Asplenium serratifolium (Aspleniaceae), a New Fern Species from Central Vietnam Based on Morphological and Molecular Evidence

Abstract

Asplenium serratifolium (Aspleniaceae), a new fern species from central Vietnam, is described and illustrated. The new species is characterized by plants 10-18 cm tall, laminae pinnatipartite, lobe margins entire or with shallow teeth, and veins simple or forked. Molecular phylogenetic analysis based on five plastid markers (atpB, rbcL, rps4, rps4-trnS, and trnL-F) indicate that the new species is closely related to A. ensiforme.

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Asplenium serratifolium (Aspleniaceae), a New Fern Species
from Central Vietnam Based on Morphological and Molecular
Evidence
Author(s): Ke-Wang Xu, Liang Zhang, Ngan Thi Lu, and Li-Bing Zhang
Source: American Fern Journal, 108(3):65-75.
Published By: The American Fern Society
https://doi.org/10.1640/0002-8444-108.3.65
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American Fern Journal 108(3):65–75 (2018)
Published on 16 October 2018
Asplenium serratifolium (Aspleniaceae), a New Fern
Species from Central Vietnam Based on Morphological
and Molecular Evidence
KE-WANG XU
State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources,
School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510275, China;
Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299, U.S.A.
e-mail: xukw6@mail2.sysu.edu.cn
LIANG ZHANG
Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming, Yunnan 650201, China. e-mail: 549133356@qq.com
NGAN THI LU
CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization and
Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province,
Chengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu, Sichuan
610041, China; University of Chinese Academy of Sciences, Beijing 100049, China;
Department of Biology, Vietnam National Museum of Nature, Vietnam Academy of Science and
Technology, 18th Hoang Quoc Viet Road, Ha Noi, Vietnam. e-mail: vnmngan@gmail.com
LI-BING ZHANG*
Missouri Botanical Garden, 4344 Shaw Blvd., St. Louis, Missouri 631110, U.S.A.;
Chengdu Institute of Biology, Chinese Academy of Sciences: e-mail: Libing.Zhang@mobot.org
ABSTRACT.—Asplenium serratifolium (Aspleniaceae), a new fern species from central Vietnam, is
described and illustrated. The new species is characterized by plants 10–18 cm tall, laminae
pinnatipartite, lobe margins entire or with shallow teeth, and veins simple or forked. Molecular
phylogenetic analysis based on five plastid markers (atpB,rbcL,rps4,rps4-trnS, and trnL-F)
indicate that the new species is closely related to A. ensiforme.
KEY WORDS.—Asplenium ensiforme, pinnatipartite, fern phylogeny, Quang Tri Province
The largest fern genus, Asplenium L. (Aspleniaceae), is sub-cosmopolitan
and comprises more than 700 species (Lin and Viane, 2013), of which 44
species are known to occur in Vietnam (Pham-Hoang, 1999). During fieldwork
to survey ferns and lycophytes in central Vietnam in November 2014, two
collections of a species of Asplenium were obtained from Quang Tri Province.
The pinnatipartite lamina of this species distinctly differs from that of any
other Asplenium known in Asia (Pham-Hoang, 1999; Lin and Viane, 2013;
Lindsay and Middleton, 2012). Based on the investigation of morphological
characters, literature, specimen comparisons, and phylogenetic analysis, we
found that this species of Asplenium is new to science (Pham-Hoang, 1999; Lin
* Corresponding Author

and Viane, 2013; Lindsay and Middleton, 2012). We here describe this new
species as Asplenium serratifolium.
MATERIAL AND METHODS
Materials and taxon sampling.—We sequenced one individual from each of
the two known populations of the new species, both from the Bac Huong Hoa
Nature Reserve, Quang Tri, Vietnam. A total of 26 additional species of
Asplenium were included in the molecular analyses based on morphological
similarities and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_
TYPE¼BlastSearch) search results of the rbcL sequence of the new species
with default settings in BLAST. Three important references, Schneider (2004),
Schneider et al. (2017), and Ohlsen et al. (2015), were also consulted in
choosing the ingroup taxa. Species of Hymenasplenium Hayata were used as
outgroups because there are only two genera in the family generally recognized
(Ohlsen et al., 2015).
Morphological study.—The morphological description of the new species
was based on the holotype and a paratype deposited in the Missouri Botanical
Garden herbarium (MO). Measurement of the characteristics of roots, stipes,
rachises, scales, and indusia was conducted using a micrometer under a
dissecting microscope. Herbarium investigations were carried out at CDBI,
MO, PHH, SYS, and VNMN and by using online images in the Chinese Virtual
Herbarium (http://www.cvh.org.cn) provided by various Chinese herbaria and
on those in JSTOR Global Plants (https://plants.jstor.org/) provided by herbaria
worldwide.
DNA extraction, PCR amplification and sequencing.—Total genomic DNA
was extracted from silica-gel-dried leaves using the modified 23CTAB
procedure of Doyle and Doyle (1987). DNA sequence data were obtained
from five plastid markers: atpB,rbcL,rps4,rps4-trnS intergenic spacer, and
trnL-F intergenic spacer. Primer sequences and PCR procedures followed Xu et
al. (2018). Amplified fragments were purified with TIANquick Mini
Purification Kits (TIANGEN). Purified PCR products were sequenced by
TSINGKE Biological Technology (Guangzhou, China). DNA sequences of 23
Asplenium species were downloaded from GenBank. All sequences used in
this study, together with their GenBank accession numbers, are listed in
Appendix I.
Sequence alignment and phylogenetic analysis.—Sequences were edited
and assembled using Sequencher V.4.14 (GeneCodes Corporation, Ann Arbor,
Michigan, USA). Sequence alignment and adjustment were conducted
manually with Bioedit (Hall, 1999). Phylogenetic relationships were
analyzed through maximum parsimony (MP), maximum likelihood (ML),
and Bayesian inference (BI). MP tree searches were carried out for each data
matrix in PAUP v. 4.0b10 (Swofford, 2002). Parsimony jackknife (JK) analyses
(Farris et al., 1996) were conducted using PAUP with the removal probability
set to approximately 37%, and ‘‘jac’’ resampling emulated. Two hundred
replicates were performed from different random addition sequence trees, with
66 AMERICAN FERN JOURNAL: VOLUME 108, NUMBER 3 (2018)

100 TBR searches per replicate and a maximum of 100 trees held per TBR
search. ML tree searches and ML bootstrapping (BS) were performed using the
web server RAxML-HPC2 on XSEDE v.8.2.10 on the CIPRES web server with
1,000 bootstrap replicates (Miller, Pfeiffer, and Schwartz, 2010). Bayesian
inference was conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck,
2003) on CIPRES (Miller, Pfeiffer, and Schwartz, 2010). Two runs of four
Markov chain Monte Carlo chains were conducted, each beginning with a
random tree and sampling one tree every 1000 generations of 10,000,000
generations. Convergence among chains was checked using Tracer ver. 1.4
(Rambaut and Drummond, 2007), and the first 25% was discarded as burn-in to
ensure that stationarity had been reached. The remaining trees were used to
calculate a 50% majority-rule consensus topology and posterior probabilities
(PP).
jModelTest 2 (Guindon and Gascuel, 2003; Posada, 2008; Darriba et al.,
2012) was used to select the best-fitting likelihood model. The Akaike
information criterion (Akaike, 1974) was used to select among models (Table
1), following Pol (2004) and Posada and Buckley (2004).
RESULTS AND DISCUSSION
The alignment of the combined plastid DNA datasets (atpB: 1,239 bp, rbcL:
1,252 bp, rps4 and rps4-trnS intergenic spacer: 1,030 bp, trnL-F intergenic
spacer: 384 bp) was 3,905 characters long (details concerning the datasets
analyzed and statistics for the resulting trees are shown in Table 1) of which
535 were parsimony-informative and 388 were variable and parsimony-
uninformative. In our plastid phylogeny, the three phylogenetic analyses (BI,
ML, and MP) revealed similar topologies, with minor differences at some
weakly supported branches, and all three trees indicated that the new species
formed a strongly supported clade (MLBS: 100%, BIPP: 1, MPJK: 100%).
The two samples of the new species had identical DNA sequences, which
were different from all other sampled species. In addition, the phylogenetic
analyses indicate that the new species is closely related to A. ensiforme
Wallich ex Hooker & Greville (Hooker and Greville, 1828; Fig. 1). Morpholog-
ically, A. serratifolium can be easily distinguished from A. ensiforme in having
plants 10–18 cm tall, laminae pinnatipartite, and lobe margins entire or with
shallow teeth, whereas A. ensiforme has plants 30–45 (–65) cm tall, laminae
TABLE 1. Best-fitting models, data matrices, and tree statistics for each of the analyses. Missing
data include missing sequences, uncertain bases (N, R, Y, V, etc.), and gaps (-).
Locus Selected model # accessions # missing (%) # chars.
Plastid atpB gene TIM1þG 6 84.5 1239
Plastid rbcL gene TIM1þG 37 2.1 1252
Plastid rps4 gene & rps4-trnS spacer TPM1ufþIþG 18 63.5 1030
Plastid trnL intron & trnL-F spacer TVMþG 18 62.4 384
Simultaneous TIM1þIþG 37 50.4 3905
XU ET AL.: ASPLENIUM SERRATIFOLIUM SP. NOV. FROM VIETNAM 67

simple and entire to repand. Detailed comparison of the two species is listed in
Table 2.
Only two populations of the new species in the Bac Huong Hoa Nature
Reserve of Vietnam were found, while the related species Asplenium
ensiforme is widely distributed in Asia (Lin and Viane, 2013).
FIG. 1. The phylogenetic position of Asplenium serratifolium based on five plastid markers (atpB,
rbcL,rps4,rps4-trnS,trnL-F). The numbers associated with branches are maximum likelihood
bootstrap support (MLBS), Bayesian posterior probability (BIPP), followed by maximum parsimony
jackknife support (MPJK). ‘‘-’’ indicates MLBS ,85%, BIPP ,0.9%, or MPJK ,80%. The major
clades (either new or following Ohlsen (2015)) are indicated on the right.
TABLE 2. Morphological comparison between Asplenium serratifolium and A. ensiforme.
Character A. serratifolium A. ensiforme
Plant height 10–18 cm 30–45(–65) cm
Petiole length 1–6 cm 5–8(–15) cm
Rhizome scales 2–4 30.08–0.15 mm Ca. 6 31mm
Lamina Pinnatipartite Simple
Lobe margins Entire or serrated Entire to repand
Distribution Central Vietnam Southeast Asia
Veins Simple or forked Once-forked
68 AMERICAN FERN JOURNAL: VOLUME 108, NUMBER 3 (2018)

TAXONOMIC TREATMENT
Asplenium serratifolium Li Bing Zhang & K.W. Xu, sp. nov. (Figs. 2, 3). TYPE:
VIETNAM. Quang Tri: Huong Hoa District, Bac Huong Hoa Nature
Reserve, Huong Viet Commune, Ta Rung Village, 1250 m, 168480N,
1068350E, 22 Nov 2014, Li-Bing Zhang, Liang Zhang & Ngan Thi Lu 7631
(holotype: MO!; isotypes: CDBI!, VNMN!).
Diagnosis.—Asplenium serratifolium somewhat resembles A. ensiforme by
its stipes sulcate adaxially, midribs raised abaxially and sulcate adaxially,
obscure veins, and sori in rows on two sides of midrib separately. The former
has plants 10–18 cm tall, laminae pinnatipartite, lobe margins entire or with
shallow teeth, and veins simple or forked, while the latter has plants 30–45(–
65) cm tall, laminae simple, entire to repand, and veins once-forked.
Plants perennial, evergreen, 10–18 cm tall. Rhizomes erect, short, apices
densely scaly; scales brown to dark brown, lanceolate, 2–6 30.18–0.5 mm,
margins entire. Fronds simple and caespitose, herbaceous, yellowish green or
grayish green when dry, subglabrous; stipes stramineous, sulcate adaxially,
rounded to carinate abaxially, 1–6 cm, bases densely covered with scales,
becoming subglabrous above, scales clathrate, brown, lanceolate, 2–4 30.08–
0.15 mm, margins entire; laminae pinnatipartite, lanceolate, 5–12 30.6–1.8
cm, bases reduced and decurrent on stipes, apices acute to acuminate, widest
at middle part of laminae, margins of lobes entire or serrate, teeth shallow and
obtuse. Midribs stramineous, adaxially sulcate, raised abaxially, sparsely
scaly, scales brown, lanceolate, ca. 0.08 30.8 mm, entire, glabrous; veins
obscure, simple or forked. Sori linear, (0.1–) 0.2–0.5 cm, starting close to
midribs at an angle of 258–458on acroscopic veinlets; indusia yellowish green
or stramineous and becoming brown when dry, linear, papery, margins entire,
opening toward midribs, persistent.
Geographical distribution.—Asplenium serratifolium is known from two
locations within the Bac Huong Hoa Nature Reserve, Quang Tri, central
Vietnam.
IUCN Red List category.—Only two populations and fewer than 50
individuals of Asplenium serratifolium are known. We did not find any
other populations in the course of our three expeditions (from 2013–2015) to
collect Vietnamese pteridophytes. As a result, a CR-Critically Endangered
category is suggested based on current information available and following
IUCN (the International Union for Conservation of Nature and Natural
Resources) guidelines (IUCN, 2015).
Etymology.—Based on the Latin prefix, serrati-, serrate, and the Latin suffix,
-folium, leaf, referring to the saw-toothed laminae of the new species.
Additional specimens examined (paratypes).—VIETNAM. Quang Tri:
Huong Hoa District, Bac Huong Hoa Nature Reserve, Huong Viet Commune,
Ta Rung Villige, Sa Mu Pass, elev. 1300 m, 168480N, 1068350E, 21 Nov 2014, Li-
Bing Zhang, Liang Zhang & Ngan Thi Lu 7614 (CDBI!, MO!, VNMN!).
XU ET AL.: ASPLENIUM SERRATIFOLIUM SP. NOV. FROM VIETNAM 69

FIG.2. Asplenium serratifolium sp. nov. A and B. Habit. C. Plant. D. Abaxial lamina. E. Sulcate
stipe, adaxial view. F. Stipe scales. G. Adaxial lamina showing the sulcate midrib. H. Portion of
abaxial lamina; red arrow shows the obscure, forked veins.
70 AMERICAN FERN JOURNAL: VOLUME 108, NUMBER 3 (2018)

ACKNOWLEDGMENTS
This research was partially supported by grants from the National Natural Science Foundation of
China (#31628002) and the National Geographic Society of the USA to L.-B.Z., funding from the
China Scholarship Council (201706380093) and the Zhang Hong-Da (Chang Hung-Ta) Science
Foundation at Sun Yat-sen University to K.-W.X. We thank Lei Jiang for the illustration and Chris
Haufler, Carl Rothfels, and other two anonymous reviewers for helpful comments.
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APPENDIX I. SPECIES USED IN MOLECULAR PHYLOGENETIC ANALYSES,THEIR VOUCHERS,
AND GENBANK ACCESSION NUMBERS.‘‘—’’ MEANS MISSING DATA.
Asplenium antiquum Makino, P22088 (WELT), Unknown: rbcL EU240033,
atpB —, rps4 & rps4-trnS EU240020, trnL-F EU240028 (Shepherd et al., 2008).
Asplenium ceterach L., Van De Riet 707 (EB), Unknown: rbcL HQ676494,
atpB —, rps4 & rps4-trnS —, trnL-F HQ676516 (de Groot et al., 2011).
Asplenium coenobiale Hance, TNS: 763925 (TNS), Kochi, Japan: rbcL
AB574855, atpB —, rps4 & rps4-trnS —, trnL-F — (Ebihara et al., 2010).
Asplenium ensiforme Wall. ex Hook. & Grev., TNS: 763995 (TNS), Miyazaki,
Japan: rbcL AB574856, atpB —, rps4 & rps4-trnS —, trnL-F — (Ebihara et al.,
2010). Asplenium ensiforme Wall. ex Hook. & Grev., Nogami et al. 8 (KYO),
Mie, Japan: rbcL AB014689, atpB —, rps4 & rps4-trnS —, trnL-F — (Murakami
et al., 1999). Asplenium ensiforme Wall. ex Hook. & Grev., Fukuoka et al. 93-
T619 (KYO), Doi Inthanon, Thailand: rbcL AB014709, atpB —, rps4 & rps4-
trnS —, trnL-F — (Murakami et al., 1999). Asplenium griffithianum Hook.,
Fujita et al., Yaku-1204 (FU), Kagoshima, Japan: rbcL AB574857, atpB —, rps4
& rps4-trnS —, trnL-F — (Ebihara et al., 2010). Asplenium griffithianum
Hook., Murakami J93-001 (TI), Kagoshima, Japan: rbcL AB013252, atpB —,
rps4 & rps4-trnS —, trnL-F —(Murakamiet al., 1999a). Asplenium
XU ET AL.: ASPLENIUM SERRATIFOLIUM SP. NOV. FROM VIETNAM 73

griffithianum Hook., Murakami J93-001 (KYO), Kagoshima, Japan: rbcL
AB014688, atpB —, rps4 & rps4-trnS —, trnL-F — (Murakami et al., 1999a).
Asplenium interjectum Christ, Lu SG/J 12 (PYU), Yunnan, China: rbcL
AY545480, atpB —, rps4 & rps4-trnS AY725043, trnL-F AY725038 (Li and Lu,
2006). Asplenium laciniatum D. Don, Cheng s.n. (BM), China: rbcL AY549747,
atpB —, rps4 & rps4-trnS AY549801, trnL-F AY549851 (Schneider et al., 2005).
Asplenium lushanense C. Chr., Lu SG/D 21 (PYU), Yunnan, China: rbcL
AY545481, atpB —, rps4 & rps4-trnS AY725042, trnL-F AY725033 (Li and Lu,
2006). Asplenium nidus L., U.C. Botanical Garden 68.0392 (UC), Madagascar:
rbcL AF525270, atpB —, rps4 & rps4-trnS AY549807, trnL-F AF525246 (Pinter
et al., 2002). Asplenium pekinense Hance, Viane 10031, Unknown: rbcL
GU929864, atpB —, rps4 & rps4-trnS —, trnL-F — (Unpublished data).
Asplenium pekinense Hance, Lu SG/C 67 (PYU), Yunnan, China: rbcL
AY545479, atpB —, rps4 & rps4-trnS AY725040, trnL-F AY725037 (Li and
Lu, 2006). Asplenium prolongatum Hook., Cult. in NYBG (UC), New York, US:
rbcL AY549752, atpB —, rps4 & rps4-trnS AY549813, trnL-F AY549856
(Schneider et al., 2005). Asplenium pseudowilfordii Tagawa, Nogami 14
(KYO), Nara, Japan: rbcL AB014696, atpB —, rps4 & rps4-trnS —, trnL-F —
(Murakami et al., 1999). Asplenium pulcherrimum (Baker) Ching ex Tardieu,
Ke-Wang Xu TTJ-GZ-015 (SYS), Guizhou, China: rbcL MH109142, atpB
MH109136, rps4 & rps4-trnS MH109148, trnL-F (Unpublished data). Asple-
nium sarelii Hook., TNS:769194 (TNS), Mie, Japan: rbcL AB574873, atpB —,
rps4 & rps4-trnS —, trnL-F — (Ebihara et al., 2010). Asplenium sarelii Hook.,
Nogami & Oohora 11, Mie, Japan: rbcL AB014693, atpB —, rps4 & rps4-trnS —,
trnL-F — (Murakami et al., 1999). Asplenium scortechinii Bedd., Fan 01144,
Hainan, China: rbcL MH509434, atpB —, rps4 & rps4-trnS —, trnL-F — (this
study). Asplenium serratifolium Li Bing Zhang & Ke-Wang Xu, Zhang et al.
7631 (CDBI,MO,VNMN),QuangTri,Vietnam:rbcL MH509432, atpB
MH509430, rps4 & rps4-trnS MH509435, trnL-F MH509437 (this study).
Asplenium serratifolium Li Bing Zhang & Ke-Wang Xu, Zhang et al. 7614
(CDBI, MO, VNMN), Quang Tri, Vietnam: rbcL MH509433, atpB MH509431,
rps4 & rps4-trnS MH509436, trnL-F MH509438 (this study). Asplenium
simaoense Ke-Wang Xu, Li Bing Zhang & W. B. Liao, Ke-Wang Xu XKW317-
1(SYS), Yunnan, China: rbcL MH109137, atpB MH109131, rps4 & rps4-trnS
MH109143, trnL-F MH109149 (Unpublished data). Asplenium tenuicaule
Hayata, TNS: 765223 (TNS), Yamagata, Japan: rbcL AB574878, atpB —, rps4 &
rps4-trnS —, trnL-F — (Ebihara et al., 2010). Asplenium tenuifolium D. Don,
Lei Jiang JL00361 (SYS), Yunnan, China: rbcL MH109141, atpB MH109135,
rps4 & rps4-trnS MH109147, trnL-F MH109153 (Unpublished data). Asple-
nium varians Wall. ex Hook. & Grev., Fraser-Jenkins 10046-10047 (BM),
China: rbcL AY300147, atpB —, rps4 & rps4-trnS AY549802, trnL-F AY300094
(Schneider et al., 2004). Asplenium wilfordii Mett. ex Kuhn, TNS: 763478
(TNS), Kagoshima, Japan: rbcL AB574883, atpB —, rps4 & rps4-trnS —, trnL-F
— (Ebihara et al., 2010). Asplenium wrightii D. C. Eaton ex Hook., Cranfill
TW040 (UC), Taiwan: rbcL AY549730, atpB —, rps4 & rps4-trnS AY549766,
trnL-F AY549833 (Schneider et al., 2005). Asplenium wrightioides Christ, Lu
74 AMERICAN FERN JOURNAL: VOLUME 108, NUMBER 3 (2018)

SG/J13 (PYU), Yunnan, China: rbcL AY725031, atpB —, rps4 & rps4-trnS
AY725044, trnL-F AY725032 (Li and Lu, 2006). Asplenium yoshinagae
Makino, Lu SG/A4 (PYU), Yunnan, China: rbcL AY725030, atpB —, rps4 &
rps4-trnS AY725045, trnL-F AY725036 (Li and Lu, 2006). Asplenium
yunnanense Franch., Lu SG/D 22 (PYU), Yunnan, China: rbcL AY545482,
atpB —, rps4 & rps4-trnS AY725041, trnL-F AY725034 (Li and Lu, 2006).
Asplenium yunnanense Franch., Fraser-Jenkins 10044-10045 (BM), China:
rbcL AY300149, atpB —, rps4 & rps4-trnS AY549803, trnL-F AY300096
(Schneider et al., 2004). Hymenasplenium cheilosorum (Kunze ex Mett.)
Tagawa, Schater 55 (GOET), Yunnan, China: rbcL JF832071, atpB —, rps4 &
rps4-trnS —, trnL-F — (Rothfels et al., 2012). Hymenasplenium excisum (C.
Presl) S. Lindsay, Brownsey & Perrie FIJI 190 (WELT), Fiji: rbcL KP774884,
atpB —, rps4 & rps4-trnS —, trnL-F — (Ohlsen et al., 2015). Hymenasplenium
obliquissimum (Hayata) Sugimoto, Murakami & Cheng 94-M 1811, Yunnan,
China: rbcL AB016186, atpB —, rps4 & rps4-trnS —, trnL-F — (Murakami et al.,
1998). Hymenasplenium unilaterale (Lam.) Hayata, Hemp 18 (BM), Kenya:
rbcL AF240652, atpB —, rps4 & rps4-trnS —, trnL-F — (Pinter et al., 2002).
XU ET AL.: ASPLENIUM SERRATIFOLIUM SP. NOV. FROM VIETNAM 75