American Journal of Botany 89(11): 1756–1763. 2002.

NOVEL MORPHOLOGY IN ENTEROMORPHA

(ULVOPHYCEAE) FORMING GREEN TIDES1
JAANIKA BLOMSTER,2,5 SAARA BÄCK,3 DAVID P. FEWER,4
MIKKO KIIRIKKI,3 ANNAMAIJA LEHVO,3 CHRISTINE A. MAGGS,2,6
AND MICHAEL J. STANHOPE2,7

2
School of Biology and Biochemistry, Queen’s University, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern
Ireland, UK; 3Finnish Environment Institute, P.O. Box 140, 00251 Helsinki, Finland; and 4Department of Microbiology, P.O. Box 56,
University of Helsinki, 00014 Helsinki, Finland

‘‘Green tides’’ are vast accumulations of unattached green macroalgae associated with eutrophicated marine environments. They
have major ecological and economic impacts globally, so an understanding of their origin and persistence is required in order to make
management decisions. Blooms predominantly consist of two common fouling genera of the Ulvales, Ulva (distromatic sheets) and
Enteromorpha (monostromatic tubes). In the Baltic Sea and elsewhere green tides have increased over the last few decades. On the
west coast of Finland, summer blooms consist of monostromatic sheets resembling Monostroma (Codiolales). We identified these as
Enteromorpha intestinalis by comparative analyses of rDNA internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 sequences, the first
time monostromatic sheets have been found in the genus Enteromorpha. Ordinary attached E. intestinalis sporulated freely in culture,
but the sheets reproduced only by cell regeneration into typical tubular thalli. The ITS sequences were identical to those of attached
E. intestinalis populations in southwestern Finland, but differed by two substitutions from other Baltic sequences. We infer that this
bloom originated from local attached populations and now reproduces clonally by fragmentation. This study provides further evidence
of radical changes in gross morphology of green algae under eutrophicated conditions and the need for molecular identification.

Key words: Baltic Sea; Chlorophyta; Enteromorpha intestinalis; green tides; ITS sequences; monostromatic; morphology; phy-
logenetic analysis; Ulvophyceae.

‘‘Green tides’’ are a type of harmful algal bloom. They are
vast accumulations of green macroalgal biomass that occur
under suitable hydrographic conditions in eutrophicated areas
(Schories and Reise, 1993; Fletcher, 1996; Hernández et al.,
1997; Valiela et al., 1997; Raffaelli, Raven, and Poole, 1998).
Astonishing quantities of material (up to 27 kg wet mass/m2)
are cast up or drift in shallow water, constituting a major nui-
sance in sheltered bays around the world (Fletcher, 1996). Del-
eterious ecological effects include the uncoupling of biogeo-
chemical cycles in sediments from those in water columns
(Valiela et al., 1997), a negative impact on seagrass beds due
to shading, and disruption of feeding by wading birds (Raf-
faelli, Raven, and Poole, 1998). The costs of mitigation are
high, involving the removal of about 108 kg of seaweed per
annum from recreational beaches of Atlantic France alone
(Dion and Le Bozec, 1996). Because of their economic impact,
green tides have been the focus of a large number of ecological
studies in Europe, North America, and Australia (reviewed by
Fletcher, 1996; Valiela et al., 1997; Raffaelli, Raven, and
Poole, 1998).
The great majority of blooms are reported to consist of
members of just two genera of the Ulvophyceae, Ulva and
Enteromorpha (Fletcher, 1996). These are among the world’s
most common fouling algae, which are used as model organ-
1
Manuscript received 28 August 2001; revision accepted 31 May 2002.
The authors thank Jim Provan (Belfast), Hillary Hayden (Seattle), and Elina
Leskinen (Helsinki), for helpful discussions and unpublished information.
This study was supported by awards to JB from the Walter and Andrée de
Nottbeck Foundation and the Oskari Huttunen Foundation.
5
Current address: Division of Systematic Biology, P.O. Box 7, University
of Helsinki, 00014 Helsinki, Finland.
6
Author for reprint requests (e-mail: c.maggs@qub.ac.uk).
7
Current address: Bioinformatics, GlaxoSmithKline, 1250 S. Collegeville
Road, Collegeville, PA 19426, USA.
1756
isms in studies of spore bioadhesion (Stanley, Callow, and
Callow, 1999; Callow et al., 2000). Their life histories consist
of morphologically similar haploid and diploid phases, both of
which reproduce prolifically by haploid or diploid asexual zoo-
spores formed by mitotic division of vegetative cells (van den
Hoek and Mann, 1996). Sexual reproduction involves fusion
of opposite mating types of haploid gametes, which can also
develop parthenogenetically into adult thalli. Ulva and Enter-
omorpha differ markedly in their general morphology (flat bi-
layered blades vs. hollow tubes a single cell in thickness; Bur-
rows, 1991). Although molecular phylogenetic analysis has re-
cently revealed that the two genera are not distinct evolution-
ary entities (Tan et al., 1999), both names are used here
pending taxonomic revision.
A better understanding of the origin and persistence of green
tide blooms is desirable in order to address the problems they
cause. Even simple taxonomic identification is confounded be-
cause the unattached algae are often morphologically atypical
(Malta, Draisma, and Kamermans, 1999). As yet there have
been very few studies concerning any aspects of the molecular
ecology of green tides. However, in conjunction with life-his-
tory studies, use of appropriate molecular markers can both
identify the algae and provide important information concern-
ing the origins and dynamics of the blooms (Coat et al., 1998;
Malta, Draisma, and Kamermans, 1999). Such information is
required to inform management decisions. Proposed remedia-
tion measures typically involve altering ‘‘bottom-up’’ controls,
i.e., reducing nutrient supply (Valiela et al., 1997).
The Baltic Sea, because of its enclosed nature, is prone to
eutrophication that has favored the formation of green tides.
The Gulf of Finland is one of the most eutrophic areas (Lap-
palainen and Pönni, 2000), receiving 76 3 105 kg of phos-
phorus and 138 3 106 kg of nitrogen annually (Pitkänen et
November 2002] BLOMSTER ET AL.—NOVEL MORPHOLOGY IN
Fig. 1. Map of study area at Olkiluodonvesi, near the Eurajoki river mouth, showing its location on the west coast of Finland, Baltic Sea (inset), with
distribution of the green tide algae (solid symbols, green tide present; open symbols, no green tide observed) in surveys in 1992–1997.
al., 1998). A green tide has occurred there over a large area
on the west coast of Finland every summer between 1992 and
2000. This bloom consists of free-floating single-layered
sheets that superficially resemble Monostroma, a genus of the
Codiolales (Ulvophyceae) not closely related to Ulva and En-
teromorpha (Ulvales) and rarely regarded as a green tide-
forming green alga (Fletcher, 1996).
To resolve the identity of this morphologically unusual
bloom and, if possible, determine its origins, we made com-
parative analyses of rDNA internal transcribed spacer 1 (ITS1)
and ITS2 spacers and the 5.8S gene. This molecular marker
was chosen because ITS sequences are available for a large
number of green algal species, including several that form
green tides, and can often reveal microevolutionary processes
(Blomster, Maggs, and Stanhope, 1998, 1999) as well as pro-
viding definite taxonomic identification (Blomster, Maggs, and
Stanhope, 1998, 1999; Coat et al., 1998; Tan et al., 1999;
Blomster et al., 2000). We also sought by culture studies to
determine the life history of this bloom-forming alga and ex-
amine whether its unusual morphology might be linked to the
green tide environment.
MATERIALS AND METHODS
Study site—The monostromatic sheets were found growing free-floating in
1992–2000 at Olkiluodonvesi (Fig. 1), a sheltered location near the Eurajoki
river mouth on the west coast of Finland (618109 N, 218309 E). In the densest
patches, the algae formed 5–15 cm thick mats in depths of less than 2 m,
over an area of sandy and soft sediment bottoms with reed beds. In deeper
water the distribution was patchy (Bäck, Lehvo, and Blomster, 2000). The
salinity is 4–5 practical salinity units (PSU) throughout the year in this tideless
area. During the winter, ice cover of up to 1 m can last for a maximum of 4
mo, and algal distribution in winter was surveyed by dredging through holes
drilled through the ice.
ENTEROMORPHA 1757
Culture studies—Bloom-forming algae were collected from the field in
1993, examined under dissecting and compound microscopes, and initially
maintained in an aquarium under ambient laboratory conditions. Cultures
were isolated from small samples of the algae using Repli-dishes (Sterilin)
with 24 wells each filled with 3 mL enriched autoclaved seawater at 5–6 PSU
(Hällfors and Hällfors, 1992). When unialgal, as determined by careful mi-
croscopic examination, isolates were grown in enriched artificial seawater in
50-mL tissue culture bottles, with a monthly change of medium, under stan-
dard conditions of 158C in a 12 h light : 12 h dark photoperiod. In attempts
to induce reproduction, a series of transfers between a wide range of culture
conditions was undertaken. All aspects of the culture environment were varied
sequentially. First, the enriched medium was replaced with autoclaved 5–6
PSU sea water without nutrient additions, then the salinity was reduced by
using autoclaved tap water. The temperature was changed to 58, 108, 158, or
208C, and the daylength was changed from 12 h : 12 h to 18 h : 6 h. Desic-
cation of the sheets under damp conditions was followed by rehydration in
medium. The final culture treatment involved leaving the medium unchanged
for 2 mo, then replacing it, which induced new growth that was transferred
to fresh bottles.
Following the discovery that the monostromatic sheets could give rise to
thalli resembling Enteromorpha intestinalis, attached E. intestinalis thalli were
collected from Hanko, Tvärminne, Finland (598509 N; 248159 E) in September
1995 and from Espoo, Haukilahti, Finland (608109 N; 248459 E) in April 1996.
Cultures were grown under the standard conditions described above.
Cultured material for DNA extraction was preserved in silica gel; subsam-
ples were pressed for herbarium specimens and photographed. Voucher spec-
imens were prepared and deposited in the Ulster Museum, Belfast (F11947),
and the Natural History Museum, London, UK.
DNA extraction and sequencing—DNA of the monostromatic sheets was
extracted from silica gel-preserved tissue of three individual thalli collected
in June 1997 and from a culture. Other algal material for DNA extraction was
collected from various locations in the Atlantic and the Baltic. Collection data
and GenBank accession numbers can be found at the AJB Supplementary
1758 AMERICAN JOURNAL
Figs. 2–4. Field-collected and cultured material from green tides in Fin-
land. 2. Herbarium specimens of monostromatic sheet-like green seaweed
from green tide at Olkiluodonvesi, Finland, on 5 June 1996. 3. Bloom-forming
sheet-like green alga grown in culture has given rise from callus-like tissue
to unbranched tubular outgrowths indistinguishable from Enteromorpha in-
testinalis. 4. Tubular green alga in culture, showing small cells with 1–3 py-
renoids (arrow).
Data web site (http://ajbsupp.botany.org/v89/). Fifty-milligram samples were
ground to a fine powder in liquid nitrogen and DNA was phenol chloroform
extracted using a modified total genomic DNA extraction protocol for algal
material (Blomster, Maggs, and Stanhope, 1999). Polymerase chain reaction
(PCR) amplification and sequencing of the ribosomal DNA cistron including
the complete ITS1, 5.8S, and ITS2 regions were performed using primers
OF BOTANY [Vol. 89
described in Blomster, Maggs, and Stanhope (1998). The PCR-amplified prod-
ucts were directly sequenced using dideoxy chain-termination methodology
with dye-termination reactions of 30 pmol primer and 400 ng template DNA
in 20 mL reactions. The cycle sequencing reactions were carried out in a cycle
of initial denaturation at 968C for 1 min, followed by 25 cycles of 968C for
50 s, 628C for 4 min, and 508C for 20 s. The reactions were loaded on Perkin-
Elmer ABI 373A or ABI 377 automated sequencers. The cistron was se-
quenced on both strands with each reaction repeated at least three different
times in different sequence runs.
Data analysis—The ITS1, ITS2, and 5.8S sequences were aligned with 21
sequences of other monostromatic sheets (Monostroma grevillei), distromatic
sheets (Ulva species), and tubular green algae (Enteromorpha species). The
sequence alignments were constructed using ClustalW Multiple Alignment
option (Thompson, Higgins, and Gibson, 1994) within the BioEdit Sequence
Alignment Editor 4.8.10. The alignment was perfected by eye using BioEdit
(Hall, 1999). Because of the high sequence divergence in some parts of the
alignment between different genera in the study, only the most conserved
regions (506 base pairs [bp] of the total 610 bp) were included in this analysis.
The data were analyzed using maximum parsimony (MP), neighbor-joining
(NJ), and maximum-likelihood (ML) methods with PHYLIP 3.5c (Felsenstein,
1993). The robustness of the MP and NJ phylogenetic hypotheses was tested
by bootstrapping (Felsenstein, 1985) with 1000 replications of the data. The
number of most parsimonious trees was determined by randomizing the input
order ten times. The distance matrix, also used for NJ analysis, used maximum
likelihood distances with a transition : transversion ratio of 2.0 and empirical
base frequencies. Determination of the highest likelihood tree was made with
the expected transition : transversion ratio at 2.0. A significance test was car-
ried out between a user-defined constrained tree and the MP tree using the
method proposed by Templeton (1983). All trees were unrooted, because the
taxa included are so distantly related that potential outgroup sequences could
not be aligned with them. For examination of the relationships among samples
of Enteromorpha intestinalis, a complete alignment of all E. intestinalis se-
quences was constructed.
RESULTS
Morphology and life history—The bloom consisted of
bright green, fragile sheets of irregular shape (Fig. 2). Just
after the ice melted in March–April the sheets were 2–4 3 2–
4 cm and enlarged rapidly to a maximum of about 15 cm
across. Blades were one cell thick, composed of cells in short
rows or irregularly arranged, loosely embedded in a gelatinous
matrix that included rhizoidal cells. Cells were 5–14 mm di-
ameter, irregularly rounded to elongate, vacuolate, with hood-
shaped chloroplasts and 1–2 pyrenoids. No specialized repro-
ductive structures were observed. The algae occasionally be-
came free-floating in the water column causing massive
blooms (Bäck, Lehvo, and Blomster, 2000). They persisted
throughout the entire year in small quantities and survived for
4 mo under the ice as small fragments.
Under the standard culture conditions, the blades continued
to grow and reproduction was not observed. None of the at-
tempts to induce sporulation over a 2-yr period was successful.
Finally, after 2 mo in old culture medium, the majority of the
cells died, leaving only a few alive. When these mostly dead
thalli were transferred to fresh medium in clean culture tubes,
the surviving cells grew into callus-like tissue that rapidly
gave rise to hollow tubular thalli up to 2–4 cm in length (Fig.
3). These tubular thalli closely resembled Enteromorpha in-
testinalis and never branched or reverted to the monostromatic
sheet form under any of the culture conditions. Cells were 4–
10 mm in diameter, rectangular to rounded-polygonal, arranged
in short rows or rosettes, and contained 1–3 pyrenoids (Fig.
4).
November 2002] BLOMSTER ET AL.—NOVEL MORPHOLOGY IN ENTEROMORPHA 1759

Fig. 5. Maximum likelihood-based tree for ITS1, 5.8S, and ITS2 sequences of the monostromatic sheet-like green alga with its tubular derivative and other
Enteromorpha, Ulva, and Monostroma samples. Branch lengths are drawn proportional to the amount of sequence change. Bootstrap support (percentage of
1000 replications) for maximum parsimony (above) and neighbor-joining analyses (below) is indicated at nodes.

Attached E. intestinalis collected from two localities in Fin-
land always sporulated freely under the standard culture con-
ditions, producing abundant viable spores. In most cases vir-
tually the whole thallus was converted into spores after a rel-
atively short cultivation period.
Phylogenetic analyses—The sequence alignment of 506 bp
contained 254 variable sites. The most likely ML tree (Ln
likelihood 5 22774.0) supported the monophyly of sheet-like
samples (including the tubular culture) and E. intestinalis sam-
ples (Fig. 5). In the parsimony analysis 89 most parsimonious
trees (MPTs) of length 575 steps were found. In all of these
trees the field-collected sheet-like samples and the cultured
tubular thalli were grouped with E. intestinalis samples from
the Baltic and Atlantic and separated from the Monostroma
samples, Ulva samples, and other Enteromorpha samples.
Bootstrap support for the assemblage of the sheet-like algae,
including their tubular derivatives, and E. intestinalis samples
was 98% (NJ) to 100% (MP) (Fig. 5). A user-defined tree that
constrained the monophyly of all monostromatic sheets, i.e.,
grouped the Baltic sheet-like samples with Monostroma, added
27 steps to the MPT. It was 702 steps in length and was sig-
nificantly longer than the most parsimonious trees.
Sequence divergence among E. intestinalis samples, includ-
ing the sheet-like accessions, was low, 0.0–2.2% (Table 1).
Sequence divergence between E. intestinalis and other species
included in the study ranged from 9.2% to 48.2%, the highest
being between E. intestinalis and Monostroma grevillei (Table
1). Sequence divergence between E. intestinalis and other En-
teromorpha species ranged from 9.2 to 16.6% and between E.
intestinalis and Ulva species from 9.3 to 14.7% (Table 1).
Phylogenetic analysis of the complete alignment of all E.
intestinalis sequences including those of the monostromatic
sheets, rooted with E. compressa, failed to resolve relation-
ships among the sequences (not shown). There was very little
geographic structure and none of the nodes had congruent
bootstrap support of over 65% for MP, NJ, and ML analyses.
This reflected the lack of variable positions in the ingroup
alignment (Fig. 6). Most of these resulted from length varia-
tions in two probable mononucleotide microsatellite repeats.
A poly (G)n at positions 79–83 was either 4 or 5 bases in
length. A poly (C)n at positions 103–110 was 6, 7, or 8 bases
long, with a transversion to T at one site in two of the samples.
It caused serious problems during sequencing, presumably due
to slippage in PCR amplification, and resulted in some unde-
termined bases in this region. Apart from microsatellite repeat
length variations, there were very few variable nucleotides,
most of which are autapomorphies for sample 512 from North-
ern Ireland. The only synapomorphies were two transversions
(T to G) in the 5.8S gene of Baltic 1 and Baltic 2, which are
published sequences of 14 Baltic specimens (termed E. intes-
tinalis/compressa ‘‘haplotype 1’’ and ‘‘haplotype 2’’ in Les-
kinen and Pamilo, 1997) and two transversions (both C to T)
for Baltic 1 and Ireland 611. There were no unique synapo-
morphies for the four bloom sequences (monostromatic sheets
and tubular culture). Sheet 1 differed at both putative micro-
satellite loci from sheets 2 and 3.
DISCUSSION
Identity of the Baltic bloom—All methods of phylogenetic
analysis showed unequivocally that the monostromatic sheet-
like green alga forming green tides on the west coast of Fin-
land was Enteromorpha intestinalis (Fig. 5). Although only
unrooted trees are presented here because we included the dis-
tantly related species Monostroma grevillei, in published root-
ed trees E. intestinalis forms a clade with E. compressa and
Ulva lactuca (Tan et al., 1999). Sequence divergence of 0–
1760 AMERICAN JOURNAL OF BOTANY [Vol. 89

TABLE 1. Divergence matrix of the alignable, conserved regions of ITS1, 5.8S, and ITS2 showing Jukes and Cantor distances for the samples of
monostromatic sheet and other Enteromorpha, Ulva, and Monostroma samples. The sample names, geographic location, and collection details
are as listed at http://ajbsupp.botany.org/v89/Sheet1–3 5 ‘Monostromatic sheet’ 1–3; Tubular 5 Tubular culture from monostromatic sheet;
Eint 5 Enteromorpha intestinalis; BS1, BS2 5 Baltic haplotypes 1, 2; Ecom 5 Enteromorpha compressa; Upseudo 5 Ulva pseudocurvata;
Eproc 5 Enteromorpha procera; Eflex 5 Enteromorpha flexuosa; Eprol 5 Enteromorpha prolifera; Emus 5 Enteromorpha muscoides; Uarm
5 Ulva armoricana; Ulact 5 Ulva lactuca; Uoliv 5 Ulva olivascens; Urotu 5 Ulva rotundata; Uscan 5 Ulva scandinavica; Eloides 5
Enteromorpha intestinaloides; MonA1, A2 5 Monostroma grevillei, Atlantic; MonBS 5 Monostroma grevillei, Baltic.

Sample Sheet1 Sheet2 Sheet3 Tube Eint512 Eint611 Eint585 Eint1BS Eint2BS Ecom543 Ecom598 Upseudo

Sheet2 0.006
Sheet3 0.002 0.000
Tube 0.000 0.000 0.000
Eint512 0.018 0.018 0.016 0.014
Eint611 0.004 0.011 0.007 0.002 0.022
Eint585 0.000 0.000 0.000 0.000 0.018 0.004
Eint1BS 0.006 0.013 0.009 0.005 0.025 0.006 0.007
Eint2BS 0.004 0.011 0.007 0.005 0.022 0.009 0.004 0.002
Ecom543 0.100 0.108 0.103 0.099 0.118 0.105 0.102 0.108 0.106
Ecom598 0.100 0.108 0.103 0.099 0.118 0.105 0.102 0.108 0.106 0.000
Upseudo 0.095 0.102 0.098 0.099 0.113 0.100 0.097 0.103 0.101 0.004 0.004
EprocBS 0.136 0.144 0.140 0.129 0.147 0.138 0.136 0.130 0.130 0.133 0.133 0.133
EflexBS 0.132 0.140 0.136 0.131 0.149 0.137 0.135 0.129 0.127 0.129 0.129 0.129
Eprol 0.123 0.131 0.126 0.117 0.128 0.128 0.123 0.131 0.128 0.125 0.125 0.125
Emus623 0.153 0.162 0.158 0.142 0.166 0.155 0.156 0.159 0.159 0.180 0.180 0.178
Uarm 0.135 0.144 0.140 0.123 0.147 0.138 0.138 0.141 0.141 0.166 0.166 0.163
Ulact 0.093 0.100 0.096 0.102 0.114 0.098 0.095 0.101 0.098 0.095 0.095 0.090
Uoliv 0.195 0.205 0.201 0.204 0.204 0.200 0.199 0.204 0.201 0.225 0.225 0.218
Urotu 0.103 0.110 0.106 0.107 0.118 0.108 0.102 0.111 0.108 0.092 0.092 0.087
Uscan 0.132 0.140 0.136 0.119 0.143 0.134 0.134 0.137 0.137 0.173 0.173 0.171
Eloides 0.092 0.099 0.095 0.092 0.112 0.092 0.094 0.095 0.097 0.079 0.079 0.074
MonA1 0.444 0.456 0.435 0.402 0.476 0.440 0.456 0.454 0.454 0.467 0.467 0.469
MonA2 0.447 0.455 0.434 0.402 0.480 0.443 0.459 0.458 0.458 0.472 0.472 0.473
MonBS 0.449 0.457 0.436 0.402 0.482 0.445 0.461 0.460 0.460 0.473 0.473 0.474

2.2% among E. intestinalis samples is comparable to the levels
found within other monospecific groupings in the Ulvales,
such as Atlantic E. compressa (0–2.3%; Blomster, Maggs, and
Stanhope, 1998; Tan et al., 1999) and E. muscoides (0–0.6%;
Blomster, Maggs, and Stanhope, 1999). Blooms of single-lay-
ered sheet-like green algae observed in the Baltic Sea have
previously been identified as members of the genus Monostro-
ma in the Codiolales, not closely related to Ulva and Entero-
morpha (Ulvales). At Inre Verkviken in the northern Aland
Islands, such loose-lying sheets were attributed to Monostroma
balticum (Mathiesen and Mathiesen, 1992), whereas in the
Askö area of eastern Sweden similar springtime blooms were
identified as Monostroma grevillei (Wallentinus, 1976, p. 98,
1979). It is possible that Monostroma does form blooms in
the Baltic, but previous reports may represent misidentifica-
tions of E. intestinalis based on the aberrant morphology. Tax-
onomic identification has important implications for under-
standing the dynamics of these green tides, because the haplo-
diploid life history of Monostroma involves a microscopic
‘‘codiolum’’ phase that grows endophytically within other al-
gae, in contrast to the isomorphic life history of Ulva and
Enteromorpha.
Origin and persistence of the bloom—With the exception
of mononucleotide repeats, all four sequences of the bloom-
forming Baltic E. intestinalis were identical. Substitutions as-
sociated with two probable microsatellite mononucleotide re-
peats are likely to be phylogenetically misleading, because
such loci exhibit high levels of bidirectional mutation and
hence homoplasy (Provan, Powell, and Hollingsworth, 2001).
Published 5.8S gene sequences for attached Baltic populations
of E. intestinalis, including three samples from southwestern
Finland, exhibited two unique T to G transversions (Fig. 6;
Leskinen and Pamilo, 1997). We could infer from this that the
blooms were not derived from attached populations at nearby
sites. However, these positions are otherwise invariant in a
large alignment of green algal 5.8S sequences (H. Hayden,
University of Washington, unpublished data). As T to G is the
rarest nucleotide substitution (Nei and Kumar, 2000) and com-
pressions due to GC-rich template DNA almost invariably lead
to incorrectly determined Gs, sequencing error may be in-
volved. If this is taken as a working assumption, and putative
microsatellite variation is ignored, sequences of our bloom-
forming E. intestinalis correspond to Leskinen and Pamilo’s
(1997) haplotype 2. Even when the mononucleotide repeats
are included, the ITS1 and ITS2 sequences for two of the three
bloom samples were identical to their haplotype 2, which was
obtained from three sites in southwestern Finland, as well as
in Sweden. The ITS1 and ITS2 sequences of Leskinen and
Pamilo’s (1997) haplotype 1, which they found only in Swed-
ish samples, were identical to one of our Atlantic sequences
(611) from a freshwater stream in Ireland.
The identical ITS1 and ITS2 sequences of bloom-forming
E. intestinalis and attached populations in southwestern Fin-
land are consistent with the bloom having originated from typ-
ical tubular thalli that occur in the local area (Keskitalo and
Ilus, 1987), rather than by introduction of an existing sheet-
like form from a different geographical area. This is also sup-
ported by our findings that the thalli derived from isolated cells
of the sheets were hollow tubular plants. We found no evi-
dence that sheet-forming E. intestinalis reproduces by spores,
either in the field or in culture, unlike the attached populations.
Instead, the presence of small fragments throughout the winter
shows that the bloom persists vegetatively and reproduces by
November 2002] BLOMSTER ET AL.—NOVEL MORPHOLOGY IN ENTEROMORPHA 1761

TABLE 1. Extended.

EprocBS EflexBS Eprol Emus623 Uarm Ulact Uoliv Urotu Uscan Eloides MonA1 MonA2

0.054
0.036 0.050
0.122 0.123 0.116
0.101 0.102 0.092 0.070
0.135 0.102 0.110 0.141 0.134
0.203 0.199 0.187 0.199 0.214 0.166
0.121 0.107 0.110 0.127 0.120 0.041 0.183
0.106 0.104 0.092 0.068 0.013 0.134 0.211 0.125
0.123 0.103 0.104 0.131 0.118 0.058 0.206 0.053 0.119
0.449 0.461 0.456 0.476 0.482 0.434 0.491 0.432 0.496 0.473
0.462 0.474 0.469 0.481 0.488 0.439 0.501 0.438 0.502 0.478 0.004
0.454 0.466 0.461 0.482 0.488 0.439 0.497 0.437 0.502 0.479 0.002 0.002

fragmentation. Monostromatic sheets may originate infre-
quently from attached tubular thalli, then reproduce clonally.
Survival in a culture of a few cells in otherwise dead thalli
(‘‘propagules’’) has previously been observed in E. intestinalis
(Burrows, 1959). She showed that this was an effective meth-
od of vegetative propagation in conditions unfavorable for the
survival of delicate zoospores, such as extreme cold or marked
salinity changes. Normal tubular thalli were never observed in
association with the monostromatic sheets under the Baltic
bloom conditions. In culture, however, once tubular thalli had
Fig. 6. Compressed alignment of ITS1, 5.8S, and ITS2 sequences of algal
samples identified in analyses shown in Fig. 5 as Enteromorpha intestinalis.
The first three sequences were from Olkiluodonvesi green tide samples
(‘‘sheets 1–3’’) and the fourth was from an additional green tide sample grown
clonally in culture into a tubular thallus (‘‘cultured tube’’). ‘‘Baltic 1’’ and
‘‘Baltic 2’’ are published sequences from attached E. intestinalis samples
(Leskinen and Pamilo, 1997; E. Leskinen, University of Oulu, personal com-
munication). The final three sequences are from the British Isles. All positions
with variable nucleotides are shown, numbered from the start of ITS1, and
those associated with probable mononucleotide microsatellite repeats (Gs or
Cs) are shaded.
formed by regeneration, they did not revert to the monostro-
matic sheet form.
A similar situation was found in the Veerse Meer (Nether-
lands), where unattached green tide Ulva scandinavica popu-
lations very rarely reproduce by spores. Instead they overwin-
ter buried in the sediment (Kamermans et al., 1998; Malta,
Draisma, and Kamermans, 1999), which is consistent with a
rare origin of the blooms from attached populations. For the
blooms in Finland and the Netherlands, removal of biomass
might therefore be an appropriate ‘‘top-down’’ mitigation mea-
sure. In contrast, in Brittany, France, green tides and local
attached populations of Ulva armoricana reproduced freely by
spores (Coat et al., 1998; Dion, de Reviers, and Coat, 1998).
The planktonic spore stage in their life history makes them
vulnerable to benthic filter feeders; Ruiz (1999) has presented
evidence to suggest that such green tides can be reduced by
high densities of cultured oysters.
Green tides and morphological plasticity in the Ulvales—
Flexibility of form in Enteromorpha intestinalis, such as the
formation of branches in normally unbranched plants, has long
been recognized (Reed and Russell, 1978). However, this is
the first report of a monostromatic morph in this genus despite
numerous physiological and ecological studies (Poole and Ra-
ven, 1997). Aberrant phenotypes of green algae are frequently
associated with green tides. In the Veerse Meer, three different
morphological species of Ulva were shown to have been in-
duced in conspecific samples (Malta, Draisma, and Kamer-
mans, 1999). Tan et al. (1999) collected distromatic sheet-like
Ulva pseudocurvata samples from a 4 km long estuary in
Aberdeenshire, Scotland, characterized by extensive green
tides (Raffaeli, Raven, and Poole, 1998). These samples
1762 AMERICAN JOURNAL
formed a strongly supported clade with samples of tubular E.
compressa (Fig. 5). The sequence divergence within this clade
ranged from only 0.42% to 1.7%, comparable with levels with-
in other monospecific groupings of Enteromorpha and Ulva.
Partly distromatic unbranched Enteromorpha linza and highly
branched tubular E. procera samples with very different mor-
phologies were likewise closely related (Tan et al., 1999).
Morphological changes in green algae are induced by al-
tered nutrient supply in eutrophicated conditions (Valiela et al.,
1997), as well as by salinity changes (Reed and Russell, 1978).
Aberrant growth forms are believed to have better potential
for survival in the prevailing conditions (e.g., rapid nutrient
uptake when only one cell layer thick, with a high surface-to-
volume ratio; Valiela et al., 1997). This may also explain in
part why Enteromorpha and Ulva remain the main compo-
nents of green tides (Poole and Raven, 1997). This novel mor-
phology of E. intestinalis indicates that the contribution of this
species to biofouling and green tides may have been under-
estimated previously. Further work is needed to establish the
causal relationship between green tide conditions and radical
changes in morphology in E. intestinalis. Accurate identifica-
tion of the causal agents of green tides is an essential prereq-
uisite to understanding the processes that contribute to green
tides, particularly as increasing pressure on coastal marine en-
vironments could result in further novel forms in this mor-
phologically plastic green algal species.
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