The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia SPP.)

RESEARCH ARTICLE
The Use of DNA Barcoding in Identification

and Conservation of Rosewood (Dalbergia
spp.)
Ida Hartvig1*, Mihaly Czako2, Erik Dahl Kjær1, Lene Rostgaard Nielsen1, Ida Theilade3
1 Forest Genetics and Diversity, Department of Geosciences and Natural Resource Management, University
of Copenhagen, Frederiksberg, Denmark, 2 Department of Biological Sciences, University of South
Carolina, Columbia, South Carolina, United States of America, 3 Global Development, Department of Food
and Resource Economics, University of Copenhagen, Frederiksberg, Denmark
* ihla@ign.ku.dk
Abstract
The genus Dalbergia contains many valuable timber species threatened by illegal logging
and deforestation, but knowledge on distributions and threats is often limited and accurate
species identification difficult. The aim of this study was to apply DNA barcoding methods to
support conservation efforts of Dalbergia species in Indochina. We used the recommended
OPEN ACCESS
rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia,
Citation: Hartvig I, Czako M, Kjær ED, Nielsen LR,
Theilade I (2015) The Use of DNA Barcoding in
and tested their discrimination ability with both traditional distance-based as well as different
Identification and Conservation of Rosewood model-based machine learning methods. We specifically tested whether the markers could
(Dalbergia spp.). PLoS ONE 10(9): e0138231. be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and
doi:10.1371/journal.pone.0138231
to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding mark-
Editor: Helge Thorsten Lumbsch, Field Museum of ers to 14 samples of unknown identity. In general, we found that the barcoding markers dis-
Natural History, UNITED STATES
criminated among Dalbergia species with high accuracy. We found that ITS yielded the
Received: June 19, 2015 single highest discrimination rate (100%), but due to difficulties in obtaining high-quality
Accepted: August 26, 2015 sequences from degraded material, the better overall choice for Dalbergia seems to be the
Published: September 16, 2015
standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and ampli-
fied well. The distance-based method TaxonDNA showed the highest identification rates
Copyright: © 2015 Hartvig et al. This is an open
access article distributed under the terms of the
overall, although a more complete specimen sampling is needed to conclude on the best
Creative Commons Attribution License, which permits analytic method. We found strong support for a monophyletic Dalbergia oliveri and encour-
unrestricted use, distribution, and reproduction in any age that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchi-
medium, provided the original author and source are
nensis was successfully identified, and a species-specific assay can be developed from the
credited.
data generated in this study for the identification of illegally traded timber. We suggest that
Data Availability Statement: All sequences are
the use of DNA barcoding is integrated into the work flow during floristic studies and at
available from GenBank and accession numbers for
each sequence are provided in S1 Table. national herbaria in the region, as this could significantly increase the number of identified
specimens and improve knowledge about species distributions.
Funding: This work was part of Ida Hartvig's PhD
project funded by a University of Copenhagen
scholarship. The authors received no specific funding
for this work.
Competing Interests: The authors have declared

that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0138231 September 16, 2015 1 / 24

Barcoding of Dalbergia
Introduction
Conservation of threatened species is an essential part of reaching the target of the Convention
on Biological Diversity 2020 on improving the status of global biodiversity [1]. The first crucial
step in conserving and managing threatened species is correct identification and delimitation
of the target species [2]. Identification of plant species traditionally relies on morphological
characters of especially reproductive parts, such as flowers and fruits, which for trees can be
time consuming to access and only present during parts of the year. Accurate identification in
species-rich or taxonomically complex groups also typically requires expert knowledge, which
is not always available, especially in tropical areas [3, 4]. Correct taxonomical identification of
endangered tropical tree species is thus often difficult. For threatened species, whose trade is
regulated by the Convention on International Trade of Endangered Species (CITES), correct
identification is crucial for the enforcement of the regulations and future conservation of the
species. The identification process can be problematic, especially if similar non-threatened spe-
cies also appear in the trade, and if only parts of the plant are being traded (e.g. wood).
A potential method to meet these identification challenges is DNA barcoding, which is the
identification of species by a short universal DNA sequence, that exhibits a sufficient level of
variation to discriminate among species [5]. The key advantage of DNA barcoding is that once
a solid reference database has been established, the method does not require expert taxonomic
knowledge in order to identify specific samples. Further, identification can be done with small
tissue samples from virtually any part of the organism, does not require reproductive material,
and the identification process is in general fast and reproducible. A limitation of the method is
that no single universal DNA region that can be used across all taxonomic groups have been
identified. While many DNA barcoding studies in animals have achieved high rates of species
discrimination using a single region, COI (see e.g. [6–8]), for plants it has proven necessary to
use a combination of regions to obtain sufficient discrimination success [9–12]. Further, within
taxonomic groups it is not always possible to discriminate between recently diverged species
(e.g. [13]). However, since the introduction in 2003, the method is now widely applied in plant
studies. First of all DNA barcoding can be used as a tool for identifying species that are difficult
to identify based on morphological characters, or be used as a supporting taxonomic tool in
delimitation and description of problematic species [14, 15]. The technique can also prove
valuable for accurate species identification as the important first step in conservation plans for
threatened species [16]. An important use of DNA barcoding is in wildlife forensics, where it
has shown ability to discriminate CITES-species from similar non-threatened species [17, 18].
Apart from identifying target species, DNA barcoding can also be applied in floristics. Con-
structing a DNA barcoding reference library of entire local floras can allow for fast and effective
floristic analyses without expert knowledge [19, 20], or even be a method of estimating species
richness in a taxonomically poorly known flora [21]. There is also a huge potential for applica-
tion of DNA barcoding to the vast collections at herbaria and Natural History Museums,
which could serve as excellent reference databases and help identify the many un-identified
specimens present in most collections, as well as identify new collections [22]. This would con-
tribute significantly to improved knowledge about distribution patterns of each species.
DNA barcoding thus has the potential of becoming an important supporting tool for con-
servation and biodiversity assessments in areas with a high number of plant species, a shortage
of expert taxonomists, and limited descriptions of the flora.
In the present study we address the applicability of DNA barcoding to support conservation
efforts of Dalbergia species in Cambodia, Laos and Vietnam (Indochina in the narrow sense).
As part of the Indo-Burma hotspot, the Indochina area is characterized by high levels of
endemic biodiversity under threat of extinction [23] and the flora of many areas remains yet to

be fully explored (see e.g. [24]). DNA barcoding could contribute to generate knowledge on
taxonomy and distribution of threatened species, and help increase identification rates in flo-
ristic investigations.
The pantropical genus Dalbergia L.f. (Fabaceae: Papilionoideae) is an example of a taxo-
nomic plant group that is in need of better identification tools in order to set up proper conser-
vation guidelines. The genus consists of shrubs, lianas, and trees with around 250 species in
total, and Indochina represents one of its centers of diversity with approximately 30–45 species
[25–27]. A number of Dalbergia trees possesses high-quality timber known as e.g. rosewood,
blackwood, cocobolo or palisander and the wood is used for construction works, fine furniture,
music instruments etc. [27]. Examples of economically important species include African
blackwood (D. melanoxylon), Brazilian rosewood (D. nigra) and Thailand rosewood (D.
cochinchinensis). Many Dalbergia species are also used in traditional medicine for various pur-
poses, and have been subject to phytochemical studies [28]. Overexploitation and illegal log-
ging have severely decreased population sizes of many of the species and several timber species
have been included in CITES, including D. nigra and D. cochinchinensis. Fifty-six species of
Dalbergia are currently listed in the endangered categories at the IUCN red list [29], although
this list is in great need of update, also taxonomically.
The timber of the Dalbergia species is often difficult to identify by wood anatomy alone [30,
31], which limits the enforcement of CITES regulations, as timber identification is a technical
requirement for monitoring and controlling trade. It has recently been shown that barcoding
has the potential to overcome this limitation, as it was possible to discriminate between two
Dalbergia timber species that were morphologically indistinguishable [31]. Many species of
Dalbergia in the Indochina area are also not easily recognized in the field, even with fruits or
flowers present (J. F. Maxwell, pers. comm.). The complications are increased by the lack of a
complete updated taxonomic revision, and although a regional revision exists for the Indochina
area, synonyms and old names are still in use. An example is the threatened D. oliveri, which
produces valuable timber and is subject to illegal logging in Indochina [32, 33]. In the latest
revision of Dalbergia in Indochina from 1997, Niyomdham et al. [25] included D. bariensis, D.
dongnaiensis, D. mammosa and D. duperreana as synonyms for D. oliveri. This has been
accepted to some extent (e.g.[34]) although the name D. bariensis is still widely used in Cambo-
dia. Also, in the first and only attempt of a molecular phylogeny for the whole genus Vatanpar-
ast et al. [35] maintained D. dongnaiensis as a separate species, and the synonyms also still exist
on the IUCN red list as threatened in variable categories [29]. Solving taxonomic issues like
this is essential for establishing effective conservation plans for the many threatened Dalbergia
species in Indochina.
We apply DNA barcoding methods to 31 Dalbergia species, with a focus on species from
the Indochina region. We use the coding plastic regions rbcL and matK, as recommended by
Plant Working Group of the Consortium for the Barcode of Life (CBOL) as the core barcode
for land plants [12]. As these markers not always yield high discrimination rates, we also apply
the highly variable ITS region, which seems to be a valid supplement to the core barcode [36,
37].
We also address the efficacy of different analytic approaches of DNA barcode data. As a
supplement to the standard distance-based methods of evaluating barcode performance [38,
39], we apply a machine learning approach, using the programs BLOG and WEKA [40, 41]. In
a comparison of different barcoding analyses methods, Van Velzen et al. [42] found that the
character-based BLOG had the highest identification rate over similarity and tree-based meth-
ods. WEKA was used by Weitschek et al. [43] to apply four different supervised classifiers on
DNA barcode data, and they found that these methods could be used with success on

barcoding datasets, reaching higher classification success than more classical barcoding meth-
ods as neighbor-joining (NJ), BLAST and nearest-neighbor.
The specific aims of this study are to i) establish a reference library for Dalbergia using the
recommended rbcL+matK+ITS barcode, ii) use this barcode to infer on the taxonomy of the
sampled Dalbergia species, iii) test the discrimination ability of the chosen markers, using both
traditional distance-based methods as well as machine learning-based approaches, iv) apply
the method on unidentified Dalbergia samples, including cambium/bark and wood samples, to
explore how the method could be used in situations where identification by morphological
characters is uncertain or not possible at all. We have a special focus on the threatened timber
species D. oliveri and D. cochinchinensis in order to address the taxonomic confusion around
D. oliveri, and the potential application of DNA barcoding for CITES identification of D.
cochinchinensis. We discuss the findings and relate it to requirements needed before this tool
can be incorporated as a standard procedure in e.g. CITES enforcement, identification of speci-
mens at local herbaria or in floristics.
Materials and Methods

Sampling of specimens
A total of 95 Dalbergia and two Machaerium Pers. specimens were included in the study.
For the majority of samples, tissue material for DNA extraction was obtained from herbaria
(AAU, C, E, K and L) or from a living collection belonging to one of the authors (M. Czako,
University of South Carolina, USA). A few samples were obtained from field studies in Cambo-
dia, Laos and Vietnam, with the necessary permissions from relevant authorities (Cambodia:
Forestry Administration, Ministries of Agriculture, Forestry and Fisheries, Laos: Ministry of
Science and Technology and provinces of Khammouane and Bolikhamsay, and Vietnam: Cen-
ter for Biodiversity and Biosafety, Institute of Agricultural Genetics, and Cat Tien National
Park). One sample was obtained from a commercial company. All D. cochinchinensis samples
were obtained prior to the inclusion of the species in CITES. S1 Table gives voucher informa-
tion for all samples.
Specimens were checked for synonyms and possible misidentifications, which led to new
names for eight of the specimens (see S2 Table for details). Naming of specimens from Indo-
china followed Niyomdham et al. [25], except for D. foliacea Wall. ex. Benth, which is not an
accepted name in either Genbank [44] nor The Plant List database [45], and D. foliacea was
therefore included in D. rimosa Roxb. following Gagnepain [46] and Thothathri [47].
After this treatment, the 95 Dalbergia specimens represented 31 species (Table 1) and 14
unidentified specimens. Four species were from America, two from Africa (incl. Madagascar),
and the remaining were from Asia, primarily Indochina (Table 1). The selection covered 19 of
the 29 species treated in Flora of Cambodia, Laos and Vietnam [25]. Whenever possible, spe-
cies were represented by two to five specimens, but for 11 species, sequences were only
obtained successfully for one specimen. Dalbergia oliveri and D. cochinchinensis were repre-
sented by eight and six specimens, respectively. The two Machaerium species (M. lunatum (L.
f.) Ducke and M. salvadorense (Donn.Sm.) Rudd) were included as outgroup for the phyloge-
netic analysis [48].
Molecular methods
Total genomic DNA was extracted from leaves, twigs, seeds, pods, cambium/bark or sapwood
samples, using slightly modified CTAB protocols [53] (full protocols can be obtained from the
authors).

Table 1. List of Dalbergia species included in this study.
Taxon Continent Habit No. of

specimens
D. assamica Benth Asia Tree[25] 3
D. benthamii Prain Asia Climbing shrub[49] 1
D. cana Graham ex Kurz Asia Tree[25, 47] 2
D. candenatensis (Dennst.) Prain Asia Climbing shrub/woody liana[25, 47] 4
D. cochinchinensis Pierre Asia Tree[25] 6
D. cultrata Graham ex Benth. Asia Tree[25, 47] 4
D. dyeriana Prain ex Harms Asia Woody liana[25, 49] 1
D. ecastaphyllum (L.) Taub. America Scandent shrub/robust shrub/small tree[50] 2
[25]
D. entadoides Pierre Asia Climbing shrub 1
D. hancei Benth. Asia Woody liana[25, 49] 2
D. horrida (Dennst.) Mabb. var. glabrescens (Prain) Thoth. & Asia Climbing shrub/woody liana[25, 47] 1
Nair.
D. hupeana Hance Asia Tree[49] 2
D. lanceolaria Linne F. Asia Tree[25, 47] 3
[47]
D. latifolia Roxb. Asia Tree 1
D. melanoxylon Guill. & Perr. Africa Tree/shrub[51] 1
D. mimosoides Franch. Asia Shrub[49] 1
D. miscolobium Benth. America Shrub/small tree[50] 1
D. monetaria L.f. America Scandent tree/shrub/robust liana[50] 2
D. nigrescens Kurz Asia Tree[25] 4
D. odorifera T.C. Chen Asia Tree[49] 2
D. oliveri Gamble ex Prain Asia Tree[25, 47] 7
[25, 47] [25]
D. ovata Graham ex Benth. Asia Small tree /liana-like shrub 2
D. pinnata (Lour.) Prain Asia Climbing shrub/small tree[25, 47] 3
D. rimosa Roxb. Asia Climbing shrub[25, 47, 49]/erect shrub[47, 49]/tree[25, 5
49]
D. sericea G. Don Asia Tree[25, 47] 1

D. sissoo Roxb. Asia Tree[47] 3
[25, 49] [49]
D. stipulacea Roxb. Asia Climbing shrub/woody liana /small tree 4
D. subcymosa Ducke America Climbing shrub[50] 1
D. trichocarpa Baker Africa Tree[52] 2
D. velutina Benth. Asia Climbing shrub[25, 47]/woody liana[25] 4
D. volubilis Roxb. Asia Climbing shrub[25, 47]/woody liana[25] 2
doi:10.1371/journal.pone.0138231.t001
PCR amplifications of rbcL, matK and ITS were carried out on a Gene Amp 2700 (Applied
Biosystems, USA) with Qiagen Taq PCR Master Mix kit (Qiagen, Sweden), using the manufac-
turer’s instructions, except that the reaction volume was increased to 50 μL. Primer sequences
are listed in S3 Table. The PCR conditions for rbcL was 94°C for 3 min, followed by 30 cycles of
94°C for 1 min, 54°C for 1 min and 72°C for 1min, and a final step at 72°C for 10 minutes. For
matK, the PCR conditions were 1 min at 94°C, 30 cycles of 94°C for 30 s, 54°C for 20 s and
72°C for 50 s, followed by 72°C for 5 min. For ITS, the PCR conditions were 5 min at 94°C, 35
cycles of 94°C for 1 min, 53°C for 1 min and 72°C for 1min, followed by 72°C for 7 minutes. If
samples did not amplify with these conditions, annealing temperature was lowered by 1–2°C
and number of cycles increased to 35 (for rbcL and matK) or 40 (for ITS).
PCR products were purified and sequenced by Macrogen Inc. (Seoul, Korea), using the
same primers as for amplification.

Data analysis
Sequences were edited and assembled in Geneious 6.0.5 to 7.1.7 (Biomatters Inc., USA). All
sequences were deposited in Genbank [44] and given accession numbers (S1 Table). The edited
sequences for each gene were aligned separately with Clustal W [54] as implemented in Gen-
eious 7.1.7 (default settings), and trimmed for primer sequences in both ends. After initial
alignment with Clustal W, the alignment was manually adjusted, if needed, following the prin-
ciples described in Kelcher & Clark [55]. Alignments can be obtained from the author by
request.
Alignments for the three barcoding regions were concatenated in Mesquite [56] to produce
different combinations of datasets with variable number of taxa included for the different
analyses.
Tree-based analyses (Maximum parsimony and Neighbour joining)

To infer on the taxonomy of the sampled Dalbergia species, a phylogenetic analysis were con-
ducted on the rbcL+matK+ITS dataset, including all known Dalbergia specimens and the two
Machaerium species, but excluding the 14 unknown Dalbergia sp. (n = 83).
The analysis was performed in PAUP vers. 4.0b10 [57] using parsimony as the optimality
criterion. Uninformative characters were excluded, gaps were treated as missing data and all
characters were equally weighted and unordered. The two Machaerium specimens were
defined as outgroup species.
An initial heuristic search was carried out with 1000 replicates, holding maximum 10 trees
at each step. Random stepwise addition was used for the starting tree in each replicate and
branch swapping was performed by tree-bisection-reconnection (TBR). Branches with maxi-
mum length equal to zero were collapsed. A second search was performed by TBR swapping
on the trees found in the first round, holding up to 15000 trees and swapping to completion.
Branch support was assessed by bootstrapping [58], using 2000 bootstrap replicates and the
same settings as in the heuristic search, but with only 10 random stepwise addition sequences
per replicate. Only groups that appeared in >50% of the trees were retained.
As a method of assigning the unknown Dalbergia specimens to species, a Neighbour-joining
(NJ) tree was constructed using the rbcL+matK+ITS dataset and included all known and
unknown Dalbergia samples, but excluded the Machaerium specimens (n = 95).
The NJ tree was constructed in PAUP , using uncorrected p-distance [59] as genetic dis-
tance measure and setting negative branch lengths to zero. No outgroup was used. If the Dal-
bergia sp. specimen in question was found within a cluster consisting exclusively of two or
more specimens of the same known species, the Dalbergia sp. was accepted as member of that
particular species. Otherwise, it was regarded as not identified/unknown.
Barcoding analyses
We compared the identification success of different barcoding analyses using the programs
TaxonDNA, BLOG and WEKA. We performed the analyses on all combinations of barcode
regions, alone or in combination of two or three regions, giving a total of seven barcodes
(Tables 2 and 3). Only Dalbergia specimens represented by two or more specimens were
included, and specimens that failed amplification for one of the three regions, were excluded
from any analysis concerning the given region. This meant that numbers of specimens and spe-
cies varied between the barcoding datasets evaluated (Table 2). Identification of the unknown
Dalbergia was conducted with the rbcL+matK+ITS barcode data set only, except for two sam-
ples (Dalbergia sp. Cambodia1 and Dalbergia sp. Thailand5), where the rbcL+matK barcode
was used because of amplication failure of ITS.

Table 2. Characteristics of the seven barcodes for Dalbergia spp. evaluated in this study.
Barcode No. of specimens/No. of Alignment length Mean intraspecific distance Mean interspecific distance
species (bp) (range) (range)
rbcL 70/21 607 0.0010 (0–0.0070) 0.0068 (0–0.0181)
matK 68/21 775 0.0019 (0–0.0144) 0.0141 (0–0.0310)
ITS 56/17 662 0.0134 (0–0.0650) 0.1110 (0–0.1887)
rbcL+matK 67/21 1382 0.0013 (0–0.0084) 0.0110 (0.0008–0.0246)
rbcL+ITS 55/17 1269 0.0077 (0–0.0351) 0.0600 (0–0.1041)
matK+ITS 53/17 1437 0.0072 (0–0.0334) 0.0570 (0.0021–0.0930)
rbcL+matK 52/17 2044 0.0055 (0–0.0251) 0.0420 (0.0015–0.0686)
+ITS
Intra- and interspecific distances calculated using uncorrected p-distances between all sequence pairs.
Distance-based analyses (TaxonDNA)

Uncorrected p-distances between all sequence pairs were calculated in TaxonDNA/SpeciesI-
dentifier 1.7.8 [38] and used to calculate mean and range of intraspecific and interspecific dis-
tances for the seven barcodes. Uncorrected p-distances was chosen over the otherwise widely
used K2P distance measure because there is no need for a complex model for distance measures
when analyzing closely related sequences [59], and K2P seems not to be the first choice if any
models should be applied [60, 61].
The ideal barcode for species identification should exhibit a ‘barcode gap’, where the mini-
mum interspecific distance is larger than the maximum intraspecific distance within any spe-
cies [39, 62]. However, as coalescent depth may vary among species, a global overlap between
intra-and interspecific distances might not interfere with identification success [63]. A more
accurate way is thus to evaluate the local barcoding gap, and for each species plotting the dis-
tance to the nearest non-conspecific against the distance to the furthest conspecific (e.g. [64,
65]). Hence, to evaluate the presence or absence of a local barcode gap, the maximum intraspe-
cific and minimum interspecific distances for each sequence were found using the èxtreme
pairwise’ function in TaxonDNA. For each species, the maximum intraspecific distance was
then plotted against the minimum interspecific distance, with a 1:1 slope representing no local
barcoding gap.
Table 3. Specimen identification rates in % (correctly identified/misidentified/not identified) for Dalbergia spp. using six different classification
methods, for each of the seven barcodes.
Barcode TaxonDNA BLOG Naïve Bayes SMO J48 Jrip

rbcL 40/6/54 43/9/48 63/37/0 60/40/0 54/6/0 44/56/0
matK 81/13/6 81/5/24 79/21/0 81/19/0 56/4/0 57/43/0
ITS 89/0/11 65/23/12 88/12/0 93/7/0 61/39/0 52/48/0
rbcL+matK 87/10/3 86/5/9 78/22/0 88/12/0 63/37/0 55/45/0
rbcL+ITS 89/4/7 65/6/29 85/15/0 93/7/0 65/35/0 62/38/0
matK+ITS 100/0/0 65/12/23 87/13/0 94/6/0 60/40/0 57/43/0
rbcL+matK+ITS 100/0/0 65/6/29 83/17/0 94/6/0 60/40/0 46/54/0
TaxonDNA: Best close match results. Not identified rates are summed over the “Ambiguous” and “No match” categories, see S4 Table for details. BLOG:
percentage correct classification for test file, using 90% slicing at species level. Naïve Bayes = Bayesian, SMO = Support vector machine, J48 = decision
tree, Jrip = rulebased, all four classification methods in WEKA, tested with 10-fold crossvalidation. See materials and methods for details on the analyses.

TaxonDNA was also used for evaluating the specimen identification success for the seven
barcodes using the `best close match’ function of the program. This method is equivalent to
the nearest neighbor-method [42], and compares each sequence (query) to all other sequences
(references) in the dataset, and assigns the query to the species with the reference sequence
with the lowest distance to that query sequence. If multiple species have equally small distance
matches, the result is considered ambiguous. If the distance to the most similar sequence(s) is
outside a certain threshold level, the query sequence is classified as no match. The threshold
used in these analyses was computed for each dataset during the pairwise distance analyses,
and can be seen in S4 Table.
The Dalbergia sp. specimens were tested against the rbcL+matK+ITS barcode (rbcL+matK
for the two samples that lacked the ITS sequence) using the `query against sequences’ function
and applying same conditions as in the `best close match’ analysis. If the distance to the nearest
reference sequences was above the threshold used in the `best close match’ analysis, the speci-
men was considered not identified.
Machine-learning approach (BLOG and WEKA)

We explored the feasibility of the recently published machine-learning approaches to assign
specimens to species [40, 43], in order to compare them with the traditional methods described
above. DNA barcoding analysis can be viewed as a classification problem: given a reference
data set composed of DNA barcode sequences of known species, and a query data set with
sequences of unknown species, how can the unknown sequences be recognized as a given spe-
cies present in the reference data set? [40, 43]. This problem can be addressed by a machine
learning approach, where a classification model (different models can be applied) is build
based on the reference dataset, where after the classification model is applied to the query data
set [43]. The query data set can contain unknown or known species, where the latter will allow
for verification of the classification model. If only one dataset is provided, then it can be ran-
domly divided into reference and query data set to test the efficacy of the model [43].
Two different programs were used to apply different classification models: BLOG[40],
which is a program specifically developed to handle DNA barcode data, and WEKA [41],
which is a software package of machine learning tools for classification, association and cluster-
ing problems.
BLOG provides a diagnostic and character-based method, which formulates a set of classifi-
cation rules that identifies the species in terms of location of key diagnostic nucleotides (e.g., if
position 432 = A, and position 615 = T, then the specimen is classified as Dalbergia ovata).
BLOG uses the supplied training data set to compute the classification rules, and these rules are
then applied on both the training set and the test set, and identification success for both data
sets are reported. The seven barcode datasets in this study were tested with a single file input,
which was then subject to a 90% slicing, meaning that 90% of the dataset was allocated to train-
ing set, and 10% was allocated to test set. The slicing is done within species-level, so for each
species, BLOG will allocate 90% of the specimens to the training set, and 10% to the test set.
For identification of the unknown Dalbergia spp., all known specimens were included in the
training data, and the Dalbergia sp. specimens were used as test data.
WEKA in the same way as BLOG works with train and test data either supplied separately
or created in the program. WEKA was used to test the four classifiers used by Weitschek et al.
[43]: Naïve Bayes [66], SMO [67], C4.5 (J48) [68] and Jrip [69]. Naïve Bayes is a Bayesian-
based classifier using estimator classes. A posteriori probabilities of the species identity are eval-
uated based on the observed data and a priori probabilities [43]. Naïve Bayes does not provide
the investigator with a readable model, so the specific assignment of specimens to species

cannot be checked manually. SMO is the WEKA version of the function-based method Sup-
port Vector Machines (SVM) [67]. SMO converts the reference data into objects in multi-
dimensional vectors and then defines an optimal hyperplane that separates the classes with the
largest minimum distance. Objects from the query set can then be classified according to the
separating hyperplane. SMO usually performs with high classification accuracy, but does not
produce a classification model that can be directly read by the investigator [43]. J48 is the
WEKA implementation of the decision tree algorithm C4.5 [68], which produces a simple tree
structure, where non-terminal nodes represent tests on one or more attributes (here nucleotide
type at specific locations). The terminal branches give the results of the decision based on the
test. The advantage of the decision-tree method is that the output model is easily read as a set
of rules composed of sequence positions and nucleotide compositions. The drawback is that it
is very sensitive to variations in the training data[43]. Jrip is the implementation of the rule-
based classification method RIPPER (Repeated Incremental Pruning to Produce Error Reduc-
tion)[69]. An initial set of rules for each class is generated, and then optimized k times. In the
same way as BLOG, this method has the advantage that it produces a set of logic rules for each
species in the dataset, that can be examined and manually applied to test species if desired [43].
All four classification methods in WEKA were run on all seven data sets, using a single
input file and testing with 10-fold cross-validation. For identification of the Dalbergia sp., only
the SMO classifier was used, as this performed best overall (Table 3). The dataset with Dalber-
gia sp. was loaded as test data set, and a saved model for the SMO and the rbcL+matK+ITS bar-
code (rbcL+matK for the two samples that lacked the ITS sequence) was loaded. The function
`re-evaluate model on current test set`was then chosen, selecting outcome predictions as
output.
Results
Sequences
The rbcL region was successfully sequenced for 96 samples, matK for 94 samples, while ITS
was only sequenced for 79 samples (due to low sequencing success of the herbarium specimens,
most likely due to DNA degradation and fungal contamination). All specimens were success-
fully sequenced for at least two of the three barcoding regions (See S1 Table for details).
For rbcL, this resulted in a length of 607 bp, and no length variation was observed for any of
the samples.
The matK sequences were variable in length, the longest being 851bp, partly due to reading
difficulties in both ends of the sequence (primarily because of a long poly-A-sequence). The
final alignment was therefore trimmed to 775 bp in order to reduce missing data at the ends.
Alignment included one 6-bp insertion for all three D. lanceolaria-specimen, one 3-bp inser-
tion shared for a group consisting of D. cochinchinensis, D. ovata and D. latifolia specimens,
and a 9-bp deletion for the outgroup species Machaerium lunatum. All sequences were
reverse-complemented to follow standard annotation of the matK region.
ITS sequences varied in length, and many sequences were short due to problems with
obtaining high quality reads. The ITS5/ITS4 primers used in this study [36] lie in the conserved
flanking regions of 18S and 26S, respectively, and the sequences were trimmed to contain only
ITS1, 5.8S and ITS2. After trimming, the longest observed sequence was 616 bp for D.
cochinchinensis (628 bp for outgroup Machaerium). The alignment including all 79 sequences
obtained for ITS was 677 bp and included numerous insertions/deletions. If the removal of
specimens from the dataset left blank positions in the alignments because their sequences had
insertions not found in any other specimens, these blank positions were removed before further
analyses. Excluding the Machaerium samples, the alignment used for NJ tree therefore

consisted of 666 bp. For the barcoding analysis, where only Dalbergia species with at least two
specimens where included, the alignment was 662 bp. For the parsimony analysis, including
the Macherium samples, but excluding the 14 unknown Dalbergia, the alignment was 675 bp.
Taxonomy and phylogenetics of Dalbergia species based on parsimony

analysis
The rbcL+matK+ITS dataset for parsimony analysis included 2057 characters, of which 303
were parsimony informative. ITS contributed the most to this number, with 207 informative
characters, while rbcL+matK combined had 96 informative characters.
The maximum parsimony analysis resulted in 8,330 equally parsimonious trees, with a tree
length of 906 steps. A second round with TBR swapping on these trees produced 15,000 trees,
still with a tree length of 906, of which a strict consensus tree was constructed (Fig 1). The con-
sistency index (CI) was 0.470 and the retention index (RI) was 0.812. The strict consensus tree
was not well resolved, showing several basal polytomies, which makes inferences on intragene-
ric relationships difficult. However, a well-supported group consisting of D. candenatensis, D.
pinnata and D. velutina (including D. benthamii) was found at the base of the tree, along with
the American species D. ecastaphyllum and D. monetaria which were 100% supported as sister
species. At the large polytomy, two well-supported groups formed, D. cochinchinensis, D. ovata
and D. latifolia (98% bootstrap support), and D. oliveri together with D. cana (87%). Dalbergia
subcymosa was not found to be closely related with the other Brazilian species D. miscolobium,
but to the Malagasy D. trichocarpa. These two seemed related to a group consisting of D. sissoo
and a D. rimosa/D. entadoides/D. odorifera-complex. The resolution was much higher at the
tips of the tree. Sixteen of the 21 species represented by two or more specimens were supported
as monophyletic with bootstrap values above 60. The two focus species D. cochinchinensis and
D. oliveri, was found well-resolved with bootstrap values of 100% and 97%, respectively. While
D. odorifera in itself was monophyletic, it was nested within a polyphyletic D. rimosa group,
also containing D. entadoides. An otherwise well supported cluster of D. velutina specimens
included D. benthamii, and was therefore not found monophyletic. The two specimens of D.
hancei, which lack ITS sequences, did not cluster together, but were unresolved at the basal
polytomy. D. assamica and D. hupeana were in the same unresolved group, and could not be
separated by the data.
Distance analysis and barcoding gap for rbcL, matK and ITS barcodes
Mean intra- and interspecific genetic distances of evaluated DNA barcodes are shown in
Table 2.
The rbcL region had the lowest values for mean intra- and interspecific distances (p-dis-
tances of 0.001 and 0.007, respectively), while it was twice as high for the matK region, and
more than 10-fold higher for ITS. Combined barcodes had intermediate values, where the rbcL
+matK had the lowest values, and the highest values were found for the rbcL+ITS barcode
(probably due to the fact that this was the shortest of the combined regions, and ITS contrib-
uted by far the most to the total number of differences between sequences). Four species had
no intraspecific variation for the combined rbcL+matK+ITS barcode (D. cultrata, D. ecasta-
phyllum, D. monetaria and D. trichocarpa). There was an overlap between the observed intra-
and interspecific distances for all evaluated barcodes.
Using rbcL and matK alone, as well as in combination, most species failed to exhibit the bar-
code gap (Fig 2). The use of ITS greatly improved this picture. The best results were found for
the matK+ITS barcode, while the added information of rbcL did not seem to increase the reso-
lution. Dalbergia rimosa and D. velutina are the two species found under the line for the matK


Fig 1. Strict consensus tree resulting from maximum parsimony analysis showing the relationship of Dalbergia species using the rbcL+matK+ITS
barcode. Tree length = 906, CI = 0.470, RI = 0.812. Numbers above branches are bootstrap support values; values below 50% are not shown. Monophyletic
species with bootstrap values above 60% are given in bold. Species are colored regarding to continent: black: Asia, green: America, red: Africa.
doi:10.1371/journal.pone.0138231.g001
+ITS and rbcL+matK+ITS barcode, which means that no barcoding gap was observed for these
species for any barcode.
Comparison of discrimination methods and barcode regions for

specimen identification
We evaluated the discrimination ability of all seven possible combinations of the three barcod-
ing regions included in this study using TaxonDNA, BLOG and four different classification
methods implemented in WEKA. The rates of correctly identified, misidentified and not iden-
tified specimens for each dataset and method are shown in Table 3.
Averaged over all discrimination methods, the ITS barcode had the highest correct identifi-
cation rates (78%), and rbcL had the lowest (53%). The single highest correct identification rate
was obtained for the matK+ITS and rbcL+matK+ITS barcode using TaxonDNA, reaching
100% identification success.
TaxonDNA and SMO were the methods that performed best on average cross all seven
tested barcodes (both 86% correct identification), and second-best was the Naïve Bayes method
(82%). The rule-based Jrip and the decision tree method J48 generally had low correct identifi-
cation rates, and especially for the combined barcodes, where the other methods showed high
identification rates. TaxonDNA performed better than SMO and Naïve Bayes on the barcodes
including the ITS region, however the SMO and Naïve Bayes gave markedly better results on
the rbcL data set. While barcodes including ITS generally had the highest correct identification
rates within each method, BLOG found the highest correct identification rate for rbcL+matK
(86%), and only 65% correct identification for all barcodes involving ITS.
While TaxonDNA and BLOG returned some non-identified results, the four WEKA meth-
ods classified all specimens (correctly or wrongly) which meant that the rate of misidentifica-
tion was relatively lower for TaxonDNA and BLOG than that of the WEKA methods. Thus
TaxonDNA showed the average lowest misidentification rates of the six methods (6%), while
BLOG had the second lowest at 10% and the SMO method had an average 15% misidentifica-
tion rate.
Identification of unknown specimens

The 14 Dalbergia sp. specimens were tested against all known Dalbergia specimens using Tax-
onDNA, BLOG and the SMO classifier in WEKA, as well as by examining their position on a
NJ tree. While identification in TaxonDNA, SMO and BLOG required several specimens per
species, and the reference data therefore only included the 21 species represented by two or
more specimens, the NJ tree included all Dalbergia species, and thus might give different
results.
For nine of the 14 specimens, all four identification methods yielded the same results
(Table 4). SMO assigned names to all samples, while BLOG and TaxonDNA did not find any
matches for the Dalbergia sp. Thailand2 and Dalbergia sp. Thailand 3, respectively. The NJ
method failed to assign names to five specimens. TaxonDNA, BLOG and SMO returned the
same results for the Dalbergia sp. Thailand5 and Dalbergia sp. Thailand7 (D. velutina and D.
rimosa, respectively), while no certain identification could be done from the NJ tree (Fig 3).
This was most likely due to the presence of D. benthamii and D. entadoides in the NJ dataset,
which were not included in the barcoding dataset. At the NJ-tree the Dalbergia sp. Thailand3


Fig 2. Presence/Absence of barcode gaps in Dalbergia spp. for the seven barcodes. Minimum interspecific vs. maximum intraspecific uncorrected p-
distances (%) for the single (a, b, c) and combined (d, e, f, g) barcodes. Each data point represents one or several species, since some species have identical
values of intraspecific and interspecific distances. Species that fall above the 1:1 line exhibit a barcode gap.
was placed together with D. mimosoides, but with only one specimen represented, identifica-
tion cannot be justified.
The cambium/bark and wood samples included in the test of unknown specimens were
assigned to D. cochinchinensis and D. cultrata, respectively (Dalbergia sp. Cambodia3 and Dal-
bergia sp. Laos3, Table 4).
Discussion
Taxonomy and phylogeny of South-East Asian Dalbergia species
To our knowledge, this analysis represents the first attempt of a molecular phylogeny focused
on South-East Asian Dalbergia. Vatanparast et al. [35] did a molecular phylogeny based on ITS
sequences from 64 Dalbergia species covering the entire pantropical distribution, but apart
from this, classifications within Dalbergia are based on morphology [47, 70, 71]. While our
data generally gives a high resolution at species level, the basal part of the phylogeny-tree is
Table 4. Identification of Dalbergia sp. specimens, based on rbcL+matK+ITS barcode.
Tissue Putative TaxonDNA (within NJ-within BLOG SMO

identification threshold) cluster
Dalbergia sp. Leaf D. nigrescens D. nigrescens Not identified D. nigrescens D. nigrescens
Cambodia1a
Dalbergia sp. Leaf unknown D. lanceolaria D. lanceolaria D. lanceolaria D. lanceolaria
Cambodia2
Dalbergia sp. Cambium/ D. cochinchinensis D. cochinchinensis D. D. D.
Cambodia3 Bark cochinchinensis cochinchinensis cochinchinensis
Dalbergia sp. Leaf D. oliveri D. oliveri D. oliveri D. oliveri D. oliveri
Cambodia4
Dalbergia sp. Laos1 Leaf D. oliveri D. oliveri D. oliveri D. oliveri D. oliveri
Dalbergia sp. Laos2 Leaf D. stipulacea D. stipulacea D. stipulacea D. stipulacea D. stipulacea
Dalbergia sp. Laos3 Sapwood D. cultrata D. cultrata D. cultrata D. cultrata D. cultrata
Dalbergia sp. Leaf unknown D. oliveri D. oliveri D. oliveri D. oliveri
Thailand1 (Herbarium)
Dalbergia sp. Leaf unknown D. candenatensis Not identified Not classified D. pinnata
Dalbergia sp. Leaf unknown outside threshold Not identified D. stipulacea D. cultrata
Dalbergia sp. Leaf unknown D. rimosa D. rimosa D. rimosa D. rimosa
Dalbergia sp. Leaf unknown D. velutina Not identified D. velutina D. velutina
Thailand5a (Herbarium)
Dalbergia sp. Leaf unknown D. rimosa D. rimosa D. rimosa D. rimosa
Dalbergia sp. Leaf unknown D. rimosa Not identified D. rimosa D. rimosa
a
only rbcL+matK was used because of lack of amplification success for ITS. NJ = Neighbour Joining. SMO = Support vector machine method, as
implemented in WEKA. Putative identification based on morphological characters, except for Dalbergia sp. Laos3, where is it based on declared identity
from commercial company (See S1 Table for details on origin of samples).

Fig 3. Neighbour-joining tree based on the rbcL+matK+ITS barcode, including test Dalbergia sp. specimens. Uncorrected p-distance was used as
distance measure.

largely unresolved (Fig 1), making it difficult to evaluate the congruence of molecular data with
the morphological classifications. We failed to obtain ITS sequences for specimens of D.
benthamii, D. hancei, D. horrida, D. mimosoides and D. volubilis and this could be the reason
why these specimens are placed at basal polytomies or group with specimens from other spe-
cies, as ITS in general show the highest discrimination ability (Table 3).
Our analysis only includes few non-Asian species, but the fact that neither the American
species nor the African (including Malagasy) species are found together (Fig 1), supports a
multiple long-distance dispersal theory as suggested by Vatanparast et al. [35].
Although the resolution in the basal parts of the tree is low, a few well-supported groups are
seen, which are consistent with findings in previous studies. The apparent sister relationships
between D. monetaria and D. ecastaphyllum [35, 48] has also been found by Vatanparast et al.
[35] and Ribeiro [48]. We also find D. pinnata and D. candenatensis as sister species, which is
consistent with the results by Vatanparast et al. [35]. The two latter species are placed as sister
to D. velutina (including one D. benthamii specimen). This pinnata/candenatensis/velutina
group is recognized to be morphologically similar by Niyomdham et al. [25].
D. cochinchinensis is highly supported as monophyletic and together with D. latifolia and D.
ovata forms a well-supported group. Prain [71] placed these three species in the same section,
Miscolobium, and Vatanparast et al. [35] also found D. cochinchinensis and D. latifolia to be
closely related. Young individuals of D. cochinchinensis can be difficult to distinguish from D.
ovata (F. Adema, pers. comm., see also S1 Table), as they have similar leaf morphology, and
Niyomdham et al. [25] also recognizes the morphological similarity between D. cochinchinensis
and D. ovata.
We find the timber species D. oliveri to be well supported as monophyletic. The specimen
sampling (n = 8) in this study includes a specimen originally identified as D. dongnaiensis (D.
oliveri Thailand3, see also S1 Table) and two specimens from Cambodia, where the name D.
bariensis is used. We therefore strongly encourage that the name D. oliveri is used consistently
across the distribution range, as suggested by Niyomdham et al. [25].
Dalbergia oliveri can be confused with D. lanceolaria (personal observations), especially if
fruits and/or flowers are not present. This study show that barcoding markers can accurately
distinguish these two species and that each is supported as monophyletic species (Fig 1). In the
analysis of Vatanparast et al. [35], a small clade consisting of two D. oliveri, one D. dongnaiensis
and one D. lakhonensis is present. According to the Niyomdham et al. revision [25], D. dong-
naiensis should be treated as D. oliveri, and D. lakhonensis as D. lanceolaria. Three other D. lan-
ceolaria specimens in their analysis occur together in another clade, affiliated with D.
assamica/D. balansae, a relationship we also find (Fig 1). Given the difficulties in species identi-
fication in Dalbergia, and the number of specimens found to be misidentified in this study
alone (S1 Table), it seems possible that the D. lakhonensis might be a misidentification. If the
true identity of this specimen is D. oliveri, then the Vatanparast et al. analysis [35] actually also
shows support for the D. oliveri sensu Nioyomdham.
We treat D. balansae as a synonym for D. assamica [25, 49], and the position of D. assamica
China2 is probably due to missing data for ITS. Vatanparast et al. [35] maintain D. balansae as
a separate species, but it is found in the same clade as D. assamica, which could further support
the synonymy of D. balansae.
Missing data could however not account for the fact that D. assamica and D. hupeana could
not be separated from each other, and that D. rimosa appeared paraphyletic with both D. enta-
doides and D. odorifera nested within it. It might be due to inadequate resolution of the barcod-
ing data for these taxa, but as the discrimination ability in this study was very high, we find it
likely that these results might be indicative of taxonomic uncertainty around these taxa.

We find three separate clades with D. rimosa specimens, each with bootstrap values of 96 or
100, and one of them including D. entadoides. This could suggest that D. rimosa should per-
haps be split into several separate species. Further studies including additional DNA regions
and a more complete taxon sampling are needed to properly solve these issues, as well as to
generally increase the resolution of the phylogeny.
However, we find that the molecular data for the Indochinese species of Dalbergia corre-
sponds well with the morphological revision by Niyomdham et al. [25], and thus encourage to
use this revision for correct naming of species in the Indochina area.
Which barcoding regions provide the best identification of species in

Dalbergia?
The barcoding markers generally discriminated well among Dalbergia specimens in this study,
although the results varied greatly over barcoding regions and analysis methods. We found
that ITS had the highest efficiency in identification of specimens in Dalbergia, alone or in com-
bination with matK. The rbcL barcode had the lowest discriminatory power and as a single-
locus barcode cannot be recommended for identification of Dalbergia specimens. ITS also
showed greater ability than rbcL and matK to produce the `barcode gap’ (Fig 2). The superior
discriminatory power of ITS over plastid barcoding regions is consistent with the results of
other recent genus-level studies, e.g. for Euphorbia [17], Lamium [64], Populus [72] and Lysi-
machia [73]. However, Yu et al. [31] used the chloroplast markers rpoC1 and trnH-psbA as
well as ITS to discriminate between Dalbergia odorifera and D. tonkinensis, and found that
trnH-psbA discriminated 100% between the two species, whereas ITS and rpoC1 yielded incon-
sistent results.
It should be noted that the analyses for ITS in this study were conducted on a smaller data-
set. Four species had only one or no sequences obtained for ITS and thus did not fulfill the cri-
teria for inclusion in barcoding comparisons. Excluding these specimens from the rbcL+matK
analysis as well increases the identification rate from 87% to 92%. It can thus not be ruled out
that a smaller data set contributes partly to the difference in identification rates between the
rbcL+matK and ITS regions, an issue experienced in other studies as well [64, 73]. A general
concern with the use of ITS is the frequent problems with obtaining high-quality sequences
due to low sequence recovery and fungal contaminations [37], which also proved difficult in
this study.
Another issue with ITS, and to some extent matK, is the challenge with proper sequence
alignment. All analysis methods used in this study require aligned sequences, which mean that
a new alignment must be made each time a new sequence is introduced to the dataset. Further,
alignment of ITS sequences is not straight-forward and most often needs to be manually
adjusted after initial alignment with alignment algorithms such as Clustal [54]. A solution
could be the application of alignment-free analysis methods, such as BLAST [74] or BRONX
[75], but it could also be seen as a reason to choose regions that require no or little alignment
work, such as rbcL or matK in this study.
If applying DNA barcoding for Dalbergia as a method of identification of herbarium speci-
mens, or for identification of timber samples for CITES control, the matK region, although not
as powerful as the ITS for identification, seems a good choice, showing both high sequence
recovery and high identification rates. The matK region has proven a useful barcode in other
studies of Fabaceae [15, 76], although some report difficulties with amplification and sequenc-
ing [77]. The matK could be supplemented with rbcL, as this seems to increase the discrimina-
tion ability slightly (Table 3).

For fresh tissue samples, a two-locus barcode of matK+ITS yields optimal discrimination
and rbcL then seems unnecessary, as it does not enhance the discrimination ability of the data.
As an alternative to the three loci tested in this study, it might be worth considering the
intergenic spacer trnH-psbA. Generally high discrimination ability of this marker have been
acknowledged by the CBOL Plant Working group [12] and Yu et al. [31] found it to be efficient
on two Dalbergia species, why it could be worth testing on a larger sample of Dalbergia species.
Which analytic methods provide the best identification of species in

Dalbergia?
TaxonDNA and SMO were the two methods that gave the highest correct identification rates;
however TaxonDNA had the single highest rates and SMO performed better at the rbcL dataset
with low variation. The decision-tree method J48 and the rule-based Jrip did not yield impress-
ing results for any of datasets. Weitchek et al. [43] also found J48 and Jrip to be less efficient
than the other machine learning methods, although not as low performing as in this study.
Comparisons between the different identification methods used in this study should however
be interpreted with caution, as the sampled number of specimens per species is not optimal
and the different methods have not been tested in the same way. While the four WEKA classifi-
cation methods were tested with 10-fold cross-validation, the Taxon-DNA is a nearest-neigh-
bour method, and is tested with leave-one-out validation (LOO). Cross-validation or LOO is
currently not implemented in BLOG, and the 90% / 10% testing of the data is sensitive to data
containing species represented by only two specimens. In fact, species represented only by two
specimens were involved in the majority of the misidentifications and none-identifications in
BLOG, for the analysis including ITS (D. assamica, D. cana, D. ecastaphyllum, D. lanceolaria,
D. monetaria, D. nigrescens). For BLOG and the WEKA classification methods, the authors rec-
ommend at least four specimens per species for optimal performance. As this criterion is only
met for a few species in this study, our dataset might not give justice to these methods, and it
might be part of the explanation why the Jrip and J48 methods show low performance across
datasets. The rule-based BLOG also does not seem very well suited for imperfect datasets as the
present, as “a complete reference library of polymorphisms for each species is required in the
training set to avoid false negatives” [40]. This is a general weakness of the method, as it is not
always possible to sample all variation within the species, and also not necessary if other meth-
ods are capable of handling such variation, such as TaxonDNA or the SMO function. The
problem with species only represented by two specimens, as well as the fact that any ITS
sequence in the test set is likely to harbor variation not seen in the train set, probably explain
why BLOG has low identification success for any barcode combination including ITS (65%,
Table 3).
For TaxonDNA, it cannot be ruled out that the low number of specimens in some species
could possibly cause an underestimation of intraspecific distances. If including more specimens
to the dataset would result in higher maximum intraspecific distances, this would reduce the
barcoding gap, and perhaps also affect discrimination rates by the Best Close Match method.
Whether any effect would be positive or negative would dependent on chance and how well the
total intraspecific variation is already covered in the dataset.
The potential bias by sample sizes set aside, our results seem to support TaxonDNA as a
highly workable and accurate method. TaxonDNA is very simple to use and understand, and
has been accepted as a standard method to evaluate barcodes in many studies (e.g. [17, 64, 73,
78–80]).
An optimal DNA barcode classifier would not only yield high correct identification rates,
but should also show a minimum level of misidentifications. As reference databases are rarely

perfect in real situations, ability to detect specimens not covered in the reference database and
return “not identified” results is a desirable feature for a DNA barcode classifier. This aspect
favours TaxonDNA, as well as BLOG, over the WEKA classification methods as they are cur-
rently implemented. However, if dealing with a dataset with low variation, our data indicates
that the Naïve Bayes or SMO method might be used with greater success rates than Tax-
onDNA. Under some circumstances, BLOG could be the method of choice, as it produces a set
of rules to characterize each species in terms of nucleotides at particular positions. This feature
might be desirable e.g. in designing species-specific assays to be used in CITES enforcement.
Efficiency of the DNA barcodes to identify unknown Dalbergia

specimens
We found that the different methods generally converge on their results on identification on
unknown specimens, and the putative ID (based on morphological observations) for six of the
samples were confirmed. So, can we trust the proposed identity of the specimens using these
barcoding markers? Including the high discrimination results found in this study, the answer
would be “yes”. However, rather than expecting the barcoding data to unravel the universal
truth, it should be regarded critically, the quality of the reference database needs to be taken
into account, and the data seen as a supplement to morphological identification.
The NJ tree has a few more non-identified or ambiguous results, which is probably due to
the fact that more species were included in the NJ tree than in the other analyses. This stresses
the importance of adequate taxon sampling: not including all relevant species in the reference
database can lead to possible misidentifications. This is particularly true for the SMO method,
which assigned names to all the query sequences. TaxonDNA shows the advantage of a “not
identified” category, as it does not identify specimens if the distance to the nearest reference-
sequence is outside the threshold computed for that particular dataset. This could prevent
many cases of misidentifications, and instead highlight issues with missing taxa in the reference
database. For the specimens that were not classified or had divergent results, it could help
guide the morphological identification process, and also serve as valuable reference data which
might lead to identification once the database is augmented.
Application of barcoding tools in conservation of Dalbergia

Barcoding tools can support on-going conservation measures of the species in several ways.
Species delimitation and identification is the first critical step in an accurate assessment of dis-
tribution, population abundance and threats of target species. In the present study we e.g.
found that the rbcL+matK+ITS barcode markers accurately identified D. oliveri as monophy-
letic in accordance with Niyomdham et al. (1997) [25]. Hence, the IUCN red list assessment
for this species can now be updated, including the data from the synonyms D. bariensis, D.
mammosa, and D. dongnaiensis, to gain a more accurate assessment of the conservation status
of this species.
The application of DNA methods to verify species identity and origin of internationally
traded timber has attracted increasing interest in recent years as part of global systems to sup-
port sustainable forestry and especially reduce illegal logging [81, 82]. Several studies has
shown ability of DNA barcoding to effectively discriminate threatened from common species,
such as among Euphorbia species from Madagascar [17], and among timber species in the
mahogany family (Meliaceae) [18]. Yu et al. [31] showed that barcoding markers had the
potential to discriminate between the precious timber species D. odorifera and the closely
related, but less valuable species D. tonkinensis.

Enhancing the applicability for direct use in CITES enforcement, genus or species-specific
assays independent on expensive sequencing and reference databases can be developed. Exam-
ples of specific assays that have been developed include a probe-based real-time PCR assay tar-
geted to discriminate between Gonostylus and non-CITES genera in Thymeleaceae [83], and a
PCR/restriction enzyme assay to discriminate between Swietenia and non-CITES genera in
Meliaceae [84]. Specific assays can also target shorter fragments of DNA, which increases the
probability of amplification of degraded DNA from wood samples [85].
Illegal logging represents a huge threat to the Dalbergia timber species in Indochina. Given
the high discrimination success rates found in this study, Dalbergia seems an ideal candidate for
using DNA barcoding for identification of traded timber. The present study provides a database
to which sequences from new samples could be matched, as well as necessary sequences from
which a species-specific assay for testing of D. cochinchinensis/not D. cochinchinensis could be
designed. Such an assay could be used in CITES enforcement and also in local control measures.
This study was able to identify wood samples which shows that DNA barcoding has the potential
to be applied as a timber identification tool in Dalbergia, although it is likely that the process
stage of the timber will have an influence on the ability to extract DNA samples of adequate qual-
ity, as well as whether the samples are sapwood or heartwood [31, 85, 86].
Future floristic investigations can benefit from the reference database established for Dalber-
gia in Indochina. We suggest future botanical collections in the region to include leaf samples
in silica gel, for subsequent DNA extraction, sequencing and comparison to the reference data-
base. Presently, DNA facilities are not available in national herbaria in the region, but due to
the simplicity of the barcoding approach the DNA work can easily be outsourced at moderate
costs. The obtained sequences can be matched to the database by herbarium staff. At first, the
database will have significant amounts of missing data, but this problem will decrease as the
use of this method increases and the population of verified sequences grow. Local herbaria
have only recently been revitalized after years of political unrest in the region and often suffer
from shortage of staff. Barcoding can speed up the identification process of collections, includ-
ing sterile material, and increase knowledge on species distributions and abundance. This will
allow more detailed assessment of threatened species that can guide conservation efforts and
priorities at national and regional levels.
Supporting Information
S1 Table. Voucher information and Genbank accession numbers for the 93 Dalbergia and
two Machaerium specimens included in the present study.
(DOCX)
S2 Table. Corrections of identification for Dalbergia herbarium specimens.
(DOCX)
S3 Table. Primers used for amplification and sequencing in DNA barcoding of Dalbergia.
(DOCX)
S4 Table. Detailed results of identification success of Dalbergia specimens using the `best
match’ and `best close match’ methods in TaxonDNA/SpeciesIdentifier 1.7.7.
(DOCX)
Acknowledgments
Thanks to the staff at following herbaria for permission to sample plant material for DNA
extraction: AAU, C, E, L, K.

So Thea, Somsanith Bouamanivong and Hoa Thi Tran are sincerely thanked for facilitating
the collection of field samples in Indochina.
Frits Adema and the late James Franklin Maxwell provided knowledge on morphological
characteristics and identification of specimens.
Trine Abrahamsen is thanked for help with data analyses using machine learning methods
and
Conny Bruun Asmussen Lange for help on phylogenetic analysis.
So Thea, Somsanith Bouamanivong, Hoa Thi Tran and Conny Bruun Asmussen Lange pro-
vided useful comments on earlier versions on the manuscript.
Author Contributions
Conceived and designed the experiments: IH EDK LRN IT. Performed the experiments: IH.
Analyzed the data: IH. Contributed reagents/materials/analysis tools: IH MC. Wrote the paper:
IH MC EDK LRN IT.
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The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia SPP.)

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RESEARCH ARTICLE

The Use of DNA Barcoding in Identification

Competing Interests: The authors have declared

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Materials and Methods

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Table 1. List of Dalbergia species included in this study.

Taxon Continent Habit No. of

D. sericea G. Don Asia Tree[25, 47] 1

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Tree-based analyses (Maximum parsimony and Neighbour joining)

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Distance-based analyses (TaxonDNA)

Barcode TaxonDNA BLOG Naïve Bayes SMO J48 Jrip

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Machine-learning approach (BLOG and WEKA)

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Taxonomy and phylogenetics of Dalbergia species based on parsimony

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Comparison of discrimination methods and barcode regions for

Identification of unknown specimens

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Table 4. Identification of Dalbergia sp. specimens, based on rbcL+matK+ITS barcode.

Tissue Putative TaxonDNA (within NJ-within BLOG SMO

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Which barcoding regions provide the best identification of species in

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Which analytic methods provide the best identification of species in

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Efficiency of the DNA barcodes to identify unknown Dalbergia

Application of barcoding tools in conservation of Dalbergia

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