Bull. Natl. Mus. Nat. Sci., Ser. B, 40(1), pp. 1–13, February 20, 2014

Species Diversity of the Genus Ulva (Ulvophyceae, Chlorophyta)
in Japanese Waters, with Special Reference toUlva tepida Masakiyo et S. Shimada sp. nov.
Yuka Masakiyo* and Satoshi Shimada
Division of the Natural/Applied Sciences, Graduate School of
Humanities and Sciences, Ochanomizu University,
Otsuka 2–1–1, Bunkyo-ku, Tokyo 112–8610, Japan* E-mail: masakiyo.yuka@gmail.com(Received 21 October 2013; accepted 18 December 2013)

Abstract In order to understand the exact species diversity of Japanese Ulva and Umbraulva(Ulvophyceae, Chlorophyta), nrITS2 region was sequenced from 164 specimens of Ulva andUmbraulva collected in Japanese waters from deep seawater around 30 m depth, coastal areas,
brackish water to freshwater. Phylogenetic analysis revealed that Japanese Ulva fall into 29
distinct clades that composed of 17 known species and 12 species possessing novel sequences.
Japanese Umbraulva was classiﬁed into four species, two of which possessed novel sequences.
Ulva sp. 5 was morphologically similar to Ulva clathratioides Kraft et al. in gross morphology andnumber of pyrenoids in cells, and there was only 1 bp difference between them. Ulva sp. 12 hadmembranous thalli with rhizoids extending the outside on the cell layer in the basal portion, acharacteristic reported only from Ulva sublittoralis Segawa. Ulva tepida Masakiyo et S.Shimada
sp. nov. (Ulva sp. 3) is characterized by tube-like thalli with simple or radial branching in the basal
region, chloroplasts covering the outer wall of cells and having 1–5 pyrenoids (one, 15.6%; two,
54.1%; three, 25.9%; four, 3.7%; and ﬁve, 0.7%) in the middle region.
Key words : ITS2, Japan, molecular phylogeny, morphology, Ulva, Ulva tepida.

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., 2009a). One hundred and ninety-one species are now included in
the genus (Guiry and Guiry, 2013), of which 21
species are currently recognized in Japan(Horimoto et al., 2011).

Many molecular phylogenetic studies of thegenus Ulva over the decade have been conducted
all over the world (Shimada et al., 2003, 2007a,
2007b, 2008; Horimoto et al., 2011; Loughnane
et al. 2008; Kraft et al., 2010; OʼKelly et al.,2010; Mareš et al., 2011; Wolf et al., 2012). Several of these reports indicated that several undescribed Ulva species exist not only in the Japanese waters including brackish and freshwater
habitats but also in the world (Shimada et al.,2007a, 2008; OʼKelly et al., 2010; Horimoto etal., 2011; Wolf et al., 2012).
The genus Ulva is one of the best members forstudying molecular mechanisms of adapted evolution, because Ulva includes closely related species distributed in different environmental condi-

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Yuka Masakiyo and Satoshi Shimada

tions. Recently, Ichihara et al. (2009b) isolated
genes potentially involved in adaptation or tolerance to freshwater conditions in a freshwater
Ulva species, U. limnetica Ichihara et Shimada.
Li et al. (2012) reported RNA-seq analysis using
Ulva prolifera O.F.Müller under low light intensity and low temperature condition. Zhang et al.
(2012) sequenced the transcriptome of Ulva linza
L. under low temperature, high temperature, highlight, high salt and UV-B stress. Moreover, Xu etal. (2012) reported that both C3 and C4 cycles
may function under normal conditions in U. prolifera, and C4 photosynthesis may play a moresigniﬁcant role under stress conditions.
The genus Umbraulva (Ulvales, Ulvophyceae)
is characterized as lacking lutein but possessing
siphonaxanthin (Bae and Lee, 2001). The special
photosynthetic pigment, siphonaxanthin, canabsorb green light (maximum absorption:
540 nm) (Kageyama et al., 1977), and the habitatof Umbraulva species is restricted to deep seawater (Bae and Lee, 2001). Umbraulva is alsointeresting in terms of adapted evolution fromshallow to deep seawater habitats, concerted withthe evolution of photosynthetic pigment.
In this study, to understand species diversity
for Ulva and Umbraulva in Japanese waters,from different habitats in deep seawater around
30 m depth, brackish water to freshwater as wellas coastal area, we carried out phylogenetic analysis of the nuclear-encoded internal transcribedspacer 2 (nrITS2). Moreover we conducted morphological observations of Ulva sp. 3, Ulva sp. 5
and Ulva sp.12 to clarify their taxonomical status.
Materials and Methods
Materials used in this study were collectedfrom various localities in Japan between 1999
and 2012. Collection details are shown in Table
1. Total DNA was extracted by using the DNeasy
Plant Mini Kit (QIAGEN, Valencia, CA, USA)
following the protocol of the manufacturer. The
region selected for PCR ampliﬁcation and automated sequencing was the nrITS2 region. The
following primer pairs were used: ITS3

(5′-CTCTCAACAACGGATATCT-3′)-ITS4(5′-TCCTCCGCTTATTGATATGC-3′) or -U26SR (5′-TGATATGCTTAAGTTCAGC-3′) (Coat etal., 1998; Malta et al., 1999; Shimada et al.,2007a).
PCR ampliﬁcation was run on a thermocycler(Veriti® 96-Well Thermal Cycler, Applied Biosystems, CA, USA) and the proﬁle of the reactions consisted of one initial denaturation of 1
min at 95°C followed by 45 cycles of denaturation of 15 sec at 95°C, primers annealing of
15 sec at 50°C and extension of 90 sec at 68°C,
terminated by a ﬁnal hold at 4°C. The presenceof the PCR-ampliﬁed products was visualized
under UV light after agarose gel electrophoresisand staining with ethidium bromide. PCR-ampliﬁed products of ITS2 region were cleaned using
the QIAquick PCR Puriﬁcation Kit (QIAGEN).
We sequenced the nrITS2 region from 191
samples collected in this study and downloaded
from GenBank (Table 1). The ITS2 sequences(336 bp) were aligned by eye with their secondary structure using the mFOLD program (Zuker,1989). The maximum likelihood (ML) analysis
was performed by Treeﬁnder version October
2008 (Jobb et al., 2004) and then bootstrap value
was conducted with 1000 replicates. The best-ﬁt
models for each partition were selected using
Akaike Information Criterion (Akaike, 1974) byusing Kakusan 4 (Tanabe, 2011). The Jobb 2008
model (J2) with gamma distribution (J2-Gamma)
was selected.
Field samples of Ulva sp. 3, Ulva sp. 5 andUlva sp. 12 and cultured samples of Ulva sp. 3
collected from Kanagawa Pref. and Ulva sp. 5collected from Chiba Pref. were used for morphological observations. The morphologicalcharacters used in this study were as follows:
thallus form, texture and color; and shape, size,
number of pyrenoids and chloroplast position of
the cells. Voucher herbarium specimens aredeposited in the phycological herbarium of the
National Museum of Nature and Science (TNS).
To understand the life history of Ulva sp. 3
and Ulva sp. 5, we used the punching methods
described by Hiraoka and Enomoto (1998), andUlva tepida Masakiyo et S. Shimada sp. nov.
Table 1.
SpeciesUlva arasakii
Ulva armoricana

Specimens of Ulva and Umbraulva used in this study
Sample#63Locality, collection date

Shizugawa, Miyagi Prefecture, Japan, 1999/5/11
Brittany, France#33
Miyajima, Hiroshima Prefecture, Japan, 1999/7/10
Ulva brisbanensis
Brisbane River in the city center of Brisbane, Australia, (R)Ulva californica
La Jolla, California, USA.
MK-77
Mikawa Bay, Tahara, Aichi Prefecture, Japan, 2005/7/8
Ulva clathrata
Backstrand, Tramore, Ireland
Ulva clathratioides
Yacht and Anglersʼ Club, Williamstown, Australia
Ulva compressa
Portaferry, Strangford Lough, Northern Ireland
NY053
Hakodate, Hokkaido Prefecture, Japan, 2004/4/30
Ulva cylindraceae
ScotlandUlva fasciata
China#1Usa, Kochi Prefecture, Japan, 1999/7/1
Ulva ﬂexuosa#C47
Oshoro, Hokkaido Prefecture, Japan, 2000/6/25
Ulva ﬂexuosa ssp. ﬂexuosa
SwedenUlva ﬂexuosa ssp. paradoxa
Czech Republic
Ulva ﬂexuosa ssp. Pilifera
Czech Republic
Ulva intestionalis
Karlskrona, Sweden
NY091
Utoro, Hokkaido Prefecture, Japan, 2004/8/15
Ulva intestinaloides
Dunbar, EastLothian, Scotland
Ulva lactuca
Sweden#C208
Nemuro, Hokkaido Prefecture, Japan, 2002/4/19
Ulva limnetica
P36
Omoto River, Ishigaki Island, Okinawa Prefecture, Japan,
2005/5/10 (R)Ulva linza
Ythan, Estuary, Aberdeenshire, Scotland
NY112
Ishikari, Hokkaido Prefecture, Japan, 2004/8/19
Ulva lobata
Newport. OR, USA.
Ulva meridionalis
RH010
Todoroki River, Ishigaki Island, Okinawa Prefecture, Japan,
2009/2/16 (R)Ulva ohnoi
KA43Tosa, Kochi Prefecture, Japan, 2000/2/12
Ulva pertusa
China
#12
Usa, Kochi Prefecture, Japan, 1997/5/1
Ulva prolifera
Ythan, Estuary, Aberdeenshire, Scotland
E21
Shimanto, Kochi Prefecture, Japan, 2001/2/26 (R)Ulva proliferoides
Lighthouse Reef, Point Lonsdale, Australia
Ulva reticulata
South Korea
6_2,6_3,6_6
Azaou, Kume Island, Okinawa Prefecture, Japan, 2006/6/13
Ulva rigida
Skara Brae, Orkney, Scotland
Ulva scandinavica
Seal Rock, Oregon, USA.
Ulva spinulosa#54
Fubenhama, Kochi Prefecture, Japan, 1999/4/29
Ulva stenophylla
Seattle, WA, USA.
Ulva stenophylloides
Lighthouse Reef, Point Lonsdale, Australia
Ulva taeniata
Seal Rock, Oregon, USA.
Ulva tanneri
South Point Cabrillo, Monterey
Kobe, Hyogo Prefecture, Japan, 2000/5/25
Ulva sp. 1
TT005,006 (SAP 102990,102991) Ara River, Onna, Okinawa Prefecture, Japan, 2005/5/10 (R)TT013 (SAP 102992)
Fukuchi River, Higashi, Okinawa Prefecture, Japan,
2005/5/10 (R)my922,923
Shin River, Onna, Okinawa Prefecture, Japan, 2012/5/16(R)
Ulva sp. 2
NY117 (SAP 100136)
Rumoi, Hokkaido Prefecture, Japan, 2004/8/19
Ulva sp. 3
TT001 (SAP 102993)
Hohtoku River, Tomigusuku, Okinawa Prefecture, Japan,
2005/5/9 (R)TT012 (SAP 102995)
Kise River, Nago, Okinawa Prefecture, Japan, 2005/5/10(R)
TT017 (SAP 102996)
Ufuguimu River, Motobu, Okinawa Prefecture, Japan,
2005/5/10 (R)TT002 (SAP 102994)
Yuhhi River, Naijou, Okinawa Prefecture, Japan, 2005/5/9(R)
RH038 (SAP 108357)
Todoroki River, Ishigaki Island, Okinawa Prefecture, Japan,
2009/2/16 (R)my167,173,663,713,721,734,939, Enoshima Island, Fujisawa, Kanagawa Prefecture, Japan,
940,964,965,974,975,978,980, 2011/2/21, 8/3, 10/13, 2012/9/31
1007–1009
my860,862
Onna, Okinawa Prefecture, Japan, 2012/3/9
my829
Kyann, Okinawa Prefecture, Japan, 2012/3/8
my915
Yagachi, Okinawa Prefecture, Japan, 2012/5/17
my828
Kyann, Okinawa Prefecture, Japan, 2012/3/8
Ulva sp. 4
NY131–133,145,my817,818,890 Small stream, Ohdo, Itoman, Okinawa Prefecture, Japan,(SAP 102997)
2004/9/28, 2012/3/8, 5/16 (R)Accession no.
AB097650
AB097660
AB097661
EU933972
AY260560
AB280867
AJ234307
EU933967
AJ234302
AB275827
AJ234308
AB735264
AB097663
AB097644
HM447564
HM447561
HM447579
AB097643
AB275836
AJ234303
AJ234311
AB097652
AB425969
AJ000203
AB275804
AY260563
AB598807
AB116934
AB735262
AB097653
AJ234304
AB298320
EU933975
AB735368
AB904759
AJ234319
AJ234318
AB097666
AY260569
EU933977
AJ234320
AY422519
AY260556
AB298454
AB298456
AB298456
AB275846
AB298462(TT001)(TT001)
AB298463
AB598809
AB904766(my1008)
AB298463
AB298463
AB904767
AB298464

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Yuka Masakiyo and Satoshi Shimada
Table 1.
SpeciesUlva sp. 5

Ulva sp. 5
Ulva sp. 6
Ulva sp. 7
Ulva sp. 8
Ulva sp. 9
Ulva sp. 10
Ulva sp. 11
Ulva sp. 12
Umbraulva amamiensis
Umbraulva japonica
Umbraulva olivascens
Umbraulva sp. 1

Umbraulva sp. 2

SampleRH039–042 (SAP 108358)

Continued
Locality, collection dateTodoroki River, Ishigaki Island, Okinawa Prefecture, Japan,
2009/2/16 (R)my690,693,696
Flower park, Minamibousou, Chiba Prefecture, Japan,
2011/9/9
my807
Kise River, Okinawa Prefecture, Japan, 2012/3/9 (R)Tenjin Island, Yokosuka, Kanagawa Prefecture, Japan,
my876–883
2012/4/24
C740
Iheya Island, Okinawa Prefecture, Japan, 2007/2/14
my867,868
Yagachi, Okinawa Prefecture, Japan, 2012/3/10, 5/17
NY056 (SAP 100110)
Shinori, Hakodate, Hokkaido Prefecture, Japan, 2004/4/30
zk1_4,57,59,96,98,101,113,119 offshore, Mage Island, Kagoshima Prefecture, Japan,(SAP 106391,106392)
2007/3/7, 2008/5/15, 7/23, 7/24 (D)zk19,20,55,58,69,76,90,94,111 shore, Mage Island, Kagoshima Prefecture, Japan,(SAP 106393, 106394)
2007/12/4, 2008/5/15, 6/11, 7/23, 7/24 (D)zk1_8,3_1,3_2,3_4,4_1,4_2,4_ offshore, Mage Island, Kagoshima Prefecture, Japan,
3,5,38–43 (SAP 106389,106390) 2007/3/7, 2008/4/24 (D)zk1_3,56,74,92,114 (SAP
offshore, Mage Island, Kagoshima Prefecture, Japan,
106385,106386)
2007/3/7, 2008/5/15, 6/11, 7/23, 7/24 (D)#C100
Kaifu, Tokushima Prefecture, Japan, 2000/6/1#C14
Shimoda, Shizuoka Prefecture, Japan, 2000/3/13
Carna, County, Galway, Ireland
zk2,6_1,6_2,6_3,6_4,14,15_1,15_
2,22–37,48,49,51,53,64,67,70,71, offshore, Mage Island, Kagoshima Prefecture, Japan,
75,81,83,84,86,88,89,129,133 2007/3/7, 7/9,10, 12/4,5, 2008/4/24, 5/15, 6/11, 7/24 (D)(SAP 106387,106388)
tateyama, K70,71
Tateyama, Chiba Prefecture, Japan, 2011/7/29

Accession no.
AB598808
AB904758(my690)(my690)AB904760
AB904761
AB281135
AB904762
AB904763
AB904764
AB904765
AB097640
AB097638
AJ234322
AB904756
AB904757

Results2–15). In this study, Ulva sp. 1 in Shimada et al.
(2007a) was renamed to Ulva sp. 8. Ulva spp.
1–7 possess Enteromorpha like tubular thalli(Figs. 2–8), and Ulva spp. 8–12 have two layered
membranous thalli (Figs. 9–13), and Umbraulva
spp. 1–2 were olive green in color (Figs. 14, 15).
Among the 14 undescribed species, Ulva spp. 1,
3, 4 and 5 (four species) were collected fromriver habitats (Ulva spp. 3, 5 grow in the sea aswell as river), and Ulva spp. 9, 10, 11, 12 andUmbraulva sp. 1 were distributed in deep seawater (Table 1). There was 1 bp difference between
Ulva sp. 5 and U. clathratioides (from Australia).
The 28 samples were included in Ulva sp. 3
clade with 98% bootstrap support, and there were
1–2 bp differences in this clade.

Molecular phylogenetic analyses
The maximum likelihood tree of nrITS2
sequences with the J2-Gamma model is shown in
Fig. 1. Sequence divergences ranged from 0.4%
among conspeciﬁc samples to 26.1% among themost distantly related species. Fifteen of our
nrITS2 sequences did not match GenBank data
and were thus provisionally referred to as anundescribed Ulva or Umbraulva species (Figs.

Morphological observations
Ulva sp. 3
Field material: Well-developed thalli from
Kanagawa Pref. (Fig. 16) grow up to 7.3 cm inheight, and up to 2.0 mm in diameter, are lightgreen in color. The thalli are tubular, and radiallybranch in base (Fig. 17). While, well-developed
thalli from Okinawa Pref. (Fig. 18) grow up to
11.5 cm in height, and up to 8.0 mm in diameter,

obtained zoids. Released zoids were cultured at
22.5°C with a 12 : 12 h light : dark (LD) cycle
under ﬂuorescent light at 100 μmol m−2 s−1 in
Provasoli Enriched Seawater (PES) medium(Provasoli, 1968) using 30 psu artiﬁcial seawater(Sea Life, Marine Tech Co., Tokyo, Japan).
Cultured thalli were artiﬁcially induced to release
zoids again using the punching method, andreleased zoids were cultured under the same
conditions as mentioned above. When the thalliof Ulva sp. 5 glowed up to around 3 cm,
we changed light intensity from 50 or
200 μmol m−2 s−1. After two weeks, the chloroplast position of the cells was observed.

Ulva tepida Masakiyo et S. Shimada sp. nov.

Fig. 1. Phylogenetic tree of maximum likelihood (ML) analysis inferred from the nuclear-encoded ITS2.
Numerals at internal nodes are bootstrap value＞50% for 1000 replicates.

5

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Yuka Masakiyo and Satoshi Shimada

Figs. 2–15. Undescribed Ulva and Umbraulva species from various localities in Japan (wet habit before press).
2. Ulva sp. 1 (my923 in Table 1). 3. Ulva sp. 2 (NY117 in Table 1). 4. Ulva sp. 3 (my978 in Table 1). 5. Ulva
sp. 4 (my890 in Table 1). 6. Ulva sp. 5 (my690 in Table 1). 7. Ulva sp. 6 (C740 in Table 1). 8. Ulva sp. 7(my868 in Table 1). 9. Ulva sp. 8 (NY056 in Table 1). 10. Ulva sp. 9. 11. Ulva sp. 10. 12. Ulva sp. 11. 13.
Ulva sp. 12. 14. Umbraulva sp. 1. 15. Umbraulva sp. 2 (K71 in Table 1).

are light to yellowish green in color. The tubular
thalli gradually widen from basal region to upper
region. In the upper basal region, cells are(10.0)–14.5±0.8–(20.3) μm in length and (6.4)–
10.6±0.7–(16.0) μm in width; in the middle
region, those are (10.9)–14.9±0.4–(19.8) μm inUlva tepida Masakiyo et S. Shimada sp. nov.

7

Figs. 16–21. Field material of Ulva tepida sp. nov. (Ulva sp. 3). 16. Holo-type specimen collected at EnoshimaIsland, Fujisawa, Kanagawa Prefecture, Japan (my1008: wet habit before press). 17. The base region. 18.
Sample collected at Onna, Okinawa Prefecture, Japan (my862: wet habit before press). 19. Surface view of theupper basal region of thallus. 20. Surface view of the middle region of thallus. 21. Surface view of the upper
region of thallus.

length and (7.0)–11.0±0.8–(16.1) μm in width;and in the upper region, those are (9.9)–14.3±0.7–(22.2) μm in length and (6.4)–
9.9±0.6–(13.1) μm in width. Chloroplasts cover
the outer wall of cells. Each cells include 1–5
pyrenoids in the upper basal region (one, 19.2%;
two, 40.8%; three, 27.5%; four, 10.8%; and ﬁve,1.7%), in the middle region (one, 15.6%; two,
54.1%; three, 25.9%; four, 3.7%; and ﬁve, 0.7%)
and in the upper region (one, 10.5%; two, 47.0%;
three, 33.5%; four, 8.5%; and ﬁve, 0.5%) (Figs.
19–21). In the upper and middle region, cells insurface view are rectangular or square. In theupper basal region, some cells are elliptical inshape.
Cultured thalli: Well-developed cultured thalli
grow up to 10.5 cm in height, and up to 0.5 mm
in diameter and are medium green in color (Fig.
22). The main axis radially branches from basal

region to middle region. Branchlets sometimes
branch again. Main axis is covered with many
short branchlets from basal region to upper
region (Fig. 23). Rhizoidal cells bear tubular
extensions on the inside of the cell layer in thestipe in the longitudinal sections (Fig. 24). In theupper basal region, cells are (14.9)–20.4±2.9–(32.4) μm in length and (5.0)–10.6±0.6–(20.1) μm in width; in the middle region, thoseare (13.6)–17.6±1.8–(22.4) μm in length and(9.1)–13.8±1.1–(18.6) μm in width; and in theupper region, those are (10.3)–17.2±1.2–(27.0) μm in length and (7.6)–12.3±1.5–(16.5) μm in width. Chloroplasts cover the outerwall of cells. Each cell includes 1–5 pyrenoids inthe upper basal region (one, 8.4%; two, 37.0%;
three, 41.2%; four, 12.2%; and ﬁve, 1.2%), in the
middle region (one, 15.1%; two, 55.0%; three,
27.5%; four, 1.9%; and ﬁve, 0.5%), and in the

8

Yuka Masakiyo and Satoshi Shimada

Figs. 22–33. Cultured sample of Ulva tepida sp. nov. (Ulva sp. 3). 22. Fifteen weeks cultured thallus. 23.
Branchlets of main axis of Fig. 22. 24. Transverse section of basal part of thallus, showing rhizoidal cells bearing tubular extensions on the inside of the cell layer. 25. Surface view of the upper basal region of thallus. 26.
Surface view of the middle region of thallus. 27. Surface view of the upper region of thallus. 28. Biﬂagellate
zoid. Allows indicate ﬂagella. 29. Settled zoid. 30. Five-day-old-germling. 31. Ten-day-old uniseriate germling. 32. Twenty-Seven-day-old erect ﬁlaments. 33. Blanching thallus cultured for 45 days.

Ulva tepida Masakiyo et S. Shimada sp. nov.

9

Figs. 34–38. Ulva clathratioides (Ulva sp. 5 ). 34. The base region of Fig. 6. 35, 36. Surface view of the middle
region of thallus collected from Chiba Pref. (Fig. 35) and Okinawa Pref., Japan (Fig. 36). 37, 38. Surface viewof the middle region of thallus cultured under ﬂuorescent light at 50 μmol m−2 s−1 (Fig. 37) and 200
μmol m−2 s−1 (Fig. 38).

upper region (one, 14.8%; two, 50.2%; three,
32.8%; four, 2.2%; and ﬁve, 0%) (Figs. 25–27).
Cells in surface view are rectangular or square.
Life history: Examined thalli (n＝6) released
2-ﬂagellate zoids (Fig. 28) that showed negative
phototaxis and are 8.7±0.8×4.6±0.6 μm.
These zoids attached to the substratum (Fig. 29),
and the ﬁrst cell division occurred within 4–6
days (Fig. 30). The germinated spores developed
into uniseriate ﬁlaments (Fig. 31) and gave rise
to tube-like thalli (Fig. 32). Branching wasobserved from thalli which were cultured for
40–45 days (Fig. 33). Sexual reproduction was
not observed in this study.
Ulva sp. 5
Field material: Well-developed thalli grew up
to 5.0 cm in height and up to 1.0 mm in diameter,
were tubular, and branching in the basal region
and light to medium green in color (Fig. 34). In
the upper basal region, cells were (11.1)–
20.6±4.4–(35.4) μm in length and (9.1)–
15.5±3.7–(30.4) μm in width; in the middle
region, those were (10.7)–18.3±1.1–(30.0) μm
in length and (7.6)–13.4±1.2–(18.5) μm in width(Fig. 35); and in the upper region, those were
(10.8)–18.0±2.3–(33.5) μm in length and (8.1)–
13.1±0.6–(24.3) μm in width. Each cell included
1–4 pyrenoids in the upper basal region (one,38.9%; two, 41.1%; three, 14.9%; and four, 5.1%of cells examined), in the middle region (one,65.6%; two, 33.2%; and three, 1.2%), and in theupper region (one, 39.2%; two, 56.8%; and three,4.0%). Cells in surface view were polygonal,
rectangular or square. In the thalli collected from
Chiba Pref., chloroplasts usually covered theouter wall of cells and gathered to cell wall in theupper and middle region (Fig. 35). The chloroplasts of thalli collected from Okinawa Pref.
were narrow in shape, and lied against a sidewall (Fig. 36).
Cultured thalli: Cultured thalli were originallycollected from Chiba Pref. Thalli cultured underﬂuorescent light at 50 μmol m−2 s−1 have chloroplasts usually covered the outer wall of cells.
However, at 200 μmol m−2 s−1, some chloroplasts
lied against a side wall (Figs. 37, 38).
Ulva sp. 12
Field material: Well-developed thalli grew up
to 15 cm in height, and up to 15 cm in width,obovate or elliptical in shape with small holes,
and were light green in color, fragile, easily torn,
and often split in the upper portion. Some thalli
possessed lobes and lanceolate in shape withsmooth margin. In the upper basal region and the
middle region, cells are 28–45 μm in length and
20–32 μm in width; and in the upper region,

10

Yuka Masakiyo and Satoshi Shimada

those are 26–35 μm in length and 18–30 μm in
width. Chloroplasts cover the outer wall of cells(Fig. 39). Each cell includes 1–3 pyrenoids in the
middle region (one, 38.0%; two, 49.0%; andthree, 13.0%), and in the upper region (one,45.0%; two, 50.0%; and three, 5.0%). In theupper region, the thallus is 33–63 μm thick, andcells in the transverse section are square. In the
middle region, the thallus is 96–150 μm thick(Fig. 40). Moreover cells are rectangular andlength of cell is 2–3 times longer than width. In
the basal region, the thallus is 76–115 μm thickand cells are square. Rhizoidal cells bear extensions on the outside of the cell layer in the transverse sections (Fig. 41).
Discussion
Ulva clathratioides L.G.Kraft, Kraft et R.F.Waller
Ulva sp. 5)[Figs. 6, 34–38]
In the original description, U. clathratioides is30–50 mm long, and has the tubular thallibranching in base, cells in surface view aresquare or rectangular, 20–35 μm in length and
10–25 μm in width in the upper basal region, andeach cell has 1–4 pyrenoids (Kraft et al., 2010).
These morphological characters matched thoseof Ulva sp. 5. Kraft et al. (2010) reported thatchloroplasts of U. clathratioides are narrow andlie against a side wall, however, the chloroplasts
position of Ulva sp. 5 was differed depend on thelocality. Our culture experiments indicated thatchloroplasts position of Ulva sp. 5 is not ﬁxed.
Therefore, in molecular phylogenetic and morphological similarities, we identiﬁed Ulva sp. 5
as Ulva clathratioides. It means that U. clathratioides is reported from Japan for the ﬁrst time
(Japanese name: Taretsu-aonori-modoki).
Ulva sublittoralis Segawa (Ulva sp. 12)[Figs. 13, 39–41]
The thallus of U. sublittoralis is obovate toelongate in shape, has rarely lobes, up to 1 mlong, and is 35 μm thick in the upper region and
130–270 μm thick in the middle region (Yoshida,1998). Ulva sp. 12 has similar morphologicalcharacters. Moreover, Segawa (1938) reported
that rhizoidal cells of U. sublittoralis bear extensions on the outside of the cell layer. This morphological character is only observed in U.
sublittoralis (membranous) and U. limnetica(tubular) (Segawa, 1938; Ichihara et al., 2009a).
Rhizoidal cells of Ulva sp. 12 bear extensions on
the outside of the cell layer. Therefore, we identiﬁed Ulva sp. 12 as Ulva sublittoralis.
Ulva tepida sp. nov. (Ulva sp. 3)
The phylogenetic and morphological differences between Ulva sp. 3 and other species ofUlva provide a basis for the establishment of a
new species, Ulva tepida Masakiyo et S.Shimada.

Figs. 39–41. Ulva sublittoralis (Ulva sp. 12). 39, 40. Middle parts of thallus. 39. Surface view. 40. Transverse
section. 41. Transverse section of basal part of thallus.

Ulva tepida Masakiyo et S. Shimada sp. nov.

Description
Ulva tepida Masakiyo et S.Shimada, sp. nov.
[Figs. 4, 16–33]
Tube-like thalli are up to 11.5 cm in height and
up to 8 mm in diameter, right to yellowish green
in color. Axes are simple or radially branching in
the basal region, which gradually widen frombasal region to upper region. Rhizoidal cells arebearing tubular extensions on the inside of cell
layer in the base of the thallus. Cells in surface
view of ﬁeld samples are arranged in longitudinal or lateral lows, square or rectangular, and9.9–22.2×6.4–16.1 μm in the middle and upper
regions. Chloroplasts cover the outer wall ofcells and have 1–5 pyrenoids (one, 15.6%; two,
54.1%; three, 25.9%; four, 3.7%; and ﬁve, 0.7%)
in middle region.
TYPE: TNS-AL 183379 (Fig. 16) in TNS(National Museum of Nature and Science, Tsukuba, Japan). Leg. Yuka Masakiyo.
Type locality: Enoshima Island, Fujisawa,
Kanagawa Pref., Japan.
Etymology: The speciﬁc epithet means ʻwarmʼ
in Latin.
Japanese name: “Natsu-aonori”.
Ulva tepida can be distinguished morphologically from other Enteromorpha-like species ofUlva thalli as follows. In gross morphology, Ulva
simplex (K.L. Vinogradova) Hayden et al. mostly
has spirally twisted stripe (Koeman and van den
Hoek, 1982b). Ulva torta (Mertens) Trevisan andU. ralfsii (Harvey) Le Jolis are ﬁliform, Ulva
kylinii (Bliding) Hayden et al. is very long andnarrow thalli, Ulva hendayensis Dangeard et Parriaud are unbranched (Bliding, 1963; Koeman
and van den Hoek, 1982b, 1984; Brodie et al.,2007). In the number of pyrenoid per cell, Ulva
radiata (J.Agardh) Hayden et al., U. compressa,
U. intsetinalis var. intestinalis Bliding, U. intsetinalis var. asexualis Bliding, U. intestinaloides(Koeman et Hoek) Hayden et al., U. prolifera, U.
linza, U. prolifera subsp. gullmariensis (Bliding)
Taskin, U. pseudolinza (Koeman et Hoek)
Hayden et al. and U. proliferoides Kraft et al.
have one pyrenoid per cell (90%＞); Ulva

11

ﬂexuosa has one pyrenoid in up to 50% of thecell; and U. jugoslavica Bliding has 1–2 pyrenoids in up to 85% of the cell (Bliding, 1963;
Koeman and van den Hoek, 1982a, 1984; Kraft
et al., 2010); U. adriatica Bliding, U. ﬂexuosa
subsp. paradoxa (C.Agardh) Bliding, U. ﬂexuosa
subsp. ﬂexuosa Wulfen, and U. brisbanensis
Kraft et al. has 1–3 pyrenoids per cell. In
U. ﬂexuosa subsp. pilifera (Kützing) Wynne, U.
clathrata (Roth) C.Agardh, U. ﬂexuosa subsp.
biﬂagellate (Bliding) Bliding, U. multiramosa
Bliding, U. ﬂexuosa subsp. linziformis (Bliding)Bliding and U. aragoensis Bliding, more than
two pyrenoids per cell were observed. Moreover,the percent of cells possessing 3 pyrenoids distinguishes U. tepida (25.9%) from U.clathratioides(1.2% to 14.9%) and U. meridionalis (3.1–3.4%)(Horimoto et al., 2011). Besides number of pyrenoids, U. tepida is distinguished from species
whose chloroplasts lie against a side wall or are
variable in position.
Consequently, Japanese Ulva and Umbraulva
were classiﬁed into 29 species (including nine
undescribed species, one newly described species, one species newly reported to Japan and
one rare already described species), and four species (including two undescribed species), respectively. In the check list of Japanese seaweeds(Yoshida and Yoshinaga, 2010), 19 species ofUlva and two species of Umbraulva werereported. And, recently freshwater and brackish
water Ulva are newly described (Ichihara et al.,2009a, Horimoto et al., 2011), and thus totally 21
Ulva and two Umbraulva species are recognizedin Japanese waters. Among them, we could notinclude Ulva clathrata (Roth) C.Agardh, Ulva
conglobata Kjellman and Ulva fenestrata Postels
et Ruprecht in our phylogenetic analysis.
Ulva clathrata is originally collected fromintertidal zone by Roth (Agardh et al., 1811).
But, original material was lost and Hayden et al.
(2003) registered neotype collected from Fehman
Island, Baltic Sea, Sweden. And in Japan, Okamura (1902) reported this species from Matsushima, Miyagi Pref. Japan. We widely investigated Japanese Enteromorpha-like Ulva, but

12

Yuka Masakiyo and Satoshi Shimada

there was no identical sequence with U. clathlata
reported from Europe. Kang and Lee (2002)
revealed that Ulva conglobata and Ulva pertusa
Kjellman belong to one clade in the phylogenetic
tree inferred from ITS2. Ulva fenestrata isreported from Kamchatka Peninsula, Siberia by
Silva et al. (1996). However, Yoshida (1998)
commented that this species has many pores andthick of thallus are thin than that of Ulva pertusa,
but Ulva fenestrata resembles Ulva pertusa.
Therefore, existence of U. clathrata, U. conglobata and U. fenestrata is unclear.
Habitats of the undescribed 12 species wereconcentrated in river and deep sea about 30 m
depth of warm regions, for example, Okinawa
and Kagoshima Pref. The species distributed insuch a habitat might be suitable for studying
adapted evolution. We should collect more samples from these sites and conduct molecular anddetailed morphological analyses to understandspecies diversity of Ulva and Umbraulva inJapan, and this will in turn facilitate studies ofmolecular mechanisms of adapted evolution.
Acknowledgments
The help of Dr. Verbruggen, H (the Univ. Melbourne), with stimulating discussion, constructive criticism and valuable suggestions on an
early version of the manuscript was greatly
appreciated. We also thank Dr. Masanori Hiraoka(Kochi Univ.), Dr. Ryuta Terada (Kagoshima
Univ.), and Dr. Masayuki Uchimura (IDEA Consultants, Inc.) for collecting materials.
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