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Received: 14 February 2024
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Accepted: 2 July 2024
DOI: 10.1002/inc3.60
RESEARCH ARTICLE
Rapid DNA barcoding‐based fern and lycophyte
inventories of protected areas—A pilot study
to introduce a simple but effective protocol
Hongmei Liu
1
|Yarong Chai
1,2
|Harald Schneider
1
1
Center for Integrative Conservation and
Yunnan Key Laboratory for Conservation of
Tropical Rainforests and Asian Elephants,
Xishuangbanna Tropical Botanical Garden,
Chinese Academy of Sciences, Mengla,
Menglun, Yunnan, China
2
School of Tea & Coffee, Pu'er University,
Pu'er, Yunnan, China
Correspondence
Hongmei Liu, Center for Integrative
Conservation and Yunnan Key Laboratory
for Conservation of Tropical Rainforests
and Asian Elephants, Xishuangbanna
Tropical Botanical Garden, Chinese
Academy of Sciences, Mengla, Menglun
666303, Yunnan, China.
Email: liuhongmei@xtbg.ac.cn
Funding information
Yunnan Province Science and Technology
Department, Grant/Award Number:
202101AS070012; Yunnan Revitalization
Talent Support Program “Innovation
Team”Project, Grant/Award Number:
202405AS350019; 14th Five‐Year Plan of the
Xishuangbanna Tropical Botanical Garden,
Chinese Academy of Sciences,
Grant/Award Number: E3ZKFF8B01
Abstract
Recording inventories of species conserved in protected areas is a
key step to evaluate the effectiveness of Kunming Montreal Global
Biodiversity Framework (KM‐GBF) targets, such as the expansion of
protected areas. The application of DNA barcoding facilitates the
rapid production of enables to obtain rapid inventories with reduced
reliance on taxon experts. These inventories aim not only to confirm
existing records but also to minimize gaps in our knowledge of
the distribution and taxonomy of species targeted for conservation
through the implementation of protected areas. This pilot study
introduces a simplified DNA barcoding pipeline as a reliable tool for
recording fern and lycophyte species occurring in protected areas.
The pipeline emphasizes limited and/or short training requirements,
reducing the input required from taxon experts and maximizing
shared benefits between conservationists and taxonomists. Despite
using a single DNA barcoding region, 78% of the accessions were
unambiguously identified to the species level. This applied approach
not only confirmed previous records but also identified several
previously overlooked species, either as newly recorded species
conserved in the protected area or as species new to science. The
pilot project effectively documented known species diversity and
identified gaps in our taxonomic knowledge by discovering previ-
ously unknown and locally rare taxa. This rapid assessment enhances
productive exchanges between conservation practitioners and taxon
experts, with substantial benefits for both parties.
KEYWORDS
DNA barcoding, GBF targets, inventories, land plants, protected areas, taxonomic
expertise
Plain language summary
Recording the biodiversity conserved in protected areas is a crucial
cornerstone to assess their effectiveness. Unfortunately, assessing the
diversity within protected areas has been a major challenge due to the
scarcity of taxonomists who can provide reliable identification. Here, we
introduce a simple DNA barcoding approach that can be established and
carried out by conservation practitioners with minimal involvement of
taxonomic experts. Notably, the records obtained through this approach
Integrative Conservation. 2024;3:196–211.196
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wileyonlinelibrary.com/journal/incon
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© 2024 The Author(s). Integrative Conservation published by John Wiley & Sons Australia, Ltd on behalf of Xishuangbanna Tropical Botanical
Garden (XTBG).
Editor‐in‐Chief: Binbin Li
Handling Editor: Alison Nazareno
can confirm the occurrence of previously recorded species, identify new
species, and even detect gaps in our taxonomic knowledge. These multiple
benefits were demonstrated through a pilot project that recorded the fern
species diversity in a small conservation area in southwest China, utilizing
asimplified DNA barcoding approach to minimize dependence on
taxonomic experts.
1|INTRODUCTION
Protected areas, such as national parks and forest
reserves, are the cornerstone of global efforts
to conserve species threatened by anthropogenic
transformations of their environments including
deforestation, urbanization, and global climate
warming (Pacifici et al., 2020; Williams et al., 2022).
As a consequence, expansion of protected areas
has been set as one of the targets of the
Kunming‐Montreal Global Biodiversity Framework
(Hughes, 2023;Watsonetal.,2023). Evaluation of the
effectiveness of already established or intended
protected areas requires reliable inventories of the
species conserved in the area protected (Rodrigues
& Cazalis, 2020). However, current inventories often
focus only on a few iconic organisms, such as large
mammals, birds, and selected tree genera, leaving
significant gaps in the data for the vast number of
taxa contributing to the ecosystems (Rodrigues &
Cazalis, 2020). Consequently, there is insufficient
evidence to support the assumption that protected
areas effectively slow the decline of species diver-
sity across all branches of the tree‐of‐life (Justin
Nowakowski et al., 2023). Besides the lack of first
records, another major challenge is the absence of
repeated occurrence confirmations necessary to
enable the evaluation of the effectiveness of applied
management strategies. To overcome these chal-
lenges, effective management of protected areas
thus requires to set targets that aim to achieve
periodic documentation of all or at least a broad
range of organisms occurring in these areas.
Unfortunately, this task is complicated by mapping
the biodiversity, especially repeated recording has to
tackle the scarcity of taxonomic expertise for many
lineages of organisms (Engel et al., 2021;Wheeler
et al., 2012). Given this limited availability of taxon
experts for most lineages of the tree‐of‐life, assess-
ments of the biodiversity in protected areas require
the employment of methodologies that minimize the
need for expert input while avoiding compromising
the essential quality of species identification. Here,
we addressed specifically the utilization of DNA
barcoding that presents a viable solution to these
challenges by facilitating rapid species inventories
with minimal taxonomic expertise. Since its intro-
duction, DNA barcoding has proven valuable in
conservation biology despite certain challenges
require to be taken into account (e.g., Liu et al., 2014;
Pereira et al., 2021; Song et al., 2023). This study
reports on a pilot project designed to establish a
DNA barcoding workflow with specific objectives:
(1) generating DNA sequences with limited costs
and technological requirements, (2) simultaneously
assessing and creating a reference data set, and
(3) establishing an effective feedback loop between
conservation practitioners and taxon experts.
DNA barcoding was introduced to leverage
advancements in DNA sequencing technologies
for achieve reliable species identification without
the constant need for taxon experts involvement
at every step (Hebert et al., 2003). Since its
introduction, substantial efforts were not only
taken to establish barcoding for all organisms,
including land plants (e.g., CBOL Plant Working
Group, 2009; Kress, 2017), and has evolved to
incorporate to expand from the CBOL concept
towards more flexible frameworks by integrating
new DNA technologies such as metabarcoding
(Ruppert et al., 2019). The successes and chal-
lenges of DNA barcoding for plants can be
illustrated by its application to identify ferns. It
has been utilized in a wide range of applications
such as to improve quality control of fern frag-
ments in the medicinal plant trade (Ma et al., 2010),
to tackle the identification of fern gametophytes
(Li et al., 2009; Nitta et al., 2017; Nitta &
Chambers, 2021; Schneider & Schuettpelz, 2006),
to document genetic differentiation among closely
relatedfernspecies(Liuetal.,2018;Wang
et al., 2016), and to enhance documentation of
fern diversity in various habitats, from temperate
(de Groot et al., 2011) to tropical regions (Ebihara
et al., 2010; Nitta et al., 2020;Trujillo‐Argueta
et al., 2021). These studies had in common the
requirement to utilize reference data sets established
under the guidance of taxon experts. Some well‐
curated DNA barcode libraries are available, such as
Practitioner points
•This highly simplified DNA barcoding
protocol can be easily applied to address
the species identification bottleneck.
•Communicating results with experts is
crucial for confirming new species records
and possibly identifying species that may
be new to science.
•DNA barcoding improves the documenta-
tion of known species while also expand-
ing the number of rare species known to
occur in protected areas.
LIU ET AL.
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for woody plants in tropical and subtropical China
(Jin et al., 2023)andflowering plants of northwest-
ern China's arid regions (Song et al., 2023). However,
the broader application of DNA barcoding for
monitoring plant diversity in protected areas
requires simultaneous inventory assessments and
the creation of DNA reference barcoding libraries.
Here, we describe a pipeline to apply DNA
barcoding to record the species diversity in
protected areas, specifically focusing on the fern
and lycophyte diversity of the Green Stone Forest
Fragment that belongs to Menglun Sub‐Nature
Reserve of the Xishuangbanna National Forest
Reserve, located in southwest China. Some infor-
mation about the fern diversity of this fragment has
been assessed by employing historical records—
available in the form of herbarium specimens—
in a previous study on the fern and lycophyte
diversity of the Xishuangbanna Dai Autonomous
Prefecture (Chen et al., 2022). Ferns and lycophytes
are not only common in tropical forest habitats but
are also considered excellent ecological indicators
for evaluating such ecosystems (Della, 2022).
The surveyed protected area is known to host
local endemics, such as Cyrtomium latifalcatum
S. K. Wu & Mitsuta (Wu & Mitsuta, 1985;Zhang
et al., 2013). The pilot project is set up with the
specific aim to be applicable without substantial
training efforts and with access to laboratories
providing only basic facilities for DNA extraction
and DNA barcode amplification. Therefore, the
number of DNA regions employed was restricted
toasingleregion,thechloroplastgenome‐based
rbcL gene. This gene has been the work‐horse of
fern and lycophyte phylogenetics since the earliest
studies applied molecular tools to improve our
understanding of their phylogenetic relationships
(Nitta et al., 2022). As a consequence, more than
20,000 rbcL sequences of ferns and lycophytes
have been deposited in public databases such
as GenBank (https://www.ncbi.nlm.nih.gov/). To
demonstrate the simplicity of the core parts of
the pipeline, collecting surveys and molecular lab
work were carried out by an intern student from a
local university. The input from experts with
extensive taxonomic knowledge was restricted to
several essential steps, such as assembling a
reference library and quality control. A specific
target of the pipeline has been the establishment
of feedback mechanisms to address taxonomic
challenges.
Specifically, the results of the study were eval-
uated to satisfy the following requirements: (1) most
species previously recorded for the protected area
were recovered through unambiguous DNA iden-
tification; (2) species gaps in the reference library
were successfully addressed by obtaining new
sequences, especially of local endemics; and (3)
short‐comings of current species treatments were
successfully detected, including the discovery
of accessions that may represent species new to
science.
FIGURE 1 Workflow of the DNA barcoding procedure. Blue = data preparation and processing carried out by conservation
practitioners; green = data processing carried out by taxon experts; brown = data analysis steps. The initial reference data set is
updated by incorporating newly generated sequences (AS) and by updating the taxonomy (UT). PCR, polymerase chain reaction; VI,
expert verified identifications.
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2|MATERIALS AND METHODS
2.1 |Study site
The pilot project employed a workflow designed
to enable contributors with limited training in
plant taxonomy and molecular biology to perform
most of the data production, thereby reducing
the dependence on experienced taxonomists and
optimizing feedback between conservation practi-
tioners and taxonomists (see Figure 1). Transect
surveys of the Green Stone Forest Fragment
(21.91022409° N, 101.28119972° E) within the Men-
glun Sub‐Nature Reserve of the Xishuangbanna
National Forest Reserve, located in the southern
part of the Xishuangbanna Dai Autonomous Pre-
fecture (Yunnan, China), were carried out repeat-
edly by students with minimal knowledge of the
target plant lineage, without the involvement of
taxon experts. The role of the experts was mini-
mized to providing initial guidance for conducting
surveys, training in the molecular laboratory,
and identifying the accessions using traditional
morphology‐based approaches. All ferns and lyco-
phytes were collected to (1) obtain voucher speci-
mens and (2) obtain materials for DNA extraction.
In addition, all collected samples were imaged in
the field. Voucher specimens were generated using
standard protocols and deposited in the research
laboratory at the Xishuangbanna Tropical Botanical
Garden, CAS. Pinnae or lamina fragments were
removed, dried in silica gel, and stored in the
laboratory. The remaining material was preserved
for future studies. All images taken were trans-
ferred to an image database. Future surveys will
only require the collection of leaf fragments,
minimizing the impact on rare species by avoiding
the collection of whole plants.
2.2 |DNA
extraction, polymerase chain reaction
(PCR) amplification, and sequencing
Whole genomic DNA was extracted using commer-
cially available DNA extraction kits (Tiangen
Biotech Co.) following the manufacturer's protocol
(see File S1 for further details). The obtained DNA
was then used to set up PCR reactions to amplify
a single DNA fragment, a process commonly
employed in DNA barcoding and molecular phylo-
genetics of these plants, specifically the chloroplast
genome region rbcL (see File S1). The PCR products
were sent to Sangon Biotech Co., Ltd for Sanger
sequencing. The obtained sequences were as-
sembled and stored using widely available and
freely accessible software tools, such as BioEdit
7. 1 ( https://thalljiscience.github.io; Hall, 1999). All
sequences were integrated into a single database in
the commonly used nexus format. The pipeline
of DNA extraction, PCR amplification, and DNA
sequencing was summarized in File S1. All newly
generated sequences were then checked for taxon
identity using DNA Blast (https://blast.ncbi.nlm.nih.
gov/Blast.cgi) to obtain a list of accessions with
highly similar DNA sequences (see BLASTN query
procedure; https://blast.ncbi.nlm.nih.gov/Blast.cgi?
PAGE_TYPE=BlastSearch). The reported list com-
prised information about the scientific name (genus,
species), GenBank accession number, and several
statistical values, particularly measures of sequence
similarity, such as the percentage identity (Per. Ident)
that enabled a ranking of the accessions with
similarities of up to 100%. Any potential contamina-
tion and mismatch sequences were identified and
filtered out by this first quality control step.
2.3 |DNA barcodes reference library
A reference database was assembled for the same
DNA region. For each species, all available rbcL
sequences were downloaded from the GenBank
database (https://www.ncbi.nlm.nih.gov/), which is
freely accessible to the public. To avoid generating
excessively large reference databases, up to three
sequences per species were maintained, emphasiz-
ing accessions with recorded voucher information
and relatively long reads (>1200 bp). These three
accessions were filtered from all accessions availa-
ble in early 2024 using the following criteria.
Sequences generated early days of Sanger sequenc-
ing, thus before the year 2000, were avoided if
possible. Specific attention was given to ensuring
the exclusion of misidentified accessions. All
sequences of a single region were assembled into
a single nexus matrix and aligned using widely used
and freely available alignment tools, with manual
adjustment as necessary to handle poorly edited
sequences. This part of the process was carried out
using Mesquite 3.81 (https://www.mesquiteproject.
org). Instead of focusing only on species recorded
from the Menglun Sub‐Nature Reserve of the
Xishuangbanna National Forest Reserve, the as-
sembled data set included all fern and lycophyte
species previously recorded in the Xishuangbanna
Dai Autonomous Prefecture (Chen et al., 2022), with
some alterations reflecting recent taxonomic prog-
ress. The classification applied followed PPGI (2016)
with minor alterations.
2.4 |Sequence alignment and
phylogenetic reconstruction
The matrix containing the newly generated
sequences was merged with the reference matrix.
The alignment of all sequences of the combined
matrix was checked and adjusted manually using
Mesquite. This matrix was then used for phyloge-
netic analyses employing PhyML (http://www.atgc-
montpellier.fr/phyml/; Guindon et al., 2010). The
optimal maximum likelihood hypothesis was re-
covered by applying automatic model selection via
LIU ET AL.
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199
SMS, with the Bayesian Information Criterion
selected (Lefort et al., 2017), and a starting tree
obtained with BioNJ (Gascuel, 1997). Standard
bootstrap analysis was conducted with up to 1000
replicates. The generated phylogenetic hypotheses
were visually inspected using freely available tools
designed to visualize phylogenetic trees, namely,
Figtree 1.4.4 (http://tree.bio.ed.ac.uk/software/
Figtree/) and ITOL v6 (https://itol.embl.de/itol.cgi;
Letunic & Bork, 2021). The results were interpreted
using the following protocol (see Figure 1). Acces-
sionsnestedincladesformedbyreference
sequences identified as a single species were
considered unambiguously identified. Clades
formed by accessions representing more than one
species were considered ambiguous. Accessions
forming lineages not including any reference
sequence were considered unknown. These acces-
sions were assumed to represent either non-
sampled species or species potentially new
to science. All voucher specimens were identified
by an experienced professional. These identifica-
tions were then compared to those obtained
through the DNA barcoding process. The following
categories were considered: (1) Identical identifica-
tion, (2) conflicting identification, (3) species ambig-
uous and unplaced in the DNA barcoding but
identified using morphology, and (4) species
unplaced in the DNA barcoding and also unknown
to science as of February 2024. To clarify the
taxonomic status of accessions belonging to Cate-
gory 4, all rbcL sequences of close relatives
available in GenBank (February 2024) were down-
loaded and assembled into a matrix as described
above. The phylogenetic analyses were run as
previously described. This procedure was specifi-
cally carried out for the genera Cyrtomium J. Sm.,
Hymenasplenium Hayata, Leptochilus Kaulf., and
Asplenium L., which are associated with several
closely related species complexes occurring in
China and adjacent regions.
3|RESULTS
The reference data set included 382 out of the 434
fern and lycophyte species recorded to occur in the
Xishuangbanna Dai Autonomous Prefecture (see
Table S1), resulting in a taxon coverage of 88.0%.
Taxa without any published DNA sequences in
public databases are primarily local endemics
in Xishuangbanna, namely, Bolbitis confertifolia
Ching, C. latifalcatum S. K. Wu & Mitsuta, Poly-
stichum paradeltodon L. L. Ziang, Pteris menglaen-
sis Ching, and Pteris undulatipinna Ching besides.
Additionally, some Yunnan endemics such as Crepi-
domanes chui Ching & P. S. Chiu, Diplazium quad-
rangulatum (W. M. Chu). Z. R. He, and Lomagramma
yunnanesis Ching. The reference data set did include
some Xishuangbanna endemics, such as Arachni-
odes pseudoasssamica Ching and Leptochilus
mengsongensis M. X. Zhao. Considering historical
records specific to the Menglun Sub‐Reserve, the
reference data set covered 197 out of 183 species,
achieving a coverage of 92.3%.
In total, 70 accessions were sampled during
surveys and incorporated into the DNA barcoding
test sample (Table 1). Employing the phylogenetic
hypotheses generated (Figure 2), 55 accessions
(78.6%) were unambiguously identified, whereas nine
accessions (12.9%) were ambiguously identified (see
Tab l e 1). Finally, five accessions (7.1%) were unplaced,
and one accession (1.4%) carried an incorrect acces-
sion number. In total, the survey recovered 53 species
belonging to 26 genera, 14 families, and five orders
(Table 1). Ambiguous identified accessions included
Angiopteris helferiana C. Presl (accession CYR35,
CYR41, CYR45), Adiantum ⨯meishanianum F. S . H s u
ex Yea C. Liu & W. L. Chiou (accession CYR24),
Asplenium humbertii Tardieu(accessionCYR23),
Christella jinghongensis (Ching) A. R. S. M. &
S. E. Fawc. (accession CYR40), Leptochilus flexilo-
bus (Christ) Liang Zhang & Li Bing Zhang (acces-
sion CYR57), Lygodium flexuosum (L.) Sw. (accession
CYR72), and Pteris venusta Kunze (accession CYR04).
The unplaced species included C. latifalcatum (acces-
sion CYR21), a local endemic that was missing in the
reference data set. Four accessions were unplaced
because they formed independent clades in the
genera: Asplenium (accession CYR17), Hymenasple-
nium (accession CYR12, CYR59), and Leptochilus
(accession CYR2) (see Figure 2).
4|DISCUSSION
In total, the study analyzed 70 accessions using the
DNA barcoding protocol, resulting in the identifica-
tion of 41 species previously recorded for the
Menglun Sub‐Nature Reserve, and nine species that
were newly recorded. Among the newly recorded
species, Asplenium humbertii Tardieu was docu-
mented for the first time in the Xishuangbanna
Dai Autonomous Prefecture. Additionally, four ac-
cessions required further study due to unresolved
species identities. The DNA barcodes determined
their generic relationship but did not match them
with previously described species, suggesting the
presence of three species potentially new to science
(see Table 1, New spec). Two of these accessions
were identified as a sister pair within the genus
Hymenasplenium, while the other two were placed
in the genera Asplenium and Leptochilus. Further-
more, the study provided the first DNA sequences
for the local endemic C. latifalcatum.
The pilot project demonstrated the effectiveness
of a straightforward DNA barcoding approach,
utilizing a single plastid‐based DNA fragment, such
as rbcL, to reliably identify approximately 75%
of the accession recovered. The success rates of
DNA barcoding in this study were comparable
to previous reports on the DNA barcoding of ferns
and lycophytes in Japan (Ebihara et al., 2010) and
French Polynesia (Nitta et al., 2017). Unfortunately,
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TABL E 1 Summary of the identifications obtained utilizing the minimalistic DNA barcoding approach.
Lineage Species ID DNA BAR CAT Acc. nr. Record status
1.1 Selaginella bodinieri Hieron. Unambiguous CYR26 New record
1.1 Selaginella helferi Warb. Unambiguous CYR34 Confirmed
1.1 Selaginella picta A. Br. ex Baker Unambiguous CYR51, CYR67 Confirmed
1.1 Selaginella repanda (Desv. Ex Poir.) Spring Unambiguous CYr14 Confirmed
2.1 Angiopteris helferiana C. Presl Ambiguous (4) CRR35, CYR41,
CYR45
Confirmed
2.2 Crepidomanes latealatum (Bosch) Copel. Unambiguous CYR05 Confirmed
2.3 Lygodium circinnatum (N. L. Burm.) Sw. Unambiguous CYR32 Confirmed
2.3 Lygodium flexuosum (L.) Sw. Ambiguous (6) CYR72 Confirmed
2.4 Microlepia rhomboidea (Wall. Ex Kunze) Prantl Unambiguous CYR48 Confirmed
2.5 Adiantum caudatum L. Unambiguous CYR13, CYR64 Confirmed
2.5 Adiantum menglianense Y. Y. Qian Unambiguous CYR11 New record
2.5 Adiantum ×meishanianum F. S. Hsu ex Yea C. Liu &
W. L. Chiou
Ambiguous (3) CYR24 New record
2.5 Antrophyum wallichianum M. G. Gilbert & X. C. Zhang Unambiguous CYR30 New record
2.5 Pteris arisanensis Tagawa Unambiguous CYR10 Confirmed
2.5 Pteris biaurita L. Unambiguous CYR52 Confirmed
2.5 Pteris ensiformis N. L. Burm. Unambiguous CYR33 Confirmed
2.5 Pteris venusta Kunze Ambiguous (1) CYR04 Confirmed
2.6 Asplenium humbertii Tardieu Ambiguous (2) CYR23 New record
2.6 Asplenium nidus L. Unambiguous CYR20 CYR63 Confirmed
2.6 Asplenium saxicola Rosenst. Unambiguous CYR25 Confirmed
2.6 Asplenium spec. Unplaced CYR17 New spec
2.6 Hymenasplenium excisum (C. Presl) S. Lindsay Unambiguous CYR46 Confirmed
2.6 Hymenasplenium spec. unknown Unplaced CYR12, CYR59 New spec
2.6 Anisocampium cuspidatum (Bedd.) Yea C. Liu,
W. L. Chiu & M. Kato
Unambiguous CYR06 Confirmed
2.6 Diplazium alatum (Christ) R. Wei & X. Z. Zhang Unambiguous CRY53 Confirmed
2.6 Diplazium simile (W. M. Chu) R. Wei & X. C. Zhang Unambiguous CRY36, CYR50 New record
2.6 Abacopteris nudata (Roxb.) S. E. Fawc. & S. R. Sm. Unambiguous CYR60 Confirmed
2.6 Christella dentata (Forssk.) Brownsey & Jermy Unambiguous CYR38 Confirmed
2.6 Christella jinghongensis (Ching ex K. H. Shing)
A. R. Sm. & S. E. Fawc.
Ambiguous (5) CYR40 Confirmed
2.6 Christella parasitica (L.) H. Lév. Unambiguous CYR39 Confirmed
2.6 Christella subelata (Baker) Holttum Unambiguous CYR37 Confirmed
2.6 Reholttumia truncata (Poir.) S. E. Fwac. & A. R. Sm. Unambiguous CYR47 Confirmed
2.7 Davallia griffithiana Hook. Unambiguous CYR31 Confirmed
2.7 Bolbitis scandens W. M. Chu Unambiguous CYR42, CYR70 New record
2.7 Cyrtomium latifalcatum S. K. Wu & Mitsuta Unplaced CYR21 Confirmed
2.7 Nephrolepis falciformis J. Sm. Unambiguous CYR08 Confirmed
2.7 Drynaria bonii Christ Unambiguous CYR03, CYR29 New record
2.7 Lepisorus carnosus (J. Sm.) C. F. Zhao, R. W. & X. C. Zhang Unambiguous CRY15 Confirmed
2.7 Lepisorus zippelii (Blume) C. F. Zhao, R. Wei, & X. C. Zhang Unambiguous CYR65, CYR68 Confirmed
2.7 Leptochilus flexilobus (Christ) Liang Zhang & Li Bing Zhang Ambiguous (6) CYR56, CYR57 New record
(Continues)
LIU ET AL.
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201
some common challenges of DNA barcoding were
encountered, including unplaced species and
ambiguous identifications. Ambiguous identifica-
tion is a recurring issue due to DNA barcodes
often failing to differentiate species complexes as a
consequence of hybridization, polyploid speciation,
and slow mutation rates. For instance, the latter
process has been reported for the marattioid genus
Angiopteris Hoffm. (Lehtonen et al., 2020; May
et al., 2021). Our results are consistent with the
expectation that low mutation rates result in failed
species identification when utilizing only rbcL
sequences. Hybridization led to the ambiguous
identification of accession CYR24 as Adiantum ×
meishanianum F. S. Hsu ex Yea C. Liu & W.
L. Chiou (Shang et al., 2016). DNA barcodes utilizing
genes from the uniparentally inherited chloroplast
genome will fail to provide unambiguous identifi-
cation of taxa arising from hybridization or poly-
ploidization (Liu et al., 2018). The other ambigu-
ously identified accessions are currently unplaced
and require further study. For example, three out
of the four accessions belonging to the genus
Christella H. Lev. were identified unambiguously
as Christella dentata (Forssk.) Brownsey & Jermy,
Christella parasitica (L.) H. Lev., and Christella
subelata (Baker) Holttum, whereas the fourth
accession was ambiguously identified according
to the rbcL sequences as Christella jaculosa (Christ)
Holttum or Christella jinghongensis (Ching ex K. H.
Shing) A. R. Sm. & S. E. Fawc. Both species are
known to occur in Xishuangbanna (Li et al., 2013)
and were represented by a single rbcL sequence in
the reference data set. Considering morphological
diagnostic characteristics (Li et al., 2013), the acces-
sion was identified as C. jinghongensis.Despite
significant progress in the morphological diagnostics
of this genus (Li et al., 2013), the species identity
of specimens utilized to generate DNA sequences
requires verification. Attention to taxonomic ambigu-
ities is especially crucial in cases where only one
rbcL sequence is available. Accessions belonging to
Lepisorus sect. Lemmaphyllym (C.Presl)C.F.Zhao,
R. Wei & X. C. Zhang (Zhao et al., 2020) exemplify
the need for further taxonomic clarification in south-
ern Yunnan (Wei & Zhang, 2013; Zhao et al., 2020).
Accessions identified as Leptochilus flexilobus illus-
trate the need for continued study of the confusing
taxonomy of these ferns (Zhang et al., 2019).
4.1 |New records
During the survey, accessions were encountered that
were identified as A. humbertii (see Lin & Viane, 2013).
This species has previously been recorded from
limestone rocks in southeastern Yunnan, as well as
in Guangxi, Hainan Island, Laos, Thailand, and
Vietnam. It is considered to form a species complex
with Asplenium antrophyoides Christ and Asplenium
grevillei Wall. ex Hook. & Grev., which requires further
study (Dong, 2011; Lin & Viane, 2013;Wei&
TABL E 1 (Continued)
Lineage Species ID DNA BAR CAT Acc. nr. Record status
2.7 Leptochilus spec. unknown Unplaced CYR02 New spec
2.7 Microsorum cuspidatum (D. Don) Tagawa Unambiguous CYR28 Confirmed
2.7 Microsorum punctatum (L.) Copel. Unambiguous CYR27 Confirmed
2.7 Pyrrosia nuda (Giesenh.) Ching Unambiguous CYR22 Confirmed
2.7 Pyrrosia nummulariifolia (Sw.) Ching Unambiguous CYR16 Confirmed
2.7 Pteridrys cnemidaria (Christ.) C. Chr. & Ching Unambiguous CYR09, CYR49,
CYR55
Confirmed
2.7 Tectaria devexa (Kunze) Copel. Unambiguous CYR19 Confirmed
2.7 Tectaria fauriei Tagawa Unambiguous CYR43 Confirmed
2.7 Tectaria fuscipes (Wall. ex Bedd.) C. Chr. Unambiguous CYR44, CYR62,
CYR66
Confirmed
2.7 Tectaria herpetocaulos Holttum Unambiguous CYR07, CYR58 Confirmed
2.7 Tectaria impressa (Fee) Holttum Unambiguous CYR18, CYR71 Confirmed
2.7 Tectaria quinquefida (Baker) Ching Unambiguous CYR61 Confirmed
2.7 Tectaria zeylanica (Houtt.) Sledge Unambiguous CYR01 Confirmed
Note: Columns report: lineage (linear clade number); species according to expert identification and DNA barcoding identification; ID DNA BAR CAT =
Identification of DNA barcoding category as unambiguous, ambiguous, unplaced, confused sample (numbers see below); Acc. Nr. = Accession Number
(number of the accessions collected in surveys of the Green Stone Forest Fragment); Record Status = species presented records for the Menglun Sub‐
Nature Reserve of which the Green Stone Forest Fragment is part of as previously recorded (confirmed), recorded for the first time (new record), or a
perhaps a species new to science (New spec). Causes of ambiguous DNA barcoding identifications: (1) lack of differentiation among Pteris heteromorpha,
Pteris subquinata, and Pteris venusta; (2) lack of differentiation between Asplenium antrophyoides and Asplenium humbertii; (3) shared chloroplast DNA
between Adiantum ×meishanianum and its maternal parent Adiantum malesianum; (4) lack of differentiation among species of Angiopteris; (5) lack of
differentiate between Christella jaculosa and Christella jinghongensis in the two available rbcL sequences; and (6) lack of differentiation among Lygodium
flexuosum and Lycodium salicifolium in rbcL.
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Dong, 2012). Cytological and DNA sequence data are
available for only two of these three species: the
tetraploid A. antrophyoides and the hexaploid A.
humbertii (Dong, 2011; Schneider et al., 2017;Xu
et al., 2020). The sample from the Green Stone Forest
Fragment was nested in a clade comprising acces-
sions of both species but failed to resolve the
differentiation between the two species. Therefore,
further studies are required to clarify the status of
these accessions. Here, we accept the identification
based on the treatment of these ferns in the Flora of
China (Lin & Viane, 2013). As a consequence, the
range of A. humbertii now includes not only south-
eastern Yunnan but also southwestern Yunnan.
4.2 |New genetic resources of the local
endemic C. latifalcatum
C. latifalcatum is currently known only from
Xishuangbanna, Yunnan (Wu & Mitsuta, 1985;
Zhang et al., 2013). Previous records support
occurrences of this local endemic in the Mengla
Sub‐Nature Reserve (Chen et al., 2022). The
generated phylogenetic hypothesis recovered this
species as part of a clade comprising species
knowntooccurinsouthernChina(Figure3).
Several of these species are local endemics, such
as the tetraploid Cyrtomium chingianum P. S .
Wang and the diploid Cyrtomium guizhouensis
FIGURE 2 Phylogenetic placement of Green Stone Forest accessions in the phylogenetic hypothesis generated based on the
reference DNA barcoding reference data set of ferns and lycophytes occurring in Xishuangbanna Day Autonomous Prefecture. The
visualized phylogenetic hypothesis was generated using PhyML 3.0. Branch length corresponded to the estimated amount of
substitution events. Green Stone Forest accessions and their branches were marked in Red. Clades providing unambiguous
identification of Green Stone Forest accessions were marked in blue, while clades providing ambiguous identification of Green Stone
Forest accessions were marked in brown.
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H. S. Kung & P. S. Wang. All species of this clade
are known to occur in limestone rocks (Zhang
et al., 2013). The clade also shows notable varia-
tion in ploidy levels, including diploids, triploids,
and tetraploids, as well as sexual and apomictic
reproduction (Lu et al., 2006; Zhang et al., 2013).
Similar to other fern genera occurring on karst
formations in southern China and northern Viet-
nam, several species new to science have been
discovered in recent years, such as Cyrtomium
calcis LiangZhang,N.T.Liu&LiBingZhang
(Lu et al., 2023)andCyrtomium remotipinnum Yan
Liu & H. J. Wei (Nong et al., 2023). The majority
of the species related to C. latifalcatum share
narrow distribution ranges due to their ecological
specialization to karst formations. Therefore, these
species are vulnerable to extinction threats caused
by anthropogenic activities and require special atten-
tion. C. latifalcatum is considered to be endangered
(Chen et al., 2022).
FIGURE 3 Phylogenetic placement of the local endemic Cyrtomium latifalcatum in the phylogeny of the genus Cyrtomium based
on all rbcL sequences available from GenBank in February 2024, plus two accessions of Cyrtomium calicos kindly shared by the
authors of this new species. The visualized phylogenetic hypothesis was generated using PhyML 3.0. Branch length corresponded to
estimated number of substitution events, whereas triangles represented the estimated number of substitution events, whereas
triangles represent the bootstrap values as given in the legend. The accession of C. latifalcatum was marked in red. Green branches
marked the clade in which this species was found to be nested. Other species in this clade were printed in bold.
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4.3 |Unplaced accessions
Accessions recovered as unplaced species require
further exploration because they highlight recording
gaps that are not restricted to the lack of documen-
tation of already known species but represent
species putatively new to science. Each of these
cases will be discussed more extensively, although
we are not attempting to resolve the taxonomic
issues here.
One accession (accession CYR17) was found to
form an independent clade sister to the Asplenium
coenobiale complex (Figure 4). This complex
comprises currently five known species (Jiang
et al., 2011; Siqi et al., 2019; Xu et al., 2022). Two
widespread species, namely, A. coenobiale Hence
and Asplenium pulcherrimum (Baker) Ching, have
been recognized since the exploration of Southeast
Asian fern diversity about 100 years ago. In the last
20 years, only three local endemics have been
reported: Asplenium cornutissimum X. C. Zhang &
R. H. Jiang from Guangxi in 2011, Asplenium
maguanense S. Q. Liang, R. Wei & X. C. Zhang
from southeastern Yunnan in 2019, and Asplenium
danxiaense K. W. XU from Guangdong in 2022.
These new discoveries arguably indicate the need
for a comprehensive investigation into the diversity
of these ferns in southern China and adjacent
tropical regions, focusing specifically on Karst
and Danxia outcrops, as these species have been
reported from such habitats (Fu et al., 2022; Jiang
et al., 2011; Siqi et al., 2019; Xu et al., 2022). The
taxonomic status of accession CYR17 is currently
under scrutiny using additional samples obtained
in the Menglun region.
Other notable discoveries from our survey
were the two accessions (CYR12, CYR59) nested
in Hymenasplenium (Figure 5). The taxonomy of
the Old World representatives of this genus
has undergone substantial revisions in recent
years. In particular, the concept of Hymenasple-
nium unilaterale as a Paleotropical species has
been dismissed. Previously rejected species have
been reinstated, and several new species have
been described (e.g., Chang et al., 2018,2022;
Lin & Viane, 2013;Xuetal.,2018;Zhang
et al., 2021). In addition to the taxonomic revisions
within the H. unilaterale complex, eight species
of this genus have been documented to occur in
the Xishuangbanna Dai Autonomous Prefecture:
Hymenasplenium apoganum (N. Kurak. & Hatan.)
Nakaike, Hymenasplenium cheilosorum (Kunze
ex Mett.) Tagawa, Hymenasplenium excisum
(C. Presl) S. Linds., H. laterepens N. Murak. & Cheng
ex Y. Fen Chang & K. Hori, Hymenasplenium
obscurum (Blume) Tagawa, Hymenasplenium obtu-
sidentatum Y. Fen Chang & G. Cheng Zhang, and
Hymenasplenium pseudobscorum Viane. Notably,
two of these species were introduced with type
specimens collected from this area, namely, H.
laterepens (Chang et al., 2018)andH. obtusidentatum
(Chang et al., 2022). The two accessions recovered as
an independent clade are morphologically distinct
from all these species but resemble somewhat
collections reported in a cytological study on Hyme-
nasplenium in Xishuangbanna (Cheng & Murakami,
1998). These authors reported a diploid of Hymenas-
plenium as Hymenasplenium latipinnum, but this
name was not properly published. Thus, Lin & Viane
(2013) reported these samples as Hymenasplenium
subnormale (Copel.) Nakaike, yet pointed out clear
differences between the specimens from Yunnan and
those from the type locality in the Philippines. These
specimens require further comparative study to
clarify their taxonomic status.
Recovering the accession CYR2 of Leptochilus
not fitting to any published species (Figure 6)
aligns with recent progress in the taxonomic
studies of this primarily southeast Asian genus
(Wei et al., 2023;Yuetal.,2024; Zhang et al., 2019;
Zhao et al., 2017). These studies not only intro-
duced several new species but also questioned
previous taxonomic treatments of the genus. The
accession CYR2 clustered in a clade (Figure 6)
containing accessions of an unknown species,
namely, Zhang et al. 6377 (GenBank accession
number MH768422), Zhang et al. 7365 (GenBank
accession number MH768436), and Zhang et al.
6711 (GenBank accession number MH768459).
These three collections were obtained in central
and northern Vietnam (Zhang et al., 2019). There-
fore, our accessions extend the range of this clade
from Vietnam to southwest Yunnan.
4.4 |DNA barcoding‐based inventories
As stated in the introduction, this study was
designed as a pilot to explore the benefits and
challenges of utilizing DNA barcoding to record
the species diversity of protected areas with
minimal involvement of taxonomists with exper-
tise in fern taxonomy. Surprisingly, the surveys
carried out by inexperienced students recorded
common species as expected but also identified
rare taxa. In particular, the recovery of C. latifalca-
tum is of importance. Repeated surveys documen-
ted a total of six individuals of this local endemic
in the Green Stone Forest Fragment, which is its
type locality (Wu & Mitsuta, 1985). Recovering
this species confirmed its continued presence in
its type locality and will also facilitate the estab-
lishment of effective conservation measurements
for its protection. Given the small number of
individuals recovered, there is a need to develop
a comprehensive program to protect this species.
Such a program should include recording crucial
evidence required including assessments of
ecological preferences, assessing demography,
and genetic diversity, and collecting spores for
ex situ reproduction followed by reintroduction
(Wall et al., 2024).
An important lesson from this study was
the confirmation of the concept's effectiveness.
LIU ET AL.
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205
Non‐Taxon expert collection was found to be effective,
although feedback loops with taxonomists are essen-
tial to take full advantage of the evidence obtained.
The recovery of new records and species new to
science also highlighted the significant gaps in our
records of species occurring in protected areas. In the
case of the Green Stone Forest Fragment, this result
may appear surprising given the history of plant
diversity exploration in southern Yunnan (see Chen
et al., 2022) and the proximity of this fragment to the
core research center of the Xishuangbanna Tropical
Botanical Garden. However, it is consistent with the
ongoing discovery of new species in this region, such
as Hymenasplenium laterepens N. Murak. & Cheng ex
Y. Fen Chang & K. Hori (Chang et al., 2018), Leptochilus
mengsongensis M. X. Zhao (Zhao et al., 2017)and
Polystichum menglaense Z. L. Liang & Li Bing Zhang
(Liang et al., 2021). Furthermore, recent studies have
provided evidence supporting the assumption that
these diversity‐rich tropical forests are home to a
disproportional frequency of rare fern and lycophyte
species (Cicuzza, 2021). The approach introduced here
has the potential to overcome hurdles that have
hampered efforts to assemble comprehensive inven-
tories of protected areas, even for those that are easily
accessible.
FIGURE 4 Phylogenetic placement of Green Stone Forest accession recovered as an unplaced Asplenium sample based on all
rbcL sequences available at GenBank in February 2024. The sampling generated included accessions of species belonging to several
Asplenium species complexes occurring exclusively or predominantly in Southeast Asia, including representatives of the Asplenium
coenobiale complex (clade in green), Asplenium exiguum complex (clade in dark purple), Asplenium fugax complex (clade in blue),
Asplenium sarellii complex (clade in brown), and Asplenium interjectum (clade in orange). The visualized phylogenetic hypothesis
was generated using PhyML 3.0. Branch length corresponded to the estimated number of substitution events, whereas triangles
represented the bootstrap values of ⩾95%. The Green Stone Forest accessions were marked in red, including their branches.
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5|CONCLUSIONS
Our pilot study demonstrated the effectiveness of a
simplified DNA barcoding approach to obtain rapid
assessments of the fern and lycophyte diversity in
protected areas. sampled accessions were success-
fully identified, although some ambiguous identifi-
cations and unplaced accessions still require expert
intervention. A significant challenge is the limited
availability of reliable identified rbcL sequences
for many species. Only 33.4% of the species were
represented with the three targeted reference
sequences, while incomplete representation was
achieved for 16.1% with two sequences, 38.5% with
one sequence, and 12.0% of the species had no
available DNA sequence. Thus, the simultaneous
FIGURE 5 Phylogenetic placement of Green Stone Forest accessions belonging to the genus Hymenasplenium in the phylogeny of
the genus Hymenasplenium based on all rbcL sequences available at GenBank in February 2024. The accession CYR46 Hymenasplenium
excisum was recovered to be nested in a clade comprising only accessions identified as H. excisum. The two accessions, CYR12 and
CYR59, formed an independent clade that did not include any accession previously published. The visualized phylogenetic hypothesis
was generated using PhyML 3.0. Branch length corresponded to the estimated number of substitution events, whereas triangles
represented the bootstrap values as given in the legend. Green Stone Forest accessions were marked in red. Clades including species
previously reported in the Xishuangbanna Dai Autonomous Prefecture were marked in color: blue clades included Hymenasplenium
chei lo sorum, brown clades included H. excisum,Hymenasplenium obscurum,Hymenasplenium obtusidentatum,andHymenasplenium
pseudobscorum, green clades included Hymenasplenium apoganum, and purple clades included Hymenasplenium laterepens.
LIU ET AL.
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207
assembly of reference data alongside inventories
is arguably the only realistic approach, but this
procedure requires the involvement of taxon experts.
Furthermore, the inventory illustrates the incomplete-
ness of our current understanding of both the spatial
distribution of species—evidenced by new records—
and the taxonomy of plants occurring in species‐rich
areas like southern Yunnan. Lastly, the effectiveness
of inventories can be further enhanced by linking
them to simultaneously established image reference
data sets, providing conservation practitioners with
an additional toolset for species identification. The
combination of DNA barcoding and digital imaging
holds the promise of overcoming limitations specific
to each approach, thereby ensuring the quality of the
identifications obtained.
AUTHOR CONTRIBUTIONS
Hongmei Liu: Conceptualization; data curation; identi-
fication; formal analysis, methodology; investigation;
FIGURE 6 Phylogenetic placement of Green Stone Forest accessions belonging to the genus Leptochilus in the phylogeny based
on all rbcL sequences available at GenBank in February 2024. The accession CYR2 was nested in a clade comprising three accessions
previously published as species unknown, namely, MH768422, MH768436, and MH768450. The specimens based on accessions
collected in Vietnam (Zhang et al., 2019). The two accessions identified as Leptochilus flexilobus were nested in a clade comprising
accessions identified as this species but also other species. The visualized phylogenetic hypothesis was generated using PhyML 3.0.
Branch length corresponded to the estimated number of substitution events, whereas triangles represented the bootstrap values as
given in the legend. Green Stone Forest accessions were marked in red. The clade comprising CRY2 was marked in green, and the
accessions of this clade were printed in bold. The clade comprising CRY56 and CRY57 was marked in blue, and all accessions identified
as Leptochilus flexilobus (Christ) Liang Zhang & Li Bing Zhang were printed in bold.
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project administration; writing—original draft;
writing—review and editing. Ya r o n g C h a i : Data cura-
tion; investigation. Harald Schneider: Conceptualiza-
tion; validation; identification; writing—original draft;
writing—review and editing.
ACKNOWLEDGMENTS
The authors are grateful to the management of the
Menglun subnature reserve of the Xishuangbanna
National Forest Reserve for the permission to
survey the Green Stone Forest Fragment. The
authors acknowledge the financial support by the
Yunnan Province Science and Technology Depart-
ment (202101AS070012), Yunnan Revitalization
Talent Support Program “Innovation Team”Project
(202405AS350019), and 14th Five‐Year Plan of the
Xishuangbanna Tropical Botanical Garden, Chinese
Academy of Sciences (E3ZKFF8B01). The authors
would like to thank several team members who
involved and helped in the field. The molecular
work was supported by Institutional Center for
Shared Technologies and Facilities of Xishuang-
banna Tropical Botanical Garden, Chinese Academy
of Sciences (CAS).
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
DATA AVAI L AB IL IT Y S TATEME N T
All newly generated DNA sequences were depos-
ited in GenBank. The reference data set is available
from the request to the corresponding author. All
other accumulated data were summarized in the
supporting information files.
ETHICS STATEMENT
The research does not involve any experiments
involving animals or humans.
ORCID
Hongmei Liu http://orcid.org/0000-0003-
2594-9258
Harald Schneider http://orcid.org/0000-0002-
4548-7268
REFERENCES
CBOL Plant Working Group. (2009) A DNA barcode of land
plants. Proceedings National Academy of Sciences of the
United States of America, 106, 12794–12797. Available from:
https://doi.org/10.1073/pnas.0905845106
Chang, Y., Hori, K., Murakami, N., Cao, L., Lu, S. & Schneider, H.
(2018) Validation of Hymenasplenium laterepens (Asplenia-
ceae): evidence from morphology and molecular analyses.
Phytotaxa, 374, 277–290. Available from: https://doi.org/10.
11646/phytotaxa.374.4.1
Chang, Y., Zhang, G., Wang, Z. & Cao, L. (2022) Molecular and
morphological evidence reveals a new fern species of
Hymenasplenium (Aspleniaceae) from south and south-
western China. PhytoKeys,211,93–106. Available from:
https://doi.org/10.3897/phytokeys.211.90363
Chen, K., Khine, P.K., Yang, Z. & Schneider, H. (2022) Historical
plant records enlighten the conservation efforts of ferns and
lycophytes' diversity in tropical China. Journal for Nature
Conservation, 68, 126197. Available from: https://doi.org/10.
1016/j.jnc.2022.126197
Cheng, X. & Murakami, N. (1998) Cytotaxonomic study of
genus Hymenasplenium (Aspleniaceae) in Xishuangbanna,
Southwestern China. Journal of Plant Research,111,495–500.
Available from: https://doi.org/10.1007/BF02507784
Cicuzza, D. (2021) Rare pteridophytes are disproportionately
frequent in the tropical forest of Xishuangbanna, Yunnan,
China. Acta Oecologica, 110, 103717. Available from: https://
doi.org/10.1016/j.actao.2021.103717
de Groot, G.A., During, H.J., Maas, J.W., Schneider, H., Vogel, J.C.
& Erkens, R.H.J. (2011) Use of rbcL and trnL‐Fas a two‐locus
DNA barcode for identification of NW‐European ferns: an
ecological perspective. PLoS One,6,e16371.Availablefrom:
https://doi.org/10.1371/journal.pone.0016371
Della, A.P. (2022) Ferns as ecological indicators. In: Marimutho, J.,
Fernandez, H., Kumar, A. & Thangaiah, S. (Eds.) Ferns:
biotechnology, propagation, medicinal uses and environ-
mental regulation. Berlin: Springer, pp. 587–601. Available
from: https://doi.org/10.1007/978-981-16-6170-9_25
Dong, S.Y. (2011) Taxonomic studies on Asplenium sect.
Thamnopteris (Aspleniaceae) I: cytological observations.
American Fern Journal, 101, 156–171. Available from:
https://doi.org/10.1640/0002-8444-103.3.156
Ebihara, A., Nitta, J.H. & Ito, M. (2010) Molecular species
identification with rich floristic sampling: DNA barcoding
the pteridophyte flora of Japan. PLoS One,5,e15136.
Available from: https://doi.org/10.1371/journal.pone.0015136
Engel, M.S., Ceríaco, L.M.P., Daniel, G.M., Dellapé, P.M., Löbl, I.,
Marinov, M. et al. (2021) The taxonomic impediment: a
shortage of taxonomists, not the lack of technical ap-
proaches. Zoological Journal of the Linnean Society, 193,
381–387. Available from: https://doi.org/10.1093/zoolinnean/
zalb072
Fu, L., K. Monro, A. & Wei, Y. (2022) Cataloguing vascular plant
diversity of karst caves in China. Biodiversity Science, 30,
21537. Available from: https://doi.org/10.17520/biods.
2021537
Gascuel, O. (1997) BIONJ: an improved version of the NJ
algorithm based on a simple model of sequence data.
Molecular Biology and Evolution, 14, 685–695. Available
from: https://doi.org/10.1093/oxfordjournals/molbev.a025808
Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W.
& Gascuel, O. (2010) New algorithms and methods to
estimate maximum‐likelihood phylogenies: assessing the
performance of PhyML 3.0. Systematic Biology, 59, 307–321.
Available from: https://doi.org/10.1093/sysbio/syq010
Hall, T.A. (1999) BioEdit: a user‐friendly biological sequence
alignment editor and analysis program for Windows 95/98/
NT. Nucleic Acids Symposium Series, 41, 95–98. Available
from: https://doi.org/10.1021/bk-1999-0734.ch008
Hebert, P.D.N., Cywinska, A., Ball, S.L. & deWaard, J.R. (2003)
Biological identifications through DNA barcodes. Proceedings
of the Royal Society of London. Series B: Biological Sciences,
270, 313–321. Available from: https://doi.org/10.1098/rspb.
2002.2218
Hughes, A.C. (2023) The post‐2020 global biodiversity frame-
work: how did we get here, and where do we go next?
Integrative Conservation,2,1–9. Available from: https://doi.
org/10.1002/INC3.16
Jiang, R.H., Zhang, X.C. & Liu, Y. (2011) Asplenium cornutissi-
mum (Aspleniaceae), a new species from karst caves in
Guangxi, China. Brittonia, 63, 83–86. Available from: https://
doi.org/10.1007/s12228-010-9139-z
Jin, L., Shi, H.Y., Li, T., Zhao, N., Xu, Y., Xiao, T.W. et al. (2023) A
DNA barcode library for woody plants in tropical and
subtropical China. Scientific Data, 10, 819. Available from:
https://doi.org/10.1038/s41597-023-02742-7
Justin Nowakowski, A., Watling, J.I., Murray, A., Deichmann, J.L.,
Akre, T.S., Muñoz Brenes, C.L. et al. (2023) Protected areas
slow declines unevenly across the tetrapod tree of life.
Nature, 622, 101 –106. Available from: https://doi.org/10.1038/
s41586-023-06562-y
Kress, W.J. (2017) Plant DNA barcodes: applications today and in
the future. Journal of Systematics and Evolution, 55,
291–307. Available from: https://doi.org/10.1111/jse.12254
LIU ET AL.
|
209
Lefort, V., Longueville, J.E. & Gascuel, O. (2017) SMS: smart
model selection in PhyML. Molecular Biology and Evolution,
34, 2422–2424. Available from: https://doi.org/10.1093/
molbev/msx149
Lehtonen, S., Poczai, P., Sablok, G., Hyvönen, J., Karger, D.N. &
Flores, J. (2020) Exploring the phylogeny of the marattia-
lean ferns. Cladistics, 36, 569–593. Available from: https://
doi.org/10.1111/cla.12419
Letunic, I. & Bork, P. (2021) Interactive Tree Of Life (iTOL) v5: an
online tool for phylogenetic tree display and annotation.
Nucleic Acids Research, 49(W1), W293–W296. Available
from: https://doi.org/10.1093/nar/gkab301
Li, F.W., Tan, B.C., Buchbender, V., Moran, R.C., Rouhan, G.,
Wang, C.N. et al. (2009) Identifying a mysterious aquatic
fern gametophyte. Plant Systematics and Evolution, 281,
77–86. Available from: https://doi.org/10.1007/s00606-009-
0188-2
Li, Z.Y., He, Z.R. & Zhang, X.C. (2013) A taxonomic revision of
Cyclosorus subgenus Cyclosoriopsis (Thelypteridaceae)
from China. Journal of Systematics and Evolution, 51,
609–638. Available from: https://doi.org/10.1111/jse.12013
Liang, Z.L., He, Z.R. & ZHang, L.B. (2021) Polystichum dong-
chuaense and P. menglaense (subg. Haplopolystichum;
Dryopteridaceae), two new ferns from Yunnan, China.
Phytotaxa, 479, 114–120. Available from: https://doi.org/10.
11646/phytotaxa.479.1.9
Lin, Y.X. & Viane, R. (2013) Aspleniaceae. In: Wu, Z.Y., Raven, P.H.
& Hong, D.Y. (Eds.) Flora of China (Pteridophytes), vol. 203.
Beijing: Science Press; St. Louis: Missouri Botanical Garden
Press, pp. 267–316.
Liu, H.M., Russell, S.R., Vogel, J. & Schneider, H. (2018) Inferring
the potential of plastid DNA‐based identification of derived
ferns: a case study on the Asplenium trichomanes aggre-
gate in Europe. Plant Systematics and Evolution, 304,
100 9–1022. Available from: https://doi.org/10.1007/s00606-
018-1529-9
Liu, J., Yan, H.F., Newmaster, S.G., Pei, N., Ragupathy, S. &
Ge, X.J. (2014) The use of DNA barcoding as a tool for the
conservation biogeography of subtropical forests in China.
Diversity and Distributions, 21, 188–199. Available from:
https://doi.org/10.1111/ddi.12276
Lu, J.M., Cheng, X., Wu, D. & Li, D.Z. (2006) Chromosome study
of the fern genus Cyrtomium (Dryopteridaceae). Botanical
Journal of the Linnean Society, 150, 221–228. Available
from: https://doi.org/10.1111/j.1095-8339.2006.00462.x
Lu, N.T., Wei, H.J., Vuong, L.D., Toan, L.C., Zhang, L.B. &
Zhang, L. (2023) Cyrtomium calcis sp. nov. and six new
records of the shield fern family (Dryopteridaceae) from
Vietnam. Phytotaxa, 583, 16. Available from: https://doi.org/
10.11646/phytotaxa.583.1.2
Ma, X.Y., Xie, C.X., Liu, C., Song, J.Y., Yao, H., Luo, K. et al. (2010)
Species identification of medicinal pteridophytes by a DNA
barcode marker, the chloroplast psbA‐trnH intergenic region.
Biological & Pharmaceutical Bulletin,33,1919–1924. Availa-
ble from: https://doi.org/10.1248/bpb.33.1919
May, M.R., Contreras, D.L., Sundue, M.A., Nagalingum, N.S.,
Looy, C.V. & Rothfels, C.J. (2021) Inferring the total‐evidence
timescale of marattialean fern evolution in the face of model
sensitivity. Systematic Biology, 70, 1232–1255. Available
from: https://doi.org/10.1093/sysbio/syab020
Nitta, J.H. & Chambers, S.M. (2022) Identifying cryptic fern
gametophytes using DNA barcoding: a review. Applications
in Plant Sciences, 10, e11465. Available from: https://doi.org/
10.1002/aps3.11465
Nitta, J.H., Ebihara, A. & Smith, A.R. (2020) A taxonomic and
molecular survey of the pteridophytes of the Nectandra
cloud forest reserve, Costa Rica. PLoS One, 15, e0241231.
Available from: https://doi.org/10.1371/journal.pone.
0241231
Nitta, J.H., Meyer, J.Y., Taputuarai, R. & Davis, C.C. (2017) Life
cycle matters: DNA barcoding reveals contrasting commu-
nity structure between fern sporophytes and gametophytes.
Ecological Monographs, 87, 278–296. Available from: https://
doi.org/10.1002/ecm.1246
Nitta, J.H., Schuettpelz, E., Ramírez‐Barahona, S. & Iwasaki, W.
(2022) An open and continuously updated fern tree of life.
Frontiers in Plant Science, 13, 909768. Available from:
https://doi.org/10.3389/flps.2022.909768
Nong, S.Y., Liu, Y., YAN, K.J. & Wei, H.J. (2023) Cyrtomium
remotipinnum (Dryopteridaceae), a new species from karst
area in Guangxi, China. Phytotaxa, 607, 133–141. Available
from: https://doi.org/10.11646/phytotaxa.607.2.2
Pacifici, M., Di Marco, M. & Watson, J.E.M. (2020) Protected
areas are now the last strongholds for many imperiled
mammal species. Conservation Letters, 13, e12748. Availa-
ble from: https://doi.org/10.1111/CONL.12748
Pereira, C.L., Gilbert, M.T.P., Araújo, M.B. & Matias, M.G. (2021)
Fine‐tuning biodiversity assessments: a framework to pair
eDNA metabarcoding and morphological approaches.
Methods in Ecology and Evolution, 12, 2397–2409. Available
from: https://doi.org/10.1111/2041-210X.13718
PPGI. (2016) A community‐derived classification for extant lyco-
phytes and ferns. Journal of Systematics and Evolution,54,
563–603. Available from: https://doi.org/10.1111/jse.12229
Rodrigues, A.S.L. & Cazalis, V. (2020) The multifaceted
challenge of evaluating protected area effectiveness.
Nature Communications, 11, 5147. Available from:
https://doi.org/10.1038/s41467-020-18989-2
Ruppert, K.M., Kline, R.J. & Rahman, M.S. (2019) Past, present,
and future perspectives of environmental DNA (eDNA)
metabarcoding: a systematic review in methods, monitor-
ing, and applications of global eDNA. Global Ecology and
Conservation, 17, e00547. Available from: https://doi.org/10.
1016/j.gecco.2019.e00547
Schneider, H. & Schuettpelz, E. (2006) Identifying fern gameto-
phytes using DNA sequences. Molecular Ecology Notes,6,
989–991. Available from: https://doi.org/10.1111/j.1471-8286.
2006.01424.x
Schneider, H., Liu, H.M., Chang, Y.F., Ohlsen, D., Perrie, L.R.,
Shepherd, L. et al. (2017) Neo‐and paleopolyploidy
contribute to the species diversity of Asplenium—the most
species‐rich genus of ferns. Journal of Systematics and
Evolution, 55, 353–364. Available from: https://doi.org/10.
1111/jse.12271
Shang, H., Wang, Y., Zhu, X.F., Zhao, G.H., Wang, F.H., Lu, J.M.
et al. (2016) Likely allopatric origins of Adiantum ×
meishanianum (Pteridaceae) through multiple hybridiza-
tions. Journal of Systematics and Evolution, 54, 528–534.
Available from: https://doi.org/10.1111/jse.12205
Siqi, L., Xianchun, Z. & Ran, W. (2019) Integrative taxonomy
resolved species delimitation in a fern complex: a case
study of the Asplenium coenobiale complex. Biodiversity
Science, 27, 1205–1220. Available from: https://doi.org/10.
17520/biods.2019316
Song, F., Li, T., Yan, H.F., Feng, Y., Jin, L., Burgess, K.S. et al.
(2023) Plant DNA barcode library for native flowering plants
in the arid region of northwestern China. Molecular Ecology
Resources, 23, 1389–1402. Available from: https://doi.org/10.
1111/1755-0998.13797
Trujillo‐Argueta, S., del Castillo, R.F., Tejero‐Diez, D., Matias‐
Cervantes, C.A. & Velasco‐Murguía, A. (2021) DNA barcod-
ing ferns in an unexplored tropical montane cloud forest
area of southeast Oaxaca, Mexico. Scientific Reports,11,
22837. Available from: https://doi.org/10.1038/s41598-021-
02237-8
Wall, W.A., Just, M.G., Huskins, S.D. & Hohmann, M.G. (2024)
Enhancing rare plant population predictions through demo-
graphic modeling of seed predation, dispersal, and habitat
suitability. Plant Ecology, 225, 63–74. Available from: https://
doi.org/10.1007/s11258-023-01376-4
Wang, F.H., Lu, J.M., Wen, J., Ebihara, A. & Li, D.Z. (2016)
Applying DNA barcodes to identify closely related species
of ferns: a case study of the Chinese Adiantum (Pterida-
ceae). PLoS One, 11, e0160611. Available from: https://doi.
org/10.11371/journal.oone.0160611
Watson, J.E.M., Venegas‐Li, R., Grantham, H., Dudley, N.,
Stolton, S., Rao, M. et al. (2023) Priorities for protected area
expansion so nations can meet their Kunming‐Montreal
210
|
INTEGRATIVE CONSERVATION
Global Biodiversity Framework commitments. Integrative
Conservation,2,140–155. Available from: https://doi.org/10.
1002/INC3.24
Wei, H.J., Huang, Y. & Chen, B. (2023) Leptochilus scandens
(Polypodiaceae), a new microsoroid fern species from
Guangdong, China. Phytotaxa, 618, 79–85. Available from:
https://doi.org/10.11646/phytotaxa.618.1.7
Wei, L.L. & Dong, S.Y. (2012) Taxonomic studies on Asplenium
sect. Thamnopteris (Aspleniaceae) II: spore morphology.
Nordic Journal of Botany, 30, 90–103. Available from:
https://doi.org/10.1111/j.1756.2011.01224.x
Wei, X.P. & Zhang, X.C. (2013) Species delimitation in the fern
genus Lemmaphyllum (Polypodiaceae) based on multivariate
analysis of morphological variation. Journal of Systematics
and Evolution,51,485–496. Available from: https://doi.org/10.
1111 / j s e . 12 0 1 9
Wheeler, Q.D., Knapp, S., Stevenson, D.W., Stevenson, J.,
Blum, S.D., Boom, B.M. et al. (2012) Mapping the biosphere:
exploring species to understand the origin, organization and
sustainability of biodiversity. Systematics and Biodiversity,
10, 1–20. Available from: https://www.tandfonline.com/doi/
full/10.1080/14772000.2012.665095
Williams, D.R., Rondinini, C. & Tilman, D. (2022) Global protected
areas seem insufficient to safeguard half of the world's
mammals from human‐induced extinction. Proceedings of
the National Academy of Sciences of the United States of
America, 119, e2200118119. Available from: https://doi.org/
10.1073/pnas.2200118119
Wu, S.K., & Mitsuta, S. (1985) Two new species of cyrtomioid ferns
from limestone area of Yunnan. Acta Phytotaxonomica et
Geobotanica,36,22–26.
Xu, K.W., Lin, C.X., Guo, J.Q., Zhou, X.X., Liao, W.B. & Mao, L.F.
(2022) Asplenium danxiaense sp. nov. (Aspleniaceae,
Aspleniineae), a new tetraploid fern species from Guang-
dong, China, based on morphological and molecular
data. European Journal of Taxonomy, 798, 162–173. Availa-
ble from: https://doi.org/10.5852/ejt.2022.798.1679
Xu, K.W., Zhang, L., Rothfels, C.J., Smith, A.R., Viane, R.,
Lorence, D. et al. (2020) A global plastid phylogeny of the
fern genus Asplenium (Aspleniaceae). Cladistics, 36, 22–71.
Available from: https://doi.org/10.1111/cla.12384
Xu, K.W., Zhou, X.M., Yin, Q.Y., Zhang, L., Lu, N.T., Knapp, R.
et al. (2018) A global plastid phylogeny uncovers extensive
cryptic speciation in the fern genus Hymenasplenium
(Aspleniaceae) from Asia. Phytotaxa, 358, 1–25. Available
from: https://doi.org/10.1016/j.ympev.2018.05.021
Yu, Z.Y., Liang, Z.L., Hu, Y.P., Liang, Z.L., He, H.L., Li, J.H.
et al. (2024) Leptochilus brevipes (Polypodiaceae), a new
fern species from southeastern Yunnan, China based on
morphological and molecular evidence. Phytotaxa, 634,
143–152. Available from: https://doi.org/10.11646/phytotaxa.
634.2.4
Zhang, G.C., Hong, H.F., Chen, G.H., Lu, S.G. & Chang, Y.F. (2021)
Species delimitation of Hymenasplenium obliquissimum
group (Aspleniaceae) in southwestern China. Phytotaxa,
480, 29–44. Available from: https://doi.org/10.11646/
phtytaxa.480.1.3
Zhang, L., Lu, N.T., Zhou, X.M., Chen, D.K., Knapp, R., Zhou, L.
et al. (2019) A plastid phylogeny of the Old World fern genus
Leptochilus (Polypodiaceae): implications for cryptic speci-
ation and progressive colonization from lower to higher
latitudes. Molecular Phylogenetics and Evolution, 134,
311–322. Available from: https://doi.org/10.1016/j.ympev.
2019.01.013
Zhang, L.B., Wu, S.G., Xiang, J.Y., Xing, F.W., He, H., Wang, F.G.
et al. (2013) Dryopteridceae. In: Wu, Z.‐Y., Raven, P.H.
& Hong, D.Y. (Eds.) Flora of China (Pteridophytes), vols. 2–3.
Beijing: Science Press; St. Louis: Missouri Botanical Garden
Press.
Zhao, C.F., Wei, R., Zhang, X.C. & Xiang, Q.P. (2020) Backbone
phylogeny of Lepisorus (Polypodiaceae) and a novel
infrageneric classification based on the total evidence from
plastid and morphological data. Cladistics, 36, 235–258.
Available from: https://doi.org/10.1111/cla.12403
Zhao, M.X., Xiang, J.Y., Geekiyanage, N., Sun, H.Y., Myo, K.M.,
Dian, R.N. et al. (2017) Leptochilus mengsongensis
(Polypodiaceae), a new species of fern from SW China.
Phytotaxa,317,144–148. Available from: https://www.
biotaxa.org/Phytotaxa/article/view/phytotaxa.317.2.7
SUPPORTING INFORMATION
Additional supporting information can be found
online in the Supporting Information section at the
end of this article.
How to cite this article: Liu, H., Chai Y. &
Schneider, H. (2024) Rapid DNA barcoding‐
based fern and lycophyte inventories
of protected areas—a pilot study to
introduce a simple but effective protocol.
Integrative Conservation,3,196–211.
https://doi.org/10.1002/inc3.60
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