TAXONOMIC REEXAMINATION OF CHARA GLOBULARIS (CHARALES,

CHAROPHYCEAE) FROM JAPAN BASED ON OOSPORE MORPHOLOGY AND rbcL GENE

SEQUENCES, AND THE DESCRIPTION OF C. LEPTOSPORA SP. NOV.
1
Hidetoshi Sakayama
2,3
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku,
Tokyo 153-8902, Japan
Fumie Kasai
Microbial Ecology Section, Environmental Biology Division, National Institute for Environmental Studies,
16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Hisayoshi Nozaki
Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Makoto M. Watanabe
Institute of Biological Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan
Masanobu Kawachi
Microbial Ecology Section, Environmental Biology Division, National Institute for Environmental Studies,
16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Mikao Shigyo
Department of General Systems Studies, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku,
Tokyo 153-8902, Japan
Jun Nishihiro, Izumi Washitani
Department of Ecosystem Studies, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku,
Tokyo 113-8657, Japan
Lothar Krienitz
Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhutte 2, D-16775 Stechlin-Neuglobsow, Germany
and Motomi Ito
Department of General Systems Studies, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku,
Tokyo 153-8902, Japan
Chara globularis Thuillier (=f. globularis sensu R. D.
Wood) is a widespread species of the genus and
inhabits fresh- and brackish-water environments. In
an attempt to reexamine the taxonomic status of
C. globularis collected from Japan, we reassessed
vegetative and oospore morphology of Japanese
material and herbarium specimens originating from
Europe (including the type specimen) and con-
ducted molecular phylogenetic analyses based on
rbcL gene sequences. Although the other vegetative
morphologies were consistent with the description
of C. globularis f. globularis sensu R. D. Wood, we
identied two types of branchlets within the
Japanese materials: one has elongate end segments
(EL type), and the other has short end segments
(SH type) corresponding to the type material. More-
over, the oospore wall of the EL type was different
from that in the SH type. The oospores of the EL
type were dark brown to reddish brown and had a
spongy pattern with the pusticular elevations on the
fossa wall, whereas the fossa wall of the SH type
was black with a granulate to papillate or ne pustic-
ular pattern. In addition, our sequence data
demonstrated that these two types are separated
phylogenetically from each other. Therefore, we
1
Received 31 January 2008. Accepted 26 January 2009.
2
Author for correspondence: e-mail charales@mail.goo.ne.jp.
3
Present address: Department of Biology, Graduate School of
Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan.
J. Phycol. 45, 917927 (2009)
2009 Phycological Society of America
DOI: 10.1111/j.1529-8817.2009.00700.x
917
describe the EL type as a new species, C. leptospora
sp. nov.
Key index words: Chara; Charales; Charophyceae;
Charopsis; morphology; new species; oospores;
rbcL gene; scanning electron microscopy; tax-
onomy
Abbreviations: BI, Bayesian inference; BS, boot-
strap value; CI, consistency index; JC, Jukes
Cantor; ME, minimum evolution; ML, maximum
likelihood; MP, maximum parsimony; OTU, oper-
ational taxonomic unit; PP, posterior provability;
rbcL, the gene encoding the LSU of RUBISCO; RI,
retention index; TBR, tree-bisection-reconnection
The genus Chara (Charales, Charophyceae) is
characterized by having unforked whorled branch-
lets, in which the female reproductive organs (oogo-
nium) are positioned above the male organs
(antheridium), stipulodes occurring below the
branchlets, and one tier of coronal cells (cronula)
in the oogonia (Wood 1965, Bryant and Stewart
2002). In most species, the main axes and branch-
lets are covered with cortical cells. This genus inhab-
its fresh- and brackish-water environments and has a
worldwide distribution (Wood 1965).
C. globularis is a widespread species, and a large
number of infraspecic taxa have been recognized
(Groves and Bullock-Webster 1924, Zaneveld 1940,
Imahori 1954, Wood 1962, 1965). Wood (1962,
1965) reduced 29 taxa previously described as inde-
pendent species to infraspecic taxa of C. globularis
because he treated differences in sexuality and some
morphological characters (e.g., axial cortical cells,
spine cells, stipulodes, and oospores) as variations
within a single species. According to Wood (1962,
1965), C. globularis f. globularis can be distinguished
from other infraspecic taxa of C. globularis in
having regularly triplostichous main axes where the
primary and secondary rows of the cortical cells are
developed equally, a globular spine-cell, a corticated
and unincurved whorled branchlet with globular
posterior bract cells, and two tiers of obscure stipul-
odes. Imahori and Kasaki (1977) and Han et al.
(1994) treated C. globularis f. globularis sensu Wood
(1962, 1965) as a variety of this species (Table S3 in
the supplementary material). John et al. (1990)
examined oospore morphology of some infraspecic
taxa of C. globularis using SEM and provided some
new and taxonomically important characters,
suggesting that these infraspecic taxa should be
classied as separate species. Recent molecular
phylogenetic studies also demonstrated the distinct
status of some infraspecic taxa of C. globularis pro-
posed by Wood (1962, 1965) (McCourt et al. 1999,
Meiers et al. 1999). Based on combined analyses of
SEM oospore morphology and molecular phylogeny,
our recent taxonomic studies of the genus Nitella
demonstrated the distinct status of many species
that had been reduced to infraspecic taxa by Wood
(1965) (Sakayama et al. 2002, 2004a,b, 2005, 2006,
Sakayama 2008). However, no such combined analy-
ses have been carried out for taxonomic studies of
species within the genus Chara.
Recently, we collected 11 specimens of C. globularis
from Japan. These could be classied into the type
form of C. globularis based on the classication of
Wood (1962, 1965) (Table S3), although variations
were recognized in oospore and branchlet morpho-
logies. To reexamine the taxonomic status of
Japanese material of C. globularis, in this study, we
examined vegetative and oospore morphology in
detail and performed molecular phylogenetic analy-
ses based on rbcL gene sequences in both eld-
collected and cultured material of this alga.
Moreover, herbarium specimens originating from
Europe (including the lectotype specimen) of
C. globularis f. globularis sensu Wood (1962, 1965)
were also examined to clarify their taxonomic status.
Consequently, we found at least two distinct types
within this entity, and one of them is described as a
new species, C. leptospora sp. nov.
MATERIALS AND METHODS
Culture and morphological methods. The localities from which
material was collected are shown in Table S1 (in the
supplementary material). The methods for eld collection,
culture, and LM and SEM were essentially the same as those
in our previous studies (Sakayama et al. 2002, 2004b), except
in the following six respects. The materials analyzed in the
present study were compared with all of the taxa reduced to
C. globularis sensu Wood (1962, 1965) and several taxa
described after Wood (1965) (Table S3). The thalli of 11
samples of the SH and EL types of C. globularis [characterized
by having short (SH) and elongate (EL) end segments,
respectively] examined in this study were preserved as dried
specimens, and they were deposited in the Department of
Botany, National Science Museum, Tsukuba, Japan. Their
culture strains were also established and deposited in the
Microbial Culture Collection, National Institute for Environ-
mental Studies, Tsukuba, Japan. Because morphology of eld-
collected and cultured material is essentially consistent with
each other, morphological analyses of thallus and oospores
were conducted using both eld-collected and cultured
material. LM observations of thalli and oospores were made
using a VHX-100 microscope (Keyence, Tokyo, Japan) and a
LEICA DM LB microscope (Leica, Tokyo, Japan), respec-
tively. SEM observations of oospores were made using an
S-4800 scanning electron microscope (Hitachi, Tokyo, Japan)
at 510 kV. The terms used to describe SEM oospore
morphology were based on those of Wood (1965), John
et al. (1990), Leitch et al. (1990), and Faegri and Iversen
(1989).
Because of the absence or immaturity of the oospore in the
specimens or strains, ve strains (S023, S024, S025, S026, and
S149) were excluded from the SEM analysis. To elucidate the
correct taxonomic status of the Japanese material of
C. globularis f. globularis, the lectotype specimen of C. globularis
originating from Europe [L0054649; deposited at the National
Herbarium of the Netherlands (L), Leiden], which was
selected by J. S. Zaneveld (Dec. 15, 1939) (see Zaneveld 1940,
p. 191), and followed by Wood (1965), was examined (Table S2
918 HIDETOSHI SAKAYAMA ET AL.
in the supplementary material). Because the thallus of the
lectotype specimen lacked oospores, the European specimen
[PC0074031; deposited at the Museum National dHistoire
Naturelle (PC), Paris], which R. D. Wood annotated as the
representative material of C. globularis f. globularis, as well as the
specimen selected as the neotype, which has been super-
seded by the rediscovery of the lectotype specimen (see Wood
1965, p. 174), was used to examine the vegetative and SEM
oospore morphology and designated here as the epitype of
C. globularis to serve as an oospore of the lectotype specimen
referred to above (Table S2).
Molecular phylogenetic analysis. Preparation of total DNA,
amplication of DNA by PCR, direct sequencing of the PCR
products, and alignment of the rbcL gene sequences were
essentially as described previously (Sakayama et al. 2002,
2004a,b, 2005, 2006). Nine species of the genera Nitella and
Tolypella (Table S1) were selected as an outgroup because
recent phylogenetic studies demonstrated that the tribe Char-
eae is monophyletic within the Charales (McCourt et al. 1996,
1999, Karol et al. 2001, Sakayama et al. 2002, 2004b, Sanders
et al. 2003, Sakayama 2008). Samples representing identical
sequences were treated as a single operational taxonomic unit
(OTU). The aligned data set of the rbcL gene sequences
representing 34 OTUs (EMBL-Align database accession num-
ber ALIGN_001269) was subjected to unweighted maximum-
parsimony (MP) analysis using PAUP* 4.0b10 (Swofford 2002).
The MP trees were constructed employing a heuristic search
with the stepwise addition of 100 random replications using the
tree-bisection-reconnection (TBR) branch-swapping algorithm.
A bootstrap analysis (Felsenstein 1985) was carried out based
on 1,000 replications of the simple heuristic search (full
heuristic type with the TBR branch-swapping algorithm).
Following the guidelines for constructing a topology using
the distance method (Nei and Kumar 2000), we selected JC
distances (Jukes and Cantor 1969) to construct minimum-
evolution (ME) trees. For the same alignment used in the MP
analysis, a distance matrix was calculated using the JC method
(Jukes and Cantor 1969) in PAUP* 4.0b10. Based on a heuristic
search using the stepwise addition of 100 random replications
(with the TBR branch-swapping algorithm), ME trees were
constructed, again using PAUP* 4.0b10; the robustness of
lineages was tested by a bootstrap analysis with 1,000 replica-
tions of the simple heuristic search (full heuristic type with
TBR branch-swapping algorithm) using PAUP* 4.0b10. The
likelihood ratio test was applied to select an appropriate
substitution model in the maximum-likelihood (ML) analysis
using MODELTEST 3.06 with a default individual alpha value
of 0.01 (Posada and Crandall 1998). Using the same alignment
data, a bootstrap analysis of an ML method (with the
GTR + G + I model selected by MODELTEST 3.06, Posada
and Crandall 1998) was carried out using PAUP* 4.0b10 based
on 100 replications of the simple heuristic search (full heuristic
type with the TBR branch-swapping algorithm). Using the
same alignment data and evolutionary model as the ML
analysis, the Bayesian inference (BI) method was carried out
using MrBayes v3.0b4 (Huelsenbeck and Ronquist 2001). Four
simultaneous Markov chains were run for 1,000,000 genera-
tions, sampling every 100 generations, for a total of 10,000
trees. The rst 1,000 trees (10%) from the run were ignored as
burn-in. Based on the remaining 9,000 trees, we obtained
the posterior probabilities (PPs) for each node of the tree
using MrBayes v3.0b4.
RESULTS
Thallus morphology. The 11 Japanese strains of
C. globularis f. globularis sensu Wood (1965), newly
examined (Table S1), were monoecious and bright
to dark green (Fig. 1a). They exhibited rudimentary
stipulodes (Fig. 1d), triplostichous main axes where
the primary and secondary rows of the cortical cells
were developed equally (Fig. 1e), globular spine
cells (Fig. 1e), and corticated branchlets consisting
of eight to 10 segments where the terminal segments
lack cortical cells and consist of one or two cells
(Fig. 1, b and c). The oogonia and antheridia
formed together at the lowest one to four branchlet
nodes (Fig. 1a). The oogonia were bright yellow to
orange, about 4801,070 lm long (including cronu-
la) and 190550 lm wide (Fig. 1, f and g). The
antheridia were orange and 410532 lm in dia-
meter. These morphologies are consistent with those
of the type specimen as well as the taxonomic con-
cept of C. globularis f. globularis of Wood (1962,
1965). However, the present morphological examin-
ations of these strains identied two groups (SH and
EL types) that are different from each other in the
end segment of whorled branchlets (Figs. 1 and 2).
In the strains S001, S006, S023, S025, S026, S149,
and S151, the end segments of whorled branchlets
were short and <650 lm long (SH type) (Fig. 2, a
and b). Conversely, the remaining four strains
(S024, N01, TAN136, and S154) exhibited elongate
end segments, which were up to 3,700 lm long
(EL type) (Fig. 1, b and c). In the EL type, the
terminal two to four segments of the branchlet occa-
sionally lacked cortical cells (Fig. 1, ac). The two
European specimens (L0054649 and PC0074031),
including the lectotype specimen, had the short end
segments of the branchlet (Fig. 2c); therefore, the
SH type of Japanese material essentially corresponds
to the type material of C. globularis.
Oospore morphology. The oospores of the European
specimen (PC0074031) designated here as the epi-
type of C. globularis (Table S2) and Japanese material
consisting of six strains (S001, S006, S151, TAN136,
S154, and N01) were examined under both LM and
SEM, and their characteristics are summarized in
Table 1. Under LM, the EL type of C. globularis
(strain TAN136, S154, and N01) was distinguished
from the SH type (S001, S006, and S151) based on
the overall shape and color of oospores (Figs. 36).
Oospores of three strains of the EL type were dark
brown to reddish brown, long ellipsoid, and 560
690 lm long (Fig. 4a). Conversely, in the SH type,
oospores of the European specimen (PC0074031)
and three of the strains (S001, S006, and S151) were
black, ellipsoid, and 660775 lm long (Figs. 5a and
6a). However, the differences in oospore wall orna-
mentation between these two types were ambiguous
under LM (Fig. 3, Table 1). The oospore wall of the
EL type exhibited a nely granulate ornamentation,
whereas the SH type of the European specimen
(PC0074031) and Japanese strains (S001, S006, and
S151) had smooth to nely granulate and coarsely
granulate patterns, respectively.
CHARA LEPTOSPORA SP. NOV. 919
On the basis of the SEM observations, in contrast,
these two types were distinguished clearly from each
other, by the differences in the fossa wall patterns
(Figs. 46, Table 1). The fossa wall of the EL type
had an obscure or prominent pusticular pattern com-
posed of irregularly fused and nodulated elements
and minute openings, which formed a spongy texture
on the surface of the fossa wall (Fig. 4, bd). Con-
versely, in the SH type, oospores of the European
specimen and three Japanese strains had ne pusticu-
lar and granulate to papillate patterns, respectively,
on the fossa wall (Figs. 5, bd; 6, bd). Although the
fossa wall patterns of the European specimen
(PC0074031) were not exactly the same as those of
the Japanese strains of the SH type, both European
and Japanese plants of the SH type were clearly dis-
tinguished from those of the EL type by lacking the
spongy texture of the fossa wall (Figs. 46).
Fig. 1. Thallus of the EL type
of Chara globularis (TAN136). (a)
Part of thallus consisting of a
main axis and whorled branchlets
with reproductive organs. (b, c)
Terminal part of whorled branch-
lets, showing elongate end seg-
ments, which lack cortical cells
and consist of one or two cells.
(d) Node of the main axis, from
which whorled branchlets (above)
and rudimentary stipulodes
(below) occur. (e) Part of the
main axis with globular spine
cells and triplostichous cortical
cells, where the primary and
secondary rows of the cortical
cells are developed equally. (f, g)
Branchlet node with an oogo-
nium (above), an antheridium
(below), and bracteoles bract cells.
ES, end segments; NO, nodes; ST,
stipulodes; SC, spine cells; BR,
bracteoles.
Fig. 2. Terminal part of whor-
led branchlets of the SH type of
Chara globularis, showing short
end segments, which lack cortical
cells and consist of one or two
cells. (a, b) Strain S006. (c) Lecto-
type specimen. ES, end segments.
920 HIDETOSHI SAKAYAMA ET AL.
Table 1. Oospore morphology of the two types of Chara globularis f. globularis sensu Wood (1965) examined in this study.
SH type (=C. globularis)
EL type (= C. leptospora)
(Figs. 3a and 4; Table S1)
European specimen
(PC0074031; Figs. 3c and 6;
Table S2 in the supplementary material)
Japanese material
(Figs. 3b and 5; Table S1 in the
supplementary material)
Color Black Black Dark brown to reddish brown
Shape Ellipsoid Ellipsoid Long ellipsoid
Length 725755 660775 560690
Width 421461 420450 310410
Number of striae 1113 1213 1113
Fossa wall structure
Under LM Smooth to nely granulate Coarsely granulate Finely granulate
Under SEM Fine pusticular Irregular granulate to papillate Spongy with pusticular elevations
Ribbon-like structure Wide and ne nodular Narrow and rough nodular Wide and ne nodular
Claw-like structure Rudimentary Developed Rudimentary
Fig. 3. Oospore wall ornamentations of the EL (strain TAN136) and SH (strains S006 and PC0074031) types of Chara globularis, LM.
(a) Part of fossa wall, showing nely granulate ornamentation (TAN136). (b) Part of fossa wall, showing coarsely granulate ornamentation
(S006). (c) Part of fossa wall, showing smooth to nely granulate ornamentation (PC0074031).
Fig. 4. Oospores of the EL type of Chara globularis (TAN136) from Japan, SEM. (a) Oospore with 13 spiral ridges, from which the rib-
bon-like structures arise. (b) Part of fossa wall, showing obscure or prominent pusticular ornamentation. (c) Detail of fossa wall, showing
spongy pattern with pusticular elevations. Note the minute openings on the surface of the fossa wall form spongy pattern, from which pus-
ticular elevations arise. (d) Close view of fossa wall, showing spongy pattern with pusticular elevations. Note the pusticular elevations are
composed of irregularly fused and nodulated elements and have a depression at the tip. (e) Part of ribbon-like structure, showing ne
nodular pattern. (f) Close view of ribbon-like structure, showing ne nodular pattern. Note the nodular elements are irregularly arranged
and fused directly.
CHARA LEPTOSPORA SP. NOV. 921
Fig. 5. Oospores of the SH type of Chara globularis (S001) from Japan, SEM. (a) Oospore with 1213 spiral ridges, from which the rib-
bon-like structures arise. (b) Part of fossa wall, showing irregular granulate to papillate ornamentation. (c) Detail of fossa wall, showing
irregular granulate to papillate pattern. (d) Close view of fossa wall, showing granules or papillae on the surface of the fossa wall. Note
the granules or papillae are irregularly arranged and occasionally fused. (e) Part of ribbon-like structure, showing rough nodular pattern.
(f) Close view of ribbon-like structure, showing rough nodular pattern. Note the nodular elements are irregularly arranged and fused
directly or connected by bridge-like strands.
Fig. 6. Oospores of the SH type of Chara globularis (PC0074031) from Europe, SEM. (a) Oospore with 1112 spiral ridges, from which
the ribbon-like structures arise. (b) Part of fossa wall, showing ne pusticular ornamentation. (c) Detail of fossa wall, showing ne pusticu-
lar pattern. (d) Close view of fossa wall, showing pusticular elevations on the surface of the fossa wall. Note the elevations occasionally
have a prominent pore at the tip. (e) Part of ribbon-like structure, showing ne nodular pattern. (f) Close view of ribbon-like structure,
showing ne nodular pattern. Note the nodular elements are irregularly arranged and fused directly or connected by bridge-like strands.
922 HIDETOSHI SAKAYAMA ET AL.
Molecular phylogenetic analyses. MP analyses of the
rbcL sequences yielded 16 equally parsimonious
trees (length = 558, CI = 0.6541, RI = 0.8051), one
of which is shown in Figure 7. The phylogenetic
relationships we report here for the tribe Chareae
are essentially consistent with the previous rbcL
phylogeny (McCourt et al. 1999), except for rela-
tionships concerning the 21 strains newly analyzed
here (Fig. 7).
Of the 11 strains of the Japanese C. globularis, the
SH type strains (S001, S006, S023, S025, S026, S149,
and S151) had identical rbcL sequences and formed
a robust clade with strains F124C and X751, sup-
ported by 83% bootstrap values (BS) and 1.00 PP
Fig. 7. One of the 16 MP trees based on 1,194 base pairs (with 190 potentially parsimony-informative characters) of the rbcL genes of 29
strains representing 11 species of the genus Chara (C.); ve species of the genera Lamprothamnium (Lamp.), Nitellopsis (Npsis.), and Lychno-
thamnus (Lych.); and nine outgroup species of the genera Nitella (N.) and Tolypella (T.) (Table S1 in the supplementary material). The MP
trees were found by PAUP* 4.0b10, based on a heuristic search using the stepwise addition of 100 random replications. The tree is 558 steps
long with CI of 0.6541 and RI of 0.8051. Branch lengths are proportional to the nucleotide changes, which are indicated by the scale bar
below the tree. Numbers above branches are bootstrap values (50% or more) based on 1,000 replications of the MP (left) and ME (right)
analyses. Branches resolved with 50% or more bootstrap values (based on 100 replications) or with 0.95 or more posterior probabilities
(based on 9,000 trees) by ML (left, based on GTR + G + I mode) or BI (right, based on GTR + G + I mode) analyses, respectively, are also
shown by numbers under the branches. Samples representing identical sequences in 1,194 base pairs are treated as a single OTU. Two
subgenera, Chara and Charopsis, are shown. BI, Bayesian inference; ME, minimum evolution; MP, maximum parsimony; ML, maximum likeli-
hood; OTU, operational taxonomic unit. The 14 strains of C. globularis f. globularis sensu Wood (1965), consisting of 11 Japanese strains (SH
and EL types) newly examined and three strains previously examined, are indicated by boldface.
CHARA LEPTOSPORA SP. NOV. 923
in the MP, ME, ML, and BI analyses. However, the
rbcL sequences of the four EL type strains (S024,
TAN136, N01, and S154) were the same as that in
strain X692. The EL type strains and strain X692
were separated phylogenetically from the other
strains of C. globularis.
The species of the genera Chara and Lamprotham-
nium formed a robust clade (with 100% BS and 1.00
PP in the MP, ME, ML, and BI analyses), in which
three species of the genus Lamprothamnium resolved
as monophyletic (with 100% BS and 1.00 PP in the
MP, ME, ML, and BI analyses), whereas monophyly
of the genus Chara was supported only by the MP
analysis with 65% BS. The genus Chara was com-
posed of four lineages: (1) C. connivens, C. globularis,
C. tomentosa, C. vulgaris, and C. polyacantha; (2)
C. longifolia; (3) C. zeylanica and C. rusbyana; and
(4) C. braunii, C. australis, and C. gymnopitys. Within
lineage 1, C. connivens and C. globularis, or C. vulgaris
and C. polyacantha formed robust clades.
DISCUSSION
Taxonomic reexamination of Chara globularis.
Although the other vegetative morphologies were
not signicantly different from each other, the
SH type of C. globularis f. globularis could be clearly
distinguished from the EL type by the difference in
end segment morphology (Figs. 1 and 2). The two
European specimens (L0054649 and PC0074031)
and seven strains of the SH type of C. globularis
(S001, S006, S023, S025, S026, S149, and S151) had
a short ecorticate end segment (Fig. 2). Conversely,
the whorled branchlet of the EL type (S024,
TAN136, N01, and S154) usually had an elongate
end segment (Fig. 1, b and c).
Moreover, the EL type of C. globularis was clearly
distinguished from the SH type of C. globularis based
on the SEM oospore morphology and molecular phy-
logeny (Figs. 47, Table 1). Oospores of the SH type
strains were black and ellipsoid and had irregularly
arranged granules papillae or ne pusticular eleva-
tions on the fossa wall (Figs. 5, ad; 6, ad). Con-
versely, oospores of the EL type strains were dark to
reddish brown and long ellipsoid and had a spongy
pattern with the pusticular elevations composed of
irregularly fused and nodulated elements that formed
minute openings on the fossa wall (Fig. 4, ad). In
addition, our rbcL gene sequence data demonstrated
that the EL type strains are separated phylogenetically
from the clade composed of three lineages of
C. globularis including the SH type strains (Fig. 7).
Although C. globularis f. globularis with the EL type
of branchlets was not reported by Wood (1965)
(Table S3), C. globularis f. capensis (= C. capensis)
has elongate end segments very similar to those in
the EL type of C. globularis f. globularis (Wood and
Imahori 1964). However, the EL type strains can
be distinguished from C. globularis f. capensis by the
fossa wall patterns of oospores (John et al. 1990)
(Figs. 3 and 4). Under the LM, the EL types of
C. globularis f. globularis exhibit a nely granulate
oospore wall ornamentation (Wood and Imahori
1964, Wood 1965; Fig. 3a). Conversely, the oospore
wall ornamentation of C. globularis f. capensis is
smooth (John et al. 1990). Moreover, SEM oospore
analyses revealed the detailed differences between
these two species. The EL type strains have obscure
or prominent pusticular elevations consisting of
fused and nodulated elements on the fossa wall
(Fig. 4, bd), whereas the fossa wall of C. globularis
f. capensis has a roughened pattern formed by
dimple-like depressions, pits, and perforations
(John et al. 1990).
On the other hand, Su et al. (1990) described a
variety, C. globularis var. beijingensis, from China
(Table S3). This taxon is similar to the EL type
of C. globularis, because the terminal three to ve
segments of the branchlet lack cortical cells. How-
ever, C. globularis var. beijingensis is distinguished
clearly from the EL type of C. globularis by the differ-
ences in stipulodes and spine cells (Su et al. 1990,
Han et al. 1994; Fig. 1). In C. globularis var. beijingen-
sis, an upper tier of stipulodes is elongated, and
spine cells are developed. Conversely, the EL type of
C. globularis has rudimentary stipulodes and globular
spine cells (Fig. 1, d and e). Therefore, the EL type
strains should be established as an independent
species from C. globularis f. globularis sensu Wood
(1962, 1965) and its related taxa.
Our SEM analysis also demonstrated that oospores
of the SH type of C. globularis collected from Japan
could be distinguished from those of the European
specimen (PC0074031) by differences in the fossa
wall patterns and ribbon-like structures (Figs. 5 and 6).
Oospores of the European specimen had a ne
pusticular pattern in the fossa wall and ne nodular
elements on the ribbon-like structure (Fig. 6, bf).
Conversely, the fossa walls and ribbon-like structures
of Japanese material of the SH type had an irregular
granulate to papillate pattern and a rough nodular
pattern, respectively (Fig. 5, bf). According to Zane-
veld (1940), C. globularis [= C. globularis f. globularis
sensu Wood (1962, 1965)] is classied into two varie-
ties, var. globularis (synonym: C. hedwigii) and var.
capillaceae (synonym: C. fragilis), on the basis of the
overall appearance of thallus. If the taxonomic system
of Zaneveld (1940) is followed, both of the SH and
EL types of Japanese material can be classied into
C. globularis var. capillaceae. Based on hybridization
analyses, Proctor (1971) suggested that C. globularis
[= C. globularis f. globularis sensu Wood (1962, 1965)]
is composed of many lineages that are isolated
reproductively from each other. Therefore, further
morphological and molecular analyses of the SH
types of C. globularis, using plants collected from
localities throughout the world (especially from
Europe), are needed to fully resolve its phylogenetic
and taxonomic relationships. Because the present
study clearly demonstrated the distinct status of the
924 HIDETOSHI SAKAYAMA ET AL.
EL type of C. globularis from the SH type based on
vegetative and oospore morphology and molecular
phylogeny, the EL type of C. globularis was described
as a separate species.
In the previous taxonomic studies (Imahori 1954,
Kasaki 1964, Imahori and Kasaki 1977), C. globularis
is distributed throughout Japan. Kasaki (1964) classi-
ed two varieties of C. globularis (var. globularis and
var. hakonensis) from Japan on the basis of differences
in vegetative morphology. Furthermore, he reported
two types of var. globularis from Japan; one has a
black-colored oospore, the other exhibits a chestnut-
brown-colored oospore. These two types clearly
correspond to the SH and EL types, respectively, of
C. globularis f. globularis resolved in the present study.
Imahori (1954) also reported a large number of
morphological variations, including the two types of
oospores within C. globularis.
The EL type of C. globularis and C. connivens X214
formed a robust clade from which two other strains
of C. connivens (JRM and X774) are separated in our
phylogenetic analyses (Fig. 7). According to Wood
(1965), C. connivens (= C. globularis f. connivens) can
be clearly distinguished from the EL type of
C. globularis by having incurved whorled branchlets
with an SH type end segment and a dioecious thallus.
Moreover, SEM oospore morphology of C. connivens
is clearly different from that in the EL type of
C. globularis. In C. connivens, the oospores have a
papillate fossa wall pattern composed of elongated
projections and lack a ribbon-like structure on the
spiral ridges (John et al. 1990). Conversely, oospores
of the EL type of C. globularis exhibit an obscure or
prominent pusticular pattern on the fossa wall and
have a ribbon-like structure (Fig. 4). However, oosp-
ores of the three strains of C. connivens (X214, JRM,
and X774) have not been examined by SEM. Further
examination of SEM oospore morphology combined
with molecular phylogenetic analyses using a large
number of strains of these species of Chara through-
out the world (including the type material of
C. connivens) are necessary to construct their natural
taxonomic system.
Phylogenetic relationships of the two subgenera of
Chara. Wood (1965) subdivided the genus Chara
into two subgenera, Chara and Charopsis, based on the
differences in stipulodes and whorled branchlets. The
subgenus Chara is characterized by having two tiers of
stipulodes and corticated branchlet segments,
whereas the subgenus Charopsis has one tier of stipul-
odes and lacks cortical cells in the branchlet segments.
Meiers et al. (1999) examined the nuclear 18S
rRNA gene sequences from 29 species of the genus
Chara representing all the sections and or subsec-
tions and demonstrated that the two subgenera are
polyphyletic. Subsequently, McCourt et al. (1999)
and Sanders et al. (2003) conducted phylogenetic
analyses based on the matK and rbcL gene
sequences. In these two studies, however, the phylo-
genetic relationships between the two subgenera
were unclear because only four species of the genus
Chara were analyzed. In this study, we newly exam-
ined ve Chara species consisting of two species of
the subgenus Chara (C. tomentosa and C. zeylanica)
and three species of the subgenus Charopsis (C. aus-
tralis, C. braunii, and C. gymnopitys) based on the
rbcL phylogeny (Fig. 7).
In our rbcL phylogeny, two subgenera Chara and
Charopsis were resolved as polyphyletic (Fig. 7), sup-
porting the phylogenetic relationship inferred from
the nuclear gene sequences (Meiers et al. 1999).
However, phylogenetic relationships among species
disagree in terms of chloroplast and nuclear phylog-
enies. For example, nuclear DNA phylogeny demon-
strated the close relationship between C. tomentosa
and C. longifolia (Meiers et al. 1999). Conversely, in
our chloroplast DNA tree, C. tomentosa formed a
robust clade with C. connivens, C. globularis, C. vulgaris,
and C. polyacantha, and C. longifolia occupied the
basal position of this clade (Fig. 7). Therefore, fur-
ther molecular phylogenetic analyses using a larger
number of species of the genus Chara and more
variable DNA markers are needed to resolve the
phylogenetic relationship within the genus.
CONCLUSION
The present study demonstrated that the com-
bined analysis of the SEM oospore morphology and
molecular phylogeny is a powerful tool for recogniz-
ing natural species of C. globularis and its related
species. Although 29 infraspecic taxa have been
recognized worldwide within C. globularis by Wood
(1962, 1965) (Table S3), we analyzed only a single
form (f. globularis) of C. globularis sensu Wood
(1962, 1965) mainly collected from Japan. There-
fore, further taxonomic studies using material
collected worldwide based on SEM oospore mor-
phology combined with molecular phylogenetic
analyses must resolve natural existence of many spe-
cies that had been assigned to Chara globularis by
Wood (1962, 1965).
TAXONOMIC TREATMENTS
Chara globularis Thuillier (Fl. Env. Paris, ed. 2:
472. 1799) (Figs. 2; 3, b and c; 5; and 6).
Synonyms: C. capillacea Thuillier (Fl. Env. Paris, ed.
2, p. 474, 1799); C. fragilis Desvaux in Loiseleur-
Deslongchamps (Not. Pl. Fl. France, p. 137, 1810);
C. pulchella Wallroth (Ann. Bot., p. 184, 1815);
C. diffusa Liljeblad (Utk. Till Svensk Fl., p. 684,
1816); C. viridis Hartman (Handb. Skand. Fl., ed. 1,
p. 378, 1820); C. hedwigii C. Agardh in Bruzelius
(Obs. Gen. Char., pp. 7, 21. 1824); C. hirta Meyen
(Linnaea 2, p. 78, 1827); C. foliolata Hartman
(Handb. Skand. Fl., ed. 2, p. 297, 1832); C. fragilis
var. elongata Cosson et Germain (Fl. Env. Par.,
p. 680, 1845); C. fulcrata Ganterer (Oesterr. Char.,
p. 20, 1847); C. trichophylla Kutzing in Henriques
CHARA LEPTOSPORA SP. NOV. 925
(Contrib. Fl. Crypt. Lusit., p. 21, 1880); C. conniventi-
fragilis Hy (Soc. Bot. France, Mem. 26, p. 41, 1913);
C. globularis var. globularis (= var. hedwigii) (Zaneveld,
Blumea 4, p. 191, 1940); C. globularis var. capillacea
(Thuillier) Zaneveld (Blumea 4, p. 195, 1940);
C. globularis f. globularis (Wood, taxon 11, p. 10,
1962); C. globularis f. globularis (Wood, Monograph
of the Characeae, vol. 1, p. 172, 1965); C. globularis
var. globularis (Imahori and Kasaki, Illustrations of
the Japanese fresh-water algae, p. 767, 1977);
C. globularis var. globularis (Han et al., Flora algarum
sinicarum aquae dulcis, vol. 3, p. 237, 1994).
Lectotype: L0054649 [deposited at the National
Herbarium of the Netherlands (L), Leiden, the
Netherlands]. This lectotype specimen was originally
collected from Paris, France; selected by J. S. Zane-
veld (Dec. 15, 1939) (see Zaneveld 1940, p. 191);
and followed by Wood (1965, pp. 168 and 174).
Epitype (here designated): PC0074031 [deposited at
the Museum National dHistoire Naturelle (PC),
Paris, France]. This specimen was originally col-
lected from France. R. D. Wood (Aug. 28, 1956)
annotated this specimen as the representative mate-
rial of C. globularis f. globularis, as well as the speci-
men selected as the neotype, which has been
superseded by the rediscovery of the lectotype speci-
men (see Wood 1965, p. 174).
Japanese specimens examined in this study: TNS-AL
164551 (H. Sakayama, strain S001; collected by
H. Sakayama from Lake Tanne-to); TNS-AL 164552
(H. Sakayama, strain S006; collected by H. Sakayama
from Lake Yunoko); TNS-AL 164553 (H. Sakayama,
strain S023; collected by H. Sakayama from Lake
Nanbu-to); TNS-AL 164554 (H. Sakayama, strain
S025; collected by H. Sakayama from Lake Odanai-
numa); TNS-AL 164555 (H. Sakayama, strain S026;
collected by H. Sakayama from Lake Towada); TNS-
AL 164556 (H. Sakayama, strain S149; collected by
H. Nozaki from Lake Tsutanuma); TNS-AL 164557
(H. Sakayama, strain S151; collected by H. Sakayama
from Lake Kawaguchi) (Table S1).
Chara leptospora Sakayama, sp. nov. (Figs. 1, 3a,
and 4).
Thalli monoeci, claro-virides, ad 20 cm longi;
axibus 310630 lm diametro, internodiis ad 2.5 cm
longis, cortice triplosticho, aculeis globularibus.
Stipulodia globularia vel rudimentalia, et formantia
duo ordines. Ramuli corticati 89 in verticillo, ad
3.0 cm longi, constructi ab 89 segmentis, quorum
47 habentes corticem diplostichum; segmentum
terminale uni- vel bi-cellulare, elongatum ad
3.7 mm longum. Cellulae bracteales adaxiales et vel
bracteola 23, ad 1,100 lm longae, et cellulae
bracteales abaxiales globulares. Oogonia et antheri-
dia conjugata, formata in nodis basalibus 4 ramulo-
rum. Oogonia 4801,030 lm longa (coronulis
includentibus), 190490 lm lata et habentia 1113
strias; coronula 69107 lm longa et 133167 lm
lata. Antheridia 410450 lm diametro. Oosporae
atro-fuscae vel rufo-fuscae et ellipsoideae longe, et
habentes 1113 juga spiralia; quae 560690 lm
longa et 310410 lm lata, et 3557 lm inter fossas.
Organa taenioida orientia e quidque jugorum spira-
lium, quae 730 lm lata et bene nodulosa. Paries
fossae habens spongiosum ordinationem tumulo-
rum pusticularium obscurorum vel conspicuorum;
tumuli plerumque habentes depressionem in apice,
qui 0.51.6 lm diametro, et 0.52.7 lm inter se.
The thalli of the present materials are monoecious,
bright green, and up to 20 cm high. The axes are
310630 lm in diameter, the internodes are up to
2.5 cm long, cortexes are triplostichous, and spine
cells are globular. The stipulodes are globular or
rudimentary and in two tiers. The branchlets are
eight or nine in a whorl, up to 3.0 cm long, and com-
posed of eight to nine segments, of which the basal
four to seven segments have a diplostichous cortex;
the terminal segment is one- or two-celled, elongate,
and up to 3.7 mm long; the adaxial bract cells and or
bracteoles are two or three, up to 1,100 lm long, and
the abaxial bract cells are globular. The oogonia and
antheridia are conjoined, formed at the basal four
branchlet nodes. The oogonia are 4801,030 lm long
(including coronula), 190490 lm wide and have
1113 convolutions; the coronula are 69107 lm
long and 133167 lm wide. The antheridia are 410
450 lm in diameter. The oospores are dark brown to
reddish brown and long ellipsoid and have 1113
spiral ridges; they are 560690 lm long and 310
410 lm wide, and 3557 lm across the fossa. The rib-
bon-like structures arise from each of the spiral
ridges; they are 730 lm wide and have a ne nodu-
lated pattern. The fossa wall has a spongy pattern with
obscure or prominent pusticular elevations; the eleva-
tions usually have a depression at the apex; they are
0.51.6 lm in diameter and are located 0.52.7 lm
from each other.
Holotype: TNS-AL 164558 [deposited at the National
Science Museum (TNS), Tsukuba, Japan]. This speci-
men was collected by Y. Tanabe (Table S1) and also
maintained as the culture strain (M. Ito, strain
TAN136), which was used in Tanabe et al. (2005).
Type locality: Pond at Chiba University, Yayoi,
Inage, Chiba, Japan (Table S1).
Other specimens examined in this study: TNS-AL 164560
(H. Sakayama, strain N01; collected by J. Nishihiro
from Lake Kasumigaura, Japan); TNS-AL 164559
(H. Sakayama, strain S024; collected by H. Sakayama
from Lake Ogawara, Japan); TNS-AL 164561
[H. Sakayama, strain S154; collected by H. Sakayama
from pond at Yonago-mizutori-koen, Yonago, Tottori,
Japan (352635.52 N, 133173.12 E)] (Table S1).
Etymology: The epithet leptospora is derived from
having the narrow width of the oospore.
Habitat: The strains S024 and S154 inhabit in the
brackish water lakes, whereas the other two strains
(TAN136 and N01) are collected from the fresh-
water environments (Table S1).
926 HIDETOSHI SAKAYAMA ET AL.
We thank Prof. Masahiro Kato for the kind use of his facilities
and use of an LM, and Mr. Daisuke Ishikawa for his assis-
tance in writing the Latin diagnosis. This study was supported
in part by a Grant-in-Aid for Young Scientists (No. 18770071
to H. S.) and a Grant-in-Aid for Scientic Research
(No.19405012 and No.18370034 to M. I. and No. 17370087
to H. N.) from the Japan Society for the Promotion of
Science, and conducted as a part of the Time Capsule Project
at NIES.
Bryant, J. A. & Stewart, N. F. 2002. Order Charales. In John, D. M.,
Whitton, B. A. & Brook, A. J. [Ed.] The Freshwater Algal Flora of
the British Isles. Cambridge University Press, Cambridge, UK,
pp. 593612.
Faegri, K. & Iversen, J. 1989. Textbook of Pollen Analysis, 4th ed. John
Wiley, Chichester, UK, pp. 328.
Felsenstein, J. 1985. Condence limits on phylogenies: an approach
using bootstrap. Evolution 38:1624.
Groves, J. & Bullock-Webster, G. R. 1924. Chareae. In Groves, J. &
Bullock-webster, G. R. [Eds.] The British Charophyta, vol. 2. Ray
Society, London, pp. 129, pl. XXIXLV.
Han, F. S., Li, Y. Y., Li, Y. J., Ling, Y. J. & Wang, Y. J. 1994. Flora
algarum sinicarum aquae dulcis, vol. 3, Charophyta. Science Press,
Beijing, pp. 267 (in Chinese).
Huelsenbeck, J. P. & Ronquist, F. 2001. MRBAYES: Bayesian
inference of phylogenetic trees. Bioinformatics 17:7545.
Imahori, K. 1954. Ecology, Phytogeography and Taxonomy of the Japanese
Charophyta. Kanazawa University Press, Kanazawa, Japan, pp.
234
Imahori, K. & Kasaki, H. 1977. Class Charophyceae. In Hirose, H. &
Yamagishi, T. [Ed.] Illustrations of the Japanese Fresh-water Algae.
Uchida Rokakuho Publishing, Tokyo, pp. 761829 (in
Japanese).
John, D. M., Moore, J. A. & Green, D. R. 1990. Preliminary
observations on the structure and ornamentation of the
oosporangial wall inChara(Charales, Chlorophyta). Br. Phycol. J.
25:124.
Jukes, T. H. & Cantor, C. R. 1969. Evolution of protein molecules.
In Munro, H. N. [Ed.] Mammalian Protein Metabolism. Academic
Press, New York, pp. 21132.
Karol, K. G., McCourt, R. M., Cimino, M. T. & Delwiche, C. F. 2001.
The closest living relatives of land plants. Science 294:23513.
Kasaki, H. 1964. The Charophyta from the lakes of Japan. J. Hattori
Bot. Lab. 27:215314.
Leitch, A. R., John, D. M. & Moore, J. A. 1990. The oosporangium
of the Characeae (Chlorophyta, Charales). Prog. Phycol. Res.
7:21368.
Manhart, J. 1994. Phylogenetic analysis of green plant rbcL
sequences. Mol. Phylogenet. Evol. 3:11427.
McCourt, R. M., Karol, K. G., Casanova, M. T. & Feist, M. 1999.
Monophyly of genera and species of Characeae based on rbcL
sequences, with special reference to Australian and European
Lychnothamnus barbatus (Characeae: Charophyceae). Aust. J.
Bot. 47:3619.
McCourt, R. M., Karol, K. G., Guerlesquin, M. & Feist, M. 1996.
Phylogeny of extant genera in the family Characeae (Charales,
Charophyceae) based on rbcL sequences and morphology. Am.
J. Bot. 83:12531.
Meiers, S. T., Proctor, V. W. & Chapman, R. L. 1999. Phylogeny and
biogeography of Chara (Chlorophyta) inferred from 18S rDNA
sequences. Aust. J. Bot. 47:34760.
Nei, M. & Kumar, S. 2000. Molecular Evolution and Phylogenetics.
Oxford University Press, New York, pp. 333
Posada, D. & Crandall, K. A. 1998. Modeltest: testing the model
DNA substitution. Bioinformatics 14:8178.
Proctor, V. W. 1971. Chara globularis Thuillier (= C. fragilis Desvaux):
breeding patterns within a cosmopolitan complex. Limnol.
Oceanogr. 16:42236.
Sakayama, H. 2008. Taxonomy of Nitella (Charales, Charophyceae)
based on comparative morphology of oospores and multiple
DNA marker phylogeny using cultured material. Phycol. Res.
56:20215.
Sakayama, H., Arai, S., Nozaki, H., Kasai, F. & Watanabe, M. M.
2006. Morphology, molecular phylogeny and taxonomy of
Nitella comptonii (Charales, Characeae). Phycologia 45:41721.
Sakayama, H., Hara, Y., Arai, S., Sato, H. & Nozaki, H. 2004a.
Phylogenetic analyses of Nitella subgenus Tieffallenia (Charales,
Charophyceae) using nuclear ribosomal DNA internal tran-
scribed spacer sequences. Phycologia 43:67281.
Sakayama, H., Hara, Y. & Nozaki, H. 2004b. Taxonomic re-exami-
nation of six species of Nitella (Charales, Charophyceae) from
Asia, and phylogenetic relationships within the genus based
on rbcL and atpB gene sequences. Phycologia 43:91104.
Sakayama, H., Miyaji, K., Nagumo, T., Kato, M., Hara, Y. & Nozaki,
H. 2005. Taxonomic re-examination of 17 species of Nitella
subgenus Tieffallenia (Charales, Charophyceae) based on
internal morphology of the oospore wall and multiple DNA
marker sequences. J. Phycol. 41:195211.
Sakayama, H., Nozaki, H., Kasaki, H. & Hara, Y. 2002. Taxonomic
re-examination of Nitella (Charales, Charophyceae) from
Japan, based on microscopical studies of oospore wall
ornamentationand rbcLgene sequences. Phycologia 41:397408.
Sanders, E. R., Karol, K. G. & McCourt, R. M. 2003. Occurrence of
matK in a trnK group II intron in Charophyte green algae and
phylogeny of the Characeae. Am. J. Bot. 90:62833.
Su, W. T., Wang, Z. X. & Wang, Y. J. 1990. Some new taxa of
Charophyta in China. Acta Phytotaxon. Sin. 28:33340.
Swofford, D. L. 2002. PAUP* Phylogenetic Analysis Using Parsimony
(* and Other Methods), version 4.0b10. Sinauer Associates, Sun-
derland, Massachusetts.
Tanabe, Y., Hasebe, M., Sekimoto, H., Nishiyama, T., Kitani, M.,
Henschel, K., Munster, T., Theissen, G., Nozaki, H. & Ito, M.
2005. Characterization of MADS-box genes in charophycean
green algae and its implication for the evolution of MADS-box
genes. Proc. Natl. Acad. Sci. U. S. A. 102:243641.
Wood, R. D. 1962. New combinations and taxa in the revision of
Characeae. Taxon 11:725.
Wood, R. D. 1965. Monograph of the Characeae. In Wood, R. D. &
Imahori, K. [Eds.] A Revision of the Characeae, vol. 1. J. Cramer,
Weinheim, Germany, pp. 904.
Wood, R. D. & Imahori, K. 1964. Iconograph of the Characeae.
In Wood, R. D. & Imahori, K. [Eds.] A Revision of the Characeae,
vol. 2. J. Cramer, Weinheim, Germany, pp. IXV (Icon 1395).
Zaneveld, J. S. 1940. The Charophyta of Malaysia and adjacent
countries. Blumea 4:1224.
Supplementary Material
The following supplementary material is avail-
able for this article:
Table S1. Species and culture strains used for
the present rbcL gene phylogeny.
Table S2. Herbarium specimens of Chara globu-
laris f. globularis sensu Wood (1965) examined in
this study.
Table S3. A list of taxa that can be assigned to
Chara globularis sensu Wood (1962, 1965).
This material is available as part of the online
article.
Please note: Wiley-Blackwell are not responsible
for the content or functionality of any supporting
materials supplied by the authors. Any queries
(other than missing material) should be directed
to the corresponding author for the article.
CHARA LEPTOSPORA SP. NOV. 927