22
DNA barcoding in floral and
faunal research
S. E. MILLER
22.1 Introduction
As I write this chapter in mid 2012, the context in which floral and faunal research is
done is changing rapidly. Demand for biodiversity information, especially to under-
stand global change, is increasing. The technologies that are available for carrying
out biodiversity research and for disseminating the results are changing dramati-
cally. The funding processes and organisational cultures of the institutions that have
been the traditional homes of such research are evolving. This creates new chal-
lenges and opportunities for floral and faunal research. This chapter will focus on
the intersection of DNA barcoding with floral and faunal research, the opportunities
that DNA barcoding offers for increasing the quality and quantity of such work, and
its connectivity with related activities. I will discuss the opportunities for DNA
barcoding and then provide an example from my own research. The essay focuses
on plants and animals, but will include some reference to other organisms. Because
of the rapid evolution of the underlying technologies, and the related social
changes, this essay represents a ‘slice of time’, and I expect some of the conclusions
will be out of date before it is published. While challenges remain, I believe this is an
exciting time of renaissance for taxonomy (Miller, 2007).
A DNA barcode is a short gene sequence taken from standardised portions of the
genome, used to identify species. Being DNA based, it can be used for specimens
Descriptive Taxonomy: The Foundation of Biodiversity Research, eds M. F. Watson,
C. H. C. Lyal and C. A. Pendry. Published by Cambridge University Press.
© The Systematics Association 2015
without morphological characters necessary for traditional identifications, such as
roots or immature insects. Being a short sequence, it can be extracted from material
that has not been specially preserved, and can often be extracted from standard
museum or herbarium specimens. Being from a standardised region allows the use
of universal primers for unknown taxa and allows rapid compilation of a global
reference library. The choice of gene regions has focused on species level identi-
fication. While barcoding is not intended as a tool for higher classification research
or for population genetics research, sometimes the barcoding gene regions have
useful information at those levels (Craft et al., 2010).
22.2 The present status of DNA barcoding
While molecular diagnostics have been used for many years, DNA barcoding as a
global initiative started by Hebert et al. (2003), who, among other things, pointed
out that a standardised choice of gene region for species-level diagnosis allows
individual projects of whatever scale to contribute to a global reference library
that becomes increasingly more powerful over time. Mitochondrial Cytochrome
c oxidase I (COI) had been widely used in animals since Folmer et al. (1994) and
was quickly adopted as a community standard following Hebert et al. (2003).
Standardised gene regions for plants proved more challenging for technical rea-
sons, but are now designated as rbcL and matK, with additional research on
trnH-psbA (Hollingsworth et al., 2009). The nuclear ribosomal internal transcribed
spacer (ITS) region has been selected as a universal DNA barcode marker for Fungi
(Schoch et al., 2012). Explorations are under way in other taxa for appropriate
standardised gene regions. The Consortium for the Barcode of Life (CBOL) was
established in 2004 as an international membership organisation to help develop
barcoding methodology and set standards, such as the ‘barcode’ reserved keyword
in GenBank (Benson et al., 2012). GenBank1 and its international partner databases,
the European Molecular Biology Laboratory (EMBL) and the DNA Data Bank of
Japan (DDBJ), provide the archival database for barcode data, and the Barcode of
Life Database2 (BOLD) at the University of Guelph, Ontario, provides an informatics
workbench aiding the acquisition, storage, analysis and publication of DNA bar-
code records, as well as providing public identification tools and other services
(Ratnasingham and Hebert, 2007, 2013). At this time, BOLD includes over 1 700 000
barcode sequences from 160 000 named species and many as yet unidentified
species, and growing rapidly. Recent reviews of the state of development of barcod-
ing include Waugh (2007), Valentini et al. (2008), Casiraghi et al. (2010), Teletchea
(2010), and Kato et al. (2012).
1 http://www.ncbi.nlm.nih.gov/genbank/ 2 http://www.barcodeoflife.org/
DNA BARCOD ING AND FLORAS AND FAUNAS 297
22.3 How does barcoding change the approach
to an inventory?
DNA barcoding provides an important tool to improve the quality or speed of floral
and faunal studies, while, at the same time, such studies contribute to development
of the global sequence library that is becoming an important community resource.
The large-scale inventory of caterpillars, their hosts and their parasites in Costa Rica
has provided an excellent example of how barcoding has changed the basic
approach to an inventory project, starting with sampling, processing, identification,
analysis, and even changing the approach to publication of results (Janzen et al.,
2009; Janzen and Hallwachs 2011a, 2011b; see also Strutzenberger et al., 2010). In
addition to the changes in work flow there have been significant impacts, from
finding cryptic species to matching dimorphic males and females, which have
substantially improved the quality and depth of the inventory, but also greatly
multiplied the number of situations requiring further taxonomic work for resolu-
tion. Although the workflow issues differ between different habitats and taxa, other
studies have demonstrated the use of barcoding in inventories of diverse taxa,
including poorly known freshwater invertebrates (Zhou et al., 2009; Laforest et al.,
2013), tropical sand flies (Azpurua et al., 2010; Krüger et al., 2011), bats in Southeast
Asia (Francis et al., 2010), difficult to distinguish agricultural pest moths (Roe et al.,
2006), pollinating insects in Africa (Nzeduru et al., 2012), diverse radiations of
tropical weevils (Pinzón-Navarro et al., 2010a, 2010b; Tänzler et al., 2012), fresh-
water fishes in Africa (Swartz et al., 2008; Lowenstein et al., 2011), butterflies at
country scales (Dinca et al., 2011; Hausmann et al., 2011), amphibians in Panama
(Crawford et al., 2010) and trees in forest plots (Kress et al., 2009, 2010; Costion
et al., 2011). Perhaps even greater opportunities for improving the speed and quality
of inventories exist in the marine realm, where poorly known larval stages exist in
vast quantities, and species concepts must be compared across vast oceanic dis-
tances (Goetze, 2010; Heimeier et al., 2010; Hubert et al., 2010; Stern et al., 2010;
Plaisance et al., 2011; Ranasinghe et al., 2012). But for most efficient use of DNA
barcoding, the appropriate sampling and data management needs to be incorpo-
rated from the beginning of the fieldwork (Leponce et al., 2010; Dick and Webb,
2012; Puillandre et al., 2012).
22.4 How does barcoding change the use
of morphospecies?
Even in floral and faunal studies, in which most identifications are well-resolved to
named species, there are usually a few species designated as informal morpho-
species, because the samples available lack the diagnostic characters formorphological
298 DESCR IPT I VE TAXONOMY
identification or the state of taxonomic knowledge does not allow identification.
Traditionally, these informal designations are useful only within the local study, cannot
be verified except for examination of voucher specimens and cannot be compared in a
regional or temporal context. Regional and continental-scale comparisons are starting
to become routine with DNA barcodes (see examples below), and Fernandez-Triana
et al. (2011) recently used barcoding to compare changes in a local wasp community
over 70 years using museum specimens.
DNA barcoding provides new tools for dealing with morphospecies, beyond the
obvious facilitation of matches of unidentified specimens with named specimens
(e.g. matching immature specimens with adults, or matching males and females).
DNA barcoding provides a character-based diagnosis of the morphospecies (the
DNA sequence) that can be made public (through GenBank) and can be compared
with other species, whether identified or not. Pinzón-Navarro et al. (2010a, 2010b)
were able to integrate historic knowledge of a diverse weevil group with larvae
and adults reared from fruit in Panama, even though most of the species lacked
taxonomic names. DNA barcoding can also provide an interim taxonomic desig-
nation, by way of the sequence, the GenBank accession number or the BOLD
cluster reference number (BIN) (Schindel and Miller, 2010; Ratnasingham and
Hebert, 2013).
22.5 Contribution of barcoding to quality control
The DNA barcode standard (and the restricted keyword barcode in GenBank, EMBL
and DDBJ) bring a new standard of documentation and transparency to taxonomy,
placing not only the sequence, but the metadata about the sequence and the
voucher specimen, in the public record. This will become increasingly important
in meeting standards of national and international regulation in fields such as
agriculture, forestry and fisheries (Jones, F. C., 2008; de Waard et al., 2010; Floyd
et al., 2010; Boykin et al., 2012), as well as emerging rules for access and benefit
sharing under the Convention on Biological Diversity (Schindel, 2010). Newmaster
and Ragupathy (2010) have applied DNA barcoding to quality control on traditional
knowledge in ethnobotany, to test identifications of plants and to explore the
presence of cryptic species.
22.6 Value added from the ability to compare
regional biotas
DNA barcode data allows comparison of species concepts across geographical
boundaries. For example, the taxonomy of the insect faunas of Australia and New
Guinea were traditionally studied independently, but extensive DNA barcode
DNA BARCOD ING AND FLORAS AND FAUNAS 299
surveys of Lepidoptera in both places are now allowing comparisons of the biogeo-
graphical relationships in new depth (Holloway et al., 2009). It has always been
challenging to assess small morphological variations across geographical barriers,
such as defining the boundaries of species across islands. This was the case in the
moth Homona salaconis (Meyrick), described from the Philippines but found on
nearby islands including New Guinea (Hulcr et al., 2007), where addition of DNA
data made the difference between the species as found in the Philippines and New
Guinea clear, resulting in the recognition of the New Guinea species as Homona
auriga (Durrant) (Miller et al., 2010). The Center for Tropical Forest Science’s
(CTFS’s) global network of forest dynamic plots is now implementing barcoding
across all the tree species in the plots (early results have been reported by Kress
et al., 2009, 2010, 2012) and the National Ecological Observatory Network (NEON) is
implementing the barcoding of selected insect groups across North America
(Gibson et al., 2012).
Benefits go both ways between floral and faunal work and DNA barcoding. The
18-volume faunal work The moths of Borneo completed in 2011 (Holloway, 2011),
while based on morphology, began to use DNA data to resolve some taxonomic
problems and also pointed out taxonomic and biogeographical problems to which
others have started to apply DNA barcode data. The existence of The moths of
Borneo series itself encouraged Erik Van Nieukerken to undertake a faunal study on
moths in Borneo testing the application of DNA barcoding (E. Van Nieukerken,
pers. comm.).
While many barcoding studies to date have focused on patterns of local diversity,
including recognition of cryptic species, barcoding shows great promise for the
analysis of supposedly widespread species, and the identification of invasive spe-
cies (Smith and Fisher, 2009; Hulcr et al., 2007; Floyd et al., 2010). One of the great
values of the creation of global DNA barcode libraries is the opportunity for
unexpected matches across the world.
22.7 Opportunities for meta-analysis: phylogeography
and community phylogenies
Floral and faunal studies have always provided the raw data for biogeographical
studies, and increasingly provide the raw data for the coalescence of the fields of
ecology and evolution in phylogeography. While phylogeography might often best
be done with longer sequences and more genes, DNA barcode data offer distinct
advantages in many cases, being relatively inexpensive to produce with high
throughput methods and having the benefit of large comparative libraries for
many taxa (Craft et al., 2010). For example, Craft et al. (2010) were able to sample
COI haplotypes from 28 Lepidoptera species and 1359 individuals across 4 host
300 DESCR IPT I VE TAXONOMY
plant genera and 8 sites in New Guinea to estimate population divergence in
relation to host specificity and geography, a much larger dataset than most pre-
viously published phylogeographical analyses, which had tended to focus on single
species. The possibilities for community phylogenetic analyses using large quanti-
ties of DNA barcode data are exciting, especially in organisms such as hosts and
their parasites, and herbivorous insects and their host plants (Kress et al., 2009,
2010; Emerson et al., 2011).
22.8 Comparing trophic interactions across
multiple levels
Traditionally, understanding ecological relationships across multiple trophic levels
required time-consuming and expensive rearing or field observations. DNA bar-
coding is now being used to characterise food items from insect guts (Greenstone
et al., 2005; Jurado-Rivera et al., 2009), fish stomachs (Valdez-Moreno et al., 2012)
or mammal faeces (Bohmann et al., 2011; Clare et al., 2011; Pompanon et al., 2012),
dead hosts of parasites from their larval remains (Hrcek et al., 2011), insect para-
sitoids dissected from caterpillars (Hrcek et al., 2011), hosts from DNA remaining in
parasites (Rougerie et al., 2011) and bloodmeals in ticks (Gariepy et al., 2012).
Kaartinen et al. (2010), Hrcek et al. (2011) and Smith et al. (2012) have applied
DNA barcoding to the analysis of complex insect food webs. As techniques for
handling degraded DNA improve and DNA libraries expand, we can expect these
techniques to be widely used. In the near future, I expect that a single caterpillar
can be sampled and, in addition to identification of the caterpillar itself, DNA
barcoding will allow identification of its plant diet, its insect parasites, and its
microbial symbionts.
22.9 Identification of immature and dimorphic stages
As noted above, faunal and floral studies are plagued by specimens in immature
stages that lack the characters necessary for identification, or sexes that cannot be
matched, especially if they are dimorphic such as some insects in which the sexes
vary dramatically in size or colour (Pinzón-Navarro et al., 2010a, 2010b). Richard
et al. (2010) have used barcoding to identify earthworm juveniles in soil ecology
studies. Plants sampled only in ‘vegetative’ stages are a widespread problem in
floral studies. Even long-term studies of forest tree plots often have individual trees
that remain unidentified for years because of the duration between flowering in
many tropical trees (e.g. Lafrankie et al., 2005). The ability to identify plant roots and
shoots using DNA will dramatically change the depth of ecological analysis that is
possible (Jones, F. A., et al., 2011).
DNA BARCOD ING AND FLORAS AND FAUNAS 301
22.10 Environmental samples
One of the exciting horizons for expansion of DNA barcoding is the application of
DNA barcode libraries to the identification and quantification of species in environ-
mental samples that have been sequenced using next-generation sequencing tech-
niques (Pfrender et al., 2010; Hajibabaei et al., 2011; Baird and Hajibabaei, 2012).
Although the technique has not yet been applied widely, interesting demonstra-
tions are being published, such as the identification of frog DNA in water samples
(Ficetola et al., 2008), phylogeography of soil invertebrates (Emerson et al., 2011)
and the interesting case of insect DNA from alcohol preservative (the ‘worm’ in
mescal liquor) (Shokralla et al., 2010). Related research is finding that, in many
cases, sequences that are shorter than the standard 648 base-pair animal barcodes
have almost as much information content as the full-length barcodes, still allowing
species identifications (Meusnier et al., 2008; Lees et al., 2010) and opening greater
opportunities for the use of next-generation sequencing.
22.11 An example of the impact of DNA barcoding
in insect faunistic studies
For many years I have been involved in local inventories of Lepidoptera (butterflies
and moths), especially in Papua New Guinea (PNG) and Kenya. The work in PNG
involves the large-scale rearing of caterpillars to adults, as part of the analysis of
ecological and biogeographical patterns among host plants, caterpillars and their
parasites (e.g. Miller et al., 2003, 2013; Craft et al., 2010; Novotny et al., 2012). The
PNG research started in 1994 with parataxonomists taking the first pass at morpho-
species identifications, with the aid of a database with images as a working identi-
fication tool (Basset et al., 2000, 2004), then a review in my laboratory based on
external morphology and then extensive dissection of genitalia, to purify species
concepts and to associate the species concepts with species names based on
published revisions or type specimens. This process worked well, but involved the
preparation of large quantities of genitalic dissections and, thus, was relatively
expensive and time-consuming. I started collaborating with Paul Hebert in DNA
barcoding in 2003, but did not start processing large quantities of DNA barcode
samples until 2005. In the early stages, we used DNA barcoding to solve problems
that morphology alone could not resolve, such as matching males and females, and
clarifying species limits in polymorphic species. Adamski et al. (2010) is an example
of a hybrid product during this transitional period – we did a traditional
morphology-based analysis of African Blastobasinae moths in the early 2000s,
then tested it with DNA barcodes in the mid 2000s and then eventually published
the joint results.
302 DESCR IPT I VE TAXONOMY
In recent years we have shifted the work process to put the emphasis on DNA
barcodes, because of their accuracy and cost-effectiveness. We still use genitalic
dissections, both for delimiting species concepts and for matching with names, but
use the barcode analysis to guide which specimens should be dissected. This has
reversed the relative importance of barcodes and genitalic morphology in our work
process, allowing faster and higher quality results. Because we are able to do DNA
barcodes for more specimens than were dissected under the old system, we have
more data for species concept delimitation, and we catchmore errors in association
of individuals with species concepts (for example, poor quality specimens). Similar
projects in Costa Rica and Canada have gone through a similar transition in work
process (de Waard et al., 2009; Janzen et al., 2009; Janzen and Hallwachs 2011a,
2011b).
A collection of Lepidoptera reared from native fruits in PNG in 2008–2009 provides
an example of the new process of using barcoding. While there are some lineages of
Lepidoptera that specialise in using fruit as larval hosts, rearing Lepidoptera from fruit
is a time-consuming, messy and relatively low-yield activity, so it is rarely done on a
mass scale, but the results are interesting. In addition to the ecology of fruit feeding,
Lepidoptera in PNG being poorly known, many of the Microlepidoptera that are fruit
specialists are very poorly known globally and the PNG fauna is almost unknown.
While a few of the butterflies are well-known, even family-level identifications prove
challenging for some of the tiny moths. Thus, I was both delighted and daunted to
receive the first instalment of Lepidoptera reared fromabout 4000 lots of native fruit in
PNG, in a programme organised by Richard Ctvrtecka.
The collection had been sorted into 70 morphospecies by the parataxonomists
in PNG, and included between 1 and 20 specimens for each of the morphospecies,
with many morphospecies represented by only a few specimens. After an
initial assessment by external morphology (including a few splits in morpho-
species), we sent up to five individuals per morphospecies to the University of
Guelph for sequencing using their standard protocols (Craft et al., 2010;
Wilson, 2012). This yielded 227 sequences, covering all morphospecies,
deposited in GenBank as accession numbers GU695412–5, GU695431–2,
GU695434–66, GU695468–9, GU695504–46, GU695548–58, GU695561,
GU695575–80, GU695623–36, GU695639–701, GU695716–7, GU695720–1,
GU695745, HM376367–75, HM376381–4, HM422448–56, HM902704–15,
HQ947496–7, HQ956600–1 and HQ956613–4. The result of barcoding, confirmed
by genitalic morphology, is 79 species. Two morphospecies were represented by
unique individuals in poor condition that were shown by barcoding to belong to
existing morphospecies. Eleven morphospecies were split, in seven cases because
of unique individuals in poor condition that were incorrectly identified based on
external morphology. This confirms the high quality of the sorting of our PNG
parataxonomists, given that they are using only external morphology.
DNA BARCOD ING AND FLORAS AND FAUNAS 303
The barcode library in BOLD is becoming rich enough in many parts of the
Lepidoptera to be useful in identification of unknowns, especially North America
(Hebert et al., 2010), but also other regions such as Costa Rica and Australia. For
example, one of the species was identified as the cosmopolitan tineid pest
Phereoeca uterella, and several Phycitinae, a very difficult group taxonomically,
were immediately identified to genus because of samples from Australia that
Marianne Horak had provided to BOLD. By contrast, BOLD has been less useful
for associating weevils reared from the same fruit, because the taxonomic sampling
in BOLD remains small (some 13 000 sequences of Curculionidae in BOLD in July
2012 compared to 114 000 sequences of Geometridae, for example). But this shows
how increasingly useful BOLD will become as more projects contribute reference
sequences.
Following purification of species concepts, we have targeted genitalic dissections
to confirm the barcoding results and to allow comparison with type specimens.
About two-thirds of the species have been positively identified (either as known taxa
or as new species) through consultation with literature, the collections in
Washington, D.C. and London and assistance from colleagues. The identification
process will continue for some time, because many of the Microlepidoptera types
have never been dissected and properly placed in modern generic concepts.
22.12 Conclusions
DNA barcoding builds on the best of the traditional strengths of floral and faunal
research based on morphological identifications of voucher specimens, especially
in enhancing the quality of identifications for named species and in enhancing the
information associated with informal morphospecies designations. While DNA
barcoding can be applied ‘after the fact,’ it is most cost-effective to plan the
appropriate sampling and data management from the beginning of fieldwork.
Floristic and faunistic works have traditionally been descriptions of areas at a
given point in time, limited by the species concepts available at the time. DNA
barcodes, along with modern information management systems for other charac-
ters, allow the local species (or morphospecies) concepts to be seen as part of a
dynamic global taxonomy that can be repeatedly queried in the context of changing
questions.
Acknowledgements
I thank many colleagues and funding agencies, especially the Sloan Foundation, for
the opportunity to be part of the development of DNA barcoding through the
Consortium for the Barcode of Life and the International Barcode of Life. David
304 DESCR IPT I VE TAXONOMY
Schindel and other Consortium for the Barcode of Life colleagues helped develop
many of the ideas discussed here. The barcode library of Papua New Guinea
Lepidoptera was built from US National Science Foundation-funded research
since 1995 (including DEB 0841885) in collaboration with Vojtech Novotny,
George Weiblen and Yves Basset, and also supported by the Czech Science
Foundation, the Czech Ministry of Education, and the Grant Agency of the
University of South Bohemia. The Papua New Guinea fruit Lepidoptera fieldwork
was coordinated by Richard Ctvrtecka, and the DNA barcoding was undertaken
with assistance of Lauren Helgen and Margaret Rosati, with sequencing provided
through the Biodiversity Institute of Ontario with funding from Genome Canada
and the Ontario Genomics Institute (2008–0GI-ICI-03). The Natural History
Museum, London, provided access to type specimens. Chris Lyal provided helpful
editorial comments and tolerated my distractions.
References
Adamski, D., Copeland, R. S., Miller, S. E.,
Hebert, P. D. N., Darrow, K. and Luke, Q.
(2010). A review of African
Blastobasinae (Lepidoptera:
Gelechioidea: Coleophoridae), with new
taxa reared from native fruits in Kenya.
Smithsonian Contributions to Zoology,
630: vi + 68.
Azpurua, J., De La Cruz, D., Valderama, A.
andWindsor, D. (2010). Lutzomyia Sand
Fly diversity and rates of infection by
Wolbachia and an exotic Leishmania
species on Barro Colorado Island,
Panama. PLoS Neglected Tropical
Diseases, 4(3): e627.
Baird, D. J. and Hajibabaei, M. (2012).
Biomonitoring 2.0: a new paradigm in
ecosystem assessment made possible by
next-generation DNA sequencing.
Molecular Ecology, 21: 2039–2044.
Basset, Y., Novotny, V., Miller, S. E. and
Pyle, R. (2000). Quantifying biodiversity:
experience with parataxonomists and
digital photography in Papua New
Guinea and Guyana. BioScience,
50: 899–908.
Basset, Y., Novotny, V., Miller, S. E.,
Weiblen, G.D., Missa, O. and
Stewart, A. J. A. (2004). Conservation
and biological monitoring of tropical
forests: the role of parataxonomists.
Journal of Applied Ecology, 41: 163–174.
Benson, D. A., Karsch-Mizrachi, I., Clark, K.,
Lipman, D. J., Ostell, J. and Sayers, E.W.
(2012). GenBank. Nucleic Acids
Research, 40(D1): D48–D53.
Bohmann, K., Monadjem, A., Lehmkuhl
Noer, C., Rasmussen, M., Seale, M. R.,
Clare, E., Jones, G., Willerslev, E. and
Gilbert, M. T. (2011). Molecular diet
analysis of two African free-tailed bats
(Molossidae) using high throughput
sequencing. PloS ONE, 6(6): e21441.
Boykin, L.M., Armstrong, K. F., Kubatko, L.
and De Barro, P. (2012). Species
delimitation and global biosecurity.
Evolutionary Bioinformatics Online,
8: 1–37.
Casiraghi, M., Labra, M., Ferri, E.,
Galimberti, A. and De Mattia, F. (2010).
DNA barcoding: a six-question tour to
improve users’ awareness about the
DNA BARCOD ING AND FLORAS AND FAUNAS 305
method. Briefings in Bioinformatics,
11: 440–453.
Clare, E. L., Barber, B. R., Sweeney, B.W.,
Hebert, P. D.N. and Fenton, M. B.
(2011). Eating local: influences of
habitat on the diet of little brown bats
(Myotis lucifugus). Molecular Ecology,
20: 1772–1780.
Costion, C., Ford, A., Cross, H., Crayn, D.,
Harrington, M. and Lowe, A. (2011).
Plant DNA barcodes can accurately
estimate species richness in poorly
known floras. PloS ONE, 6(11): e26841.
Craft, K. J., Pauls, S. U., Darrow, K.,
Miller, S. E., Hebert, P. D.N.,
Helgen, L. E., Novotny, V. and
Weiblen, G.D. (2010). Population
genetics of ecological communities with
DNA barcodes: an example from New
Guinea Lepidoptera. Proceedings of the
National Academy of Science U S A,
107: 5041–5046.
Crawford, A. J., Lips, K. R. and
Bermingham, E. (2010). Epidemic
disease decimates amphibian
abundance, species diversity, and
evolutionary history in the highlands of
central Panama. Proceedings of the
National Academy of Sciences U S A,
107: 13777–13782.
de Waard, J. R., Landry, J. F., Schmidt, B. C.,
Derhousoff, J., McLean, J. A. and
Humble, L.M. (2009). In the dark in a
large urban park: DNA barcodes
illuminate cryptic and introduced moth
species. Biodiversity and Conservation,
18: 3825–3839.
de Waard, J. R., Mitchell, A., Keena, M. A.,
Gopurenko, D., Boykin, L.M.,
Armstrong, K. F., Pogue, M.G., Lima, J.,
Floyd, R., Hanner, R.M. and
Humble, L.M. (2010). Towards a global
barcode library for Lymantria
(Lepidoptera: Lymantriinae) tussock
moths of biosecurity concern. PLoS
ONE, 5(12): e14280.
Dick, C.W. and Webb, C. O. (2012). Plant
DNA barcodes, taxonomic
management, and species discovery in
tropical forests. In DNA barcodes:
methods and protocols, W. J. Kress and
D. L. Erickson (eds). New York, NY:
Springer, pp. 379–393.
Dinca, V., Zakharov, E. V., Hebert, P. D.N.
and Vila, R. (2011). Complete DNA
barcode reference library for a country’s
butterfly fauna reveals high
performance for temperate Europe.
Proceedings of the Royal Society B,
278: 347–355.
Emerson, B. C., Cicconardi, F.,
Fanciulli, P. P. and Shaw, P. J. (2011).
Phylogeny, phylogeography,
phylobetadiversity and the molecular
analysis of biological communities.
Philosophical Transactions of the Royal
Society of London B, 366: 2391–2402.
Fernandez-Triana, J., Smith, M. A.,
Boudreault, C., Goulet, H.,
Hebert, P. D.N., Smith, A. C. and
Roughley, R. (2011). A poorly known
high-latitude parasitoid wasp
community: unexpected diversity and
dramatic changes through time. PloS
ONE, 6(8): e23719.
Ficetola, G. F., Miaud, C., Pompanon, F. and
Taberlet, P. (2008). Species detection
using environmental DNA from water
samples. Biology Letters, 4: 423–425.
Floyd, R., Lima, J., de Waard, J., Humble, L.
and Hanner, R. (2010). Common goals:
policy implications of DNA barcoding
as a protocol for identification of
arthropod pests. Biological Invasions,
12: 2947–2954.
Folmer, O., Black, M., Hoeh, W., Lutz, R. and
Vrijenhoek, R. (1994). DNA primers for
amplification of mitochondrial
cytochrome c oxidase subunit I from
diverse metazoan invertebrates.
Molecular Marine Biology and
Biotechnology, 3: 294–299.
306 DESCR IPT I VE TAXONOMY
Francis, C.M., Borisenko, A. V,
Ivanova, N. V., Eger, J. L., Lim, B. K.,
Guillen-Servent, A., Kruskop, S. V.,
Mackie, I. and Hebert, P. D.N. (2010).
The role of DNA barcodes in
understanding and conservation of
mammal diversity in Southeast Asia.
PLoS ONE, 5(9): e12575.
Gariepy, T. D., Lindsay, R., Ogden, N. and
Gregory, T. R. (2012). Identifying the last
supper: utility of the DNA barcode
library for bloodmeal identification in
ticks. Molecular Ecology Resources,
12: 646–652.
Gibson, C.M., Kao, R. H., Blevins, K. K.
and Travers, P. D. (2012). Integrative
taxonomy for continental-scale
terrestrial insect observations. PLoS
ONE, 7(5): e37528.
Goetze, E. (2010). Species discovery in
marine planktonic invertebrates
through global molecular screening.
Molecular Ecology, 19: 952–967.
Greenstone, M.H., Rowley, D. L.,
Heimbach, U., Lundgren, J. G.,
Pfannenstiel and R. S., Rehner, S. A.
(2005). Barcoding generalist predators
by polymerase chain reaction: carabids
and spiders. Molecular Ecology,
14: 3247–3266.
Hajibabaei, M., Shokralla, S., Zhou, X.,
Singer, G. A. and Baird, D. J. (2011).
Environmental barcoding: a next-
generation sequencing approach for
biomonitoring applications using river
benthos. PLoS ONE, 6(4): e17497.
Hausmann, A., Haszprunar, G.,
Segerer, A. H., Speidel, W., Behounek, G.
and Hebert, P. D.N. (2011). Now DNA-
barcoded: the butterflies and larger
moths of Germany. Spixiana, 34: 47–58.
Hebert, P. D.N, Cywinska, A., Ball, S. L. and
de Waard, J. R. (2003). Biological
identifications through DNA barcodes.
Proceedings of the Royal Society B,
270: 313–321.
Hebert, P. D.N., de Waard, R. J. and
Landry, J. F. (2010). DNA barcodes for
1/1000 of the animal kingdom. Biology
Letters, 6: 359–362.
Heimeier, D., Lavery, S. and Sewell, M. A.
(2010). Using DNA barcoding and
phylogenetics to identify Antarctic
invertebrate larvae: lessons from
a large scale study. Marine Genomics,
3: 165–177.
Hollingsworth, P.M., Forrest, L. L.,
Spouge, J. L., Hajibabaei, M.,
Ratnasingham, S., van der Bank, M.,
Chase, M.W., Cowan, R. S.,
Erickson, D. L., Fazekas, A. J.,
Graham, S.W., James, K. E., et al. (2009).
A DNA barcode for land plants.
Proceedings of the National Academy
of Sciences U S A, 106: 12794–12797.
Holloway, J. D. (2011). Themoths of Borneo:
Families Phaudidae, Himantopteridae
and Zygaenidae; revised and annotated
checklist. Malayan Nature Journal,
63: 1–548.
Holloway, J. D., Miller, S. E., Pollock, D.M.,
Helgen, L. and Darrow, K. (2009).
GONGED (Geometridae of New Guinea
Electronic Database): a progress report
on development of an online facility of
images. Spixiana, 32: 122.
Hrcek, J., Miller, S. E., Quicke, D. L. J. and
Smith, M. A. (2011). Molecular detection
of trophic links in a complex insect
host–parasitoid food web. Molecular
Ecology Resources, 11: 786–794.
Hubert, N., Delrieu-Trottin, E., Irisson, J. O.,
Meyer, C. and Planes, S. (2010).
Identifying coral reef fish larvae through
DNA barcoding: a test case with the
families Acanthuridae and
Holocentridae. Molecular Phylogenetics
and Evolution, 55: 1195–1203.
Hulcr, J., Miller, S. E, Setliff, G. P.,
Darrow, K., Mueller, N.D.,
Hebert, P. D.N., Weiblen, G.D. (2007).
DNA barcoding confirms polyphagy in a
DNA BARCOD ING AND FLORAS AND FAUNAS 307
generalist moth, Homona mermerodes
(Lepidoptera: Tortricidae). Molecular
Ecology Notes, 7: 549–557.
Janzen, D.H., Hallwachs, W., Blandin, P.,
Burns, J.M., Cadiou, J.M., Chacon, I.,
Dapkey, T., Deans, A. R., Epstein, M.,
Espinoza, B., Franclemont, J., Haber, W.,
et al. (2009). Integration of DNA
barcoding into an ongoing inventory of
complex tropical biodiversity. Molecular
Ecology Resources, 9 (Suppl 1): 1–26.
Janzen, D.H. and Hallwachs, W. (2011a).
Joining inventory by parataxonomists
with DNA Barcoding of a large complex
tropical conserved wildland in
Northwestern Costa Rica. PLoS ONE,
6(8): e18123.
Janzen, D.H, Hallwachs, W., Burns, J.M.,
Hajibabaei, M., Bertrand, C.,
Hebert, P. D.N. (2011b). Reading the
complex Skipper butterfly fauna of one
tropical place. PLoS ONE, 6(8): e19874.
Jones, F. A., Erickson, D. L., Bernal, M. A,
Bermingham, E., Kress, W. J.,
Herre, E. A., Muller-Landau, H. C. and
Turner, B. L. (2011). The roots of
diversity: below ground species richness
and rooting distributions in a tropical
forest revealed by DNA barcodes
and inverse modeling. PLoS ONE,
6(9): e24506.
Jones, F. C. (2008). Taxonomic sufficiency:
the influence of taxonomic resolution on
freshwater bioassessments using
benthic macroinvertebrates.
Environmental Reviews, 16: 45–69.
Jurado-Rivera, J. A., Vogler, A. P.,
Reid, C. A.M., Petitpierre, E. and
Gómez-Zurita, J. (2009). DNA barcoding
insect–host plant associations.
Proceedings of the Royal Society B,
276: 639–648.
Kaartinen, R., Stone, G.N., Hearn, J.,
Lohse, K. and Roslin, T. (2010).
Revealing secret liaisons: DNA
barcoding changes our understanding of
food webs. Ecological Entomology,
35: 623–638.
Kato, T., Jinbo, U. and Ito, M. (2012). DNA
barcoding: a novel tool for observation
of biodiversity. In The biodiversity
observation network in the Asia–Pacific
region: toward further development of
monitoring, S.-I. Nakano (ed.). Japan:
Springer, pp. 259–266.
Kress, W. J., Erickson, D. L., Jones, F. A.,
Swenson, N.G., Perez, R., Sanjur, O. and
Bermingham, E. (2009). Plant DNA
barcodes and a community phylogeny
of a tropical forest dynamics plot in
Panama. Proceedings of the
National Academy of Science U S A,
106: 18621–18626.
Kress, W. J., Erickson, D. L., Swenson, N.G.,
Thompson, J., Uriarte, M. and
Zimmerman, J. K. (2010). Advances in
the use of DNA barcodes to build a
community phylogeny for tropical trees
in a Puerto Rican forest Dynamics Plot.
PLoS ONE, 5(11): e15409.
Kress, W. J., Lopez, I. C. and Erickson, D. L.
(2012). Generating plant DNA barcodes
for trees in long-term forest Dynamics
Plots. In DNA barcodes: methods and
protocols, W. J. Kress and
D. L. Erickson (eds). New York:
Springer, pp. 441–458.
Krüger, A., Strüven, L., Post, R. L. and
Faulde, M. (2011). The sandflies
(Diptera: Psychodidae, Phlebotominae)
in military camps in northern
Afghanistan (2007–2009), as identified
by morphology and DNA ‘barcoding’.
Annals of Tropical Medicine and
Parasitology, 105: 163–176.
Laforest, B. J., Winegardner, A. K.,
Zaheer, O. A., Jeffery, N.W., Boyle, E. E.
and Adamowicz, S. J. (2013). Insights
into biodiversity sampling strategies for
freshwater microinvertebrate faunas
through bioblitz campaigns and DNA
barcoding. BMC Ecology, 13(1): 13.
308 DESCR IPT I VE TAXONOMY
Lafrankie, J. V., Davies, S. J., Wang, L. K.,
Lee, S. K and Lum, S. K. Y. (2005). Forest
trees of Bukit Timah: population ecology
in a tropical forest fragment. Singapore:
Simply Green.
Lees, D. C., Rougerie, R., Zeller-Lukashort,
C. and Kristensen, N. P. (2010). DNA
mini-barcodes in taxonomic
assignment: a morphologically unique
new homoneurous moth clade from the
Indian Himalayas described in
Micropterix (Lepidoptera,
Micropterigidae). Zoologica Scripta,
39: 642–661.
Leponce, M., Meyer, C., Haeuser, C. L.,
Bouchet, P., Delabie, J. H. C., Weigt, L.
and Basset, Y. (2010). Challenges and
solutions for planning and
implementing large-scale biotic
inventories. InManual on field recording
techniques and protocols for all taxa
biodiversity inventories and monitoring,
J. Eymann, J. Degreef, C. Häuser,
J. C. Monje, Y. Samyn and
D. VandenSpiegel (eds). Brussels:
Belgian Development Cooperation,
pp. 18–48.
Lowenstein, J. H., Osmundson, T.W.,
Becker, S., Hanner, R. and Stiassny, M. L.
(2011). Incorporating DNA barcodes
into a multi-year inventory of the fishes
of the hyperdiverse Lower Congo River,
with a multi-gene performance
assessment of the genus Labeo as a
case study. Mitochondrial DNA,
22 (Suppl 1): 52–70.
Meusnier, I., Singer, G. A. C., Landry, J. F.,
Hickey, D. A., Hebert, P. D.N. and
Hajibabaei, M. (2008). A universal
DNA mini-barcode for
biodiversity analysis. BMC Genomics,
9: 214.
Miller, S. E. (2007). DNA barcoding and the
renaissance of taxonomy. Proceedings of
the National Academy of Science U S A,
104: 4775–4776.
Miller, S. E., Novotny, V. and Basset, Y.
(2003). Studies onNewGuineamoths. 1.
Introduction (Lepidoptera). Proceedings
of the Entomological Society of
Washington, 105: 1035–1043.
Miller, S. E., Helgen, L. E. and
Hebert, P. D.N. (2010). Clarification of
the identity of Homona salaconis
(Lepidoptera: Tortricidae). Molecular
Ecology Resources, 10: 580.
Miller, S. E., Hrcek, J., Novotny, V.,
Weiblen, G.D. and Hebert, P. D.N.
(2013). DNA barcodes of caterpillars
(Lepidoptera) from Papua New Guinea.
Proceedings of the Entomological Society
of Washington, 115: 107–109.
Newmaster, S. G. and Ragupathy, S. (2010).
Ethnobotany genomics – discovery and
innovation in a new era of exploratory
research. Journal of Ethnobiology and
Ethnomedicine, 6: 2.
Novotny, V., Miller, S. E., Hrcek, J., Baje, L.,
Basset, Y., Lewis, O. T., Stewart, A. J. A.
and Weiblen, G.D. (2012). Insects
on plants: explaining the paradox of
low diversity within specialist
herbivore guilds. American Naturalist,
179: 351–362.
Nzeduru, C. V., Ronca, S. and
Wilkinson, M. J. (2012). DNA barcoding
simplifies environmental risk
assessment of genetically modified
crops in biodiverse regions. PLoS ONE,
7(5): e35929.
Pfrender, M. E., Hawkins, C. P., Bagley, M.,
Courtney, G.W., Creutzburg, B. R.,
Epler, J. H., Fend, S., Schindel, D.,
Ferrington, L. C. Jr, Hartzell, P. L.,
Jackson, S., Larsen, D. P., et al. (2010).
Assessing macroinvertebrate
biodiversity in freshwater ecosystems:
advances and challenges in DNA-based
approaches.Quarterly Review of Biology,
85: 319–340.
Pinzón-Navarro, S., Barrios, H., Múrria, C.,
Lyal, C.H. C. and Vogler, A. P. (2010a).
DNA BARCOD ING AND FLORAS AND FAUNAS 309
DNA-based taxonomy of larval
stages reveals huge unknown species
diversity in Neotropical seed
weevils (genus Conotrachelus):
relevance to evolutionary ecology.
Molecular Phylogenetics and Evolution,
56: 281–293.
Pinzón-Navarro, S., Jurado-Rivera, J. A.,
Gomez-Zurita, J., Lyal, C. H. C. and
Vogler, A. P. (2010b). DNA profiling of
host–herbivore interactions in tropical
forests. Systematic Entomology,
35 (Suppl 1): 18–32.
Plaisance, L., Caley, M. J., Brainard, R. E. and
Knowlton, N. (2011). The diversity of
coral reefs: what are we missing? PLoS
ONE, 6(10): e25026.
Pompanon, F., Deagle, B. E.,
Symondson, W.O. C., Brown, D. S.,
Jarman, S. N. and Taberlet, P. (2012).
Who is eating what: diet assessment
using next generation sequencing.
Molecular Ecology, 21: 1931–1950.
Puillandre, N., Bouchet, P., Boisselier-
Dubayle, M. C., Brisset, J., Buge, B.,
Castelin, M., Chagnoux, S.,
Christophe, T., Corbari, L.,
Lambourdière, J., Lozouet, P.,
Marani, G., et al. (2012). New taxonomy
and old collections: integrating DNA
barcoding into the collection curation
process. Molecular Ecology Resources,
12: 396–402.
Ranasinghe, J. A., Stein, E. D., Miller, P. E.
and Weisberg, S. B. (2012). Performance
of two southern California benthic
community condition indices using
species abundance and presence-only
data: relevance to DNA barcoding. PLoS
ONE, 7(8): e40875.
Ratnasingham, S. and Hebert, P. D.N.
(2007). BOLD: the barcode of life data
system (http://www.barcodinglife.org).
Molecular Ecology Notes, 7: 355–364.
Ratnasingham, S. and Hebert, P. D.N.
(2013). A DNA-based registry for all
animal species: the barcode index
number (BIN) system. PloS ONE,
8(7): e66213.
Richard, B., Decaens, T., Rougerie, R.,
James, S.W., Porco, D. and
Hebert, P. D.N. (2010). Re-integrating
earthworm juveniles into soil
biodiversity studies: species
identification through DNA
barcoding. Molecular Ecology
Resources, 10: 606–614.
Roe, A.D., Stein, J. D., Gillette, N. E. and
Sperling, F. A.H. (2006). Identification of
Dioryctria (Lepidoptera: Pyralidae) in a
seed orchard at Chico, California.
Annals of the Entomological Society of
America, 99: 433–448.
Rougerie, R., Smith, M. A., Fernandez-
Triana, J., Lopez-Vaamonde, C.,
Ratnasingham, S. and Hebert, P. D. N.
(2011). Molecular analysis of parasitoid
linkages (MAPL): gut contents of adult
parasitoid wasps reveal larval host.
Molecular Ecology, 20: 179–186.
Schindel, D. E. (2010). Biology without
borders. Nature, 467: 779–781.
Schindel, D. E. and Miller, S. E. (2010).
Provisional nomenclature: the on-ramp
to taxonomic names. In Systema naturae
250: the Linnaean ark, A. Polaszek (ed.).
Boca Raton, FL: CRC Press, pp. 109–115.
Schoch, C. L., Seifert, K. A., Huhndorf, S.,
Robert, V., Spouge, J. L., Levesque, C. A.,
Chen, W. and the Fungal Barcoding
Consortium. (2012). Nuclear
ribosomal internal transcribed spacer
(ITS) region as a universal DNA barcode
marker for Fungi. Proceedings of the
National Academy of Science U S A,
109: 6241–6246.
Shokralla, S., Singer G. A. C. and
Hajibabaei, M. (2010). Direct PCR
amplification and sequencing of
specimens’ DNA from
preservative ethanol. BioTechniques,
48: 233–234.
310 DESCR IPT I VE TAXONOMY
Smith, M. A. and Fisher, B. L. (2009).
Invasions, DNA barcodes, and rapid
biodiversity assessment using ants of
Mauritius. Frontiers in Zoology, 6(1): 31.
Smith, M. A., Bertrand, C., Crosby, K.,
Eveleigh, E. S., Fernandez-Triana, J.,
Fisher, B. L., Gibbs, J., Hajibabeaei, M.,
Hallwachs, W., Hind, K., Hrcek, J.,
Huang, D.-W., et al. (2012). Wolbachia
and DNA barcoding insects: patterns,
potential and problems. PloS ONE,
7(5): e36514.
Stern, R. F., Horak, A., Andrew, R. L.,
Coffroth, M. A., Andersen, R. A.,
Küpper, F. C., Jameson, I.,
Hoppenrath, M., Véron, B., Kasai, F.,
Brand, J., James, E. R. et al. (2010).
Environmental barcoding reveals
massive dinoflagellate diversity in
marine environments. PLoS ONE,
5(11): e13991.
Strutzenberger, P., Brehm, G. and Fiedler, K.
(2010). DNA barcoding-based species
delimitation increases species count
of Eois (Geometridae) moths in a
well-studied tropical mountain forest by
up to 50%. Insect Science, 18: 349–362.
Swartz, E. R., Mwale, M., Hanner, R. (2008).
A role for barcoding in the study of
African fish diversity and conservation.
South African Journal of Science,
104: 293–298.
Tänzler, R., Sagata, K., Surbakti, S., Balke, M.
and Riedel, A. (2012). DNA barcoding
for community ecology – how to tackle a
hyperdiverse, mostly undescribed
Melanesian fauna. PLoS ONE,
7(1): e28832.
Teletchea, F. (2010). After 7 years and 1000
citations: Comparative assessment of
the DNA barcoding and the DNA
taxonomy proposals for taxonomists
and non-taxonomists. Mitochondrial
DNA, 21: 206–226.
Valdez-Moreno, M., Quintal-Lizama, C.,
Gómez-Lozano, R. and García-Rivas, M.
del C. (2012). Monitoring an alien
invasion: DNA barcoding and the
identification of lionfish and their prey
on coral reefs of the Mexican Caribbean.
PLoS ONE, 7(6): e36636.
Valentini, A. Pompanon, F. and Taberlet, P.
(2008). DNA barcoding for ecologists.
Trends in Ecology and Evolution,
24: 110–117.
Waugh, J. (2007). DNA barcoding in animal
species: progress, potential and pitfalls.
BioEssays, 29: 188–197.
Wilson, J. J. (2012). DNA barcodes for
insects. In DNA barcodes: methods and
protocols, W. J. Kress and D. L. Erickson
(eds). New York, NY: Springer,
pp. 17–46.
Zhou, X., Adamowicz, S. J., Jacobus, L.M.,
Dewalt, R. E. and Hebert, P. D.N. (2009).
Towards a comprehensive barcode
library for arctic life – Ephemeroptera,
Plecoptera, and Trichoptera of
Churchill, Manitoba, Canada. Frontiers
in Zoology, 6(1): 30.
DNA BARCOD ING AND FLORAS AND FAUNAS 311
Index
ABCD (Access to Biological Collections Data)
schema, 276
accuracy, GPS data, 228, 229, 230
acoustic techniques
ocean survey, 219
species records, 184, 199–200, 202–203, 204
adventitious species, 71
Africa
areas of importance for seed collection,
149, 157
coverage by existing Floras, 84, 85–86, 129–130
textual analysis of East African Floras, 47–54
uses of digitised regional Floras, 88–94
alpha diversity, 27, 45
alphabetic order, merits and problems, 72, 134
altitude
altitudinal range, as search query, 89
measurement techniques, 229
Aluka website, 89, 94
amateur enthusiasts
attitude to project contribution, 74, 186
growth of interest in dragonflies, 113, 115–116
American Museum of Natural History, 38, 187
Amsterdam,University ZoologicalMuseum, 70, 71
animation software, 175, 176
Antarctic Treaty legislation, 222
ants, 190, 200
APG (Angiosperm Phylogeny Group)
classification, 134
aphid monitoring, 196, 198
Arabian Peninsula, FAPSA floristic project,
14–15, 16, 20
ArcMap GIS programme, 149
arthropods
biodiversity, 190, 202
collection techniques, 171, 191, 192–199
disease vectors, field identification, 171,
174–177
arXiv server for e-prints, 282
Asia, southeast
forest tree functional ecology, 46–47
inadequacies of Floras, 16
tropical forest soundscape, 191
Atlas of Australian Birds (web database), 38
attraction traps, 196–197
authors
benefits of automated peer review, 281–282
editorial review of manuscripts, 78
fees for Open Access publishing, 284
intentions for their books, 58–59, 64
use of implicit information, 254
automated vs. manual traps, 198
automontage imaging systems, 175–177
AUVs (autonomous underwater vehicles),
218, 219
backup of data in field conditions, 234–235,
237, 240
bacteria, naming code, 261
baited traps, 199
barcoding
digitised herbarium specimens, 150, 157
DNA, for identification, 206, 296
globally unique identifiers (GUIDs), 241
initiative for sponges, 221
barometric measurements of altitude, 229
bats, sound recordings, 184
batteries, used for field equipment, 234–235
beetles
3D image animations, 175
arrangement in museum collections, 73
missing from first Fauna Europaea release, 70
numbers and diversity, 190, 201
Bentham, George
(and Hooker) classification system, 86
aphorisms on Flora writing, 12–13, 20