Plant Syst Evol (2009) 281:77–86
DOI 10.1007/s00606-009-0188-2
ORIGINAL ARTICLE
Identifying a mysterious aquatic fern gametophyte
Fay-Wei Li Æ Benito C. Tan Æ Volker Buchbender Æ
Robbin C. Moran Æ Germinal Rouhan Æ
Chun-Neng Wang Æ Dietmar Quandt
Received: 5 January 2009 / Accepted: 11 May 2009 / Published online: 17 June 2009
Ó Springer-Verlag 2009
Abstract Süßwassertang, a popular aquatic plant that is
sold worldwide in aquarium markets, has been long considered a liverwort because of its ribbon-like thallus. However, its antheridia are remarkably fern-like in morphology.
To corroborate the hypothesis that Süßwassertang is a fern
gametophyte and to determine its closest relative, we have
F.-W. Li (&) C.-N. Wang (&)
Department of Life Science, National Taiwan University,
Taipei 106, Taiwan
e-mail: fayweili@gmail.com
C.-N. Wang
e-mail: botwang@ntu.edu.tw
sequenced five chloroplast regions (rbcL, accD, rps4–trnS,
trnL intron, and trnL-F intergenic spacer), applying a DNAbased identification approach. The BLAST results on all
regions revealed that Süßwassertang is a polypod fern (order:
Polypodiales) with strong affinities to the Lomariopsidaceae.
Our phylogenetic analyses further showed that Süßwassertang is nested within the hemi-epiphytic fern genus
Lomariopsis (Lomariopsidaceae) and aligned very close to
L. lineata. Our study brings new insights on the unexpected
biology of Lomariopsis gametophytes—the capacity of
retaining a prolonged gametophytic stage under water. It is of
great interest to discover that a fern usually known to grow on
trees also has gametophytes that thrive in water.
B. C. Tan
The Herbarium, Singapore Botanic Gardens,
Singapore 295969, Singapore
Keywords Aquarium DNA barcoding
DNA-based identification Gametophyte Fern
Lomariopsis Lomariopsidaceae
V. Buchbender D. Quandt (&)
Plant Phylogenetics and Phylogenomics Group,
Institute of Botany, Dresden University of Technology,
01062 Dresden, Germany
e-mail: dietmar.quandt@tu-dresden.de; quandt@uni-bonn.de
Introduction
R. C. Moran
The New York Botanical Garden, Bronx,
New York, NY 10458-5126, USA
G. Rouhan
Muséum National d’Histoire Naturelle, UMR 7205,
Herbier National, CP39, 16 rue Buffon, 75005 Paris, France
C.-N. Wang
Institute of Ecology and Evolutionary Biology,
National Taiwan University, Taipei 106, Taiwan
Present Address:
D. Quandt
Nees Institute for Biodiversity of Plants, University of Bonn,
Meckneheimer Allee 170, 53115 Bonn, Germany
An aquatic plant called Süßwassertang, which means
‘‘freshwater seaweed’’ in German, has been commercially
available on the aquarium market worldwide for a number
of years (Fig. 1a). Because of its liverwort-like appearance,
it has long been considered to be a liverwort, such as Pellia
or Monoselenium. Our observation of Süßwassertang
gametangia, which are only rarely produced by the submerged thallus, suggested that this plant is not a liverwort
but a fern gametophyte. Its archegonia are fern-like in
having short necks, and the venter is immersed partly in the
thallus. The antheridia resemble those of polypodialean
ferns in that they consist of three cells: a cap cell, a ring
cell, and a basal cell (Fig. 1b; Nayar and Kaur 1971).
Although gametangia are present occasionally, sporophytes
123
78
F.-W. Li et al.
Fig. 1 Süßwassertang, its
microscopic features, and the
habit of Lomariopsis
spectabilis. a A portion of the
gametophyte thallus showing
extensive lateral branching.
Bar: 1 cm. b Side view of an
antheridium, showing a cap cell
(cc), ring cell (rc), and basal cell
(bc). Bar: 20 lm. c Scanning
electron microscope image of
developing lateral branches with
rhizoids (arrowhead) and
meristems (m) in the rounded
apex. Bar: 0.2 mm. d Ribbonlike, branched gametophyte (g)
of L. spectabilis bearing a young
sporophyte (sp) in a field of
Taiwan. It has similar
morphology with the mysterious
gametophyte. Arrowhead
Branch points. Bar: 1 cm
have never been observed in aquaria and even after
planting onto soil. It is therefore difficult to identify the
plant with certainty.
To unravel this mysterious identity, we employed a
DNA-based identification approach consisting of sequence
comparisons and phylogenetic analyses. We sequenced five
chloroplast regions [rbcL, accD, rps4–trnS, trnL intron,
and the trnL-F intergenic spacer (IGS)], of which rbcL has
already been successfully used to identify an unknown fern
gametophyte in a similar study (Schneider and Schuettpelz
2006). The widespread occurrence of rbcL in online databases for all plant lineages makes it well-suited for broadscale screening. Likewise, the analyses by Quandt et al.
(2004) indicated that the trnL-F region (intron and IGS)
has the power to relate any sequence via a database comparison on generic level in most land plant lineages. The
rapidly evolving gene rps4 (plus rps4–trnS IGS) was
included because it represents one of the broadly
sequenced regions (together with the trnL-F region and
rbcL) in seedless plants (e.g., Quandt and Stech 2003;
Schneider et al. 2004). In the approach chosen here,
BLAST results pinpointed which clade Süßwassertang
belongs to and therefore guided the taxonomic sampling
for phylogenetic inferences. To increase the phylogenetic
signal, we combined accD with rbcL in the analyses. Once
narrowed to a certain lineage, a more precise marker was
then used to resolve the position of Süßwassertang among
more recently diverged lineages. In this case, the plastid
trnL-F IGS was employed to infer inter-species affinities of
this supposedly aquatic fern gametophyte.
123
Materials and methods
Taxonomic sampling for molecular phylogeny
Taxonomic sampling was guided by the MegaBLAST
(Zhang et al. 2000) results obtained from the five regions
sequenced (rbcL, accD, rps4–trnS, trnL intron, and trnL-F
IGS). To obtain more robust confirmation of the relationship of Süßwassertang, we compiled two data sets for
phylogenetic analyses. As indicated by the BLAST results,
the first data set comprised a representative set of
sequences from two coding plastid regions, rbcL and accD,
of polypod ferns. In addition to the Süßwassertang
sequences, 32 species representing 28 fern genera were
included in the analyses. Athyrium niponicum (Mett.)
Hance and Matteuccia struthiopteris (L.) Todaro were used
as outgroups. The second matrix included all available
trnL-F IGS accessions of Lomariopsis plus sequences
obtained from two Süßwassertang accessions, L. spectabilis (Kunze) Mett., and Cyclopeltis crenata (Fée) C. Chr.
Cyclopeltis crenata and Hypodematium crenatum Kuhn ex
v. Deck. were used as outgroups for the second matrix.
DNA extraction, amplification, and sequencing
Total genomic DNA was extracted using either the Plant
Genomic DNA Mini kit (Geneaid, Taipei, Taiwan) or
the Plant Genomic DNA Purification kit (GeneMark,
Taichung, Taiwan). In some cases a modified CTAB (cetyl
trimethylammonium bromide) procedure (Wang et al.
An aquatic fern gametophyte
2004) was applied. The PCR amplifications, which followed standard PCR protocols, were performed in 50-ll
reaction volumes containing 1.5 U Taq DNA polymerase,
1.0 mM dNTPs-Mix, 109 buffer, 1.5 mM MgCl2, 10 pmol
of each amplification primer, and 1.0 ll DNA. The PCR
primers used for amplification and sequencing were: trnL-F
with primers C (or E) and F (Taberlet et al. 1991; modifications according to Quandt and Stech 2004); rps4 (plus
rps4–trnS IGS) with rps50 (Nadot et al. 1994) and trnS
(Souza-Chies et al. 1997); rbcL with NM34 (Cox et al.
2000) and M1390 (Lewis et al. 1997); accD with the newly
designed primers ‘‘FW_accDF’’ (50 -ACG TCT GTA ACA
AAT TGG TTT GAA G-30 ) and ‘‘FW_accDR’’ (50 -AAA
CTC AAC GTT CCT TCT TGC AT-30 ). The PCR products
were either directly purified using the GeneMark PCR
Clean-Up kit (Taichung, Taiwan) or cleaned via gel
extraction employing the Nucleospin PCR Purification kit
(Macherey-Nagel, Düren, Germany). Sequencing was done
with the amplification primers by Macrogen (Seoul,
Korea). In order to corroborate the results, isolation,
amplification, and sequencing of all regions were performed independently on two different samples in Taipei
and Dresden. Newly obtained sequences and other accessions from GenBank used in the analyses are summarized
in the Appendix.
Sequence alignment and phylogenetic analyses
DNA sequences were manually aligned using PhyDE0.995
(Müller et al. 2005). During manual alignment, gap
placement was guided by the identification of putative
microstructural changes following recently published concepts (Kelchner 2000; Quandt et al. 2003). Identified
inversions were positionally separated in the alignments,
but they were included as a reverse complement in the
phylogenetic analyses, as discussed in Quandt et al. (2003).
Phylogenetic reconstructions using parsimony were performed using winPAUP* 4.0b10 (Swofford 2002) in
combination with PRAP (Müller 2004). The latter program
generates command files for PAUP* that allow parsimony
ratchet searches as designed by Nixon (1999). In our study,
ten random addition cycles of 200 ratchet iterations each
were used, with 25% of the positions being randomly
double-weighted. The shortest trees collected from the
different tree islands were finally used to compute a
strict consensus tree. Heuristic bootstrap searches (BS;
Felsenstein 1985) were performed with 1000 replicates, ten
random addition cycles per bootstrap replicate, and otherwise the same options in effect as in the ratchet.
For a further measurement of support, posterior probabilities were calculated using MrBayes V3.1 (Ronquist and
Huelsenbeck 2003), applying the GTR ? C ? I model.
The a priori probabilities supplied were those specified in
79
the default settings of the program. Posterior probability
(PP) distributions of trees were created using the Metropolis-coupled Markov chain Monte Carlo (MCMCMC)
method and followed the search strategies suggested by
Huelsenbeck et al. (2001, 2002). Ten runs with four chains
(106 generations each) were run simultaneously. Chains
were sampled every ten generations, and the respective
trees were written to a tree file. Calculation of the consensus tree and of the PP of clades was performed based
upon the trees sampled after the chains converged (within
the first 250,000 generations). Consensus topologies and
support values from the different methodological approaches were compiled and drawn using TreeGraph (Müller
and Müller 2004).
Results
BLAST results of the sequenced markers
The sequences (rbcL, accD, rps4-trnS, trnL intron, and
trnL-F) obtained from two independent collections of
Süßwassertang were identical. BLAST results indicated
that Süßwassertang shares high sequence similarities to
leptosporangiate ferns and is closest to the Lomariopsidaceae (except for trnL intron), especially in terms of the
reported maximum identity (Table 1). Lomariopsis lineata
(C. Presl) Holttum was found to be the best match for the
trnL-F IGS, whereas for the three coding regions (rbcL,
accD, and rps4), L. spectabilis Mett. or L. marginata
(Schrad.) Kuhn. received the highest maximum identity
scores. Although members of the Dryopteridaceae were
among the best matches in a BLAST search using trnL,
these results are biased since this region is currently represented by only few ferns in GenBank.
Phylogenetic analyses
Phylogenetic inferences of a representative set of polypod
ferns for each of the single-gene data sets (rbcL and accD)
were congruent. The phylogenetic relationships presented
are thus based on the analyses using the combined data set.
Maximum parsimony and Bayesian inference both clearly
positioned the aquatic fern gametophyte within the fern
genus Lomariopsis (Lomariopsidaceae), a placement that
receives high branch support in the phylogenetic analyses
(BSMP = 100, PPMB = 1.0; Fig. 2).
Phylogenetic analyses of Lomariopsis based on the
trnL–F IGS (340 nt) indicated that Lomariopsis lineata
(C. Presl) Holttum is the species closest to Süßwassertang
(BSMP = 96, PPMB = 1.0; Fig. 3). The trnL-F sequences
from L. lineata and the aquatic fern gametophyte show a
97.6% similarity and share an 8-nt indel (Fig. 3) that is
123
80
F.-W. Li et al.
Table 1 Results of BLAST searches in GenBank, with only the first ten hits shown
Accessions
Description
Maximum score
Total score
Query coverage (%)
E value
Maximum identity (%)
AB232401
Lomariopsis spectabilisa
2185
2185
98
0.0
98
AY818677
Lomariopsis marginataa
2108
2108
99
0.0
96
rbcL
a
DQ054517
Cyclopeltis crenata
1808
1808
99
0.0
92
AY545489
Cyrtomium hookerianum
1735
1735
99
0.0
91
AF537233
Phanerophlebia umbonata
1727
1727
99
0.0
91
AY268885
Dryopteris dickinsii
1725
1725
99
0.0
91
AY268864
Dryopteris polylepis
1725
1725
99
0.0
91
U62032.1
Matteuccia struthiopteris
1725
1725
99
0.0
91
AB232405
AB212687
accD
Oleandra pistillaris
1725
1725
98
0.0
91
Oleandra wallichii
1725
1725
99
0.0
91
AB232429
Lomariopsis spectabilisa
959
959
100
0.0
96
AB232421
Polybotrya caudata
749
749
99
0.0
90
AB232442
Hypodematium crenatum
737
737
99
0.0
89
AB232433
Oleandra pistillaris
737
737
99
0.0
89
90
AB232432
Nephrolepis cordifolia
737
737
98
0.0
AB232431
Nephrolepis acuminataa
737
737
99
0.0
89
AB212687
Oleandra wallichii
737
737
99
0.0
89
AB232437
Gymnogrammitis dareiformis
732
732
99
0.0
89
AB232436
Goniophlebium persicifolium
732
732
99
0.0
89
AB212686
Arthropteris backleri
732
732
99
0.0
89
rps4-trnS
AY529187
Drynaria quercifolia
479
479
89
8e-132
80
AY529189
Drynaria sparsisora
473
473
89
4e-130
80
AY529183
Drynaria descensa
473
473
89
4e-130
80
AY540049
AY529186
Lomariopsis marginataa
Drynaria mollis
462
462
462
462
57
89
8e-127
8e-127
86
79
AY529181
Aglaomorpha splendens
455
455
89
1e-124
79
DQ642210
Phlebodium pseudoaureum
453
453
83
5e-124
80
AY529184
Drynaria fortunei
453
453
83
5e-124
80
AY362663
Phlebodium pseudoaureum
449
449
80
6e-123
83
DQ642221
Pleopeltis thyssanolepis
448
448
80
2e-122
83
Polystichum subacutidens
326
326
98
8e-86
78
trnL intron
AY534749
AY534748
Polystichum nepalense
311
311
98
2e-81
77
AY736356
Arachniodes tonkinensis
263
263
93
6e-67
76
AY651840
Polypodium vulgare
257
257
99
3e-65
76
AF515242
Arachniodes setifera
235
235
89
1e-58
76
AF515230
Acystopteris japonica
195
195
41
2e-46
82
AF515248
Gymnocarpium oyamense
189
189
90
1e-44
74
DQ401124
DQ480129
Microsorum novae-zealandiae
Woodsia polystichoides
176
174
176
174
93
44
9e-41
3e-40
74
80
AF514837
Rhachidosorus consimilis
171
171
90
4e-39
74
Lomariopsis lineataa
508
508
100
4e-141
97
trnL-F IGS
DQ396572
a
DQ396602
Lomariopsis sp.
DQ396589
Lomariopsis pollicina
123
427
427
100
1e-116
92
427
427
100
1e-116
92
An aquatic fern gametophyte
81
Table 1 continued
Accessions
Description
Maximum score
Total score
Query coverage (%)
E value
Maximum identity (%)
DQ396587
Lomariopsis pervilleia
427
427
100
1e-116
92
DQ396576
Lomariopsis madagascaricaa
427
427
100
1e-116
92
DQ396557
Lomariopsis boiviniia
427
427
100
1e-116
92
a
DQ396561
Lomariopsis hederacea
420
420
100
2e-114
91
DQ396594
Lomariopsis rossiia
409
409
100
4e-111
91
DQ396582
Lomariopsis muriculataa
409
409
100
4e-111
91
DQ396577
Lomariopsis manniia
409
409
100
4e-111
91
Sequence data of five plastid regions (rbcL, rps4-trnS, accD, trnL intron, and trnL-F IGS) were tested against GenBank entries. In total 6750
plastid sequences of monilophytes were recorded in GenBank on February 18 2008 (rbcL: 2385; accD: 162; rps4-trnS: 1052; trnL intron: 342
(2/3 Asplenium), trnL-F IGS: 482)
a
Members of the family Lomariopsidaceae
absent in other Lomariopsis species. Despite the strong
sequence similarity in the non-coding region of the chloroplast genome, the possibility for the fern gametophyte to
be a different species, rather than L. lineata, could not be
eliminated. Monophyly of Lomariopsis is robustly supported, in contrast to previous study by Rouhan et al.
(2007).
Interestingly, two hairpin-associated inversions were
observed in the spacer approximately 185 nt upstream of
trnF, which is different from the previously reported one
for bryophytes (Quandt and Stech 2004; Quandt et al.
2004). Inversion 1 (inv 1) is homoplastic and occurs twice
in: (1) L. recurvata, L. vestita, L. maxonii, and L. salicifolia
as well as (2) L. amydrophlebia and L. wrigthii, whereas
inversion 2 unites L. hederacea, L. muriculata, and L.
manii. The distribution of both inversions in phylogenetic
context is plotted on the tree in Fig. 3.
Morphology of the aquatic fern gametophyte
The thallus of the alleged aquatic gametophyte of Lomariopsis is ribbon-shaped, profusely branched, and onecell thick throughout, without a midrib or multicellular
cushion. Rhizoids are colorless, mostly borne as marginal
clusters. It grows indeterminately with active meristematic
cells at the rounded apex (Fig. 1c). There are no gemmae,
although small lateral branches sometimes detach from the
thallus and develop as new individuals. Archegonia and
three-celled antheridia are sparsely formed. These characters were also observed in the gametophytes of Lomariopsis spectabilis found in Taiwan (Fig. 1d), although they
do not exactly match the strap-shaped Lomariopsis gametophytes described and illustrated by Atkinson (1973).
No associated sporophyte of the Süßwassertang under
study has ever been observed. Following transplantation of
the gametophyte from water to soil, its growth rate was
reduced, and the old portions of the thallus began to die.
Unlike the gametophyte in water, rhizoids in soil-grown
gametophyte were brown, and numerous antheridia formed
along the thallus margins. However, sporophytes did not
develop under such conditions either.
Discussion
The utility of different markers in identifying
the mysterious gametophyte
The DNA-based identification approach used here shares a
similar concept with DNA barcoding, yet the latter tries to
utilize more or less universal DNA barcodes. Deciding
which barcode to be used for plants is still in progress (e.g.,
Kress et al. 2005; Chase et al. 2005, 2007; Ford et al. 2009;
Hollingsworth et al. 2009). Several of the proposed DNA
barcodes, such as the trnL intron (Taberlet et al. 2006), accD
(Ford et al. 2009), and rbcL (Schneider and Schuettpelz
2006; Kress and Erickson 2007), were employed in this study
for identifying the mysterious thallus; hence, we believe our
results could provide a guideline for the future selection of
plant barcodes, especially considering the situation of
seedless plants.
The maximum identity and E-values from the BLAST
results nicely illustrate that the trnL-F IGS is more suitable
for species identification in ferns than rbcL, as sequence
similarity for rbcL of Süßwassertang and Lomariopsis
spectabilis or L. marginata already reaches 96–98%, while
sequence identity of the trnL-F IGS of both species with
Süßwassertang is only 89%. In addition, the trnL-F IGS
amplicon is only about 600 nt compared to 2.1 kb of rbcL and
therefore easier to handle in a barcoding approach. However,
as the spacer is missing in some green algae (Quandt et al.
2004) and merely reaches 60 nt in derived mosses (Quandt
and Stech 2004), its use is limited.
123
82
F.-W. Li et al.
Fig. 2 One of two most
parsimonious trees [length 1739
steps, consistency index (CI)
0.449, retention index (RI)
0.602, rescaled consistency
index (RC) 0.270] retained by
the parsimony ratchet analysis
performed based on the
combined rbcL and accD
sequence data. This tree was
chosen as it perfectly reflects the
Bayesian inferences. The values
above the branches refer to
posterior probabilities from
Bayesian analysis, whereas
those below the branches
indicate bootstrap support
values
1.0
0.96
56
0.62
98
Crypsinus enervis
Gymnogrammitis dareiformis
Pyrrosia rasamalae
-
Grammitis reinwardtii
1.0
99
1.0
0.93
1.0
-
100
98
Colysis wrightii
Microsorum zippelii
Goniophlebium persicifolium
1.0
Loxogramme avenia
97
Davallia formosana
1.0
1.0
100
95
Araiostegia faberiana
Oleandra wallichii
1.0
1.0
100
Oleandra pistillaris
100
Tectaria phaeocaulis
69
Oleandraceae
Tectariaceae
Arthropteris backleri
1.0
75
1.0
1.0
99
100
1.0
Mysterious Gametophyte
Lomariopsis spectabilis
Lomariopsis marginata
98
Cyclopeltis crenata
0.78
82
Nephrolepis acuminata
89
1.0
88
0.84
0.96
Lomariopsidaceae
Nephrolepis cordifolia
1.0
100
0.80
Davaliaceae
Quercifilix zeylanica
1.0
100
0.79
1.0
Polypodiaceae
-
Dryopteris erythrosora
Arachniodes aristata
Polystichum fibrilloso-paleaceum
-
Ctenitis eatonii
53
Polybotrya caudata
1.0
100
1.0
0.79
97
1.0
1.0
100
81
Elaphoglossum callifolium
Teratophyllum wilkesianum
Dryopterdiaceae
Bolbitis repanda
92
Rumohra adiantiformis
1.0
1.0
100
100
Leucostegia pallida
Leucostegia immersa
Hypodematium crenatum ssp. fauriei
Athyrium niponicum
Matteuccia struthiopteris
Although more than 21,000 trnL intron sequences
(bryophytes[3000, flowering plants[18,000) are recorded
in GenBank, ferns are vastly underrepresented, with 342
records (on 2 February 2008), which rendered the database
comparison problematic. No trnL intron sequences of
Lomariopsis species or Lomariopsidaceae are recorded in
GenBank, which explains why the closest matches were
found among members of the Dryopteridaceae (Table 1).
However, similar to the coding regions, trnL also placed
Süßwassertang within polypod ferns. The reported values
from BLAST searches representing sequence divergence
indicate that the trnL intron resolves more relatively recent
divergences compared to rbcL. Likewise, BLAST searches
123
outgroup
based on the obtained rps4 (plus rps4–trnS IGS) sequence
showed only 86% maximal sequence identity of Süßwassertang with L. marginata compared to 96% found for
rbcL, indicating the higher potential of rps4-trnS in barcoding approaches compared to rbcL (Table 1). AccD
displayed a slightly higher performance than rbcL, with
96% identity to L. spectabilis (Table 1).
Therefore, if rbcL was to be chosen as the DNA
barcode, a two-step approach would be favorable. With
the similar concept, Kress and Erickson (2007) proposed
a two-locus barcode combining trnH–psbA with rbcL for
plants. This combination worked well in filmy ferns
(Nitta 2008). However, trnH–psbA is absent in black
An aquatic fern gametophyte
83
Fig. 3 One of 53 most
parsimonious trees (length 243
steps, CI 0.774, RI 0.835, RC
0.646) retained by the
parsimony ratchet analysis
performed on the trnL-F IGS
sequence data. The values
above the branches refer to
posterior probabilities from
Bayesian analysis, whereas
those below the branches
indicate bootstrap support
values. The occurrence of both
observed inversions as well as
two characteristic indels (5 and
8 nt) are indicated on the tree
-
0.99
L. boivinii
L. sp TJ2527
84
1.0
L. madagascarica
91
0.99
L. crassifolia
82
-
L. pervillei
-
L. spectabilis
-
L. manii
1.0
-
87
0.92
0.94
L. muriculata
61
1.0
L. hederacea
94
L. rossii
75
2b
Mysterious gametophyte
1.0
100
1.0
Mysterious gametophyte F042
96
-
L. lineata
-
8 nt
L. cordata
L. pollicina
1.0
L. sp TJ2444
100
L. sp GR318
1.0
1b
1.0
-
85
L. recurvata
1b
L. vestita
1b
L. maxonii
1b
L. salicifolia
1b
L. jamaicensis
1a
L. kunzeana
1a
L. amydrophlebia
1b
L. wrightii
1b
89
0.53
59
0.99
76
0.59
1.0
99
5 nt
1.0
100
2a
1b
1.0
0.88
0.77
-
100
L. japurensis
L. latipinna
55
L. prieuriana
-
L. nigropaleata
-
L. marginata
0.99
62
0.77
53
L. guineensis
L. palustris
L. longicaudata
Cyclopeltis crenata
Hypodematium crenatum
pine (Wakasugi et al. 1994) and since two copies of
trnH–psbA can be found in the Adiantum chloroplast
genome (Wolf et al. 2003), a careful investigation should
be done on whether multiple copies may or may not
mislead species identification in ferns. Regardless of the
rather conserved rbcL, Lahaye et al. (2008) proposed
matK as the prime plant barcode. However, due to the
rapidly evolving nature of ferns’ matK and a lack of
universal priming sites, especially at the 50 end (Kuo
et al., unpublished data; Wicke and Quandt, unpublished
data), it would be problematic to use matK in ferns.
Clearly, a comprehensive survey in seedless plants on
the utility of different potential barcodes is urgently
needed.
123
84
An aquatic gametophyte from an epiphytic sporophyte
Fern gametophytes are well known for their extreme tolerance to environmental stresses, such as winter cold (Sato
1982), light deficiency (Johnson et al. 2000), and desiccation (Watkins et al. 2007). As a result, gametophytes in
some cases were able to establish populations in sites that
were probably far too extreme for sporophytes by exclusively maintaining the gametophyte generation (Farrar
1967, 1990; Dassler and Farrar 1997; Rumsey et al. 1999).
Our discovery of Süßwassertang contributes another
extraordinary example. Süßwassertang, originally known
as a species of bryophytes, has been used to decorate fish
tanks. Based on the results of our study involving DNA
markers, we have identified Süßwassertang as gametophytes of Lomariopsis, an exclusively hemi-epiphytic fern
clade, and found that these gametophytes have an exceptional capability to thrive in water for years without
forming their sporophyte counterpart.
Acknowledgment The authors thank Li-Yaung Kuo for laboratory
help, Tien-Chuan Hsu for collecting Taiwanese Lomariopsis spectabilis, and Dr. Wen-Liang Chiou (Taiwan Forestry Research Institute)
for valuable comments.
Appendix
Voucher information and GenBank accession numbersa
for rbcL, accD, trnL-F and rps4 (plus rps4-trnS IGS)
sequences used in this study.
Part 1: rbcL and accD
Taxon—GenBank accessions: rbcL, accD; voucher (collection locality; herbarium) or reference.
Arachniodes aristata (G.Forst.) Tindale—AB232490,
AB232418; Tsutsumi and Kato 2006. Araiostegia faberiana (C.Chr.) Ching—AB212688*; Tsutsumi and Kato
2005. Arthropteris backleri (Hook.) Mett.—AB212686*;
Tsutsumi and Kato 2005. Athyrium niponicum (Mett.)
Hance—AB232413, AB232441; Tsutsumi and Kato 2006.
Bolbitis repanda (Blume) Schott—AB232399, AB232427;
Tsutsumi and Kato 2006. Colysis wrightii (Hook.) Ching—
AB232406, AB232434; Tsutsumi and Kato 2006. Crypsinus enervis (Cav.) Copel.—AB232407, AB232435;
Tsutsumi and Kato 2006. Ctenitis eatonii (Baker) Ching—
AB232391, AB232419; Tsutsumi and Kato 2006. Cyclopeltis crenata (Fée) C. Chr.—DQ054517?; Li and Lu
2006. Cyclopeltis crenata (Fée) C. Chr?, EU216746; F. W.
Li 568 (private garden, originally from Thailand; TAIF).
Davallia formosana Hayata—AB212704*; Tsutsumi and
Kato 2005. Dryopteris erythrosora (D.C.Eaton) Kuntze—
123
F.-W. Li et al.
AB232392, AB232420; Tsutsumi and Kato 2006. Elaphoglossum callifolium (Blume) T.Moore—AB232400,
AB232428; Tsutsumi and Kato 2006. Goniophlebium
persicifolium (Desv.) Bedd.—AB232408, AB232436;
Tsutsumi and Kato 2006. Grammitis reinwardtii Blume—
AB232398, AB232426; Tsutsumi and Kato 2006. Gymnogrammitis dareiformis (Hook.) Ching ex Tardieu and
C.Chr.—AB232409, AB232437; Tsutsumi and Kato 2006.
Hypodematium crenatum (Forssk.) Kuhn ssp. fauriei
(Kodama) K.Iwats.—AB232414, AB232442; Tsutsumi
and Kato 2006. Leucostegia immersa (Wall. ex Hook.)
C.Presl—AB232388, AB232416; Tsutsumi and Kato 2006.
Leucostegia
pallida
(Mett.)
Copel.—AB232389,
AB232417; Tsutsumi and Kato 2006. Lomariopsis marginata (Schrad.) Kuhn.—AY818677, NA; Skog et al. 2004.
Lomariopsis
sp.
(Süßwassertang)—EU216743,
EU216744; F. W. Li 569 (unknown origin; TAIF). Lomariopsis sp. (Süßwassertang) —AM946394?; F042
(unknown origin; SING). Lomariopsis spectabilis (Kunze)
Mett.—AB232401, AB232429; Tsutsumi and Kato 2006.
Loxogramme avenia (Blume) C.Presl—AB232410,
AB232438; Tsutsumi and Kato 2006. Matteuccia struthiopteris (L.) Tod.—AB232415, AB232443; Tsutsumi and
Kato 2006. Microsorum zippelii (Blume) Ching—
AB232411, AB232439; Tsutsumi and Kato 2006. Nephrolepis acuminata (Houtt.) Kuhn—AB232403, AB232431;
Tsutsumi and Kato 2006. Nephrolepis cordifolia (L.)
C.Presl—AB232404, AB232432; Tsutsumi and Kato 2006.
Oleandra pistillaris (Sw.) C.Chr.—AB232405, AB232433;
Tsutsumi and Kato 2006. Oleandra wallichii (Hook.)
C.Presl—AB212687*; Tsutsumi and Kato 2005. Polybotrya caudata Kunze—AB232393, AB232421; Tsutsumi
and Kato 2006. Polystichum fibrilloso-paleaceum (Kodama) Tagawa—AB232394, AB232422; Tsutsumi and Kato
2006. Pyrrosia rasamalae (Racib.) KH.Shing—
AB232412, AB232440; Tsutsumi and Kato 2006. Quercifilix zeylanica (Houtt.) Copel.—AB232395, AB232423;
Tsutsumi and Kato 2006. Rumohra adiantiformis
(G.Forst.) Ching—AB232396, AB232424; Tsutsumi and
Kato 2006. Tectaria phaeocaulis (Rosenst.) C.Chr.—
AB232397, AB232425; Tsutsumi and Kato 2006. Teratophyllum wilkesianum (Brack.) Holttum—AB232402,
AB232430; Tsutsumi and Kato 2006.
Part 2: trnL-F
Taxon—GenBank accessions: trnL-F; voucher (collection
locality; herbarium) or reference.
Lomariopsis: L. amydrophlebia (Sloss. ex Maxon)
Holttum—DQ396555; Rouhan et al. 2007. L. boivinii
Holttum—DQ396557; Rouhan et al. 2007. L. cordata
(Bonap.) Alston—DQ396558; Rouhan et al. 2007.
L. crassifolia Holttum—DQ396559; Rouhan et al. 2007.
An aquatic fern gametophyte
L. guineensis (Underw.) Alston—DQ396560; Rouhan
et al. 2007. L. hederacea Alston—DQ396561; Rouhan
et al. 2007. L. jamaicensis (Underw.) Holttum—
DQ396562; Rouhan et al. 2007. L. japurensis (Mart.) J.
Sm.—DQ396567; Rouhan et al. 2007. L. kunzeana (Underw.) Holttum—DQ396570; Rouhan et al. 2007. L. latipinna Stolze—DQ396571; Rouhan et al. 2007. L. lineata
(C. Presl) Holttum—DQ396572; Rouhan et al., 2007. L.
longicaudata (Bonap.) Holttum—DQ396573; Rouhan
et al. 2007. L. madagascarica (Bonap.) Alston—
DQ396576; Rouhan et al. 2007. L. mannii (Underw.)
Alston—DQ396577; Rouhan et al. 2007. L. marginata
(Schrad.) Kuhn—DQ396579; Rouhan et al. 2007.
L. maxonii (Underw.) Holttum—DQ396580; Rouhan
et al. 2007. L. muriculata Holttum—DQ396582; Rouhan
et al. 2007. L. nigropaleata Holttum—DQ396584;
Rouhan et al. 2007. L. palustris (Hook.) Mett. ex Kuhn—
DQ396585; Rouhan et al. 2007. L. pervillei (Mett.)
Kuhn—DQ396587; Rouhan et al. 2007. L. pollicina Willem. ex Kuhn—DQ396588; Rouhan et al. 2007. L. prieuriana Fée—DQ396590; Rouhan et al. 2007. L. recurvata
Fée—DQ396592; Rouhan et al., 2007. L. rossii Holttum—
DQ396594; Rouhan et al. 2007. L. salicifolia (Kunze)
Lellinger—DQ396595; Rouhan et al. 2007. L. sp.—
DQ396602; Rouhan et al. 2007. L. sp.—DQ396601;
Rouhan et al. 2007. L. sp.—DQ396603; Rouhan et al.
2007. L. sp. (Süßwassertang)—EU216745; F. W. Li 569
(unknown origin; TAIF). Lomariopsis sp. (Süßwassertang) —AM946393; F042 (unknown origin; SING).
L. spectabilis—EU216748; F.W. Li 567 (Wulai, Taiwan;
TAIF). L. vestita E. Fourn.—DQ396598; Rouhan et al.
2007. L. wrightii Mett. ex D. C. Eaton—DQ396600;
Rouhan et al. 2007.
Cyclopeltis crenata (Fée) C. Chr.—EU216746; F. W. Li
568 (private garden, originally from Thailand; TAIF).
Hypodematium crenatum (Forssk.) Kuhn—AF425122;
Smith and Cranfill 2002.
Part 3: rps4 (plus rps4-trnS IGS)
Lomariopsis sp. (Süßwassertang) — AM947063; F042
(unknown origin; SING).
a
Asterisk, The same accession as previously noted;
cross, this sequence is available in a different voucher but
the same taxon; NA, data are not available for this taxon.
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