Table Of ContentOsmunda
Molecular Evidence on
the Origin of
Xmildei (Osmundaceae)
C. TsuTsuMi*, Y. HiRAYAMA, and M. Kato
Tsukuba
305-0005, Japan
Yatabe-Kakugawa
Y.
Botanical Gardens, Graduate School
of Science, the University of Tokyo. Hakusnn.
Zhang
S.-Z
Shenzhen Luohu
Fairylake Botanical Garden, Shenzhen, Guangdong, China 518004
District,
The Osmunda
genus
Osmundaceae
of the leptosporangiate fern family has
natural hybrids Tsutsumi One Osmunda
(Kato, 2009; such
et ah, 2011).
is
Xmildei = homonym
C.Chr. 0. bipinnata Hook.,
a later of O. bipinnata
L.),
{
which became known
nearly Hong
extinct in range in Kong. However,
its this
was
hybrid found
recently Shenzhen, Guangdong, and
in Mt. Qiyun,
Jiangxi
(Zhang and
et al, 2008), less than 10 individuals are known. was also found
It
Hunan
in Zhangjiajie, (Y.-H. Yan, pers. comm.). can propagate via spores
in
It
experimental
conditions Yang, unpubl.
but uncertain
data), the
(J.-F. it is if
individuals were derived from spore propagations from independent
or
Osmunda
formations of the hybrid. Xmildei characterized by subcoria-
is
ceous, bipinnate-bipinnatifid leaves with round, entire pinnules, and
fertile
pinnae below
inserted the middle of the For the origin of O. Xmildei,
leaf.
two
possibilities were proposed Based on and
karyological
(Fig.
1).
He
morphological
analyses. suggested Xmildei
et (2006) that O.
al. a
is
hybrid of O. japonica Thunb. (subgenus Osmunda) and Ching
O. angustifolia
(subgenus Plenasium]. Zhang
et al. (2008) observed the absence of
chromosome
and
pairings meiosis
at resulting abortive spores in O. Xmildei,
and
suggested that it is a sterile Fl hybrid, but argued that the parents were O.
* Corresponding author: (e-mail: [email protected])
AMERICAN FERN VOLUME NUMBER
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102
(2012)
1
and
japonica O. vachellii Hook, (subgenus Plenasium], because O. vachellii
co-occurs with O. Xmildei, but O. angustifolia does not occur in some of the
Xmildei Gou proposed
localities of O. (Fig. et (2008) also that O.
2). al.
vachellii the maternal progenitor of O. Xmildei, based on inferences from
is
DNA
chloroplast sequence data. Under either parentage hypothesis O.
Xmildei an between Osmunda
likely intersubgeneric hybrid the subgenera
is
and Plenasium.
known
There are three more hybrids reported Osmundaceae
in (Kato, 2009).
Eastern North American O. Xruggii Tryon is O. regalis L. (subgenus Osmunda)
X
O. claytoniana L. (subgenus Claytosmundd) (Tryon, 1940; Wagner al,
et
Whetstone and and
1978; Atkinson, 1993; Li Haufler, 1994). Japanese O.
Xnipponica Makino Osmunda) x
O. japonica (subgenus O. claytoniana
is
(subgenus Claytosmunda) Sugimoto
1964), but (1979) suggested that
(Ito, is
it
an X Osmundastrum cinnamomeum
intergeneric hybrid, O. japonica
i.e., (L.)
X
Neither given molecular
C.Presl. evidence. Finally, Japanese O. intermedia
is
X
(Honda) Sugim. O. japonica O. lancea Thunb. (both in subgenus
is
Osmunda;
Tagawa, 1959; Iwatsuki, 1995; Tatuno and Yoshida, 1966; Yatabe
et 2009).
al.,
—
Samples
Materials. of three species of subgenus Plenasium
[O. vachellii, O.
Kuhn,
banksiifolia (C.Presl) O. angustifolia], the putative intersubgeneric
AMERICAN FERN JOURNAL: VOLUME NUMBER
102
1 (2012)
hybrid O. Xmildei, one species of subgenus Claytosmunda
(O. claytoniana],
Osmundastrum cinnamomeum,
and Todea
barbara T.Moore, along with
(L.)
Osmunda
three species subgenus
of (O. japonica, O. lancea, O. were
regalis],
The
collected in the field or botanical gardens. sources of materials used are
shown
Table
in
1.
DNA—
Sequencing and
of chloroplast nuclear Leaf fragments were used
for
DNA
molecular was
analysis. extracted from fresh or silica-gel-dried material
QIAGEN
using a DNeasy Mini Kit (QIAGEN, Valencia, CA) following the
DNA
manufacturer's instruction. Three nuclear markers (EST_L058,
EST_L110, EST_L258)
from
selected the expressed sequence
tag library
developed by Yatabe and
et (2009) the chloroplast locus rbcL were
al.
analyzed. Primers for amplification and sequencing, and detailed information
on shown
the three nuclear markers are in Table 2 and Table respectively.
3,
PGR DNA
was
performed using Perkin-Elmer 9700
a thermal (Applied
cycler
DNA
Biosystems, Foster, GA) with Ex Taq polymerase (TaKaRa Tokyo,
Bio,
and Ampdirect
Japan) Plus (Shimadzu,
Kyoto, Japan) in 35 denaturation,
and
annealing, elongation cycles (30 sec at 94 G; 30 sec at 50 G for the rbcL and
G
58 in the three nuclear markers; and 90 with
sec at 72 G) a final elongation
min PGR
The
step (7 at 72 G). products were purified with ExoSAP-IT (USB
OH)
corporation, Gleveland,
following the manufacturer's
instructions.
Sequencing was conducted ABI PRISM
using an 3130x1 Genetic Analyzer
(Applied Biosystems). The raw sequence data were assembled using Seqman
II
DNA
(Dnastar, Madison, WI). For the sample of O. Xmildei, PGR of the nuclear
DNA
markers was performed with PrimeSTAR Max polymerase (TaKaRa
Bio)
G
in 35 cycles (10 sec at 98 G, 5 sec at 55 and 5 sec at 72 G). The PGR products
pGEM-T
were cloned
using a Vector System (Promega, Madison, WI) and
at
I
least 10 clones were sequenced. Minor variants from single clones, presumably
sequencing errors, were observed. Therefore a consensus sequence was used
for each allele type; the differences between each consensus sequence and
the
shown
original clones are in Table The assembled sequences were aligned
4.
X
by program (Thompson
Glustal 1997) and then aligned manually.
et
al.,
—
Molecular phylogeny. Phylogenetic analyses were performed by maximum
parsimony (MP) and Bayesian Osmundaceae
analysis. Registered sequences
of
Genbank were added Maximum
in into the analyses Table parsimony
(see
1).
(MP) was PAUP*
inference conducted with 4.0bl0 (Swofford, 2002). The bases
that could not been identified were treated as unknown (N), and gaps were
treated as missing data. All characters were equally weighed and
heuristic
searches were conducted with 1000 random TBR
addition replicates involving
branch swapping. were
Bootstrap values calculated from 1000 pseudorepli-
random
each with 100 MrMod-
cates, additions. For the Bayesian analyses,
was
eltest 2.0 (Nylander, 2004) used to determine the nucleotide substitution
MGMG
model. Bayesian searches were conducted by with two independent
sets of four chains, each run for ten million generations, sampling every 100
generations by MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and
Huelsenbeck, The model GTR
2003). nucleotide selected was: rbcL, + + G;
\
HKY
GTR GTR
EST_L058, + EST_L110, + EST_L258, + The program
I; I; I.
m
iiiii!
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Jill!
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1= alS
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It
VOLUME NUMBER
102
1
EST ATAAGGTTTCGCCCTCGAAT
L058
tc:ttggagttgggagttcac
EST GATTGGAGATGGGAGATGAT
LI
10
CAGAGGTGAAACAGGGTGAA
EST TCATGGCGAGTGTGAAGAAG
L258
GGCCCTTTGGATTTAGGATA
Tracer (Rambaut and Drummond, 2009) was used to check the runs had
reached and sample was
stationarity effective size of the parameters high
all
The
(>100). first 2.5 million generations before sufficient stationary genera-
tions were discarded as burn-in periods and the rest of trees were used to
Osmundastrum cinnamomeum
calculate posterior probabilities. and Todea
barbara were used as outgroups (Yatabe et 1999; Metzgar et al, 2008).
al.,
Results
maximum
Bayesian and parsimony analyses of each dataset produced
congruent shown
topologies (Bayesian consensus Maxi-
trees in Figs. 3-6).
mum
parsimony
analyses resulted in three shortest trees of a length of 146
= = =
HI
steps (CI 0.77, 0.23, RI 0.93) for chloroplast rbcL (1227 bp) with 101
parsimony-informative characters, 78 shortest trees of a length of 47 steps (CI
= = =
HI EST_L058
0.89, 0.11, RI 0.88) for nuclear (198 bp) with 18
MP
parsimony-informative
characters, ten shortest trees of a length of 125
= = =
HI EST_L110
steps (CI 0.91, 0.09, RI 0.91) for nuclear (572 bp) with 57
parsimony-informative and two
characters, shortest trees of a length of 122
= = =
HI EST_L258
steps (CI 0.89, 0.12, RI 0.94) for nuclear (361 bp) with 59
parsimony-informative
characters.
uLg
sequenc
(blastn)
1
GenBank
hit
^
Marker
no. (bp) gion (bp) region (bpf (specie^s) (E-value)
EST_L058 FS9936B1 AYl
198 101 97 glycolate oxidase 73074
X "*)
10
(gox)
(2
hi
d
[Z
t
EST_L110 FS993713 548 148 400 EU964956
glycerol-3-
10^")
X
phosphate
(9
[Zea mays)
EST_L258 FS993861 317 79 238 ribosomal AF334838
TSUTSUMI ET ORIGIN OF OSMUNDA XMILDEI
AL.:
The
rbcL sequence Xmildei and
of O. identical that of O. vachellii also
is to
was and Blume The
similar to those of O. banksiifolia O. javanica
(Fig.
3).
Osmunda
Xmildei sequence was more
distantly related to O. angustifolia (but
with low support), and very far from O. japonica and other species of subgenus
Osmunda.
In the three nuclear markers, the O. Xmildei sample has two distinct allele
One
types (Figs. 4-6). type (Type A) had the same sequence as some plants of
O. japonica, while the other (Type B) had the same sequence as O. vachellii
(in
EST_L58
and LllO in Figs. 4 and or a sequence very similar to (in
5)
it
EST_L258
in Fig. In each of the three nuclear-marker trees, O. Xmildei was
6).
more
closely related to O. vachellii than to O. angustifolia.
Discussion
DNA
Our
trees constructed from the chloroplast gene and nuclear sequences
with monophyly Osmunda
agree previous trees in the of the three subgenera of
Gou
(Yatabe et al., 1999; et al, 2008; Metzgar et al, 2008). The rhcL
same
phylogenetic relationships of the subgenera are the as Yatabe
et
al.'s
(1999) from the same gene and Metzgar et (2008) from seven chloroplast
al.'s
loci including rbcL, and different from Gou et (2008) rbcL relationships.
al. 's
The deduced EST
relationships from the three nuclear markers are not
consistent with each other, but the relationship of the EST__L110 agrees with
Osmunda
the rbcL relationship in the subgenera and Plenasium being
sister to
each
other.
EST
All the phylogenetic trees inferred from the three nuclear markers show
Osmunda
that Xmildei has two distinct allele types, and one identical
is to
those of O. japonica, and the other formed a monophyletic clade with those
Plenasium
of species (Figs. 4-6). suggested that O. Xmildei an
It is
is
Osmunda
intersubgeneric hybrid between the subgenera and Plenasium. The
Type B Xmildei have same EST_L058, EST_L110
alleles of O. the sequences
and EST_L258
the closest sequences to those of O. vachellii, suggesting
that O.
Xmildei is most likely derived by hybridization of O. japonica and O. vachellii
AMERICAN FERN VOLUME NUMBER
JOURNAL:
102
1 (2012)
The
(Figs. 4-6). chloroplast rbcL sequence of O. Xmildei
is identical to that of
O. vachellii (Fig. suggesting that is the maternal progenitor of O. Xmildei;
3), it
hence
O. japonica is paternal. This suggested parentage agrees with Zhang
et
and Gou who
al. (2008) et al. (2008), suggested O. vachellii as the maternal
DNA
parent, based on chloroplast and Osmunda
data
distributional
data.
banksiifolia is also very closely related to O. Xmildei, however, comparative
morphology
does not support
that O. banksiifolia a parent, because has
is
it
prominently
dentate pinnae, whereas O. vachellii and O. Xmildei (and
also O.
japonica) are both distinct with entire or somewhat serrate pinnae or pinna-
segments.
This study analyzed a sample of O. Xmildei from Shenzhen, Guangdong
(Fig. 2). It has very low spore viability and a very low offspring reproduction
even
rate in carefully controlled culture conditions (Zhang
2008;
et
al.,
J.-F.
Yang
et al., unpubl. data). Considering the low reproductive ability and a few
isolated localities in southern China, possible Xmildei
it is that O. is of
multiple origins, although no molecular evidence Osmunda
available.
is
Xruggii of eastern North America (Connecticut and USA) an
Virginia,
is
