Bull. Natl. Mus. Nat. Sci., Ser. B, 37(4), pp. 155–167, December 22, 2011

Enteromorpha-like Ulva (Ulvophyceae, Chlorophyta) Growing in the
Todoroki River, Ishigaki Island, Japan, with Special Reference toUlva meridionalis Horimoto et Shimada, sp. nov.
Rika Horimoto1, Yuka Masakiyo2, Kensuke Ichihara3 and Satoshi Shimada4,*
1

Department of Ecosystem Studies, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan2
Department of Biology, Faculty of Science, Ochanomizu University,
2–1–1 Otsuka, Bunkyo-ku, Tokyo 112–8610, Japan3
Department of Biology, Faculty of Science, Toho University, Chiba 274–8510, Japan
4
Division of the Natural/Applied Sciences, Graduate School of Humanities and Sciences,
Ochanomizu University, 2–1–1 Otsuka, Bunkyo-ku, Tokyo 112–8610, Japan* E-mail: shimada.satoshi@ohca.ac.jp(Received 10 August 2011; accepted 28 September 2011)

Abstract The nuclear-encoded ITS2 rDNA region and the plastid-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbcL) were sequenced from 47 specimens ofEnteromorpha-like Ulva collected from the Todoroki River, Okinawa Prefecture. Sequence data revealed that the samples fall into four distinct clade: Ulva linza-procera-prolifera (LPP) complex
clade (1 sample), Ulva sp. 2 clade (41 samples), Ulva sp. 3 clade (1 sample), and Ulva sp. 5 clade(4 samples). Ulva meridionalis Horimoto et Shimada sp. nov. (Ulvales, Ulvophyceae) (Ulva sp. 2)
is characterized by thalli that are: (1) simple or branched, tubular, fragile and often wrinkled; (2)up to 40 cm in height and up to 1 cm in diameter; (3) middle to yellowish green in color; (4) having
an sexual reproduction via female and male gametes, and quadriflagellate zoospores; and (5)chloroplasts variable in position and including 1–4 pyrenoids. Ulva meridionalis is distinguishedfrom species with similar thalli by branching pattern, number of pyrenoids per cell, chloroplast
position of the cells, life history, and developmental pattern.
Key words : ITS2, Japan, molecular phylogeny, morphology, rbcL, Ulva, Ulva meridionalis.

Introduction
The green algal genus Ulva, including species
previously placed in the genus Enteromorpha(monostromatic tubular thalli: Enteromorphalike Ulva) (Hayden et al., 2003), is well known
for its wide distribution in marine, freshwaterand brackish environments throughout the world(Reed and Russell, 1979; Canter-Lund and Lund,
1995; van den Hoek et al., 1995; Martins et al.,
1999; McAvoy and Klug, 2005; Shimada et al.,2007b, 2008; Ichihara et al., 2009). More than
100 species are now included in the genus (Guiry
and Guiry, 2007), of which only 20 species are
currently recognized in Japan (Ichihara et al.,2009; Yoshida and Yoshinaga, 2010).
Recently, phylogenetic and phylogeographic
relationships of Japanese Ulva growing in freshwater and brackish rivers were studied by Shimada et al. (2008). Molecular phylogenetic analyses
using nuclear encoded internal transcribed spacer(ITS) region including the 5.8S rDNA were conducted and it suggested the presence of six speices: U. flexuosa, U. linza-procera-prolifera(LPP) complex, and four unidentified species inJapanese rivers (Shimada et al., 2008). Furthermore, these four unidentified species were allcollected from southern regions of Japan, andthis suggested that the diversity of Ulva species
growing in rivers might be higher than currently

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Table 1.

Specimens of Enteromorpha-like Ulva used in this study
Accession no.

SampleLocality, collection date
ITS2

LPP complex
RH008
U. meridionalis RH001–007,
RH009–037,
RH043–047
E16
Ulva sp. 3
RH038
Ulva sp. 5
RH039–042

rbcL

Todoroki River, Ishigaki Island, Okinawa Prefecture, 2009/2/16 AB598806 AB598810
Todoroki River, Ishigaki Island, Okinawa Prefecture, 2009/2/16 AB598807 AB598812
Yoshino River, Tokushima, Tokushima Prefecture, 2000/7/18
AB298457 AB598813
Todoroki River, Ishigaki Island, Okinawa Prefecture, 2009/2/16 AB598809 AB598814
Todoroki River, Ishigaki Island, Okinawa Prefecture, 2009/2/16 AB598808 AB598811

appreciated in Japanese warm regions (Shimada
et al., 2008).
In this study, we conducted a field survey inestuary of the Todoroki River, Ishigaki Island, theRyukyu archipelago of southwest Japan, where“Ulva sp. 2” was found in Shimada et al. (2008).
We carried out phylogenetic analyses of thenuclear-encoded internal transcribed spacer 2(ITS2) region, and the large subunit of the plastid-encoded rbcL gene. In addition, to clarify thestatus of the alga that was treated as “Ulva sp. 2”
in Shimada et al. (2008), we studied the morphology of field and cultured materials and determined its life history.
Materials and Methods
Materials used in this study were collectedfrom the Todoroki River (24°22N, 124º15E),
Ishigaki Island, Okinawa Prefecture, Japan. Collection details are shown in Table 1. The specimens were transported live to Ochanomizu University for culture studies. Unigal cultures were
established from zoids.
Total DNA was extracted from field or culturematerials (Table 1) using the Dneasy Plant Mini
Kit (QIAGEN, Valencia, CA, USA) following
the protocol of the manufacturer. In this study,
we used three pairs of primers: ITS3-ITS4 for
ITS2 (Malta et al., 1999); rbcLstart-R750 and
F650-rbcLend for rbcL (Shimada et al., 2003).
Polymerase chain reaction (PCR) amplificationsof the ITS2 region and rbcL gene were run on athermocycler (Veriti® 96-Well Thermal Cycler,
Applied Biosystems, CA, USA). The profile of

the reactions for ITS2 region consisted of one
initial denaturation of 1 min at 94°C followed by
35 cycles of denaturation of 15 sec at 95°C,
primers annealing of 15 sec at 50°C and extension of 30 sec at 68°C, terminated by a final holdat 4°C; and that for rbcL gene consisted of one
initial denaturation of 1 min at 94°C followed by
45 cycle of denaturation of 15 sec at 95°C,
primers annealing of 30 sec at 50°C and extension of 60 sec at 68°C, terminated by a final holdat 4°C. The presence of the PCR-amplified products was visualized under UV light after agarose
gel electrophoresis and staining with ethidum
bromide. PCR-amplified products were cleaned
using the QIAquick PCR Purification Kit (QIAGEN).
We sequenced the ITS2 region and rbcL genefrom 47 samples (Table 1). Additional rbcL sequences were generated from 3 samples previously analyzed ITS sequences in Shimada et al.
(2008). Moreover, 45 ITS2 sequences and 52
rbcL sequences were downloaded from GenBank
and included in the alignments. The ITS2 sequences (295 bp) were aligned by eye with regard
to their secondary structure using the mFOLD
program (Zuker, 1989). The rbcL sequences(1308 bp) were aligned manually because nodeletion/insertion was detected. The alignments
are available from the last author upon request.
Three species of Ulvales, Umbraulva olivascens(Dangeard) Bae et Lee, Umbraulva amamiensis(Tanaka) Bae et Lee, and Umbraulva japonica(Holmes) Bae et Lee were included in ITS2 aligment; four species of Ulvales, Ulvaria fusca(Wittrock) Vinogradova, Ulvaria obscura var.

Enteromorpha-like Ulva (Ulvophyceae, Chlorophyta)

blyttii (J.E. Areschoug) Bliding, Umbraulva
amamiensis, and Umbraulva japonica were included in rbcL aligment as out groups in eachdataset to understand the relationships of ouralga among the genus Ulva (Ichihara et al.,2009). The maximum likelihood (ML) method
was used to construct phylogenetic trees with
PAUP* 4.0 b10 (Swofford, 2002). Regions of uncertain homology in the ITS2 alignment(105–119, 144–163) and identical sequenceswere excluded from alignment. The program
MODELTEST version 3.7 (Posada and Crandall,1998) was used to find the model of rbcL sequence evolution that best fit each dataset by ahierarchical likelihood ratio test (a 0.01). Takahashi and Nei (2000) suggested that a simple
model is better when a large number of short sequences are analyzed. Thus, we used the JC
model (Jukes and Cantor, 1969) in the ITS2
analysis. When the best sequence evolution
model was determined, ML tree searches werecarried out using estimated model parameterswith following options: starting tree obtained by
stepwise addition, and tree bisection and reconnection (TBR) branch swapping algorithm. Bootstrap values (Felsenstein, 1985) based on 100 resamplings in ML and 2000 re-samplings in MP
calculated (TBR, full heuristic search option).
We formed the secondary structure of ITS2 of“Ulva sp. 2” (RH001, NY138, NY140, E16,
C591, C593), U. flexuosa Wulfen subsp. paradoxa (C. Agardh) Kraft, and Ulva sp. 3 (RH038,
TT002) and compared the contiguous 5-side 30
nucleotide positions of helix III. Secondary
structure of ITS2 was derived by applying the
ITS2 data base (http://its2.bioapps.biozentrum.
uni-wuerzburg.de/).
Field and cultured samples of “Ulva sp. 2” including sample E16 (SAP 102989) collected at
Yoshino River, Tokushima, Tokushima Prefecture(Shimada et al., 2008) were used for morphological observations. The morphological charactersused in this study were as follows: thallus form,
texture and color, shape, size, number ofpyrenoids, and chloroplast position of the cells.
Voucher herbarium specimens are deposited in

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the Herbarium of Graduate School of Science,
Hokkaido University, Sapporo (SAP 10847108355) and the Herbarium of the National Museum of Nature and Science (TNS-AL 173588),
located at Tsukuba City, Japan.
For observations on the algal life history of“Ulva sp. 2”, we used the punching methods described by Hiraoka and Enomoto (1998) and obtained zoides. Released zoides were cultured at
22.5°C, with a 12:12 h light : dark (LD) cycle
under fluorescent light at 100 m mol m2 s1 in 30
psu artificial seawater (Sea Life, Marine Tech
Co., Tokyo, Japan) supplemented with PES
medium (Provasoli, 1968). Cultured thalli wereartificially induced to release zoids again using
the punching method, and released zoids were
cultured under the same conditions as mentioned
above.
Results
Molecular phylogenetic analyses
Maximum likelihood analysis of ITS2 sequences with the JC model produced a topology
shown in Fig. 1 (ln L2174.26303). The 47
samples from the Todoroki River were resolved
into four clades, of which 41 samples were included in “Ulva sp. 2” clade with high bootstrap
values (87 ML/96MP). The ITS2 sequences of all
41 samples were identical and there were only 0–
4 substitutions (0–1.587%) in the “Ulva sp. 2”
clade. “Ulva sp. 2” was clustered with U. flexuosa subsp. paradoxa, but the bootstrap support
was less than 50% in ML analyses and 53% in
MP analyses. One sample (RH008, SAP 108356)
was included in Ulva linza-procera-prolifera(LPP) complex clade, but the bootstrap support
was less than 50% in ML analyses and 67% in
MP analyses. The other one sample (RH038,
SAP 108357) was included in Ulva sp. 3 cladewith 100% bootstrap support in both analyses.
The remaining one clade did not include GenBank samples with which they could be identified and were thus provisionally referred to as
Ulva sp. 5 (RH039–RH042, SAP 108358). The
ITS2 sequences of these four samples of Ulva sp.

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5 were identical, and Ulva sp. 5 was sister to U.
clathratioides with high bootstrap support (90ML/97MP).
Likelihood settings of the rbcL gene from thebest-fit model (GTRGI) were selected (assumed nucleotide frequencies A0.28160, C
0.15490, G0.21690, and T0.34660). ML
analysis produced the topology shown in Fig. 2(ln L4798.80231). As in the case of ITS2
analysis, the 13 samples from the Todoroki River
were resolved into four clades: “Ulva sp. 2”
clade (nine samples), LPP complex clade (onesample), Ulva sp. 3 clade (one sample), and Ulva
sp. 5 clade (two samples). “Ulva sp. 2” clade received high bootstrap support (89 ML/94MP)
and there were only 0–1 substitutions (0–0.076%)
in this clade. “Ulva sp. 2” was sister to Hawaii
Ulva sp. OTU11 with high bootstrap support (98ML/100MP). LPP complex clade was clusteredwith U. prolifera with moderate bootstrap support (60 ML/62MP). Ulva sp. 3 was sister to
Hawaii Ulva sp. OTU6 with high bootstrap support (98 ML/98MP). Ulva sp. 5 was sister to NZ
Ulva sp. 2, U. clathratioides, and Hawaii Ulva sp.
OTU5 with high bootstrap support (97ML/99MP).
Comparison of ITS2 5-side 30 nucleotide position of helix III
There existed 0–2 hemi-CBC (Compensatory
Base Change) within “Ulva sp. 2”, 6-8 hemiCBC between “Ulva sp. 2” and U. flexuosa
subsp. paradoxa, and 2–4 hemi-CBC between“Ulva sp. 2” and Ulva sp. 3. CBC was not existed in all these cases.
Morphological observations
Field material of “Ulva sp. 2”
Well-developed thalli (Figs. 3, 4) grow up to
40 cm in height, and up to 1 cm in diameter, aremedium to yellowish green in color, fragile andbreak easily. The thalli are tubular, wrinkled especially in wider parts, and simple or branched
up to second order. In the upper basal region,
cells are 9.5–22.9 m m in length (average 14.8
m m, standard deviation (SD)2.3) and 6.1–15.9m m in width (10.5 m m1.7); in the middle region, those are 8.7–20.2 m m in length (14.5 m m1.9) and 5.4–15.6 m m in width (10.5 m m1.6);
and in the upper region, those are 8.4–20.2 m m inlength (14.0 m m2.0) and 4.8–16.6 m m in width(10.1 m m1.9). Three types of chloroplast positions were observed in the upper basal region (lieagainst a side wall, giving a lattice appearance,
30% of samples; covers the outer face of cell,
30% of samples; and both types of chloroplast
positions are mixed, 40% of samples), in the
middle region (lie against a side wall, giving alattice appearance, 19% of samples; covers theouter face of cell, 35% of samples; and bothtypes of chloroplast positions are mixed, 46% of
samples), and in the upper region (lie against aside wall, giving a lattice appearance, 27% of
samples; covers the outer face of cell, 49% of
samples; and both types of chloroplast positions
are mixed, 24% of samples) (Figs. 5–7). Eachcell includes 1–4 pyrenoids in the upper basal region (one, 49.3%; two, 47.2%; three, 3.4%; andfour, 0.1% of cells examined), in the middle region (one, 48.58%; two, 48.19%; three, 3.19%;
and four, 0.03%), and in the upper region (one,50.46%; two, 46.38%; three, 3.16%; and four,
0.03%) (Figs. 5–7). Cells in surface view arepolygonal or square (Figs. 5–7).
Cultured thalli of “Ulva sp. 2”
Both samples from Ishigaki island andTokushima Prefecture are tubular, yellowish
green, with up to second order branches, andhave rhizoidal cells bearing tubular extensions on
the inside of the cell layer in the stipe in the longitudinal sections (Fig. 8). In samples from Ishigaki Island, in the upper basal region, cells are
15.7–23.0 m m in length (19.2 m m1.6) and
9.4–16.3 m m in width (12.7 m m1.4); in the
middle region, those are 12.3–22.3 m m in length(17.5 m m2.1) and 7.7–17.4 m m in width (12.0m m1.7); and in the upper region those are
14.0–22.8 m m in length (18.3 m m1.7) and 8.5–
16.7 m m in width (11.9 m m1.6). Two or 3 typesof chloroplast positions were observed in theupper basal region (covers the outer face of cell,Enteromorpha-like Ulva (Ulvophyceae, Chlorophyta)

159

Fig. 1. Phylogenetic tree of maximum likelihood (ML) analysis inferred from the nuclear-encoded ITS2.
Numerals at internal nodes are bootstrap values 50% for 100 replicates in ML and 2000 replicates inmaximum parsimony (MP) analyses (ML/MP). * 100.

25% of samples and both types of chloroplast positions are mixed, 75% of samples), in the middle
region (lie against a side wall, giving a lattice appearance, 11.1% of samples; covers the outerface of cell, 44.4% of samples; and both types ofchloroplast positions are mixed, 44.4% of samples), and in the upper region (covers the outerface of cell, 44.4% of samples and both types ofchloroplast positions are mixed, 55.6% of samples) (Figs. 9, 10). Each cell includes 1–4
pyrenoids in the upper basal region (one, 33.5%;

two, 56.6%; three, 9.5%; and four, 0.3%), in the
middle region (one, 33.6%; two, 54.8%; three,
10.4%; and four, 1.2%), and in the upper region(one, 30.2%; two, 56.4%; three, 11%. 2; andfour, 2.1%) (Figs. 9, 10). Cells in surface vieware polygonal or square (Figs. 9, 10). In sample
E16 from Tokushima Prefecture, in the upperbasal region, cells are 17.9–23.7 m m in length(20.8 m m1.5) and 9.7–16.1 m m in width (13.5m m1.6); in the middle region, those are
15.3–22.0 m m in length (18.0 m m1.5) and 7.8–

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Rika Horimoto et al.

Fig. 2. Phylogenetic tree of maximum likelihood (ML) analysis inferred from plastid-encoded rbcL gene.
Numerals at internal nodes are bootstrap values 50% for 100 replicates in ML and 2000 replicates inmaximum parsimony (MP) analyses (ML/MP). * 100.

14.1 m m in width (10.5 m m1.6); and in theupper region those are 14.1–18.9 m m in length(16.2 m m1.2) and 8.3–14.0 m m in width (11.0m m1.4). Chloroplast covers the outer face ofcell. Each cell includes 1–4 pyrenoids in theupper basal region (one, 24.5%; two, 54%; three,
19.5%; and four, 2.0%), in the middle region(one, 19.5%; two, 68.5%; three, 11.0%; and four,1.0%), and in the upper region (one, 19.0%; two,
70.5%; three, 10.0%; and four, 0.6%). Cells insurface view are polygonal or square.
Life history of “Ulva sp. 2”
Examined thalli (n22) released biflagellate

female or male gametes (Figs. 11, 12) that displayed positive phototaxis. Female gametes are
6.60.54.20.4 m m, slightly larger than maleones that are 6.30.52.90.4 m m, so thespecies is slightly anisogamous. Female and malegametes copulated and formed zygotes thatshowed negative phototaxis. Zygotes attached tothe substratum (Fig. 14), and the first cell division occurred within 2–4 days (Fig. 15). The germinated spores developed into uniseriate filaments (Fig. 16) and gave rise to tube-like thalli(Fig. 17). Neither early longitudinal divisions ofcells nor branching was observed. Developed
sporophytes released meiospores (Fig. 13) that

Enteromorpha-like Ulva (Ulvophyceae, Chlorophyta)161

Figs. 3 and 4. Samples collected at the Todoroki River, Ishigaki Island, Okinawa Prefecture (wet habit before
pressing). 3. Holotype specimen of Ulva meridionalis (SAP 108347, RH010 in Table 1). 4. Isotype specimenof Ulva meridionalis (TNS-AL 17358, RH046 in Table 1).

showed negative phototaxis and are 9.80.6
5.40.8 m m, which are larger than both female
and male gametes. Based on these observations,this new species has a sexual reproduction via
female and male gametes, and quadriflagellate
zoospores. The developmental pattern ofmeiospores and parthenogenetic gametes was
similar to that of zygotes as mentioned above.
The phylogenetic and morphological differences between our alga and other species of Ulva
provide a basis for the establishment of a new
species, “Ulva sp. 2” as Ulva meridionalis Hori-

moto et Shimada.
Description
Ulva meridionalis Horimoto et Shimada, sp.
nov.
Alga sulsuginea thallis tubulosis. rugosis, vellubricis; thalli usque ad 40 cm in altitudine et 1
cm diametro, virides ad flavo-virentes, simplices
vel ramos ordinis usque ad secondi gerentes. Cellulae rhizoideae extensiones tubulares gerentes
intus stratum cellulas in basi thalli. Cellulae a162

Rika Horimoto et al.

Figs. 5–7. Anatomy of field materials of Ulva meridionalis. 5. Surface view of middle part of thallus (chloroplasts lie against a side wall, giving a lattice-like effect). 6. Surface view of middle part of thallus (chloroplasts cover the cells). 7. Surface view of middle part of thallus (chloroplasts lie against a side wall or cover
the cells).

Enteromorpha-like Ulva (Ulvophyceae, Chlorophyta)

163

Figs. 8–10. Anatomy of cultured materials of Ulva meridionalis. 8. Rhizoidal cells. 9. Surface view of middle
part of thallus. 10. Surface view of upper part of thallus.

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Figs. 11–13.

Rika Horimoto et al.

Reproductive cells of Ulva meridionalis. 11. Female gamete. 12. Male gamete. 13. Meiospore.

Figs. 14–17. Development of Ulva meridionalis. 14. Settled zygote. 15. Four-day-old-germling. 16. Seven-dayold uniseriate germling. 17. 20-day-old filamentous sporophyte.

facie visae speciminum nativorum irregulariterdispositae, polygonae vel quadratae, 8.4–20.2
4.8–16.6 m m inpartibus mediis et superis.
Chloroplasti variabiles in positione et 1–4
pyrenoides includentes. Gametophyta dioecia,
anisogametas biflagellatas producentia; gametae
femineae 6.60.54.20.4 m m, gametae musculae 6.30.52.90.4 m m. Sporophyta meiosporas quadriflagellatas (9.80.65.40.8 m m)
producentia.
Brackish alga with tubular, winkled or lubricous thalli; the thalli up to 40 cm in height and 1
cm in diameter, green to yellowish green, simple

or branched up to second order. Rhizoidal cells
bearing tubular extensions on the inside of cell
layer in the base of the thallus. Cells in surface
view of field samples irregularly arranged, polygonal or square, 8.4–20.24.8–16.6 m m in the
middle and upper regions. Chloroplasts variable
in position and including 1–4 pyrenoids.
Gametophytes dioecious, producing biflagellate
anisogametes; female gametes 6.60.54.2
0.4 m m, male gametes 6.30.52.90.4 m m.
Sporophytes producing quadriflagellate meiospores(9.80.65.40.8 m m).
Holotype and type locality: SAP 108347 col-

Enteromorpha-like Ulva (Ulvophyceae, Chlorophyta)

lected at Ishigaki Island, Okinawa Prefecture,
Japan by R. Horimoto (isotype, TNS-AL 17358).
Etymology: The specific epithet means southin Latin.
Japanese name: Minami-aonori
Discussion
We investigated the species diversity of Enteromorpha-like Ulva growing in the Todoroki
River, using nuclear encoded ITS2 region andcholoroplast encoded rbcL gene. Four groupings:
LPP complex clade, U. meridionalis (Ulva sp. 2)
clade, Ulva sp. 3 clade and Ulva sp. 5 clade weredetected and which showed the diversity of Enteromorpha-like Ulva species in this river ishigher than reported in Shimada et al. (2008).
Ulva meridionalis can be distinguished morphologically from other Enteromorpha-likespecies of Ulva thalli as follows. In U. pseudolinza (R.P.T.Koeman et Hoek) Hayden et al.,U. linza L., U. flexuosa subsp. linziformis (Bliding) Bliding and U. stipitata var. linzoides Bliding, the two layers are loosely adnate except for
the hollow margin (Bliding, 1963; Koeman andvan den Hoek, 1982a, b, 1984). Ulva simplex(K.L. Vinogradova) Hayden et al. mostly has spirally twisted stipe (Koeman and van den Hoek,
1982b). Ulva radiata (J.Agardh) Hayden et al.
has many proliferations, thalli of U. torta(Mertens) Trevisan and U. ralfsii (Harvey) Le
Jolis are filiform, and thalli of U. kylinii (Bliding)Hayden et al. are very long and narrow (Bliding,
1963; Koeman and van den Hoek, 1982b, 1984).
Ulva compressa L., U. intsetinalis (L.) Link var.
intestinalis Bliding, U. intsetinalis (L.) Link var.
asexualis Bliding, U. intestinaloides (R.P.T.Koeman et Hoek) Hayden et al. and U. prolifera
O.F.Müller have one perenoid per cell (90%);U. clathrata (Roth) C.Agardh, U. flexuosa subsp.
biflagellate (Bliding) Bliding, U. multiramosa
Bliding and U. aragoensis Bliding have morethan one pyrenoids per cell; and U. flexuosa
subsp. pilifera (Kützing) M.J.Wynne, U. flexuosa
Wulfen, U. flexuosa subsp. paradoxa and U. hendayensis P.J.L.Dangeard et H.Parriaud have 1–5

165

or more pyrenoids per cell (Bliding, 1963; Koeman and van den Hoek, 1982a, 1984). Ulva jugoslavica Bliding has a sexual generation withsmall isogametes and an asexual generation withzoospores and U. adriatica Bliding has germings
with early longitudinal divisions of cells (Bliding, 1963).
Recently, Kraft et al. (2010) suggested the revisions to the Australian ulvaceous flora and four
new species, U. clathratioides L.G.K.Kraft, Kraft
et R.F.Waller, U. brisbanensis L.G.K.Kraft, Kraft
et R.F.Waller, U. proliferoides L.G.K.Kraft, Kraft
et R.F.Waller and U. stenophylloides L.G.K.
Kraft, Kraft et R.F.Waller were proposed as thesynthesis of both molecular and morphological
data. Ulva meridionalis can be distinguishedmorphologically from these four species. Ulva
clathratioides and U. proliferoides are densely
and irregularly radically branched, thalli of U.
brisbanensis are dark green in color, and U.
stenophylloides is bilayered.
Chloroplasts position of U. meridionalis was
not fixed. Blomster et al. (1998) and Blomster(2000) classified the position of chloroplast into
three types: type 1, central/pressed against cell
walls/filled the cell in surface view (most speciesof Ulva); type 2, apical end of the cells/hoodshape/cap-shape (U. intestinalis and U. compressa); and type 3, mobile (only one species, U.
clathrata). Therefore, U. meridionalis is the second report of type 3 chloroplast. Ulva clathrata
is characterized by large cells and the presence ofthe large number of pyrenoids (Koeman and vanden Hoek, 1984): in upper basal region, cells are(20–) 25 (–29)(14–) 16 (–19) m m; in the middle region, those are (19–) 22 (–25)(12–) 16(–19) m m; in the apical region, those are (18–) 21(–25)(12–) 15 (–18) m m. Each cell includes
3–7 pyrenoids in the upper basal region and 2–6
pyrenoids in the middle and apical regions. On
the other hand, U. meridionalis lacks such features. These morphological differences between
U. clathrata and U. meridionalis strongly support
independent species of our new alga, Ulva
meridionalis.
Sequence analyses of the ITS2 and rbcL gene

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Rika Horimoto et al.

strongly support the independent status of U.
meridionalis and its establishment as a new
species. In the rbcL analyses, 7 bp–9 bp (0.610%–
0.696%) differences were found between U.
meridionalis and most closely related entity,“OUT 11 Ulva sp.” from Hawaii (O’Kelly et al.,2010). This amount of sequence divergence inthe rbcL gene is within the range of interspecificvariation of previous studies of Ulva (Hayden
and Waaland, 2002, 2004; Shimada et al., 2003),
as well as other ulvophyceaen genera, such as
Codium (Shimada et al., 2007a) and Caulerpa(Domis et al., 2003). The sequence divergence
between Ulva sp. 3 and “OUT 6 Ulva sp.” from
Hawaii (O’Kelly et al., 2010) is 0.439% and suggests they may be conspecific. The divergence
between Ulva sp. 5 and three closely related
species, “NZ Ulva sp. 2” (Heesch et al., 2009),
U. clathratioides and “OUT 5 Ulva sp.” (O’Kelly
et al., 2010) is 0.076–0.254% and suggests theirconspecificity. However, morphological study
and/or crossing tests will be needed to clarify
these taxonomic statuses.
It is known that 5-side 30 bp of ITS2 helix III
is highly conserved nucleotide sequences anddifference of even one CBC pairing in this region
predicts total failure of crossing (Coleman,2009). Comparison of this 5-side 30 bp is usedin the genus Chlamydomonas for species classification, although it has not used in the genus
Ulva. In this study, CBC was not existed between
U. meridionalis and both U. flexuosa subsp. paradoxa and Ulva sp. 3. However, the absence ofCBC does not show the loss of mating potential
so we can not determine the species status ofUlva meridionalis in this aspect. Also because
hybridization study is not made in this group,
further study is required for using this ITS2 region for species classification of genus Ulva.
In this study, Enteromorpha-lile species in the
Todoroki River is suggested its close relation to
the entities from Hawaii, New Zealand and Australia. These close relation may be exist in “Ulvasp. 1”, “Ulva sp. 4” (Shimada et al., 2008) andother entities in uninvestigated tropics and subtropical areas. Hybridization study added to mol-ecular and detailed morphological data are requiring for better understanding of species diversity of Ulva in these locals.
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