Phylogenetic Relationships of Isoëtes (Isoëtaceae) in China as Revealed by Nucleotide Sequences of the Nuclear Ribosomal ITS Region and the Second Intron of a LEAFY Homolog

2004·5.4 MB·English

by  W Carl Taylor

American Fern Journal 94(4}:196-205 (2004)
Phylogenetic Relationships of Isoetes (Isoetaceae) in
China Revealed by Nucleotide Sequences of the
as
and
Nuclear Ribosomal ITS Region the
LEAFY
Homolog
Second
Intron of
a
W. Carl Taylor and Angel Lekschas
R.
WI
Department of Botany, Milwaukee Public Museum, Milwaukee, 53233
Wang
QiNG Feng and Xing Liu
and
Laboratory of Plant Systematics Evolutionary Biology,
Wuhan Wuhan
University, 430072. People's Republic of China
Nancy and Sara Hoot
Napier
S. B.
WI
Department of Biological Sciences, University of Wisconsin, Milwaukee, 53201
—
Abstract. Isoetes an ancient lycopod lineage with a highly conserved morphology that
is
provides few morphological characters to resolve the phylogeny of its species. Species appear to
have evolved by divergence and allopolyploidy. The basic diploids bypsopbila, tahvanensis,
I. I.
and yunguiensis and the tetraploid sinensis occur in China. Analysis of ITS sequences
J. /.
indicates that the Chinese Isoetes species are part of an Australasian clade including brevicula
7,
New Two
from Western Australia and kirkii from Zealand. distinct cloned sequences of the
/.
LEAFY
second intron of a homolog were recovered from sinensis supporting the hypothesis that
/.
One
sinensis an allotetraploid. of the sinensis cloned sequences was similar to the
is
I. /. /.
taiwonensis sequence and the other cloned sequence was similar to the L yunguiensis sequence
identifying L taiwanensis and yunguiensis as the likely parents of sinensis. Other cloned
/. 7.
sequences recovered from sinensis were recombined parts of the two distinct sequences.
7.
Morphological evidence supporting an found
allotetraploid origin of sinensis in larger
is its
7.
microspore size and intermediate megaspore texture compared taiwonensis, and
to
7. 7.
yunguiensis.
Isoetes L, a cosmopolitan genus of heterosporous lycopods containing
is
hundreds
of species. Plants usually appear as tufts of linear leaves arising from
an underground, expanded
corm-like rootstock. Ellipsoidal sporangia occur in
leaf bases. Species range evergreen aquatics ephemeral
ft-om to terrestrials.
Although
Isoetes an ancient lineage with distinctive morphology rec-
is its
ognizable in the Triassic [Retallack, 1997), few characters have been found in
highly conserved morphology
its to resolve the phylogenetic relationships of
species. Distinguished by megaspore morphology,
its their habitat preference,
and chromosome
numbers, species appear to have evolved by ecological
and
isolation genetic divergence separated populations adapted
as
to ter-
restrial or aquatic habitats and by interspecific hybridization and chromosome
when
doubling
(allopolyploidy) divergent species were dispersed into the
same
(Taylor
sites et 1993).
al.,
Interspecific hybridization and allopolyploidy are well documented for
Many
Isoetes. interspecific hybrids have been recognized by production
their
of irregular spores and confirmed by chromosome numbers (Brunton and
their
TAYLOR ET PHYLOGENY OF CHINESE ISOETES
197
AL.:
and
Britton, 1999). In several cases, interspecific hybrids their suspected
chromosome
have been by isozyme
allopolyploid derivatives verified counts,
DNA
profiles, and sequences (Taylor and Hickey, 1992; Hoot and Taylor, 2001;
A = =
Hoot polyploid ranging from 3x 33 12x 132
et ah, 2004]. series to is
known 60%
Over which chromosome
counts
for Isoetes, of Isoetes taxa, for
have been published, are polyploid (Troia, 2001). Therefore, not only there
is
documentation hybridization and allopolyploidy occur
that interspecific in
Isoetes, but there also evidence that allopolyploidy an equally important
is is
mechanism
of speciation in this genus.
Recently, herbarium, field, and laboratory studies have been conducted to
learn more about the Isoetes of China. These studies have provided an opportu-
new
nity to determine the status of historical populations, discover popula-
new
tions and taxa, and obtain live specimens from which root tips could be
DNA
harvested for chromosome counts and fresh leaves could be collected for
isolations.
Four species of Isoetes have been described for China. All are believed to be
known
rare and endangered. Isoetes hypsophilo Handel-Mazzetti from the
is
Hengduan Mountains northwestern Yunnan Province and southwestern
in
Sichuan Province. In this region, hypsophila occurs in the shallow water of
/.
m
lakes and ponds about 3600 above sea level. Isoetes sinensis T. C. Palmer has
been found about ten in and along rivers and lakes of the middle and
at sites
known
lower Yangtze River system. At present, only three populations are to
remain been from Kyushu and
in China. Isoetes sinensis has also reported the
Chubu
[Takamiya and Cheju South Korea
Districts Japan et ah, 1997] Island,
known Menghuan
(Takamiya, 2001). Isoetes taiwanensis DeVol only from
is
Lake near the foot of Zhixing Mountain in the Yangming Mountains National
Wang
and
Park, north of Taipei in northern Taiwan. Isoetes yunguiensis Q. F.
W. C. Taylor known from the Yunnan-Guizhou Plateau in southwest China.
is
In this region, plants have been recorded at four sites, but only two small
known
populations, totaling about 400 individuals, are to remain. Liu et
al.
(2002) reported that hypsophila, L taiwanensis, and yunguiensis are basic
/. /,
^ — —
2x=
and 4x
diploids (2n 22) Isoetes sinensis a tetraploid [2n 44).
is
Nucleotide sequences from the nuclear ribosomal ITS region, the chloroplast
LEAFY
atpB-rbcL spacer region, and the second intron of a homolog have been
and
used to determine phylogenic relationships of Isoetes, delimit species,
reveal an interspecific hybrid and allotetraploid derivative (Hoot and Taylor,
its
2001). Hoot et (2004) used cloning to separate homoeologous sequences of
al.
LFAFY
homolog By
the second intron of a for several Isoetes allotetraploids.
some
comparing these cloned sequences those of putative parents, of the
to
parent species could be identified.
The paper were use nucleotide sequences from the
goals of this to
LEAFY
and homolog
nuclear ribosomal ITS region the second intron of a
Chinese
determine the relationships of the Isoetes species, the
test
to; (2)
(1)
hypothesis that the tetraploid L sinensis is an allotetraploid and, if this
hypothesis identify the basic diploid parent species of
correct,
is (3)
sinensis.
/.
AMERICAN FERN VOLUME NUMBER
198 JOURNAL:
94
4 (2004)
Table Specimens sampled. Columns number-
1. indicate species, location, collector, collection
DNA
isolation number, date of collection, and herbarium acronym for location of voucher.
Collections are from Mainland China unless otherwise noted.
Isolation
Species Voucher Number
Collection
Isoetes brevicula Rock pool, summit of Lily McCarthy Rock, Western 189
&
Australia, 25 Sep 2002, W. C. Taylor N. T. Luebke
6383 (MIL)
Isoetes hypsophila Tu-er-sjan, Dao-cheng County, Sichuan Province, 01 Aug 127
Wang Hong
2001, Aing-Feng, Liu Xing, Liu Yang
Er
Xiao-Lin 2 (WH)
New
Isoetes kirkii Lake Brunner, South Island, Zealand, 27 Mar 2004, kiNZ
Woodland
W.
D. Felicity Cutten (MIL)
Er s.n.
Isoetes sinensis Xing-an-jiang, Jiande City, Zhejiang Province, 19 Oct 129, 131
Xing Pang Xin-An (WH)
2001. Liu 4
Er 3,
Isoetes tahvanensis Menghuan Lake, Yangming Mountains, Taiwan, May 78
Oiiou Wen-Liang
1998, (MIL)
s.n.
yu ng
Isoetes uicn sis Sha-shi-chong, Ping-ba County, Guizhou Province, 15 130
Aug
Xing Yang (WH)
2001, Liu &- Xiao-Lin 5
Materials and Methods
Species sampling.—Table contains number,
1 locality, collector, collection
and
location of voucher specimens for plants used in this study. Specimens were
identified to species using the original descriptions of the species (Handel-
Wang
Mazzetti, 1923; Palmer, 1927; DeVol 1972a; and by
Q. F. et al, 2002)
comparison with and
authentic type specimens. morphology
Diagnostic
for
megaspore textures was evaluated using an Olympus SZX12
stereomicroscope.
—
DNA
DNA
and was mg
isolation amplification. isolated from 20 of
silica
dried leaves from each sample by grinding
the leaf tissue, frozen in liquid
powder
nitrogen, to a with a disposable 1.5 pellet pestle (Kimble-Kontes) in
ml
a 1.5 snap-cap microcentrifuge tube (Eppendorf) and using the DNeasy®
Mini
Plant Kit (Qiagen) following the manufacturer's
protocol.
The
ITS region for all samples was amplified with the primers ITS-I (5'-
GTCCACTGAACCTTATCATTTAG-3';
Urbatsch, et oL 2000) and ITS4 (5'-
TCCTCCGCTTATTGATATGC-3';
Z^^Fr
White The
et al. 1990). intron for
samples was GATCTTTATGAA-
all amplified with the primers 30F (5'-
CAATGTGG-3')
and 1190R GAAATACCTGATTTGTAACC-3');
(5'- Nancy
S.
LEAFY
Napier PCR
designed both
primers. reaction mixtures followed
manufacturer Ready-To-Go™ PCR
protocols using Beads (Amersham
Bio-
PCR
sciences). amplification the ITS region began with
for denaturation 60
for
s at 97°C followed by 40 cycles of denaturation for 10 annealing
s at 97''C, for
and
30 s at 48^C, extension 20 72°C with added
for at 4 extension time
s s to
each and
cycle ending with a final extension of min 72T. PCR
7
at
LEAFY
amplification
for the intron began with min
denaturation 94^C
for 5
at
followed by 40 cycles of denaturation min min
for 1 at 94''C, annealing for 1 at
and
min
50^C, extension for 1 at 72''C, and ending with a final extension of 5
TAYLOR ET PHYLOGENY OF CHINESE ISOETES 199
AL.:
mm
2%
PCR
products were concentrated via electrophoresis in a
at 72''C.
agarose gel containing ethidium bromide and visualized with transilluminated
QIAquick®
Gel
(Q
—
PCR
was
Cloning and sequencing. Sequencing of the ITS products
DNA
performed on both 5' and 3' strands using the amplification primers
ITS-I and ITS4 as cited above.
LEAFYPCR
product and subsequent transformation,
Ligation of the purified
and transformed clones followed manufacturer
visualization of
cloning,
®
pGEM
protocols using the
100 X-gal plates (Teknova).
taiwanensis and L yunguiensis, eight clones
For basic diploid species,
7.
each species were sequenced. For the tetraploid species,
(colonies] for
/.
16 clones from each of two plants) were sequenced to increase
sinensis, [eight
the odds of capturing parental cloned sequences. For the outgroup species,
all
L L hypsophila, and A. Braun, four clones
brevicula E. R. L. Johnson, kirkii
/.
from each species were sequenced.
DNA
manufacturer
Cleaning and concentration of the vector followed
QIAPrep® Spin Miniprep
Kit (Q
M13F
M13R ABl Dye® Terminator Cycle v Sequencing
unction with the Big 3.1
Sequencing
(Applied Biosystems) following the manufacturer's protocol.
Kit
DNA
Iowa
products were resolved on an ABI (model 377) sequencer at the State
DNA
Ames,
and Iowa.
Sequencing Synthesis
University Facility,
Sequence alignment and phylogenetic ana/ysis.—Nucleotide sequences
Gaps were
Codes
were aligned and edited using Sequencher 4.1 (Gene Corp.).
with one multiple base
or
presence/absence characters,
additional
treated as
(Baldwin ah
gaps scored as a single character et 1995).
PAUP*
Maximum narcimnnv was conducted with
analvsis of the data
using the
=
maximum and
trees 4000 for the ITS data set
= LEAFY PAUP*
maximum was used run 500
100 data to
trees for the set.
bootstrap replicates for each data set to estimate reliability of the clades
(Felsenstein, 1995).
Results
and yunguiensis from
bypsophila, L taiwanensis
sequences o^Isoetes
ITS
/.
New
and from
from southwestern Australia, kirkii
China, L brevicula 7.
and
Zealand form an Australasian clade with other species clades previously
by Hoot and Taylor (2001). The data set analyzed consisted 16
reported
and two outgroup species with a total of 826 characters; 313
ingroup species
The
were variable and 239 were parsimony informative. ITS tree
characters
50% majority-rule consensus tree of the 39 most
bootstrap
a
illustrated is
shows
Tree topology a close relationship
parsimonious trees retained (Fig. 1).
FERN JOURNAL
200
22
Isoetes
brevicula
12
i
15 c
94
kirkii
13
CO
96
taiwanensis
18
79 w
98 1
yunguiensis ^
92
16
hypsophila
6
echinospora
81
100
d
flaccJda
76
c
engelmannii S
89 2
welanopoda
J
94
6
howellii
100
2
bolanderi
7
nutiallii
8
I
15
9.S
orcuttii
31
11
78
olympica
26
125 i
20
ion
95 abyssinica
27
histrix
14
pen
ca
SIS
20
stellenbossiensis
50%
Fig. Isoetes ITS region tree. Bootstrap majority-rule consensus tree of 39 trees resulting
1.
maximum
from parsimony analysis using heuristic search of ITS region sequence data for eighteen
basic diploid species of Isoetes. Tree length 489 steps of equally weighted nucleotide
is
= =
Numbers above
substitutions and gaps, CI [excluding uninformative characters) 0,76, RI 0.86.
number Numbers below branches
the branches are the of nucleotide substitutions. the are
bootstrap values. Major clades are labeled on the right. The tree is rooted with capensis and
7. /.
from South
stellenbossiensis Africa.
exists between taiwanensis, and yunguiensis. All species in the tree are
/, 7.
—
basic diploids [2n
22).
LEAFY
Defined by the 30F and 1190R Napier primers, the region for aligned
sequence clones of sinensis, taiwanensis, and yunguiensis was 1105
J. I. J.
bases long and included of the second intron and parts of the flanking
all
exons. All of the eight sequence clones of/, taiwanensis were 1075 bases long
and of the eight sequence clones of yunguiensis were 1072 bases long.
all
7.
Two LEAFY
of the 16 sinensis clones did not amplify for the intron.
7.
Therefore, 14 aligned, cloned sequences were compared informative
for sites.
At 15 informative sites that included 12 substitutions, two one base gaps, and
one three base gap, six cloned sequences of sinensis, each 1076 bases long,
7.
matched matched and
or nearly the cloned sequence type of taiwanensis five
7.
cloned sequences of sinensis, each 1079 bases long, matched or nearly
7.
matched the consensus cloned sequence type of yunguiensis (Table Seven
2).
7,
sinensis cloned sequences showed evidence of recombination part of the
7. i.e.,
PCR
cloned sequence matched
amplified, either the taiwanensis or the
first
7.
yunguiensis sequence but on matched sequence
type, further the other type.
7.
TAYLOR ET PHYLOGENY OF CHINESE ISOETES
AL.: 201
Table Comparison of fourteen cloned sequences of sinensis with cloned sequences of
2.
/.
I. tohvanensis and 7. }ninguiensis at fifteen informative sites. Sites are numbered sequentially
beginning with the 30F Napier primer. Isoetes sinensis cloned sequences are identified by
number-DNA
collection isolation number-clone number. Nucleotides in italic compare to those of
taiwanensis. Nucleotides in bold face compare to those of yunguiensis. Gaps are indicated by
/. /.
dashes. Sequences marked with an asterisk showed extensive recombination and were removed
from the data set before the final analysis. Clones 4-131-6 and 4-131-7 did not amplify for the
luEAFY region and are not included in the table.
CGTTCGTTAAAA
site/clone 205 289 290 357 525 549 660 741 776 827 840 865 965-967 987 1059
CGTTCGTTAAAA
--- A
taiwanensis
CGTTCGTTAAAA
-
--- A
3-129-3
CGTTCGTTAAAA
-
A
-•
3-129-7
CGTTCGTTAAAA
-
-
--- A
4-131-1
CGTTCGTTAAAA
-
--- A
4-131-3
CGTTCGTCAAAA
-
--- A
4-131-4
CGrTTTCCGG-G
-
--- G
3-129-5
CGrTTTCCGG-GATGT- ^
--- A
3-129-2*
TACCCGTTAAAA
-
3-129-1*
CACCTTCCGG-GATG-
--- A
4-131-8*
-
TACCTTCCGG-GATG-
G
4-131-2
TACCTTCCGG-GATG-
G
3-129-4
TACCTTCCGG-GATGTG
G
3-129-8
TACCTTCCGG-GATGTG
3-129-6
TACCTTCCGG-GATGT
4-131-5
G
yunguiensis
Three sequences showing evidence extensive recombination were removed
of
PAUP*.
from by
the data evaluated
set
Based on the relationships indicated in the ITS tree (Fig. brevicula,
1), I. I.
LEAFY
and L hypsophila were chosen as outgroup species to root the
kirkii,
LEAFY
was
second intron The sequenced region for brevicula 1035
tree.
/.
bases, was 1090 bases, and for hypsophila was 1095 bases.
for kirkii
it
it /.
/.
LEAFY
The second intron data set analyzed contained 16 sequences with
were and were parsimony
a of 1125 characters; 171 characters variable 54
total
informative. The data set included consensus sequences from clones of
/.
hypsophila, L L taiwanensis, and yunguiensis and cloned
brevicula,
kirkii, 1 1
I. /.
LEAFY
The second
sequences of/, sinensis. intron tree illustrated is a bootstrap
50%
majority-rule consensus tree of the two most parsimonious trees retained
Six of the L sinensis cloned sequences formed a clade with taiwanensis
(Fig.
2). /.
and cloned sequences formed a clade with L yunguiensis.
of the sinensis
five
/.
Discussion
worldwide
Although Isoetes Paleozoic in origin (Pigg, 1992), in
is
distribution, and over time, undoubtedly adapted to changing climates and
many
aquatic habitats on every continent times, the morphology
to terrestrial
of Isoetes has been remarkably conserved. Thus, morphology provides few
characters that can be used to reliably reconstruct phylogenetic relationships.
Nevertheless, pteridologists have speculated about the relationships oi Isoetes
AMERICAN FERN VOLUME NUMBER
JOURNAL:
202
94
4
(2004J
Isoetes 16
yunguiensis 5-130
3-129-4
sinensis
2
25
85
98
4-129^8
Sinensis
sinensis 4-129-6
8
2
87
86
1
sinensis 3-131-5
2
100
sinensis 3-131-2
2
taiwanensis s.n.-78
1
3-129-3
sinensis
34
100
sinensis 3-129-7
4
1
4-129-5
sinensis
85
sinensis 4-131-3
sinensis 4-131-1
sinensis 4-131-4
28
s.n.-kiNZ
kirkii
12
38
95
6383-189
brevicula
38
hypsophila 2-127
LEAFY
Fig.
2.
maximum
four trees resulting from parsimony analysis using heuristic search of LEAFY second
homolog
intron sequence data for five basic diploid and one tetraploid sinensis) species of
(/.
Isoetes. Eleven sequence clones that form a sister clade to either yunguiensis or taiwanensis
7. 7.
represent the tetraploid genome of sinensis. Tree length 183 steps of equally weighted
is
7.
= =
nucleotide and
substitutions gaps, CI [excluding uninformative
characters) 0.89, RI 0.96.
Numbers
above the branches are the number of nucleotide substitutions. Numbers below the
branches
are bootstrap values. Figures to the right of the specific epithets are the collection and
clone identification labels. The tree is rooted with hypsophila from China, 7 brevicula from
7.
New
Western Australia, and from Zealand.
kirkii
7.
species based on ecology, morphology, and biogeography. and Bmnton
Britton
(1991] reevaluated the spore morphology of taiwanensis, concluding
that
J. it
was
not related to taxa from southwestern Australia proposed by Marsden
as
New
(1979), but instead appeared to have closest from
its affinity to kirkii
/.
The
Zealand. ITS shows
tree (Fig. that both brevicula from southwestern
1]
/.
rm
a sister clade to the
members
Huang
habitat, et al. (1992) concluded that L taiwanensis probably closer to
is
asiatica than
/. it is to sinensis, but asiatica echinospora subsp. asiatica
7.
7.
(7.
(Makino) Love member
A.
is a of the echinospora species complex, a group of
/.
taxa
ITS shows
member
tree (Fig. that echinospora, a an American
2] of
clade,
7. is
only members
distantly related to of the Australasian clade, which includes
J.
DeVol
taiwanensis. mentioned
(1972b) seemed
that taiwanensis
nearer
7. to
7.
sinensis than any other Takamiya saw
species.
(2001) similarities in the spore
m
taxa
TAYLOR PHYLOGENY
ET OF CHINESE
ISOETES
AL.:
203
taiwanensis
A yunguiensis
I.
TT YY
X
2n = 22 m 2n =
22
TY
Chromosome
Doubling
sinensis
/.
TTYY
2n = 44
Hypothetical phylogeny
Fig. 3. of Isoetes sinensis involving interspecific hybridization and
chromosome
doubling of the basic diploids taiwanensis and yunguiensis. Data presented
in
/. /.
this study supports the hypothesis of an allotetraploid origin for sinensis.
/.
The
recovery of two, distinct, L£VlFy second intron sequence types from the
tetraploid sinensis supports the hypothesis that sinensis an allotetraploid
is
7.
I.
To
(Table was assumed some
clarify the results, that of the sequenced
2). it
clones recovered from sinensis were recombinations of the two distinct
/.
sequence types and three of the recombinant sequences were removed from
the
data set evaluated by PAUP*. Since recombination occurs from crossing over
between chromosomes
during meiosis, possible that the observed
is re-
it
combined
sequences were products of natural events. the recombined
If
we
sequences were would
detected the result of crossing-over during meiosis,
would
predict that identical crossover sequences be recovered as clones. All
seven of the recombinant cloned sequences from sinensis were
different,
/.
indicating that these recombined sequences more likely occurred during the
PCR
amplification reaction. Whatever their source, including recombinant
sequences and need
in a cladistic analj^sis will affect results therefore, they to be
and removed
recognized from the data set before the final analysis.
Comparison of the two distinct cloned sequences from the allotetraploid
I.
sinensis with the cloned sequences of taiwanensis and yunguiensis
indi-
/. /.
two
cates that either these basic diploid species, or closely related taxa, likely
Although
participated in the formation of sinensis (Table the L
2).
/.
yunguiensis including sinensis cloned sequences, and
clade, five the
/.
I.
taiwanensis clade, including six sinensis cloned sequences, are both well
/.
supported with high bootstrap percentages, the yunguiensis sequence
I. is
distinguished from L sinensis clones by sixteen autapomorphies and
sister
its
the L taiwanensis sequence distinguished from sister sinensis clones by
is its
I,
two autapomorphies These unique nucleotide substitutions could be
(Fig.
2].
due sequencing nucleotides of taxa different from those of the parent
to taxa,
(1]
and
continued evolution of the progenitor parent species the allotetraploid
(2]
species following allopolyploidy, or copy errors during PCR. Causes for the
(3)
autapomorphies might be determined by additional sampling and repeated
PCR
same
of the clones.
molecular morphological
In addition to the characters, characters indicate
that sinensis, L taiwanensis, and yunguiensis are distinct, but closely
/.
/.
VOLUME NUMBER
AMERICAN FERN
204 JOURNAL:
94 4 (2004)
and some
related species provide evidence supporting the allopolyploid origin
AH
of/, sinensis, three species are amphibious plants with tri-lobed rootstocks.
They have a rudimentary velum that covers only the upper edge of the
all
The
sporangium. microspores of L taiwanensis and yunguiensis range from
/.
20-26
|im in length whereas, those of/, sinensis range from 26-30 jim in length
Wang
(Britton and Brunton, 1991; et al, 2002; Palmer, 1927). The larger size of
sinensis microspores attributed increased chromosome number.
/. is to its
chromosome number
Increases in are usually accompanied by larger spore size
and
(Kott Britton, 1983). In contrast, megaspores of the tetraploid sinensis
/.
and
the basic diploid yunguiensis average about 400 |im in diameter, whereas
/.
megaspores
of the basic diploid taiwanensis average about 300
jim in
/.
Megaspore
diameter. texture appears to be the most distinctive character that
However, view
separates these three species. in of the results presented here,
the cristate to verrucate megaspores of sinensis can also be interpreted as
/.
combining
subtly the textures of the rugulate megaspores
to reticulate of
/.
taiwanensis and the megaspores
cristate to reticulate of/, yunguiensis.
Acknowledgments
The
authors thank Chiou Wen-Liang, Felicity Cutten, Gerald Gastony, Kuo Chen Meng, Neil
T.
J.
Luebke, Lytton Musselman, and Dennis W. Woodland
for providing plant specimens analyzed
in
J.
NSF
DEB-9981460
this study. grants to Sara B. Hoot and DEB-9981501 to W. Carl Taylor and the State
Key Basic Research and Development Plan of China (G200004B805) supported
parts of this study.
Literature Cited
M.
Baldwin, B. G., Sanderson, M. Porter, M. Wojciechowski, Campbell and M.
F. C.
S.
J. J,
J.
A
DoNOCHUE. The ITS DNA:
1995. region of nuclear ribosomal valuable source of evidence on
angiosperm phylogeny. Ann. Missouri Bot. Gard. 82:247-277.
Britton, D. M. and D. F. Brunton. 1991. The spores and affinities oi Isoetes taiwanensis (Isoetaceae:
Pteridophyta]. Fern Gaz. 14:73-83,
Brunton, and M. new
D. F. D. Britton. 1999. Isoetes xechtuckerii, hyb. nov., a triploid quillwort
from
northeastern North America. Canad. 77:1662-1668.
Bot.
J.
Devol, C. E. 1972a. Isoetes found on Taiwan. Taiwania 17:1-7.
A
Devol,
C. E. 1972b. correction for Isoetes taiwanensis DeVol. Taiwania 17:304-305.
An
Felsenstein, 1995. Confidence Hmits on phylogenies: approach using
the bootstrap. Evolution
J.
39:783-791.
Handel-Mazzetti,
H. 1923. Isoetes bypsophila Hand.-Mzt. Akad. Wien
Wiss,
13:95.
Hoot, and W.
S. B. C. Taylor. 2001. The utility of nuclear ITS, a LEAFY homolog intron, and
chloroplast a tpB-rbcL spacer region data in phylogenetic analyses and species delimitation in
Isoetes. Amer. Fern 91:166-177.
J.
Hoot, N. Napier and W. unknown
S. B., S. C. Taylor. 2004. Revealing or extinct lineages within
DNA
Isoetes (Isoetaceae) using sequences from hybrids. Amer. 91:899-904.
Bot.
J.
Huang, T. C, H. Chen and Ll A
L. C. 1992. palynological study of Isoetes taiwanensis DeVol.
J.
Amer. Fern 82:142-150.
J.
Kott, L. S. and D. M. Britton. 1983. Spore morphology and taxonomy
of Isoetes in northeastern
North America. Canad.
61:3140-3163.
Bot.
J.
Cytotaxonomy
Love, A. 1962. of the Isoetes echinospora complex. Amer.
Fern 52:113-123.
J.
Wang
Wang,
Liu, X., Y. Q. F. and Y. H. Guo. 2002. Chromosome numbers
of the Chinese Isoetes and
taxonomical
their significance. Acta Phytotax. Sin. 40:351-356.
TAYLOR PHYLOGENY
ET OF CHINESE ISOETES
AL.:
205
Marsden, Morphology and Taxonomy
C. R. 1979. of Isoetes in Australasia, India, north-east and
south-west Asia, China and Japan. Ph.D. thesis, Department of Botany, University of
Adelaide, Adelaide.
A
Palmer, T. C. 1927. Chinese Isoetes. Amer. Fern 17:111-113.
J.
PiGG, K. B. 1992. Evolution of Isoetalean lycopsids. Ann. Missouri Bot. Card. 79:589-612.
Retallack, G. 1997. Earliest Triassic origin of Isoetes and quillwort evolutionary
radiation.
J.
Paleontol. 71:500-521.
J.
PAUP*:
SwoFFORD, D. L. 2002. Phylogenetic analysis using parsimony (* and other methods).
Sinauer, Sunderland, Massachusetts, USA.
M. Watanabe
Takamiya, M., and O. Kanji. 1997. Biosystematic studies on the genus Isoetes
(Isoetaceae] in Japan. IV, Morphology and anatomy of sporophj^es, phytogeography and
taxonomy. Acta Phytotax. Geobot. 48:89-121.
Takamiya, M. 2001. Isoetes sinensis var. sinensis in Korea (Isoetaceae: Pteridophj^a). Fern Gaz.
16:169-176.
W. and
Taylor, C. R. Hickey. 1992. Habitat, evolution and speciation in Isoetes. Ann. Missouri
J.
Bot. Card. 79:613-622.
Taylor, W. C, N. T. Luebke, D. M. Britton, R. Hickey and D. F. Brunton. 1993. Isoetaceae, pp. 64-
}.
FNA
75, in Editorial Committee, cds. Flora of North America, North of Mexico, Volume 2.
New
Oxford University Press, York.
The
TroIa, a. 2001. genus Isoetes L. (Lycophyta, Isoetaceae]: synthesis of karyological data. Webbia
56:301-218.
Urbatsch, L. E., B. L. Baldwin and M, Donoghue. 2000. Phytogeny of the coneflowers and relatives
J.
rONA
(Heliantheae: Asteraceae) based on nuclear internal transcribed spacer (ITS) sequences
DNA
and
chloroplast 25:539-565.
restriction site data. Syst. Bot.
X
Wang, Q. F., Liu, W. C. Taylor and Z. R. He. 2002. Isoetes yunguiensis (Isoetaceae), a new basic
Novon
diploid quillwort from China. 12:587-591.
Appendix
DNA
Genbank accession numbers for the new Isoetes sequences used in this manuscript
Genbank
number
Species accession
Nuclear ribosomal ITS region sequences
AY641098
brevicula
/.
AY641099
hypsophila
I.
AY641100
kirkii
I.
AY641101
taiivanensis
I.
AY641102
yunguiensis
I.
LEAFY
second homolog sequences
intron
AY641103
brevicula
/.
AY641104
hypsophila
I.
AY641105
L
kirkii
AY641106
L
taiivanensis
AY641107
L yunguiensis
AY641108
L yunguiensis type clone)
sinensis
[I.
AY641109
sinensis taiivanensis type clone)
7. [I.