (like those ones made
available in BOLD system) is accessible, most sequences can
be assigned to species, and DNA metabarcoding would be
powerful to detect alien species and follow their spreading;
bwhen a comprehensive reference database is not available,
DNA metabarcoding could not lead to a specific identification in
all cases. In these conditions it is necessary to use public
databases (such as NCBI) that is known to contain many
taxonomic errors and under-coverage in several parts of the Tree
of Life. Under these premises it is likely to reach an
identification of molecular variability (i.e. MOTUs) only, that
is anyway the first and essential step to achieve a global
overview of the biodiversity
DNA (meta)barcoding of biological invasions 913
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cases, the continuous supply of barcodes to global
databases still offers the possibility that an unassigned
MOTU may be assigned later.
Taxonomic bias
One other limitation is the taxonomic bias that may
arise from PCR: for example, if primers are not truly
universal, then some taxa may not be amplified (or less
efficiently amplified) and not detected although pre-
sent in the sample (Taberlet et al. 2012; Clarke et al.
2014). Taxonomic bias may also impair the estimation
of relative abundances of different taxa (Porazinska
et al. 2009). New technical developments would allow
PCR-free DNA metabarcoding: for example a new
Illumina
Ò
pipeline has been developed to recover
biodiversity of bulk experimental insect samples,
showing promising results (Zhou et al. 2013). How-
ever, applications to natural samples have still to be
carried out.
DNA versus organisms
Detecting DNA is not necessarily detecting living
organisms (see Box 2for a short review about
environmental DNAs or eDNAs). This has been
recently reviewed by Darling and Mahon (2011),
who focused on the example of the potential spread of
the introduced Asian carp in the Great Lakes region:
based on eDNA analysis, the identification of the fish’s
DNA in areas where the species was not recorded yet
would not necessarily reflect recent colonization but
might be explained by various sources, even in the
absence of any fish (e.g. excrements of birds that had
eaten carp). This is a known drawback in all the
molecular diagnostic tools, but on the other side it is
also one of their strength, because it allows detecting
traces (see above). Such potential false positives (i.e.
DNA is there but not individuals are present) are likely
to occur at high frequency in highly diffusive and
dispersive habitats (like water or air). In particular,
false positives represent a serious problem when legal
consequences follow the detection of living beings,
such as the case of alien introductions. Of course, we
are aware of this important limitation, but we want to
stress two relevant aspects, the first more general, and
the second more technical. In general, it has to take
into consideration that early warning is crucial to
facing biological invasions. Most of the invasions
could have been avoided if restrictive measures were
rapidly adopted. An effective control is the result of a
well-integrated system, in which every node (scien-
tists, politicians, managers) plays a key role. An early
detection of eDNAs, even if not related to the presence
of viable organisms, is a ‘‘red flag’’ indicating that the
sample was in some way exposed to the organism (or
part of it) and it is potentially dangerous. Additional
protocols, a good level of sampling replication and a
verified sequence database are then necessary (Fice-
tola et al. 2014) but their implementation may be much
Box 2 Particular case of environmental DNA (eDNA)
In the metabarcoding approach, DNA is typically retrieved from environmental samples (i.e. water, soil, plankton samples)
composed of a complex mix of organisms. However, environmental samples also contain short DNA molecules (free, cellular
debris or particle-bound), which are released by organisms, and have been called environmental DNA, or eDNA. Because DNA
is a particularly stable molecule, it is possible to observe eDNA presence in complex matrices such as waters and soils after
months or even after several years (Dejean et al. 2011; Yoccoz et al. 2012; Thomsen et al. 2012), so that eDNA forms a
molecular signature of the inhabiting species that could be observed (Ficetola et al. 2008). The use of eDNA may allow efficient
detection of rare species in aquatic ecosystems, being in some cases more sensitive than conventional (i.e. sorting and counting)
surveys (Jerde et al. 2011; Thomsen et al. 2012), which is a key feature in the early detection of potential invaders
eDNA was first used to detect the presence at low densities of the American bullfrog (Lithobates catesbeianus), invasive in
Western Europe, through PCR of a short fragment of the cytb mtDNA using species-specific primers and through Roche 454
pyrosequencing (Ficetola et al. 2008; Dejean et al. 2012). The species was detected in 15-mL samples collected in ponds. The
same approach was used to determine the invasion front of two freshwater fishes, the silver carp and the bighead carp, in the
Great Lakes region (Jerde et al. 2011). Quantification of species density from the study of eDNA is still poorly documented. By
using quantitative PCR (qPCR) Thomsen et al. (2012) showed a positive correlation between DNA concentration and estimated
population density based on conventional monitoring, suggesting that eDNA could be used to estimate population densities,
which however still requires further investigations
DNA metabarcoding on eDNA samples using minibarcodes (Hajibabaei et al. 2006) was successfully used to survey the diversity
of aquatic systems (Thomsen et al. 2012), and could thus be applied for the early detection of introduced species, even if DNA
has been degraded
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simpler because the potential hazard is known. In an
integrated system scientists give a first warning,
politicians alert managers and the system is ready to
for a rapid answer.
From a technical point of view, the viability
problem is well known in HTS analyses (Zhan et al.
2014b). Indeed, the detection of bacterial DNA is not
enough to establish the ‘‘real presence’’ of faecal
coliforms in drinking water and the coupling with
vitality tests is mandatory. This is mainly due to the
use of a singular molecular marker, which is often not
sufficiently species-specific (i.e. the ‘‘Species’’ level is
accessible only when the reference database are
verified) (see Fig. 2and Deagle et al. 2014). More
work is necessary to improve this aspect of HTS based
researches, but the analysis of the level of expression,
possible with HTS analyses is very promising and it is
likely that more solutions will be made available in the
next future.
A further aspect that has to be taken into account is
the quantification of living beings starting from DNA
detection. This is another well-know issue in HTS
environments. For sure there are many factors affect-
ing the amount of reads of a certain species/OTU
present in the sample and there is not the possibility to
simply convert reads in number of organisms. We
would like to stress again that we are not promoting
HTS analysis as the only solution to the invasive
species. Integrated systems, with interacting tech-
niques, are necessary. HTS is the ideal method for
early warning; we should always keep it in mind.
Standardization and transferability
DNA barcoding is a standardized method of identifi-
cation. For DNA metabarcoding to be used in
biosecurity and regulation contexts, and to improve
protocols and data transferability, it would need also
some standardization, at all steps of the analytical
pipeline (i.e. sampling, DNA extraction, PCR, bar-
code, sequencing technique, sequence analysis pipe-
line and so on), which is not achieved yet (Cristescu
2014). In particular, several sequencing technologies
are available and evolve rapidly (e.g. Glenn 2011). In
addition, although it is tempting to focus on metabar-
coding based on standard DNA barcodes (e.g. cox1)
used to build reference libraries (Hajibabaei et al.
2011) many metabarcoding projects involve different
markers (Cristescu 2014) for which detection power
and PCR efficiency vary widely (Zhan et al. 2014a). A
future breakthrough in biodiversity analysis provided
by metabarcoding is thus a shift towards multiple
marker approaches, a fundamental change in modern
integrative taxonomy, species delimitation and species
identification (Carstens et al. 2013; Cristescu 2014).
Most DNA-based approaches to detect non-native
species have the advantage that they are available to
most laboratories, and that they avoid the need for
taxonomic expertise, which may be expensive or
difficult to obtain rapidly (Darling and Mahon 2011;Ji
et al. 2013). It is not sure that this advantage is still true
with DNA metabarcoding, mostly because it also
requires a high level of expertise in bioinformatics
(Coissac et al. 2012; Zhou et al. 2013; Cook et al.
2013), and homemade data analyses procedures usu-
ally require high computational power, particularly in
wide DNA metabarcoding projects. Such require-
ments are currently not available to most laboratories
and even less to management agencies. Recent
progress, however, has been made and many lab
services are now available on the market, which
perform basic bioinformatics analyses allowing non-
experts to get some results. Although a good oppor-
tunity, these basic analyses generally do not allow
extracting all the information contained in the data
generated, because they do not reach optimization and
the depth of analysis required to avoid false negatives
and biased results.
Despite the limitations stated above, DNA metabar-
coding will be a huge step forward in the study and
monitoring of biological invasions, in complement to
DNA barcoding, In particular it should be implement-
ed in early detection procedures. Integrated in
monitoring it will allow drawing the baseline of local
diversity, and detecting very early a new potential
introduction, or at least drawing some attention to
dedicate particular surveys to search for the presence
of an alien species.
DNA barcoding, biosecurity and regulations
Preventing introductions and early detection of intro-
duced species are crucial pre-requisites to respond
quickly and at lower costs to biological introductions
(Simberloff et al. 2005). Inspections, quarantine
procedures, and monitoring programs are key compo-
nents of the management of biological introductions
DNA (meta)barcoding of biological invasions 915
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and constitute the basis of biosecurity measures
(Genovesi and Shine 2011). The ease of use of DNA
barcoding, its low cost, the low level of false positive
results, all argue for its broad application in manage-
ment strategies (Darling and Mahon 2011), and the
great potential of DNA metabarcoding (low cost,
community-scale, high sensitivity; see discussion
above) makes it an essential tool for the management
of biodiversity (Ji et al. 2013). Although not routinely
yet, DNA barcoding and metabarcoding are now
implemented in some biosecurity protocols (Amaral
et al. 2013; Levy et al. 2014).
DNA barcoding represents a significant improve-
ment in providing identification tool for controls at
country borders, in quarantine procedures, trade
statistics and compliance with international certifi-
cations or legal standards. Early work by Armstrong
and Ball (2005) clearly demonstrated that DNA
barcodes improved the identification of tussock
moth (Lepidoptera) and fruit fly (Diptera) eggs and
larvae intercepted at the New Zealand border from
86 to 93 %, as compared to previous molecular
method.
DNA barcoding may further help preventing acci-
dental introductions, for example through aquaculture,
one of the most important vector in aquatic habitats
(Naylor et al. 2001), by analyzing the fauna associated
to species imported deliberately. For example, egg
capsules of the oyster drill Ocinebrellus inornatus
were identified through DNA barcoding in European
oyster beds from the Limfjord, Denmark, which led to
quarantine procedures before import of Danish oysters
in The Netherlands (Lu
¨tzen et al. 2012). Similarly, it
may help preventing the introduction of non-native
parasite species infecting deliberately introduced
hosts. For example, using ITS sequences, Moss et al.
(2007) revealed the presence of two species of
protistan parasites (Perkinsus) in oysters Crassostrea
ariakensis that were planned to be transferred from
Asia to the USA. In this regard, DNA barcoding may be
a solution to answer some requirements of internation-
al regulations. For instance, the European Council
Regulation No 708/2007 requires an authorization for
any aquaculture project concerning an alien species
(http://europa.eu/legislation_summaries/environment/
nature_and_biodiversity/l28179_en.htm) which should
include the name and characteristics of the species. In
species for which morphological identification is diffi-
cult, a certification by DNA barcoding would allow
circumventing the risk of put in the farm the wrong
species.
Similarly, monitoring the diversity transported in
ships’ ballast water and sediment would undoubtedly
gain from DNA barcoding and DNA metabarcoding in
the framework of risk assessment analyses, by allow-
ing the identification of multiple taxa (Briski et al.
2010, Briski et al. 2011a,b). This will help for
example to be in compliance with legal requirements
of the International Convention for the Control and
Management of Ships’ Ballast Water and Sediments
(IMO 2004), in particular in situations where exemp-
tions are requested.
Finally, accurate taxonomy is crucial for commer-
cial species, with numerous examples of different
species sold under the same vernacular name. DNA
barcoding is an ideal tool to solve such problems and
avoid the introduction of incorrectly named species, in
the ornamental plants trade (Ghahramanzadeh et al.
2013) or in the aquarium trade (Collins et al. 2012).
This latter study led to the estimation that up to 25 %
of commercial cyprinid fishes could be mislabeled in
the ornamental fish industry, based on morphology
alone, highlighting the need for complementary tools.
In the future eDNA might be particularly interest-
ing in terms of biosecurity surveys, especially during
quarantine measures, as it is non-destructive and may
be used after the target species have been removed (see
Box22). This approach has been tested by using
species-specific primers for a 95-bp fragment of the
coxI, that targeted one fish species in tanks that had
contained this target (both alone and in mixed
samples) under a simulated international transit sce-
nario. The target species was successfully detected at
densities well below those at which fishes are typically
transported, even within mixed samples. This ap-
proach was then successfully applied in a quarantine
facility in New Zealand in an operational biosecurity
complex (Collins et al. 2013).
Several biosecurity agencies now recognize DNA
barcoding as a powerful identification tool and
recommend and sometimes used it in operations
(Mountfort and Hayden 2006). DNA barcoding is
thus routinely used by government agencies in New
Zealand and Australia for survey purposes, for exam-
ple for the detection of the invasive ascidians Didem-
num vexillum and D. perlucidum (Smale and Childs
2012). DNA barcoding is also mentioned in some
standardized protocols for pest detection, for example
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in the FAO document ISPM N°27 (International
Standards for Phytosanitary Measures) ‘‘Diagnostic
Protocols for Regulated Pests’’, or in the United States
Department of Agriculture which uses DNA barcod-
ing to track the invasive light brown apple moth
Epiphyas postvittana in California (Floyd et al. 2010).
In New Zealand, since 2005, DNA barcoding has been
used routinely to control the presence at the border of
the highest risk insect species, fruit flies and lymantri-
id moths, particularly the pink gypsy moth Lymantria
mathura, the yellow peach moth Canocethes punc-
tiferalis and the fall web worm Hyphantria cunea
(Armstrong 2010). In Europe, the QBOL project
funded under the 7th Framework Program of the
European Union was conducted to develop DNA
barcoding as a diagnostic tool to identify quarantine
plant pests or pathogens from a wide range of taxa
(arthropods, bacteria, fungi, nematodes, phytoplasma
and viruses) (Bonants et al. 2010).
Conclusion
DNA barcoding has led to a revolution in the
identification of living beings. Its strength mostly
relies on its integrative nature and standardized
workflow. Furthermore, the development of an inter-
national database of DNA barcodes with a wide
taxonomic coverage allows the identification of
unknown specimens with no biogeographic a priori,
which allows to conduct programs of biodiversity
surveys at both local and global scales. For these
reasons DNA barcoding played and will continue to
play a pivotal role in the study and management of
biological invasions.
Its universality and standardized procedures make
it a very powerful tool to be implemented in
operational strategies for the prevention and early
detection of biological introductions. It is already used
in routine by some biosecurity agencies to monitor
some target aliens, and there is no doubt that its use
will still increase in the near future.
Similarly, it is a safe bet that DNA metabarcoding
will be used more and more, especially in early
warning systems and for studying the community in
which non-native species are introduced or estab-
lished. By assessing both the native and non-native
biodiversity simultaneously, it would be very efficient
in the detection of new species, particularly in regions
where the local diversity is well known and where
regular surveys are conducted. This would, however,
still require both technical (e.g. quantification),
analytical (bioinformatics tools), and coordination
(databases) improvements before it may be used
routinely by scientists and environment agencies.
DNA barcoding and metabarcoding should not be
considered competing approaches but as complemen-
tary tools to help in the detection and management of
nondigenous and invasive species at different steps.
Whereas DNA metabarcoding would be a key tool for
the early detection of yet-unseen aliens, DNA bar-
coding will still be valuable to confirm the identifica-
tion of individual specimens for which accurate
diagnostic is problematic with more traditional
techniques.
Acknowledgments We are grateful to Stefano Piraino, John
Darling, Esther Lubzens and Gary Carvalho for inviting us to
contribute to this special issue. The publication of this paper is
supported by CoNISMa (Italian National Interuniversity
Consortium for Marine Sciences), which received funding
from the European Community’s Seventh Framework
Programme (FP7/2007-2013) for the project VECTORS
(http://www.marine-vectors.eu). This paper stems from the In-
ternational workshop MOLTOOLS (Molecular Tools for
Monitoring Marine Invasive Species), held in Lecce, Italy, in
September 2012. We thank the two anonymous reviewers for
their comments and suggestions that improved the manuscript.
This review benefited from insights and outcomes from various
projects we would like to acknowledge: the Interreg IVa Mar-
inexus; the AXA Research Fund (AXA Marine Aliens and
Climate Change Project); the Bibliothe
`que du Vivant sequenc-
ing programme; Programmi di ricerca scientifica di rilevante
interesse nazionale 2007 (PRIN 2007): ‘‘Nuova metodica per
l’analisi della biodiversita
`: un’applicazione del pirosequen-
ziamento allo studio degli organismi del suolo’’; the following
grants funded by Fondazione Cariplo: ‘‘Dai geni all’ecosistema:
il DNA barcoding come supporto innovativo per la protezione
della biodiversita
`e l’analisi della funzionalita
`delle reti eco-
logiche’’; ‘‘Le connessioni ecologiche nelle selve castanili nel
Parco Regionale Campo dei Fiori: valutazione e sviluppo di
sistemi di gestione’’; ‘‘Il corridoio ecologico del Lambro: in-
terventi per il consolidamento e l’implementazione della con-
nettivita
`e della biodiversita
`’’; ‘‘Seminare biodiversita
`: il ruolo
dell’avifauna migratrice nell’implementazione della biodiver-
sita
`del Parco Monte Barro’’.
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