Botanical Studies (2007) 48: 117-126.

*

Corresponding author: Email: wlwang@cc.ncue.edu.tw;

Tel: 886-4-7232105, ext. 3436.

INTRODUCTION

There are six genera in the Family Characeae. Both

Chara Linnaeus and Nitella Agardh are widespread and

abundant species; both Lamprothamnium J. Groves and

Tolypella (A. Braun) A. Braun have a few species; and

both Nitellopsis Hy and Lychnothamnus (Ruprecht)

Leohardi are represented each by only one extant species.

The single extant species of Lychnothamnus, L. barbatus

(Meyen) Leonhardi, is actually a rare charophyte species

considered in decline (Casanova et al., 2003). The species

is the first charophyte declared endangered in Australia

(Garcia, 2003).

In Taiwan, freshwater algal studies have mainly

focused on microalgae (Yamagishi, 1992; Moriwaka and

Chyi, 1996; Wang and Chen, 2000; Wang et al., 2002)

and very little on macroalgae (Wu, 1999, 2001; Liu and

Wang, 2004). Reports on Characeae from Taiwan seldom

appear, except for some by Imahori (1951, 1953, 1954a,

1957) and Yang and Chiang (1978). In total, 21 species,

4 subspecies, and 10 varieties from two charalean genera

have been recorded in Taiwan (Imahori, 1951, 1953,

1954a, 1954b, 1957). Recently, observations of oospore

wall ornamentation using scanning electron microscopy

(SEM) have been used to identify the species of the

Order Charales (John and Moore, 1987; John et al., 1990;

Leitch et al., 1990; Casanova, 1991, 1997). Furthermore,

molecular phylogenetic analysis based either on the

large subunit of Rubisco (rbcL) or 18S ribosomal DNA

sequences has been carried out to resolve phylogenetic

relationships at the species or genera levels (McCourt

et al., 1996, 1999; Meiers et al., 1999). In addition,

the chloroplast-encoded atpB gene is a photosynthetic

gene that has also been used in the phylogeny of certain

algae and land plants (Lockhart et al., 1992; Wolf, 1997;

Sakayama et al., 2004a). However, Wolf (1997) and

Nozaki et al. (1999) pointed out similarities between the

divergences of the atpB gene and the rbcL gene among the

fern genera and the colonial Volvocales.

In this study, we collected three charalean algae from

southeast, northeast, and northern Taiwan, respectively.

We use SEM characters of oospores and the rbcL, atpB

gene and combine data from sequences to define the

species taxonomic relationships of our materials. Their

morphological characters are also described in detail.

MATERIALS AND METHODS

Morphological observations

Specimens were collected from southeast, northeast,

and northern Taiwan. Some specimen parts were preserved

in 95% alcohol for molecular analysis while other parts

were preserved in 4-5% formalin solution or dried as

herbarium specimens for morphological observations.

The liquid or cultural specimens were favored for detailed

examination. In LM observations, the vegetative and

reproductive structures were examined under a light

microscope at 100x and 200x magnification (Zeiss

Axioskop 2) and under a dissecting microscope (Zeiss

Stemi SV11). In SEM observations, matured oospores of

these species were cleaned before examination, and then

the tube cells were removed from the mature oospores by

a fine needle and forceps under a dissecting microscope.

Three new members of Characeae (Charales,

Chlorophyta) from Taiwan, including one endangered

monospecific genus

Jui-Yu CHOU

1

, Wei-Lung WANG

1,

*, and Jui-Sheng CHANG

2

1

Department of Biology, National Changhua University of Education, Changhua 500, Taiwan

2

Center of General Education, Yu-Da University, Miaoli 361, Taiwan

(Received September 12, 2005; Accepted May 17, 2006)

ABSTRACT

. From morphological and molecular analysis, three charalean species, Lychnothamnus barbatus

(Meyen) Leonhardi, Nitella japonica Allen, and N. inversa Imahori are recorded for the first time to the

freshwater algal flora of Taiwan. The endangered monospecific genus, Lychnothamnus (Ruprecht) Leohardi, is

the first genus recorded to the freshwater algal flora of Taiwan. In this study, we describe their morphological

characters in detail and use rbcL and atpB sequences to confirm their phylogenetic relationships.

Keywords: atpB gene; Characeae; Lychnothamnus; Nitella; rbcL gene; Taiwan.

TAxONOMy

118

Botanical Studies, Vol. 48, 2007

Selected oospores were placed for 12 h in a 10% solution

of Tween-20 detergent in water at 60¢XC (Casanova, 1991).

Then, they were sonicated for ca. 2 min (Casanova, 1997),

washed several times with distilled water, dehydrated

by Freeze Dryer (Eyela FDU-506), coated with Gold-

Paladium by a Sputter coater (Hitachi E-1010), and finally

observed with an SEM (Hitachi 2460N) at an accelerated

voltage of 20 KV. The terminology used to describe the

ornamentation patterns of charalean oospores followed

that of Wood (1965) and John and Moore (1987). Voucher

specimens have been deposited at Department of Biology,

National Changhua University of Education, Taiwan.

Phylogenetic analysis

Preparation of total DNA, amplification of DNA by the

polymerase chain reaction (PCR), and direct sequencing

of the PCR products followed the methods described by

Sakayama et al. (2002, 2004a). For phylogenetic analysis,

rbcL (1169 bp), atpB (1020 bp), and the combined

sequence data set (2189 bp) from 33 charalean operational

taxonomic units (OTUs) and three related species as

outgroup (Table 1) were each subjected to unweighted

maximum parsimony (MP) analysis using PAUP

*

4.0b10

(Swofford, 2002). The MP trees were constructed using

a heuristic search with the stepwise addition of 100

random replications and a tree bisection-reconnection

(TBR) branch swapping algorithm. Support for nodes of

the MP tree was assessed by calculating 1000 bootstrap

resamplings of the heuristic searches based on random

stepwise additions, MULTREES and TBR (Felsenstein,

1985). Based on the same alignment data, a maximum

likelihood (ML) analysis with the Hasegawa-Kishino-

Yano 85 model (Hasegawa et al., 1985) was carried out

using PAUP

*

4.0b10 to estimate "quartet puzzling support

(QPS) values," which has the same practical meaning as

bootstrap values, for internal branches of the phylogenetic

tree with 1000 "puzzling steps" (comparable to the number

of bootstrap replicates) (Strimmer and von Haeseler,

1996). We compared the pairwise distance based on the

Kimura 2-parameter method (Kimura, 1980) between the

populations of L. barbatus, N. mirabilis, and N. inversa in

Taiwan and those of other countries.

In order to test the congruence between the data sets

of the rbcL gene sequences (1169 bp) and the atpB

gene sequences (1020 bp), a partition-homogeneity test

(Farris et al., 1994) was carried out for the two data sets

based on 2000 replications of the heuristic search (with

TBR branch-swapping algorithm) using PAUP

*

4.0b10.

The P-value (0.4915) obtained here was not significant,

suggesting no conflicting phylogenetic signals between the

two data sets.

RESULTS

Morphological observations

Lychnothamnus barbatus (Meyen) Leonhardi, Lotos 13:

57, 1863. Figure 1

Synonym. Chara barbata Meyen, 1827; Charopsis

barbata (Meyen) Kutzing, 1843; Nitella barbata (Meyen)

Rabenhorst, 1847.

Specimen examined. The specimens were collected

by Liu, S. L. and Wang, W. L. from Lanyu Island (E121¢X

33¡¦ 2.10¡¦¡¦, N22¢X02¡¦ 19.38¡¦¡¦), Taitung, Taiwan, on 11 July

2003, No: NCUE-JYC-920711-1, 920711-2, 920711-3.

Diagnosis. Plants are up to 30 cm in height and light

green in color (Figure 1A). The stem axes are 550 £gm in

diameter. The internodes at the lower part of the plant are

longer than the upper ones, which are longer than branch-

lets. Spine cells are absent on the stem axes. Stipulodes are

in a single whorl and outnumber the branchlets twofold

(Figure 1B). There are 6-7 branchlets in a whorl, and each

branchlet is up to 1-3 cm high and with 2-3 articulations.

In culture, several shoots rise from one starchy bulbil at

the base of the axis. Plants are monoecious. Gametangia

arise from all the nodes of the branchlets, except at the

basal part of the whorls. At each node, one oogonium lo-

cated between two antheridia are produced (Figure 1C).

Antheridia are 200-230 £gm in diameter. The largest polar

axis (LPA) of mature oogonia are 950-1075 £gm, and the

largest equatorial diameter (LED) is 600-725 £gm. Spiral

cells show 8-10 convolutions and consist of 5 coronula

cells (Figure 1D). Oospores are brown in color, 710-850

£gm long and 500-580 £g m in width with 8-9 striae (Figure

1E). SEM observation of the mature oospore wall showed

verrucate ornamentation (Figure 1F).

Nitella japonica Allen, Bull. Torrey Bot. Club 20:

119-120, 1893. Figure 2

Synonym. Nitella furcata (Roxburgh ex Bruzelius) C.

Agardh subsp. furcata var. sieberi (A. Braun) R. D. Wood

f. japonica (Allen) R. D. Wood, 1965.

Specimen examined. The specimens were collected

by Chou, J. Y. and Wu, S. Y. from Hsinchu (E121¢X08¡¦

024¡¦¡¦, N24¢X50¡¦565¡¦¡¦), Taiwan on 21 September 2005, No:

NCUE-JYC-940921-1, 940921-2, 940921-3.

Diagnosis. Plants are greenish in color and up to

15-25 cm in height (Figure 2A). The branchlets are once-

forked with 2-3 2-celled dactyls (Figure 2B), gametangia

conjoined at all the branchlet node. Thalli are monoecious.

The antheridia are 250-280 £gm in diameter (Figure 2C),

and the oogonia are 500-600 £gm in length (incl. coronula),

400-430 £gm in width (Figure 2D). The oospores, 310-340

£gm in length, 260-300 £gm in width, are oval in face view

and have 6-7 flanged ridges (Figure 2E). Under SEM

observation, the oospore wall ornamentation makes a

papillate pattern (Figures 2F-G). The papillae extend onto

the spiral ridges and also are produced on the flanges.

Nitella inversa Imahori, Kanazawa University Press,

Japan 234 pp, 1954b. Figure 3

Synonym. Nitella furcata (Roxburgh ex Bruzelius) C.

Agardh subsp. furcata var. Sieberi (A. Braun) R. D. Wood

f. inversa (Imahori) R. D. Wood, 1965.

CHOU et al. ¡X Three new members of Characeae from Taiwan

119

Table 1. List of the charalean species or strains and related species and GenBank accession numbers used for the present

phylogenetic analysis.

Species

Strain designation and

collection information

Accession number

rbcL gene atpB gene

Chara connivens Salzmann ex A. Braun

F140, Spain

AF097161

1

AF408782

9

Lamprothamnium macropogon (A. Braun) Ophel

X695, Australia

U27534

2

AF408783

9

Nitellopsis obtusa (Desvaux in Loiseleur-Deslongchamps) J. Groves F131B, Germany

U27530

2

AF408785

9

Lychnothamnus barbatus (Meyen) Leonhardi

Croa

U27533

1

-

Aus

AF097171

1

AF408784

9

Ger

AF097172

1

-

Lanyu Island, Taiwan AY707914

3

DQ076313

3

N. gracilens Morioka

S017, Japan

AB076061

4

AB110847

5

S018, Japan

AB076062

4

AB110848

5

KINU, Japan

AB076063

4

AB110849

5

S049, Japan

AB110870

5

AB110850

5

S050, Japan

AB110871

5

AB110851

5

N. furcata (Roxburgh ex Bruzelius) C. Agardh

S003, Japan

AB076058

4

AB110842

5

S037, Japan

AB076059

4

AB110843

5

S074, Japan

AB169966

6

AB169958

6

N. inversa Imahori

S035, Japan

AB076060

4

AB110844

5

Hsinchu, Taiwan

AY804256

3

DQ119289

3

N. tumulosa Zaneveld

S058, Thailand

AB110868

5

AB110845

5

S060, Malaysia

AB110869

5

AB110846

5

N. japonica Allen

S077, Japan

AB169959

6

AB169967

6

S083, Japan

AB169960

6

AB169968

6

S077, Japan

AB169961

6

AB169969

6

Hsinchu, Taiwan

N. pseudoflabellata A. Braun

S031, Japan

AB076064

4

AB110852

5

S032, Japan

AB076065

4

AB110853

5

S016, Japan

AB076066

4

AB110854

5

N. hyalina (De Candolle) C. Agardh

S012, Japan

AB076067

4

AB110856

5

S061, unknown

AB110873

5

AB110857

5

N. spiciformis Morioka

S015, Japan

AB076068

4

AB110859

5

S055, Japan

AB110875

5

AB110860

5

N. moriokae R.D. Wood

S004, Japan

AB076069

4

AB110861

5

S052, Japan

AB110876

5

AB110862

5

Tolypella prolifera (Ziz ex A. Braun) Leonhardi

F150, France

AF097175

1

AF408787

9

Coleochaete orbicularis Pringsheim

UTEX LB 2651

L13477

7

AF408788

9

Zygnema peliosporum Wittrock

UTEX LB 45

U38701

8

AF408799

9

Klebsormidium flaccidum (Kutzing) P.C. Sliva et al.

UTEX LB 2017

L13478

7

AF408801

9

1

McCourt et al., 1999;

2

McCourt et al., 1996;

3

This study;

4

Sakayama et al., 2002;

5

Sakayama et al., 2004a;

6

Sakayama et

al., 2004b;

7

Manhart, 1994;

8

McCourt et al., 1995;

9

Karol et al., 2001;

10

Cimino and Delwiche, 2002.

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Botanical Studies, Vol. 48, 2007

Specimen examined. The specimens were collected by

Chou, J. Y. and Wu, S. Y. from Hsinchu (E121¢X08¡¦024¡¦¡¦,

N24¢X50¡¦565¡¦¡¦), Taiwan on 14 May 2004, No: NCUE-

JYC-930514-1, 930514-2, 930514-3.

Diagnosis. Plants are greenish in color and up to 8-20

cm in height (Figure 3A). Dactyls are predominantly

abbreviated and 2 (-3) celled (Figure 3B). Gametangia

are conjoined at each branchlet node, except the ultimate

node. The antheridia are 190-260 £gm in diameter

(Figure 3C), and the oogonia are 440-500 £gm in length

(incl. coronula), 300-400 £gm in width (Figure 3D). The

oospores, 302-310 £gm in length, 279-311 £gm in width, are

orbicular in face view and have 6-7 weakly flanged ridges

(Figure 3E). Under SEM observation, the oospore wall

ornamentation makes a papillate pattern (Figures 3F-G).

Molecular analyses

The topologies resolved in the rbcL-atpB combined

gene analysis were essentially the same as those in

the rbcL gene analysis (Figure 4, the tree is 767 steps

long, with CI=0.6845 and RI= 0.8328), except for some

detailed phylogenetic relationships that did not affect

our conclusion. Only one of six equally maximum

parsimonious trees (based on rbcL sequence data set¡¦s

1169 aligned characters with 271 potentially parsimony-

informative characters), was found in MP analyses, based

Figure 1. Lychnothamnus barbatus (Meyen) Leonhardi. A, Habit; B, Part of branchlets showed the single whorl stipulodes (arrow);

C, One oogonium (arrowhead) between two antheridia (arrow) arose from the node of branchlet; D, Mature oogonium arose from the

node; E, Oospore with 8-10 flanged spiral ridges under SEM observation; F, Oospore wall showed verrucate ornamentation.

CHOU et al. ¡X Three new members of Characeae from Taiwan

121

on a heuristic search using the stepwise addition of 100

random replications, and it is shown in Figure 4, in which

we show the branches supported by . 50% bootstrap

values in MP analysis and the QPS value. In our molecular

analysis, we compared the pairwise distance based on the

Kimura 2-parameter method between the populations of

L. barbatus, N. japonica and N. inversa in Taiwan and

those of other countries. All of them were below 1% in

the rbcL gene, atpB gene, and combined sequence (Table

2). In the L. barbatus pair, the pairwise distance of the

rbcL gene, atpB gene, and combined sequence between

the populations in Taiwan and Australia was 0-0.086%,

0.197%, and 0.046%, respectively. In the N. japonica pair,

no difference between the pairwise distances of the rbcL

gene, atpB gene, or the populations in Taiwan and Japan

emerged. In the N. inersa pair, the pairwise distance of the

rbcL gene, atpB gene, and combined sequence between

the populations in Taiwan and Japan was 0.086%, 0.197%,

and 0.138%, respectively.

DISCUSSION

The Characeae comprise about 80 species in the

world (Wood, 1965). A total of three genera have been

recorded in Taiwan (Imahori, 1951, 1953, 1954a, 1957;

Yang and Chiang, 1978; this study). The occurrences of

some species, such as L. barbatus, are really very rare.

After an extensive survey from Taiwan and its offshore

island, three new members of Characeae, including one

new recorded genus, Lychnothamnus, can be added to

the algal flora of Taiwan. Imahori (1954a, 1957) pointed

out that the affinities between the floras of Taiwan and

the Philippines are 61.2%. Although these countries are

not continuous geographically, from the standpoint of

phytogeography, both belonging to the Paleotropical

Floral Zone. Imahori identified those species, however,

based only on morphological characters. Taxonomic re-

examination of Characeae in Taiwan is carried out based

on SEM characters and molecular analysis. In this study,

Figure 2. Nitella japonica Allen. A, Habit; B, The 2-celled dactyl; C, The antheridium; D, The oogonium; E, Oospore with six to

seven flanged spiral ridges under SEM observation; F, Under SEM observation, the oospore wall ornamentation makes a papillate

pattern that extends onto the spiral ridges and is also produced on the flanges; G, Detail of fossa wall showed it has more than 22

papillae across the fossa.

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Botanical Studies, Vol. 48, 2007

Figure 3. Nitella inversa Imahori. A, Habit; B, The dactyl is three-celled; C, Branchlet node with conjoined antheridium (arrowhead)

and immature oogonium (arrow); D, Immature oogonia and mature oogonia (arrow); E, Oospore with six to seven flanged spiral ridges

on the surface under SEM observation; F, Part of fossa wall showed papillate pattern, with flanged spiral ridges on the surface under

SEM observation; G, Detail of fossa wall showed papillate pattern under SEM observation.

CHOU et al. ¡X Three new members of Characeae from Taiwan

123

Figure 4. One of the six equally maximum parsimonious (MP) trees based on 1169 bp in the coding regions of the rbcL gene sequence

data using 33 OTUs of the Characeae as ingroup and three related species, Coleochaete orbicularis, Zygnema peliosporum, and

Klebsormidium flaccidum as outgroup (tree length = 767 steps, CI = 0.6845, RI = 0.8328). The numbers on the branches are bootstrap

values (.50%) in MP analysis (above the branch) or QPS value (below the branch).

we found molecular analysis to also be a fast and accurate

way to identify taxonomic relationships.

Among the charalean algae, the genus Lychnothamnus

was erected based on the species Chara barbata Meyen

(1827) (Leonhardi, 1863; cf. Wood, 1965). Fossil records

make clear this genus occurred widely from the Late

Eocene to the Holocene. It was particularly divergent

and widespread in the Pliocene, but has declined since

then (Casanova et al., 2003). For the last few decades,

Lychnothamnus barbatus could not be found in Europe

or Asia, except for a population in Wallace Creek,

Queensland, Australia (McCourt et al., 1999) and one in

Lithuania (Balevicius, personal communication). It has

been enrolled in the protected list by the government

of Australia since 1997 (Garcia, 2003). Currently, L .

barbatus is considered a "Rare and Endangered Species"

in Australia (Casanova et al., 2003). Today, only five

different populations (in Croatia, Australia, Germany,

Lithuania, and Taiwan) are extant, and very few samples

are used for molecular study (McCourt et al., 1999; this

study). In this study, we found that the rbcL sequences of

L. barbatus from Taiwan are nearly identical to those of

specimens from Croatia, Australia, and Germany (data not

shown). When we compare the rbcL gene, atpB gene, and

combined sequence between the populations in Taiwan

and Australia, their sequences are closely related. The

124

Botanical Studies, Vol. 48, 2007

origin of the population in Taiwan can not be resolved

using present molecular data. We also compared the mean

oospore isopolarity index (ISI = LPA ¡Ñ 100/LED) of those

extant and fossil populations by measuring the largest

polar axis (LPA), the largest equatorial diameter (LED)

of oospores. We found the ISI index of the population

of Taiwan (ISI = 135-166) was close to the population

of Australia (ISI = 140-171), but quite different from

the population of Europe (ISI = 115-140). We suggest

the distribution of L. barbatus in Taiwan was dispersed

from Australia. Furthermore, we will try to use some

appropriate gene marker, such as the ITS regions of

nuclear ribosomal DNA, to support our hypothesis.

In this study, the materials of N. japonica agree with

the descriptions of Wood (1965) in having 2-4 forked

branchlets with 2-celled dactyl, gametangia conjoined

at all the branchlet nodes. Based on the similarity in

vegetative characters, Wood (1965) reduced N. japonica

to a form of N. furcata. However, the two species can

be clearly distinguished by differences in their oospore

wall ornamentation under SEM (Sakayama et al., 2002,

2005). The ornamentation of the former species is in

a papillate pattern (Sakayama et al., 2005; this study,

Figure 2E-G) while the later species exhibits an imperfect

reticulate ornamentation (Caceres, 1975; Mandal et al.,

1995; Sakayama et al., 2002). Though the oospore wall

ornamentation of N. japonica is similar to N. inversa,

it can be distinguished based on the number of papillae

across the fossa. The former species has more than 22

papillae across its fossa (Sakayama et al., 2005; this study,

Figure 2F) while the later species has up to 20 (Sakayama

et al., 2002; this study, Figure 3F). Additionally, we can

use phylogenetic analyses to separate these two species

(Figure 4).

The oospore wall ornamentation of N. furcata exhibits

an imperfect reticulate ornamentation (Mandal et al.,

1995; Sakayama et al., 2002), and N. inversa exhibits

papillate ornamentation (Sakayama et al., 2002).

According to Wood (1965), N. furcata f . megacarpa

(Allen) R. D. Wood (= N. megacarpa Allen) has the same

papillate ornamentation on the fossa wall as N. inversa.

However, the positioning of the antheridia in N. inversa

is unique in the infraspecific taxa of N. furcata. They are

predominantly lateral on the brachlet nodes and borne

terminally in the other taxa (Wood, 1965). Therefore,

N. inversa can be clearly distinguished from N. furcata

and its related taxa. John and Moore (1987), however,

suggested that the morphology of oospores should be used

to delineate the species of Nitella, due to its stability under

SEM observations. Both N. inversa and N. furcata exhibit

nearly identical rbcL gene sequences (Sakayama et al.,

2002). The fossa wall of N. furcata shows an imperfect

or irregular reticulate pattern formed by swollen waved

and occasionally fused ridges (Sakayama et al., 2002),

but the fossa wall of N. inversa has a papillate pattern,

with flanged spiral ridges on the surface under SEM

observation. The algae are clearly distinguished from each

other in oospore wall ornamentation, as in the position of

antheridia on the branchlet nodes (Wood, 1965; Sakayama

et al., 2002). The characteristics of N. inversa collected

from Taiwan

agreed well with those morphological

characteristics, and we suggest that they are different

species.

Many charophyte species have declined in response

to the changes in their habitats (Moore, 1991; Blindow

and Langangen, 1995; Simons and Nat, 2000). Given

the apparent pattern of decline in this family and genus,

further decline could be prevented by conserving the

natural water-regimes of the extant populations. Secondly,

conservation strategies based on protection of endangered

habitat and ecosystems are likely to benefit these species,

as they have L. barbatus in its current habitat in Australia

Acknowledgments. The authors are grateful to Mr.

Sheng-Yu Wu and Mr. Shi-Qiang Liu for their help in

field collections. Sincere thanks to Dr. Ausrys Balevicius,

Institute of Ecology, Vilnius University, Lithuania for

valuable information on the distributions of L. barbatus in

Lithuania. This study was supported by Academic Sinica

Grant APEC II (Asia Paleo-Environment Changes II) and

National Science Council Grant NSC 92-2611-M-018-001.

LITERATURE CITED

Allen, T.F. 1893. Notes on new Characeae. Bull. Torrey Bot.

Club 20: 119-120.

Blindow, I. and A. Langangen. 1995. Lamprothamnium

papulosum (Wallr.) J. Groves, a threatened charophyte in

Scandinavia. Cryptogamie Algologie 16: 47-55.

Caceres , F.J. 1975. Novedades carologicas Argentinas I. Una

nueva especie de Nitella y tres adiciones al genero para la

flora Argentina. Kurtziana 8: 105-125.

Cas an ova, M. T. 1 991. An S E M st udy of de vel opm enta l

Table 2. Pairwise distance between the populations in Taiwan and other countries of L. barbatus, N. japonica, N. inversa in the

rbcL (1169 bp), atpB (1020 bp) genes and combining (2189 bp) sequence. Each number indicates the absolute distances and

sequence divergence in parentheses based on Kimura 2-parameter method (%).

L. barbatus

N. japonica

N. inversa

rbcL gene

0-2 (0-0.086)

0 (0)

1 (0.086)

atpB gene

2 (0.197)

0 (0)

2 (0.197)

Combining sequence

1 (0.046)

0 (0)

3 (0.138)

CHOU et al. ¡X Three new members of Characeae from Taiwan

125

variation in oos pore wall ornamentation of three Nitella

species (Chrophyta) in Australia. Phycologia 30: 237-242.

Casanova, M.T. 1997. Oospore variation in three species of

Chara (Charales, Chlorophyta). Phycologia 36: 274-280.

Casanova, M.T., A. Garcia, and M. Feist. 2003. The ecology and

conservation of Lychnothamnus barbatus (Characeae). Acta

Micropalaeontologica Sin. 20: 118-128.

Cimino, M.T. and C.F. Delwiche. 2002. Molecular and

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