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Filling gaps of reference DNA barcodes in Syzygium from rainforest fragments

in Sumatra

Article  in  Tree Genetics & Genomes · February 2022

DOI: 10.1007/s11295-022-01536-z

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Tree Genetics & Genomes (2022) 18:6
https://doi.org/10.1007/s11295-022-01536-z

ORIGINAL ARTICLE

Filling gaps of reference DNA barcodes in Syzygium from rainforest

fragments in Sumatra
Ridha Wati1,2 · Fitri Yola Amandita3 · Fabian Brambach4 · Iskandar Z. Siregar2,5 · Oliver Gailing6,7 ·
Carina Carneiro de Melo Moura7

Received: 19 August 2020 / Revised: 7 December 2021 / Accepted: 29 December 2021

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Given the difficulties for rapid biodiversity assessments in understudied regions, DNA barcoding appears as a suitable alter-
native. Still, this approach relies heavily on accurate reference sequence databases for correct taxonomic assignments. In this
study, we evaluated the effectiveness of matK, rbcL, and ITS regions for the identification of Myrtaceae species with emphasis
on the megadiverse genus Syzygium from Sumatra, Indonesia; and analyzed the applicability of species-tree inference for
species assignment using barcode markers. ITS was the most variable barcode region (42.6% of variable sites), followed
by matK (25.7%), and rbcL (14.9%). In terms of assignments of sequences using the BLAST algorithm, all markers were
effective for genus-level attribution. For assignments at species rank, rbcL was able to attribute 30.15% of the samples at
the species level, followed by matK (26.47%), and ITS (17.21%). These results are largely related to the availability of refer-
ence sequences for Myrtaceae in the databases since for the 27 species analyzed in this study, only 8 species had reference
sequences for all three barcode regions available in GenBank. The species-tree inference based on the combination of matK,
rbcL, and ITS markers recovered 41% of the species as monophyletic clades with strong node support. Due to its high level
of differentiation, we recommend the ITS region as the most efficient barcode marker for the identification of Syzygium, and
the traditional core-barcodes (matK + rbcL) as add-on barcodes.

Keywords  Myrtaceae · Conservation · Species delimitation · Tropical plants · Indonesia

Communicated by V. Decroocq

2
* Iskandar Z. Siregar Department of Silviculture, Faculty of Forestry
siregar@apps.ipb.ac.id and Environment, IPB University (Bogor Agricultural
University), 16680 Bogor, Indonesia
Ridha Wati
3
ridha_w@apps.ipb.ac.id Center for Research and Development of Environmental
Quality and Laboratory, Ministry of Environment
Fitri Yola Amandita
and Forestry of Indonesia, Puspiptek Building 210,
fitri.amandita@menlhk.go.id
15314 South Tangerang, Indonesia
Fabian Brambach 4
Biodiversity, Macroecology, and Biogeography, University
fbrambach@uni-goettingen.de
of Göttingen, Büsgenweg 1, 37077 Göttingen, Germany
Oliver Gailing 5
Advanced Research Laboratory (ArLab), IPB University
ogailin@gwdg.de
(Bogor Agricultural University), 16680 Bogor, Indonesia
Carina Carneiro de Melo Moura 6
Centre of Biodiversity and Sustainable Land Use, University
carinamoura@uni-goettingen.de
of Göttingen, Göttingen, Germany
1 7
Tropical Silviculture Study Program, Graduate School Department of Forest Genetics and Forest Tree Breeding,
of IPB University (Bogor Agricultural University), University of Göttingen, Büsgenweg 2, 37077 Göttingen,
16680 Bogor, Indonesia Germany

13
Vol.:(0123456789)
 6  Page 2 of 15
Introduction
Myrtaceae is an ecologically important family of angio-
sperms and has been reported as a key element of the south
and southeast Asian tropical forests (Ray et  al. 2020).
This family is one of the most diverse plant families in
Malesia with ca. 1000 species in 31 genera (Govaerts
et al. 2021), and Indonesia is recognized as a diversity
hotspot. Indonesia is an archipelago that stretches along
the equator, with 18.108 islands, harboring highly diverse
terrestrial ecosystems (Cribb and Ford 2009). Sumatra,
located between 6° southern and 6° northern latitude and
95° to 106° eastern longitude, is one of the main islands
in western Indonesia and forms the Sundaland region
together with the Malay Peninsula on the Asian mainland,
as well as the large islands of Borneo and Java, and sur-
rounding small islands. According to the Government of
Indonesia (2003), this island also has one of the largest
lowland tropical forest areas in the world, with some of
the most important tropical rainforests which are rich in
both flora and fauna. Sumatra is a global center of vascu-
lar plant diversity with 3.000 to 5.000 species per 10.000
­km2 (Barthlott et al. 2005), although the total number of
species for the island is unknown. As in other subregions
of Sundaland, Myrtaceae are mostly represented by the
megadiverse genus Syzygium on the island with ca. 200
recorded species but estimates indicate that this represents
less than two-thirds of the true diversity of the genus in
Sumatra (SYZWG 2016). Indeed, several new species of
Syzygium have recently been discovered in Sumatra, and
the taxonomy of this group is continuously being updated
(Widodo and Chikmawati 2016; Widodo and Lucas 2018).
Myrtaceae are often dominant in the various Indonesian
forest ecosystems such as in central Sulawesi (Brambach
et al. 2017), Kalimantan (Maimunah et al. 2019), West
Java (Rosleine et al. 2006), and also in Aceh province,
Sumatra (Yulisma et al. 2018). The family provides rel-
evant ecosystems services, e.g. as a food source for pol-
linators and fruit-eating animals (Culmsee et al. 2011),
and some of its members are also economically important
(Mudiana 2016; Chandra and Mishra 2007; Purwaning-
sih and Kartawinata 2018; Kesharwani et  al. 2018). It
includes timber species rich in essential oils (Zhang et al.
2010; Barbosa et al. 2013), and that are used in furnish-
ings (Binghong et al. 2019). Additionally, several genera
of Myrtaceae exhibit pharmacological activities, and some
of them produce consumable fruits (Musthafa et al. 2017;
El-Saber Batiha et al. 2020). Unfortunately, in the last dec-
ades, natural forests in Indonesia have continued to suffer
severe deforestation and degradation and have decreased in
terms of diversity (Forest Watch Indonesia 2020). Between
2000 and 2012, most of Indonesia's deforestation and
13
Tree Genetics & Genomes (2022) 18:6
forest degradation was caused by logging (legal and ille-
gal), conversion of natural forests to agro-industrial land,
and forest encroachment by smallholder farmers (Margono
et al. 2014). As a result, the diversity of native plants from
Indonesian forests including the Myrtaceae family has also
decreased (FWI 2002; 2020) Therefore, intensive conser-
vation efforts for Indonesian biodiversity are needed.
As in many tropical plant families, the taxonomy of
Myrtaceae is largely unresolved, especially within the genus
Syzygium. The morphological identification of Myrtaceae is
considered challenging due to its large diversity and wide
distribution, and it is not always possible to observe repro-
ductive traits during field surveys which impede the identi-
fication of the specimens (Raven and Axelrod 1974; Lahaye
et al. 2008; Parmentier et al. 2013). Syzygium is a megad-
iverse genus with currently 1.193 accepted species (www.​
plant​softh​eworl​donli​ne.​org), and an estimated total number
of up to 1.800 species. About 200 of them are distributed
in Sumatra (SYZWG 2016) representing the most species-
rich genus of woody plants in the Paleotropics. Syzygium
started to diversify ca. 50 Million years (My) ago, but most
of its species arose during the recent (since 9.3 My ago)
diversification of subgenus Syzygium (Low et al. 2021). It is
therefore not surprising, that despite its richness, Syzygium is
remarkably uniform in many morphological characters, both
vegetative and reproductive. This uniformity, the sheer size
of Syzygium, and the fact that recent taxonomic revisions are
lacking for many regions where it occurs (e.g. Sumatra, Phil-
ippines, Sulawesi), make species identification extremely
challenging. Preferably, the taxonomic resolution must be
following the highest degree of ecological and genetic dif-
ferentiation, or in other words, it must reach to the lowest
taxonomic level, so that it can bring maximal and accurate
information at the species level (Bozzuto and Blackenhorn
2017). However, extra effort and care are required in large
and recently diversified families such as Myrtaceae, which
show low taxonomic resolution at morphological traits and
slowly evolving DNA regions (Bozzuto and Blackenhorn
2017; Ely et al. 2017). As a result, this lack of taxonomic
resolution can lead to problems in conservation (Soberon
and Medellín 2007; Brambach et al. 2017; Ely et al. 2017).
Therefore, molecular species identification can be employed
as a complementary approach of morphological identifica-
tion to increase the level of accuracy of plant identification
(Waugh 2007). DNA barcoding is a useful tool for rapid
biodiversity inventory, and can potentially facilitate the
establishment of suitable conservation units (Francis et al.
2010; Kress et al. 2015).
DNA barcoding can perhaps be the most recommended
approach for accurate rapid species assessment and has been
vastly applied together with morphological species identi-
fication as an integrative taxonomic approach. Strikingly,
the strongest applicability of DNA barcoding lies in the
Tree Genetics & Genomes (2022) 18:6 Page 3 of 15  6
delimitation of species, the discovery of cryptic species, and
taxonomic revisions (DeSalle and Goldstein 2019). How-
ever, its main drawback remains the availability of compre-
hensive references at the taxonomic or geographic level (Par-
mentier et al. 2013). DNA barcodes are highly reliable when
combined with the development of local barcode libraries to
determine the identity of unknown samples (Chase and Fay
2009; Kress et al. 2009).
The choice of universal DNA barcode regions with a high
taxonomic resolution is another key parameter since DNA
barcodes cannot always distinguish between closely related
species (Parmentier et al. 2013). In plants, the CBOL Plant
Working Group has recommended a core-barcode consist-
ing of two plastid coding regions, rbcL and matK. Previous
studies have proven that these chloroplast loci work quite
well in identifying flowering plant species in the Sumatran
lowland rainforest, at least up to the genus level (Amandita
et al. 2019; Moura et al. 2019), given easy accessibility of
reference sequences in NCBI (https://​www.​ncbi.​nlm.​nih.​
gov/) and BOLD (https://​www.​bolds​ystems.​org) for both
markers. Furthermore, the inclusion of additional barcode
markers is strongly recommended for better assessment at
lower taxonomic levels, especially in highly diverse regions
(Moura et al. 2019). Due to the high rate of nucleotide sub-
stitution, the relatively easy amplification, and the large
coverage in the available reference databases, the internal
transcribed spacer (ITS) region has been very successful at
species-level discrimination across flowering plants (China
Plant BOL Group et al. 2011; Feng et al. 2016; Hossein-
zadeh-Colagar et al. 2016). Consequently, the China Plant
BOL Group et al. (2011) reported that the ITS marker is an
additional candidate for plant DNA barcodes by combining
high discriminatory power and high species coverage (Hol-
lingsworth et al. 2011). Therefore, in this study, we aimed
to evaluate the efficiency of the barcode regions matK, rbcL,
and ITS for species delimitation in Myrtaceae (especially
Syzygium) from Sumatra, and emphasize species assignment
using barcode markers based on species-tree inference.
Materials and Methods
Study site, sample collection, and taxonomic
identification
The Myrtaceae samples were collected in 32 tree inventory
plots, each 0.25 ha in size (50 m × 50 m), in and around
Bukit Duabelas National Park (1°51′S 102°39′E) and Hara-
pan Rainforest (2°14′S 103°19′E) in Jambi province, Suma-
tra, Indonesia (Fig. S1). These locations contain remaining
fragments of once widespread tropical lowland rain forests
of Sumatra surrounded by agricultural land (Laumonier et al.
2010). Bukit Duabelas National Park consists of primary and
logged-over forests, while Harapan Rain Forest was once a
logging concession that is now managed by a legal entity for
ecosystem restoration in production forests.
Within each research plot, tree individuals with a diam-
eter at breast height of ≥ 10 cm were pre-identified as mor-
phospecies in the field. Subsequently, specimens were col-
lected from at least one individual of each morphospecies.
Additional specimens were taken from five 25 m ­ 2 subplots,
where all vascular plants were recorded. Herbarium speci-
mens were collected, dried, and mounted for subsequent
morphological identification at the Herbarium Bogorien-
sis (BO) and SEAMEO-BIOTROP Herbarium (BIOT) in
Indonesia.
High-quality pictures were taken in the field or in the field
facilities against a black cloth background that was used to
facilitate the identification of the 144 specimens sampled
in this study (exemplified in Fig. S2). In this study, we only
considered species identified (in the field or subsequently
during herbarium work) as Myrtaceae. For further details
regarding sampling procedure and species identification, see
Rembold et al. (2017). Fresh leaf tissue from each sample
was dried in silica gel to preserve the material before DNA
extraction and further genetic analysis.
DNA extraction, barcode amplification,
and sequencing
Genetic analysis was carried out using 144 samples for
the following species: Decaspermum parviflorum (Lam.)
A.J.Scott (N = 6), Psidium guajava Linn (N = 2), Rhoda-
mnia cinerea Jack. (N = 6), Syzygium acuminatissimum
(Blume) DC. (N = 21), S. cf. aemulum (Blume) Amshoff
(N = 2), S. cf. claviflorum (Roxb.) Wall. Ex Steud. (N = 1),
S. cf. confertum (Korth.) Merr. & L.M.Perry (N = 2), S. cf.
filiforme Chantar. & J.Parn (N = 2), S. cf. hirtum (Korth.)
Merr. & L.M.Perry (N = 1), S. cf. nervosum A. cunn. ex DC
(N = 3), S. cf. palembanicum (Miq.) P. S. Ashton (N = 3),
S. cf. scalarinerve (King) I.M. Turner (N = 2), S. cf. splen-
dens (Blume) Merr. & L.M. Perry (N = 1), S. chloranthum
(Duthie) Merr. & L.M. Perry (N = 6), S. claviflorum (Roxb.)
Wall. Ex Steud. (N = 3), S. creaghii (Ridl.) Merr. & Perry
(N = 1), S. cymosum (Lam.) DC. (N = 6), S. euneuron Miq.
(N = 2), S. fastigiatum (Blume) Merr. & L.M. Perry (N = 4),
S. glabratum (DC.) Veldkamp (N = 2), S. grande (Wight)
Walp. (N = 5), S. korthalsianum (Miq.) Miq. (N = 1), S. laxi-
florum (Blume) DC. (N = 10), S. leptostemon (Korth.) Merr.
& L.M.Perry (N = 1), S. lineatum (DC.) Merr. & L.M.Perry
(N = 11), S. maingayi Chantar. & J. Parn. (N = 2), S. malac-
cense (L.) Merr. & L.M.Perry (N = 1), S. minimum (Blume)
Airy Shaw (N = 6), S. oblatum (Roxb.) Wall. ex A. M.
Cowan & Cowan (N = 1), S. paludosum P.S. Ashton (N = 1),
S. polyanthum (Wight) Walp. (N = 3), S. polycephalum
(Miq.) Merr. & L.M.Perry (N = 1), S. racemosum (Blume)
13
 6  Page 4 of 15 Tree Genetics & Genomes (2022) 18:6

DC. (N = 3), S. samarangense (Blume) Merr. & L.M.Perry
(N = 1), S. splendens (Blume) Merr. & L.M.Perry (N = 2),
S. tetrapterum (Miq.) Chantar. & J.Parn. (N = 2), S. urceo-
latum ssp. palembanicum (Miq.) P. S. Ashton (N = 7), and
Syzygium sp. (N = 10).
The DNA was extracted from dried and healthy leaf tis-
sue of each sample using the DNeasy 96 Plant Kit (Qiagen,
Hilden, Germany) by following the manufacturer’s protocol.
The concentration of extracted DNA was examined using 1%
agarose gel electrophoresis with Lambda DNA as standard
(Analytik Jena, Jena, Germany).
The target barcode regions were amplified using specific
chloroplast markers, matK, rbcL, and the nuclear ribosomal
ITS (Table 1). For the samples that failed to be amplified
for the matK fragment using the primer pairs 1R_KIM_f
and 3F_KIM_r, another PCR reaction was conducted using
the primer pair 390_f and 990_r (Table 1). PCR products
were purified using the innuPREP Gel Extraction Kit pro-
tocol (Analytik Jena, Jena, Germany). ABI PrismTM Big
DyeTM Terminator Cycle Sequencing Ready Reaction Kit
v1.1 (Applied Biosystems) was used for the sequencing reac-
tion, following the manufacturer's protocol.
Sequence data analysis
Reverse and forward sequences were assembled using
Codon Code Aligner software (https://w ​ ww.c​ odonc​ ode.c​ om/​
align​er). Sequences were trimmed and edited if necessary
by checking each electropherogram. Consensus sequences
were generated for each sample and then used for multi-
ple sequence alignments. Different regions of nucleotide
sequences often evolve differently (https://w​ ww.m ​ egaso​ ftwa​
re.​net); therefore, variable sites and parsimony-informative
sites were calculated after the complete deletion of the miss-
ing/gap sites from all the sequences (Singh et al. 2017).
All obtained sequences were compared with reference
nucleotide sequences available in the National Center for
Biotechnology Information (NCBI) GenBank (Porter and
Hajibabaei 2018)and Barcode of Life Data Systems (BOLD)
(Lis et al. 2016) databases by using the BLAST algorithm.
The efficiency of DNA barcodes was tested by comparing
morphological-based identification and molecular assign-
ment matching the reference sequences. The percentage of
correspondence between morphological and molecular iden-
tification results was distributed into three taxonomic ranks:
family, genus, and species.
Barcoding gap and species‑tree inference
Interspecific and intraspecific genetic distances were cal-
culated and characterized using the Tamura 3-Parame-
ter + Gamma in MEGA7 (Kumar et al. 2016) for each single
and combined marker region (matK, rbcL, ITS, matK + rbcL,
matK + ITS, rbcL + ITS, and matK + rbcL + ITS). The
Tamura 3-Parameter model was estimated as the best sub-
stitution model for matK, rbcL, and ITS using MEGA 7,
taking into account differences in the transitional and trans-
versional changes and the biased content of G + C (Tamura
1992), whereas the speed of evolution between them can
be modeled by the distribution of gamma (G) (Yang 1994).
Sequences of each morphologically defined species were
grouped to estimate inter- and intraspecific genetic distances.
The frequency distribution (%) of the inter- and intraspecific
divergences of each marker was calculated and visualized in
boxplots using the package ggplot2 in R (Williamson 1989;
Gómez-Rubio 2017). Based on the intra- and interspecific
divergences of all sequences including the combined mark-
ers, the discriminatory power of each marker was tested
following previous studies by Amandita et al. (2019) and
Chen et al. (2020). Differences in the discriminatory power
of markers were detected when the interspecific genetic
distance between two taxa was significantly higher than
the intraspecific genetic distance of each separate species
(Amandita et al. 2019). The Kruskal–Wallis test was per-
formed to estimate the significance of the discriminatory
power of each marker and the significance of the mean dif-
ferences between intra- and interspecific divergences among
the markers (α = 0.05).
For the species-tree inference, we estimated Bayes-
ian Inference (BI) using the single ITS and combined

Table 1  Universal primers used
in this study
Region Primer name Primer sequences (5’—3’) References
matK 3F_KIM_f CGT​ACA​GTA​CTT​TTG​TGT​TTA​CGA​G CBOL Plant Working Group (2009)
1R_KIM_r ACC​CAG​TCC​ATC​TGG​AAA​TCT​TGG​TTC​ CBOL Plant Working Group (2009)
390_f CGA​TCT​ATT​CAT​TCA​ATA​TTTC​ Schmitz-Linneweber et al. (2001);
CBOL Plant Working Group (2009)
990_r GGA​CAA​TGA​TCC​AAT​CAA​GGC​ Gamage et al. (2006)
rbcL rbcLa_f ATG​TCA​CCA​CAA​ACA​GAG​ACT​AAA​GC Kress and Erickson (2007)
rbcLa_r GAA​ACG​GTC​TCT​CCA​ACG​CAT​ Taberlet et al. (1991)
ITS ITS1 TCC​GTA​GGT​GAA​CCT​GCG​G White et al. (1990)
ITS4 TCC​TCC​GCT​TAT​TGA​TAT​GC

13
Tree Genetics & Genomes (2022) 18:6 Page 5 of 15  6
genomic regions (matK + rbcL + ITS and matK + rbcL) in
BEAST 1.8.0 (Drummond et al. 2012), by employing the
Hasegawa, Kishino, and Yano (HKY) model, since the
Tamura 3-Parameter model was not available in Beast 1.8.0.
We selected the Yule model of branching and homogene-
ous rates among branches by choosing the strict model. BI
was run for 1­ 06 generations and sampled every 1.000 trees
using the Cipres platform (Miller et al. 2010). The posterior
probability was calculated using a burn-in of 8% in Treean-
notator 1.8.0 (Drummond et al. 2012). The delimitation of
tribes was based on Wilson (2011). Reference sequences
available in GenBank that have queries higher than 98% for
each species were selected for comparison, the details can
be found in Table S2.
Results
Characteristics of barcode markers
A total of 80 out of the 144 sampled individuals were suc-
cessfully sequenced for all targeted regions (matK, rbcL, and
ITS) potentially as results of low DNA quality and muta-
tions in primer binding sites (Table S1, S5). The ITS data-
set showed the highest proportion of variable sites (42.6%),
followed by matK (25.7%) and rbcL datasets (14.9%). The
proportion of parsimony-informative sites followed a similar
pattern, with ITS displaying the highest proportion (26.1%)
and rbcL the lowest (4.03%) (Table 2). Variation in the poly-
morphism level of each barcode marker was also detected
in the interspecific genetic distance estimation, which was
significantly different across markers (p < 0.0001, Wilcoxon
test) (Fig. S4).
Reference sequences available in the GenBank and BOLD
databases for matK, rbcL, and ITS for the Myrtaceae species
can be found in Table S5. Newly generated sequences for
the 27 morphologically identified species were compared
to these reference sequences. A total of sixty-three percent
of sequences for the species investigated in this study for
the barcodes ITS and matK, and fifty-nine percent for rbcL
Table 2  Main information, diversity parameters, and characteristics
of the three sequenced markers
Parameter matK rbcL
Variable sites (proportion) (%) 25.7 14.9
Parsimony-informative sites (pro- 10.1 4.03
portion) (%)
CG content mean (range) (%) 32.5 45.6
Length of the alignment, bp 704 595
Number of sequences 136 136
Sequencing success (%) 94.44 94.44
are not yet available in the GenBank database; while in
BOLD 89% of the species investigated in this study have
no sequence available for the regions matK and rbcL. Since
BOLD does not provide ITS sequences for plants, the survey
was only made for NCBI.
The percentage of analogous morphological and molecu-
lar identification at the species level by assigning sequences
to the reference sequence database (GenBank) was deter-
mined using the BLAST algorithm. The correspondence was
higher using rbcL (30.15%), followed by matK (26.47%),
and ITS (17.21%). At the genus level, more than 70% of the
samples had consistent morphological and molecular iden-
tifications using individual chloroplast markers combined
with ITS, whereas matK + rbcL and matK + rbcL + ITS
obtained 65.87% and 65.42%, respectively. A minor fraction
of the total sequences were classified as mislabeled samples
(Table S3).
Genetic distance and barcoding gap
Genetic distance analysis was performed using 74 out of
a total of 80 sequences of the datasets since six sequences
were not successfully identified to the species level based
on morphological characteristics (Syzygium sp.) and were
excluded to avoid bias in the results of intra- and interspe-
cific distances. Intra- and interspecific genetic distances
were significantly different regardless of the marker used
(p < 0.001, Wilcoxon signed-rank test) (Fig.  S4), which
provides evidence of barcoding gaps for all markers used
for molecular differentiation of Syzygium as shown in Fig. 1
and Fig. S4. For interspecific genetic distance, the highest
values were observed for ITS (mean 0.041 ± 0.035 SD) fol-
lowed by matK (0.018 ± 0.015) and rbcL (0.008 ± 0.006).
For the combined dataset, matK + ITS displayed the highest
mean overall interspecific genetic distance (0.030 ± 0.022)
followed by rbcL + ITS (0.025 ± 0.019) and the combination
of all three markers (0.022 ± 0.015), while matK + rbcL had
the lowest values (0.013 ± 0.009) (Fig. 1).
Since ten species only have one sequence for each spe-
cies in this study, the total number of species investigated
to assess intraspecific distance was 17 out of 27 species
for every single marker and the combined marker set. For
individual markers and their combinations, the intraspe-
cific genetic distance was very low, however, matK pre-
ITS sented the largest range (0–0.065) in comparison with the
other two barcodes. The ITS dataset showed higher mean
42.6 intraspecific genetic variation (0.004 ± 0.011) in comparison
26.1 with the matK and rbcL plastid regions (0.002 ± 0.007 and
0.001 ± 0.004, respectively). Among the combined markers,
56.7
the combination of matK + ITS datasets showed the high-
640
est mean intraspecific genetic distance, (0.003 ± 0.007);
123
followed by rbcL + ITS (0.002 ± 0.006); and matK + rbcL
85.42
(0.001 ± 0.004) (Fig. 1). The three combined markers had
13
 6  Page 6 of 15
Fig. 1  Interspecific (red) and
intraspecific (green) genetic
distances for 74 samples of
Myrtaceae from Sumatra using
the traditional barcode markers
matK, rbcL, and ITS and their
combinations. The differences
between inter- and intraspecific
distances were significant for all
markers or their combinations
according to a Wilcoxon signed-
rank test (p < 0.001)
an intermediate intraspecific genetic distance (0.002 ± 0.005)
(Fig. 1).
The discriminatory power of all markers based on multi-
ple samples per species can be considered high since more
than 70% of the species studied were successfully differen-
tiated from each other, except for the single marker rbcL
which discriminated only 63% of the species (Table S4).
The highest species discriminatory power of the three sin-
gle markers was found for ITS (78%) and followed by matK
(73%), whereas, the concatenated markers, matK + rbcL, dis-
played the lowest values for combined datasets (73%). For
all possible combined datasets for which ITS was included,
an increase in the percentage of discriminatory power was
detected with the highest value found in matK + rbcL + ITS
(89.47%). Significant differences in the discriminatory
power were found between all markers (p < 0.0001, Wil-
coxon signed-rank test).
Species‑tree inference
Phylogenetic analyses were carried out using 80 selected
samples with high-quality sequences for every three mark-
ers. The combination of the core-barcode (matK and rbcL)
and the ITS datasets produced the best-resolved tree and
displayed strong node support for at least part of the spe-
cies of Myrtaceae analyzed in this study (Fig. 2). For tribe
and genus level, the three combined barcode markers recov-
ered clades with strong node support (posterior probability,
PP ≥ 0.99). For species rank assignment, 41% of the species
analyzed in this study clustered in monophyletic clades with
13
Tree Genetics & Genomes (2022) 18:6
robust node support (≥ 0.99): Syzygium acuminatissimum,
S. fastigiatum, S. minimum, S. cf. filiforme, S. cf. scalarin-
erve, S. grande, S. chloranthum, S. cf. nervosum, S. lineatum,
Rhodamnia cinerea and Decaspermum parviflorum (Fig. 2,
Table S5). Further conclusions could not be drawn for the
species for which only one exemplar was sampled (37% of
the species). Six species appeared as non-monophyletic in
the species-tree inference: S. claviflorum, S. cymosum, S.
laxiflorum, S. racemosum, whereas, S. cf. aemulum and S.
urceolatum ssp. palembanicum with low support (< 0.50);
and therefore, the phylogenetic species identification was
considered as unresolved for these species (Figs. 2, 3, 4;
Table S5).
The overall topology of the three-loci species tree and
the ITS tree alone showed stronger node support and a bet-
ter resolution of relationships between species in Syzygieae
and Myrteae than the combined chloroplast tree. S. acumi-
natissimum, S. minimum, S. cf. scalarinerve, S. grande, R.
cinerea, and D. parviflorum were resolved as monophyletic
lineages with strong support (PP = 1.00), in all three trees. S.
chloranthum and S. fastigiatum were resolved as monophyl-
etic lineages with strong support (PP = 1.00) for the ITS and
three-loci trees (Figs. 2 and 3), and displayed node support
of 0.95 and 0.98 in the matK + rbcL tree. S. cf. filiforme,
S. lineatum, and S. cf. nervosum were unresolved based on
combined chloroplast markers. In contrast, when using the
ITS region, these same taxa (S. cf. filiforme, S. lineatum,
and S. cf. nervosum) were resolved as monophyletic line-
ages with strong support of 1.00, 0.99, and 0.75, respectively
(Fig. 3).
Tree Genetics & Genomes (2022) 18:6 Page 7 of 15  6
Fig. 2  Bayesian inference of tribes Syzygieae and Myrteae (family
Myrtaceae) based on the combined sequences of matK, rbcL and ITS
for samples collected in Sumatra, Indonesia. Posterior probability val-
Discussion
Characteristics of the barcode markers
In this study, ITS was the most polymorphic barcoding
marker in comparison to matK and rbcL in terms of total
variable sites. For an appropriate DNA barcode, a high
level of polymorphism is one of the most crucial criteria
along with primer universality (Hollingsworth et al. 2011;
China Plant BOL Group et al. 2011; Yang et al. 2012). The
ues are placed next to the nodes. Tip labels display species identifi-
cation based on morphological taxonomy, and internal IDs. Colours
represent genera Syzygium, Rhodamnia, and Decaspermum 
chloroplast region matK was reported to produce high levels
of discrimination among angiosperms species (CBOL Plant
Working Group 2009; Hajibabaei et al. 2007; Li et al. 2015),
however, matK has a lower universality than rbcL (Hollings-
worth et al. 2011; Meiklejohn et al. 2019). Furthermore,
many previous studies have reported rbcL as a useful region
for species diagnostics (CBOL Plant Working Group 2009;
Hollingsworth et al. 2009), since this marker has a stable
success rate of PCR amplification, and produces high-quality
sequences. In our study, all regions performed well with a
13
 6  Page 8 of 15
Fig. 3  Bayesian inference of tribes Syzygieae and Myrteae (family
Myrtaceae) based on ITS sequences of samples collected in Sumatra,
Indonesia. Posterior probability values are placed next to the nodes.
Tip labels display species identification based on morphological iden-
sequencing success rate of more than 90% either with matK
or rbcL, while ITS displayed a sequencing success rate of
85%. PCR or sequencing failure for some samples can be
linked to the sample conditions such as low DNA quality or
quantity or sequence variation in the primer binding sites.
ITS was proposed as a complementary marker to the core
barcodes (CBOL Plant Working Group 2009) or a core bar-
code (China Plant BOL Group et al. 2011). Many studies
have demonstrated high mutability in ITS (Kress et al. 2005;
Sass et al. 2007). However, in our study, ITS had a lower
sequencing success rate with the ITS1/ITS4 primer set as
compared to matK and rbcL, indicating that more univer-
sal primers for ITS as a plant DNA barcode may still be
needed. Yang et al. (2012) even disregarded the ITS region
for Arecaceae since the success rate for ITS bidirectional
sequencing was zero due to strong signal overlap in sequenc-
ing. The poor success rate for ITS sequencing may be due to
the primer set of ITS1/ITS4 which was initially designed for
fungi (White et al. 1990).
13
Tree Genetics & Genomes (2022) 18:6
tification, and internal IDs. Colours represent genera Syzygium, Rho-
damnia, and Decaspermum. Accession numbers of sequences down-
loaded from NCBI are displayed beside the species IDs
Therefore, it is obvious that different plant taxa can
respond in different ways to the use of ITS. In Syzygium,
both diploidy and polyploidy occur with a base chromo-
some number of n = 11 (Rye 1979). In addition, Syzygium
species are known to hybridize frequently and new species
from the Syzygium group are continuously reported (Widodo
and Chikmawati 2016; Widodo and Lucas 2018). Widodo
(2007) stated that one of the speciation processes that occur
in the genus Syzygium is characterized by hybridization
activity, and Syzygium is known as one of the large gen-
era that reveal rapid speciation (Widodo 2007; Biffin et al.
2010). Furthermore, the incomplete concerted evolution of
this nuclear multiple-copy region caused by hybridization
or other factors (Álvarez 2003) can be another possible rea-
son for the lower sequencing success rate compared with
the two chloroplast markers. Thus, the utilization of ITS for
taxonomic purposes is challenging, unless previously tested
and properly characterized. Nonetheless, a highly resolved
phylogeny and classification of genera Glycine and Flem-
ingia have been provided based on the ITS sequence (Wu
Tree Genetics & Genomes (2022) 18:6 Page 9 of 15  6
Fig. 4  Bayesian inference of tribes Syzygieae and Myrteae (family
Myrtaceae) based on matK and rbcL sequences of samples collected
in Sumatra, Indonesia. Posterior probability values are placed next to
the nodes. Tip labels display species identification based on morpho-
et al. 2013). Furthermore, the ITS region stood out as the
most variable in comparison with the traditional barcode
core datasets, and a previous study has recognized ITS as
standard DNA barcode for medical plant species diagnosis
(Chen et al. 2010), being successfully applied for Syzygium
in this study as well.
Genetic distance and discriminatory power
According to Meyer and Paulay (2005), the ideal bar-
code markers can be determined by the existence of
logical taxonomy, and internal IDs. Colours represent genera Syzyg-
ium, Rhodamnia, and Decaspermum. Information on accession num-
bers of sequences downloaded from NCBI are included in Table S2
non-overlapping distributions between intra- and interspe-
cific distances, called barcoding gaps. In several studies, an
incomplete gap was found in large-scale plant inventories
(Piredda et al. 2011; Tripathi et al. 2013; Jaén-Molina et al.
2015; Hartvig et al. 2015; Besse 2021). The lack of a barcod-
ing gap might be the result of variations of the genetic dis-
tance range across distinct plant families and highlights the
relevance of investigating phylogenetically related groups,
since the existence of a barcoding gap might be the result of
microevolutionary signals (Lahaye et al. 2008; Parmentier
et al. 2013; Bello et al. 2015). Our results showed a clear
13
 6  Page 10 of 15
and significant barcoding gap in all studied markers with an
average value of the interspecific genetic distance greater
than the intraspecific genetic distance. The visualization of
the barcoding gap by the non-overlap between boxplots of
the intra- and interspecific genetic distances estimated per
amplicon dataset and their combination confirm the suit-
ability of these barcode markers for molecular species iden-
tification in Syzygium.
The ITS marker had the highest power to distinguish
between species among the single markers (Fig.  1),
whereas the combination of matK and rbcL had the lowest
discriminatory power among the other combined markers
with a small divergence. Nonetheless, chloroplast markers
either alone or combined have a higher average interspe-
cific than intraspecific genetic distance resulting in a clear
barcoding gap. Furthermore, the three combined markers,
matK + rbcL + ITS, had the highest discriminatory power,
being able to discriminate more than 95% of the species
of Myrtaceae in this study, which is higher than the 72%
identification rate recorded by the CBOL Plant Working
Group (2009). The use of combined markers maximizes the
sequence variation and increases accuracy for molecular
identification of plants, as reported by several previous stud-
ies (Lahaye et al. 2008; Li et al. 2011; Amandita et al. 2019).
Species attribution using DNA barcodes
DNA barcoding proved to be a fairly useful method to sup-
port the morphological identification of Syzygium from
Sumatra. Our results indicate that a single DNA barcode is
not enough to determine a species correctly, and only a third
of the investigated samples could be correctly identified to
the species level. This value largely depends on the avail-
ability of reference sequences in the database, since only ca.
one-third of the species used in this study had correspondent
sequences available in GenBank (NCBI). The highest correct
percentage of sequence assignments at the species level was
found for rbcL followed by matK and ITS, which also pre-
sented the highest numbers of existing reference sequences
in GenBank. For example, S. fastigiatum, S. polyanthum, and
S. polycephalum in this study were successfully identified
to the species level using rbcL, whereas with matK and ITS
it was only up to the genus level since sequences for these
species were not yet available in the database.
So far, DNA barcoding might provide dubious species
attribution, due to its dependence on high-quality refer-
ence databases, and is therefore sometimes considered as
a conundrum for species delimitation (Packer et al. 2009;
Meiklejohn et al. 2019). However, accurate species identi-
fication using traditional methods can be time-consuming
and proves difficult due to various factors (Colpaert et al.
2005). For example, morphological identification of species
can be difficult when important parts for the identification of
13
Tree Genetics & Genomes (2022) 18:6
the plant are not available or are not well developed (flower,
fruit, etc.). Thus, an association between morphological
and molecular identification can provide consistent spe-
cies assessment results, while identification of other bar-
code regions and establishing new markers is needed for the
discrimination of recently diversified groups, as previously
discussed (Stace 2005; Newmaster et al. 2008; Packer et al.
2009; Meiklejohn et al. 2019).
Many studies have reported that combining and including
new markers can improve the accuracy of molecular identi-
fication. Thus, the nuclear internal transcribed spacer (ITS)
which was documented as a potential barcode by Kress et al.
(2005) has been used successfully to classify angiosperms
(CBOL Plant Working Group 2009), tree species in the trop-
ical cloud forest (Kang et al. 2017), various medicinal plants
(Chen et al. 2010), and other tropical plant families such as
Rutaceae (Luo et al. 2010). Additionally, ITS was success-
fully employed for the identification of Syzygium, clarifying
the classification of the species in the species-tree inference
presented in this study. Apart from ITS, the non-coding
spacer psbA-trnH can also be used as an additional marker
to improve the molecular species classification of closely
related taxa (Hollingsworth et al. 2011). Gonzalez et al.
(2009) reported that this marker achieved the best results
among other markers in Amazonian tree identification, and
it is highly recommended as a barcode marker in addition to
matK and rbcL. Nevertheless, the restricted number of refer-
ence sequences available in the databases makes the choice
of this barcode region uninviting. For the species used in
this study, only 22% of the species have existing reference
sequences for the psbA-trnH barcode region.
The highest success rate of correct identification for the
Myrtaceae here analyzed was observed at the genus level.
Several studies have also reached similar results, such as
studies on the classification of Myrtaceae based on matK by
Wilson et al. (2005), and in tropical flowering plants studied
by Amandita et al. (2019). These results show that DNA
barcoding is useful for identifying plants at least to the genus
level (Parmentier et al. 2013; Li et al. 2015). Therefore, eco-
logical and taxonomic investigations are needed to achieve
an unequivocal identification of the specimens, especially
for some taxonomic plant groups that are particularly diffi-
cult to resolve and continuously focus of systematic updates.
In this study, Syzygium acuminatissimum was not
resolved at the species level when using rbcL, but the
morphological identification results were equivalent to
the molecular identification when using matK and ITS.
Besides, the species-tree inference based on rbcL com-
bined with matK was not sufficient for distinguishing
some taxa at the species level, especially S. cf. filiforme,
and S. lineatum. Although rbcL recovered the highest
value for identification at the species level because of
its availability in the database, rbcL alone had the lowest
Tree Genetics & Genomes (2022) 18:6 Page 11 of 15  6
discrimination value among the three markers used here.
In contrast, S. cf. filiforme and S. lineatum, also S. acumi-
natissimum, S. grande, S. minimum were successfully
resolved as monophyletic clades using ITS alone or in
combination with rbcL and matK, furthermore, the com-
bination of rbcL + matK + ITS exhibited the best resolving
posterior probability. The high resolution of ITS either
individually or in combination with chloroplast markers
has also been reported in closely related groups of Lysi-
machia L. (Zhang et al. 2012), Terminalia (Mishra et al.
2017), and 141 genera of seed plants (China Plant BOL
Group et al. 2011). The poor performance of matK and
rbcL in resolving Syzygium species has largely prevented
their widespread use as a barcode in the Syzygium genus.
According to Zhang et al. (2012), the main cause of their
poor performance in closely related plant groups is the
inherently slow evolution of chloroplast genes, suggest-
ing that it is necessary to focus on more rapidly evolv-
ing regions of the chloroplast or nuclear genomes such
as intergenic spacers and introns. Therefore, the use of
phylogenetic species identification based on multi-loci
species-tree inference can be recommended as an alter-
native to reduce the bias of single barcode regions for
molecular species identification.
de Vere et al. (2012) reported that the use of distinct
markers for different taxa will result in different rates of
identification success. Discrepancies in the identification
of specimens can result from naming errors or contami-
nation of the sample, either during sample collection or
when the sample is being processed in the laboratory.
In many cases, an increase in sample size and number
per species may reveal such errors (de Vere et al. 2012),
and some precautions in laboratory protocols can mini-
mize these problems (Meiklejohn et al. 2019). Another
factor influencing the successful identification of spe-
cies using DNA barcodes is the availability of reference
sequence data from appropriate taxa in the databases. In
this study, 80 sequences consisting of 28 different species
(including Syzygium sp.) were barcoded for three regions
(matK, rbcL, and ITS) and had been uploaded in GenBank
(NCBI). Sixty-three percent of the sequences for the spe-
cies analyzed in this study were not yet available in the
databases for at least one of the markers in GenBank,
whereas in BOLD it is 89%. Samples belonging to spe-
cies that are not available yet in the databases can explain
the amount of non-assigned taxa in studies employing
the DNA barcoding approach. An accurate and complete
molecular database, especially for plant species, is far
from being achieved under current conditions (Pentinsaari
et al. 2020). Such databases are expected to be developed
in the future since many plant DNA barcode projects are
ongoing (https://​natur​alhis​tory.​si.​edu/; https://​phylo​diver​
sity.​net/​xmale​sia/; https://​www.​barco​dingl​ife.​org/).
DNA barcoding for conservation
Identification of tropical trees at least to the genus level is
useful for ecological surveys and environmental assess-
ments. According to Liu et al. (2015), genus diversity can
be very helpful as a substitute for species richness. Our
study indicates that DNA barcoding is a rapid assessment
tool for identifying tropical Sumatran plants to the genus
level, as also stated by Amandita et al. (2019) and Moura
et al. (2019). However, the most difficult challenge in tropi-
cal forests is the large number of plant species (Wright
2002; Leigh et al. 2004). Based on previous studies, the
lower latitudes have more speciation events per unit time
(Martin and McKay 2004; Mittelbach et al. 2007; Jansson
and Davies 2007; Liu et al. 2015; Massante et al. 2019).
High rates of speciation in the tropics result in more closely
related species and the consequent inherent challenges of
barcoding polytypic genera in tropical forests (Martin and
McKay 2004; Liu et al. 2015). Syzygium represents such a
large genus, with a reported number of ca. 200 species dis-
tributed in Sumatra (SYZWG 2016). Although the nuclear
ITS region alone or combined with chloroplast markers
does increase species discrimination, some species in our
study have not been resolved, and this is probably typical of
tropical floras with many closely related species. Resolution
of closely related taxa may need to depend on increasing
applications of rapid and accurate approaches such as DNA
barcoding.
Several researchers have reported the role of DNA bar-
coding in enhancing biodiversity assessments and conserva-
tion efforts (Selvaraj et al. 2013; Farooq et al. 2020). Based
on the results we obtained, DNA barcodes (rbcL, matK, and
ITS) may have a contribution to conservation actions by
providing rapid and accurate species identification without
using morphological cues. Furthermore, 74% of the species
investigated in this study were well supported in the phy-
logenetic inference, which has not been reported in several
previous studies on delimitation, evolution, and phylogeny
of Syzygium (Biffin et al. 2010; Thornhill et al. 2015; Vas-
concelos et al. 2017). Moreover, the generation of 240 new
sequences from three different markers for 28 species will
make a valuable contribution to the enrichment of the Gen-
Bank database, and the increase in the number of sequences
and taxa of the large genus Syzygium in this public database
can be a reference that supports and refines biodiversity con-
servation especially in Sumatra, Indonesia.
Conclusions
Species-tree inference based on the combination of matK,
rbcL, and ITS datasets provided a robust alternative for
phylogenetic species identification of Syzygium using
13
 6  Page 12 of 15
barcode markers, and could be implemented as an addi-
tional tool for tropical species delimitation, and therefore
aiding species identification. Additionally, species-tree
inferences facilitate the clarification of non-existent taxa
in reference sequence databases.
Finally, we conclude that DNA barcoding withstands
its use as a tool for rapid species assessment. Based on
our findings, we deduce that the ITS marker could be
applied as the main barcode marker for the identification
of Syzygium in this study, and the traditional core-barcodes
(matK + rbcL) as add-on barcode resources, which could
be potentially extrapolated for tropical plants. Neverthe-
less, individual barcode markers are rarely employed
recently, and the use of single barcode markers for molec-
ular species identification has become obsolete. We rec-
ommend firstly assessing the proper reliability of the ITS
region and the use of at least three barcode markers for
successful molecular species placement. However, truly
universal ITS primers are not available for plants, and the
use as a barcoding marker could be limited due to the pres-
ence of paralogues.
Thus, a deeper exploration of DNA barcoding in tropi-
cal plants across several plant families is essential to mark
boundaries for the use of DNA barcoding for tropical plant
species identification and the use of other markers such as
the non-coding spacer psbA-trnH to expand the informa-
tion and fill gaps in the reference databases.
Supplementary Information  The online version contains supplemen-
tary material available at https://​doi.​org/​10.​1007/​s11295-​022-​01536-z.
Acknowledgements  We thank Katja Rembold and the EFForTS
B06/B14 field and herbarium teams for sampling and management
of Myrtaceae specimens. Furthermore, we acknowledge our techni-
cians Gudrun Diederich, Larissa Kunz, and Oleksandra Dolynska for
their assistance with DNA isolation, amplification, and sequencing.
Moreover, we appreciate the Ministry of Research and, Technology
(RISTEK)/Agency for Research and Innovation (BRIN) of the Republic
of Indonesia (RISTEKDIKTI) for permitting us to perform the research
in Indonesia, and recognize the support of the German Research Foun-
dation. We thank the two anonymous reviewers for contributing with
valuable comments to our manuscript.
Author contributions  Development of concept, all authors; Sampling
preparation and specimen processing, F.Y.A., I.Z.S, C.C.M.M. and F.B;
Bioinformatics analysis, R.W., F.Y.A., and C.C.M.M.; Data curation,
R.W., F.Y.A., C.C.M.M. and F.B.; Original draft preparation, R.W. and
C.C.M.M.; Review and editing, all authors.
Funding  This research was supported by the German Research
Foundation (Deutsche Forschungsgemeinschaft, DFG) as part of the
EFForTS project (Collaborative Research Centre 990, https://​www.​
uni-​goett​ingen.​de/​effor​ts), project number 192626868.
Declarations  China Plant BOL Group, Li DZ, Gao LM, Li HT, Wang H, Ge XJ,
Conflicts of Interest  The authors declare no conflict of interest.
13
Tree Genetics & Genomes (2022) 18:6
Data Archiving Statement  All sequences used in this study have been
submitted to GenBank (NCBI), accession numbers are MT864746 to
MW854235 (details in Table S1).
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