DOI 10.

1515/bot-2012-0197 Botanica Marina 2013; 56(2): 197–205

Franciane Pellizzari*, Mariana C. Oliveira, Amanda da Silva Medeiros, Nair S. Yokoya

and Eurico C. Oliveira

Morphology, ontogeny, and phylogenetic position

of Gayralia brasiliensis sp. nov. (Ulotrichales,
Chlorophyta) from the southern coast of Brazil
Abstract: A new species, Gayralia brasiliensis (Ulotrichales, Introduction
Chlorophyta), is described from Brazil on the basis of
ontogenetic, morphological, and molecular phylogenetic
Monostromatic members of the green algal order Ulo-
data. Liberation of zooids from the monostromatically
trichales are widely distributed from temperate to tropi-
bladed thalli occurred by disintegration of the zooidan-
cal seas (Leliaert et al. 2012). Some species have economic
gium wall, releasing four biflagellate cells with prominent
importance, mainly in the food and cosmetic industries
eyespots. Fusion of zooids was not observed. After zooid
(Pellizzari et al. 2007, Pise et al. 2012). Chemicals with
attachment, cell divisions gave rise to uniseriate filaments
antiviral and anticoagulant properties were recently iso-
that developed into fan-shaped blades. Only asexual
lated from some species in the genera Monostroma Thuret
reproduction was observed; this took place through recy-
and Gayralia Vinogradova (Cassolato et al. 2008, Zhang
cling of blades by germination of zooids. Phylogenetic
et al. 2008). However, the taxonomy, biology, phylogeny,
analysis based on rDNA internal transcribed spacer (ITS)
and biogeography of this group remain largely under-
sequences showed that G. brasiliensis specimens formed a
studied. The taxonomy has undergone several nomen-
monophyletic group closely related to Monostroma nitidum
clatural changes since 1960 (e.g., Gayral 1965, Bliding
and two unidentified monostromatic species from Japan
1968, Vinogradova 1969, Tatewaki 1972). Tentative revi-
and Tanzania. The genus Monostroma was polyphyletic in
sions resulted in taxon transfers, and current species are
our analysis. ITS sequences also confirmed the presence of
mainly assigned to the genera Monostroma, Gayralia, and
a second monostromatic species of Ulotrichales in the Bra-
Protomonostroma Vinogradova. The genus Monostroma
zilian coast, Gayralia oxysperma. There were overlapping
is cosmopolitan, comprising 60 species, of which only 29
morphological and life history traits between G. brasilien-
are currently accepted taxonomically (Guiry and Guiry
sis and G. oxysperma. However, these two species were
2012). Gayralia and Protomonostroma are monospecific,
clearly divergent based on ITS sequences and ontogeny.
comprising G. oxysperma (Kützing) K.L. Vinogradova ex
Scagel, P.W. Gabrielson, D.J. Garbary, L. Golden, S.C. Lind-
Keywords: Gayralia brasiliensis sp. nov.; Gayralia
strom, J.C. Oliveira et T.B. Widdowson and P. undulatum
oxysperma; internal transcribed spacer; ontogeny;
(Wittrock) K.L. Vinogradova, respectively. Protomonos-
Ulotrichales.
troma undulatum is found in southern Argentina and Ant-
arctica (Boraso de Zaixso et al. 2003, Quartino et al. 2005,
*Corresponding author: Franciane Pellizzari, Laboratório de Oliveira et al. 2009).
Ficologia e Qualidade de Água do Mar, Universidade Estadual do Monostromatic members of the Ulotrichales vary
Paraná (UNESPAR), Campus FAFIPAR, Comendador Correa Júnior 117, little in external morphology; however, the genera differ
82203-280 Paranaguá, PR, Brazil, e-mail: francianep@yahoo.com;
in the numbers of flagella on reproductive cells, ontogeny,
franciane.pellizzari@fafipar.br
Mariana C. Oliveira, Amanda da Silva Medeiros and Eurico C.
and life history (Bast et al. 2009a). Gayralia oxysperma
Oliveira: Departamento de Botânica, Instituto de Biociências, has saccate stages before developing laminar thalli,
Universidade de São Paulo, Rua do Matão 277, CEP 05508900 while Monostroma may or may not have “Codiolum” and
São Paulo, SP, Brazil tubular stages (Gayral 1965). Protomonostroma undu-
Nair S. Yokoya: Núcleo de Pesquisa em Ficologia, Instituto de latum has a long blade with dentate margins, asexual
Botânica, Secretaria do Meio Ambiente do Estado de São Paulo,
reproduction, and quadriflagellate zooids (Boraso de
Av. Miguel Estefano 3687, CEP 04301-012 São Paulo, SP, Brazil
Eurico C. Oliveira: Departamento de Botânica, Centro de Ciências Zaixso et al. 2003).
Biológicas, Universidade Federal de Santa Catarina, Florianópolis, In Brazil, monostromatic members of the Ulotrichales
SC 88040-970, Brazil have been reported as Ulvaria oxysperma (Kützing)

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198 F. Pellizzari et al.: Gayralia brasiliensis sp. nov.
Bliding, and Monostroma oxyspermum (Kützing) Doty
(Joly 1965, Cordeiro-Marino et al. 1993, Braga 1997, Braga
et al. 1997). However, U. oxysperma and M. oxyspermum
have been synonymized with Gayralia oxysperma basis
on thallus ontogeny and flagellar ultrastructural features
(South and Skelton 2003). Furthermore, Cordeiro-Marino
et al. (1993) and Braga et al. (1997) observed two differ-
ent thallus ontogenies for the two monostromatic green
species from São Paulo populations, and they identified
these taxa as Ulvaria oxysperma and Monostroma sp.
The taxonomic criteria used for distinguishing
Gayralia and Monostroma from the South Atlantic are
controversial. Pellizzari et al. (2008) studied the ontog-
eny and morphological characters in two populations
of monostromatic green algae from mangroves and
estuaries of the Paraná coast in southern Brazil and
concluded that the populations had distinct thallus
ontogenies. However, at that time, the absence of a
Codiolum phase and sexual reproduction suggested
that both populations could be assigned to the genus
Gayralia. Further experiments were performed, and
it was verified that the morphology, life history, and
anatomy of these taxonomic complexes had overlap-
ping features that were insufficient to establish species
identities. Hayden and Waaland (2002) used a molecu-
lar approach to provide a phylogenetic framework for
the Ulotrichales and Ulvales; however, in addition to G.
oxysperma and P. undulatum, DNA sequences are avail-
able for only eight species of Monostroma.
In this work, the ribosomal internal transcribed
spacer (ITS) region, including the ITS1, 5.8S gene, and
ITS2, was sequenced as an additional tool for the iden-
tification of these monostromatically bladed species
of Ulotrichales. We also discuss morphology, thallus
ontogeny, and life history, which are commonly used
tools for specific and generic characterization in the
Ulotrichales. On the basis of these studies, we describe
the occurrence of two monostromatic members of the
Ulvophyceae on the Brazilian coast, and report a new
species of Gayralia.
Materials and methods
Morphological analysis
Specimens of Gayralia brasiliensis were collected from
mangrove pneumatophores and rocks in the outer
sectors of Paranaguá and Guaratuba bays (25°33′S,
48°26′W and 25°52′S, 48°34′W) in Paraná State, southern
Brazil (n = 10 for each population). Specimens of Gay-
ralia oxysperma were collected in the inner sector of
Antonina Bay (25°25′S, 48°42′W), Paraná State. Voucher
specimens (holotype and isotype) were deposited in the
Phycological Herbarium of São Paulo at the Instituto de
Biociências, Universidade de São Paulo (SPF) and at the
Herbarium of Instituto de Botânica (SP), Brazil (Table
1). Anatomical observations were based on fresh and
preserved thalli (in 4% formalin). The selected material
was cleaned, and sections were made with a razor blade.
Semipermanent slides of handmade sections stained
with aniline blue were mounted in aqueous 30% corn
syrup (Karo; Unilever, São Paulo, Brazil). Besides color,
shape, and size of the thalli, we analyzed the following
morphological/anatomical characters under a micro-
scope (Axioskop 2; Zeiss, Göttingen, Germany): thallus
margins; position, size, and shape of holdfast; dimen-
sion and shape of cells; and shape and number of chlo-
roplasts in frontal view.
Thallus ontogeny and life history
The specimens were transported to the laboratory in an
insulated container. Small marginal pieces were excised
from the thalli, washed in sterile seawater, and placed
on glass slides inside Petri dishes with sterile seawater
to release zooids. After 24 h, the slides were transferred
to 50-ml flasks filled with seawater enriched with 25%
strength modified Provasoli’s solution (PES/4; Oliveira
et al. 1996) at 23 ± 1°C, 20 ± 1 salinity, 14:10 h light/dark
cycle, and an irradiance of 40 ± 10 μmol photons m-2 s-1.
Germanium dioxide (1 mg l-1) was used to suppress diatom
growth when necessary. The culture medium was renewed
weekly, and cultures were maintained for 2 months.
Morphology, liberation of zooids, germination pattern,
and thallus ontogeny were observed under a photomicro-
scope (Zeiss MC 80 DX).
Molecular analysis
The ITS sequences of the nuclear ribosomal genes were
obtained from 10 specimens of Gayralia brasiliensis
from Santa Catarina, Paraná, São Paulo, Rio de Janeiro,
Espírito Santo, Bahia, and Pernambuco states and from
three specimens of Gayralia oxysperma from Paraná and
São Paulo. An additional Gayralia sp. sample collected
in Tanzania was also included in the analysis. Samples
for molecular analysis were dried in silica gel; voucher
specimen numbers and sample collection sites are
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 F. Pellizzari et al.: Gayralia brasiliensis sp. nov. 199

Table 1 Monostromatic species sequenced in this study (SPF, Institute of Biosciences Phycological Herbarium, University of São Paulo, São
Paulo, Brazil).

Species Locality, date, and collector GenBank Voucher

Accession No. specimen

Gayralia oxysperma (Kützing) PR: Antonina Bay, PR, 09 Jul 2006, F. Pellizzari KC143758 SPF56197
K.L. Vinogradova ex Scagel, P.W. SP: Lagoinha Beach, Ubatuba, SP, 07 Aug 2005, KC143759
Gabrielson, D.J. Garbary, L. Golden, M.T. Fujii
M.W. Hawkes, S.C. Lindstrom, J.C. SC: Laguna, 23 Nov 2010, E.C. Oliveira KC143760 SPF57337
Oliveira et T.B. Widdowson

Gayralia brasiliensis Pellizzari, M.C. SC: Sambaqui Point, Florianópolis, SC, 01 Jun 2006, KC143761 SPF56250
Oliveira et N.S. Yokoya sp. nov. E.C. Oliveira
PR-1: Guaratuba Bay, PR, 11 Aug 2006, F. Pellizzari KC143762 SPF56198
PR-2: Maciel (cultivation net/pneumatophore), KC143763 SPF56157
Paranaguá Bay, PR, 01 Jan 2005, F. Pellizzari KC143764 SPF56199
SP: Dura Beach, Ubatuba, SP, 14 Oct 2008, E.C. SP427739
Oliveira SPF56199
SPF57054
RJ: Marambaia Island (Cadim), RJ, 15 Aug 2006, F. KC143765 SPF56285
Pellizzari
ES: Piúna Beach, Piúna, ES, 12 Sep 2007, D. Barata KC143766 SPF57026
BA-1: Tucuruipe Beach, Trancoso, Caraíva, BA, 07 KC143767 SPF57024
May 2007, D. Barata
BA-2: Caravelas, BA, 26 Jan 2008, E.C. Oliveira KC143768 SPF57027
PE-1: Itapissuma, PE, 09 May 2007, E.C. Oliveira KC143769 SPF57025
PE-2: Itapissuma, PE, 09 May 2007, E.C. Oliveira KC143770 SPF57025
Gayralia sp. Zanzibar, Tanzania, 05 Dec 2009, E.C. Oliveira KC143771 SPF57055

States of Brazil: BA, Bahia; ES, Espírito Santo; PE, Pernambuco; PR, Paraná; RJ, Rio de Janeiro; SC, Santa Catarina; SP, São Paulo.

listed in Table 1. Total DNA was extracted (after grind-
ing in liquid nitrogen) using the DNeasy Plant Mini Kit
(Qiagen, Valencia, CA, USA) according to the manufac-
turer’s protocol. The ITS region was amplified by PCR
using the following primers: 5′-TAGGTGAACCTGCGGAA-
GGAT-3′ (Milstein and Oliveira 2005) and 5′-ATATGCT-
TAAGTTCAGCGGGT-3′ (Bellorin et al. 2002), with the fol-
lowing cycle: 94°C for 5 min; 35 × : 94°C for 30 s, 55°C for
1 min; 72°C for 2 min; and a final extension step at 72°C
for 7 min. Amplification reactions were purified using
MicroSpin S-300 columns (Amersham Bioscience, Pis-
cataway, NJ, USA) and sequenced using BigDye in ABI
Prism 3100 (Applied Biosystems, Foster City, CA, USA)
according to the manufacturer’s protocol. For each PCR,
at least four sequences were obtained, one for each
PCR primer and one for each internal primer, 5.8SF and
5.8SR (Milstein and Oliveira 2005). The full sequence
was obtained from both DNA strands. The consensus
sequences were assembled using BioEdit 7.0.4.1 software
(Hall 1999). Multiple alignments for ITS sequences were
constructed with ClustalW in BioEdit, and included
other available sequences from GenBank. Ulothrix
zonata (Z47999) was used as an outgroup. Sequences
corresponding to amplification primers were removed
from the alignments, yielding a final matrix of 33 taxa
and 566 positions.
Phylogenetic relationships were inferred with PAUP
4.0b10 (Swofford 2002) and MrBayes v.3.0b4 (Ronquist
and Huelsenbeck 2003). An appropriate evolution model
was selected using MrModeltest 2.2 under the Akaike
information criterion (Nylander 2004): GTR+I+G model
with γ distribution = 1.8998; proportion of invariable
sites = 0.4004; base frequencies, A = 0.2333, C = 0.2905,
G = 0.2497, and T = 0.2265; and rate matrix, A-C = 1.0007,
A-G = 2.7173, A-T = 1.4502, C-G = 0.3629, C-T = 4.2904, and
G-T = 1.0000. Trees were inferred using three different
methods. A neighbor-joining (NJ) tree was built with
a Tamura and Nei (1993) substitution model, and a
maximum parsimony (MP) tree was inferred by a heuris-
tic search. In both NJ and MP trees, gaps were treated as
missing data, all sites were weighted equally, and 2000
replicates of bootstraps were performed. For Bayesian
analyses (MB), we performed two runs consisting of four
Markov chains over 4,000,000 generations, sampling
every 100 generations. The initial 50,000 generations
were discarded as burn in.

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200 F. Pellizzari et al.: Gayralia brasiliensis sp. nov.
Results
Gayralia brasiliensis sp. nov. Pellizzari, M.C.
Oliveira et N.S. Yokoya
Holotype SPF56198 (11 Aug 2006; F. Pellizzari).
Isotype SPF56199, SPF56157, SP427.739 (details in Table 1).
Type locality Guaratuba Bay, Paraná State, south Brazil
(25°52′S, 48°34′W).
Iconotype Figure 1A–D.
Diagnosis Gayralia brasiliensis differs from G. oxysperma
in its habitat in the intertidal zone of Brazilian shorelines,
in having higher halotolerance than this congener, its
formation of a flat blade after a filamentous phase rather
than a tube in the early stages of ontogeny of the macro-
scopic thallus, and its ITS sequence.
Description Single foliaceous monostromatic thallus,
bright green in nature and becoming dark olive green after
drying. Liberation of zooids from specimens starts with mar-
ginal zone discoloration, disintegration of the zooidangium
A C
1 cm
30 μm
Figure 1 Gayralia brasiliensis Pellizzari, M.C. Oliveira et N.S. Yokoya (iconotype).
(A) General aspect of the thallus (8 cm tall); (B) transverse section of the thallus; (C) detail of cells in the thallus; (D) detail of basal cells.
wall, and the release of four biflagellate zooids. Zooid
fusions and a Codiolum phase were not observed; there-
fore, reproduction is asexual. After zooid release and a
short swimming period, one of the flagella attaches to the
substratum, while the zooid spins a little longer until set-
tling. After zooid attachment, cell divisions start, giving
rise to a uniseriate filament, which becomes multiseriate
without formation of tubular or saccate stages. A small
foliaceous thallus develops and expands into a monostro-
matic plantlet after 30–40 days in culture, around 300 μm
broad. The mature thallus size is usually 7 ± 2.6 cm, with
cell size and thallus thickness of ∼8 ± 3 μm and ∼25 ± 1.8 μm,
respectively. Cells are uninucleate, with a large central
vacuole, parietal chloroplast, and one or two pyrenoids.
Morphological analysis The morphology and vegetative
anatomy of Gayralia brasiliensis is characterized by a single,
expanded, laminar, monostromatic thallus and the absence
of marginal teeth (Figure 1A). The basal holdfast is discoid
(1.1±0.1 mm in diameter), formed by long rhizoidal projec-
tions. The fronds are ca. 10 cm broad, with a thickness of
25.0±1.8 μm and cell lumen of 9.0±1.0 μm (Figure 1B). Cell
D
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sizes in the transverse section are ∼7.0±1.3 μm. In frontal
view, marginal and medium cells are 11±1.7 μm, and basal
cells 12±1.8 μm in diameter. The rhizoidal cells are longer,
with a length of ∼35±1.3 μm. Cells in the upper region have
irregular polygonal shapes; in surface view, cells are in
groups of two (Figure 1C), becoming more elongate toward
the base (Figure 1D). Cells are uninucleate with a large
central vacuole, parietal chloroplast, and one or two pyr-
enoids. We observed no perforations in thalli. The thallus
of Gayralia oxysperma is pale green and smaller (Figure 2)
than that of G. brasiliensis, ∼4.2±0.7 cm in diameter and
∼15±0.7 μm in thickness. Cells in the transverse section
measure ∼6±0.4 μm; marginal cells, 6±0.4 μm; mid thallus
cells, 7±0.8 μm; basal cells, 10±1.0 μm; and rhizoidal cells,
25±2.5 μm. Cells are uninucleate with parietal chloroplasts
and single prominent pyrenoids.
Ontogeny and life history In Gayralia brasiliensis,
the fertile region occurs at the thallus margin and can
occupy 15 ± 5% of the thallus area; it is differentiated by
a yellow or pale brownish color. Zooid release begins
when the zooidangium ruptures from the mother plant
and the wall disintegrates, releasing four biflagellate
zooids per cell. Zooid fusions were not observed. After
zooid release and a short swimming period, one flagel-
lum attaches on the substratum, while the zooid spins
until settling. Then, by degeneration and loss of the
flagellum, the cellular body extends to initiate the first
cell (9.0 ± 1.58 μm in diameter) with a prominent eyespot
and positive phototropism. After the first cell divisions,
a uniseriate filament is formed (30–40 μm long). After
14 days, a multiseriate filament (45–60 μm) is formed,
and rhizoids become evident (100–150 μm) after 17 days.
After 30–40 days, a foliaceous germling with a flat fan
shape (up to 250 μm in length) is formed; we did not
observe saccate or tubular stages. Under experimental
A B
1 cm
Figure 2 Gayralia oxysperma.
(A) General aspect of thallus (1.5 cm tall); (B) adult plants in the field ( ± 3 cm tall).
F. Pellizzari et al.: Gayralia brasiliensis sp. nov. 201
conditions, reproduction occurred only by asexual recy-
cling of the gametophytic blades through germination of
biflagellate zooids, followed by several mitotic divisions.
As there was no fusion of biflagellated zooids, the Codi-
olum phase was not formed.
Spores from Gayralia oxysperma were liberated and
germinated as described for Gayralia brasiliensis; however,
they produced an intermediate saccate-like stage before
giving rise to a foliaceous blade. This stage was well devel-
oped at ∼40 days in culture, when the plantlets were about
100 μm long. The saccate stage opened up after 50 days in
culture, giving rise to a foliaceous monostromatic blade
∼200 μm in length. No sexual reproduction was observed
in the specimens of Gayralia we analyzed, and the asexual
cycle repeated successively.
Molecular phylogeny ITS sequences were obtained for
10 samples of Gayralia brasiliensis and three samples of
Gayralia oxysperma collected along the Brazilian coast
(Figure 3). An additional sequence was obtained for a
sample collected in Tanzania (Table 1). The 10 samples
of G. brasiliensis had from 99.6% to 100% identity for ITS
sequences. The three samples of G. oxysperma collected
along the south-southeastern coast of Brazil were identi-
cal and had 99.4% identity to the available G. oxysperma
sequence (AY016306) from Australia.
The different phylogenetic inferences produced
similar results (Figure 4). Specimens of G. brasiliensis
formed a well-supported clade, closely related to Mon-
ostroma nitidum and two unidentified monostromatic
species from Tanzania and from Japan. This clade was in
turn sister to a clade containing all G. oxysperma. In our
analysis taking into account the actual species circum-
scriptions, the monostromatic genus Protomonostroma is
monophyletic, Gayralia is paraphyletic, and Monostroma
is polyphyletic.
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202 F. Pellizzari et al.: Gayralia brasiliensis sp. nov.
PE
BA
ES
RJ
SP
PR
SC
Figure 3 Geographical distribution of Gayralia brasiliensis (black
arrows) and Gayralia oxysperma (white arrows) along the Brazilian
coast.
SC, Santa Catarina (27°45′S, 48°30′W); PR, Paraná (25°33′S,
48°26′W); SP, São Paulo (23°55′S, 45°47′W); RJ, Rio de Janeiro
(22°37′S, 41°44′W); ES, Espirito Santo (19°59′S, 40°04′W); BA,
Bahia (13°38′S, 38°51′W); PE, Pernambuco (9°36′S, 35°42′W).
Figure 4 Bayesian ITS analysis showing the phylogenetic relation-
ships of the new species Gayralia brasiliensis (boldface) and other
available species.
Bootstrap support values for MP (normal type) and NJ (italics) analy-
ses are shown on the branches. Bayesian posterior probabilities are
represented by branch thicknesses (as indicated in the figure).
Discussion
Morphological features, life history, thallus ontogeny, and
distinct ITS sequences support the description of the new
species Gayralia brasiliensis, which is distributed along the
Brazilian coast. The ITS sequences also support the exist-
ence of another species of monostromatic Ulotrichales in
Brazil, identified as Gayralia oxysperma. As G. brasiliensis
has some overlapping characters of morphology and life
history with G. oxysperma, several taxonomic tools were
necessary to distinguish them.
The taxonomy of ulotrichalean monostromatic algae
is complex, having several inconsistencies among differ-
ent classification tools and lack of agreement on the bio-
logical features considered as relevant for the separation
of taxa. Phenotypic variation in this group is well docu-
mented. Kida (1990) demonstrated different morpholo-
gies in Monostroma latissimum related to distinct habitats,
which is commercially recognized in Japan as “Hirohano-
hitoegusa”. Also, a taxonomic key proposed for only the
Northern Hemisphere (Wittrock 1866) has generated disa-
greement among authors (synopsis in Golden and Garbary
1984).
During the past two decades, the life history and
thallus ontogeny of Ulotrichales species have been
studied (Tanner 1981, Tatewaki et al. 1983, Lokhorst 1984,
Golden and Cole 1986). Gayralia brasiliensis has a thallus
ontogeny similar to M. latissimum and Protomonostroma
undulatum, except for the hollow saccate phase in M. latis-
simum and quadriflagellate zooids in P. undulatum. The
thallus ontogeny of G. oxysperma has a saccate intermedi-
ate phase.
Thallus ontogeny has been considered a fundamental
feature for identifying genera of the Monostroma complex
specimens with similar morphology. However, members
of the Ulvophyceae are known to have broad variation in
reproductive patterns, and deletion of one or the other
alternate generations is common (Ohno and Rebello 1995,
Bast et al. 2009a). Therefore, as the study of the thallus
ontogeny and life history were not sufficient to identify
the samples analyzed in this work, the addition of other
taxonomic tools, mainly molecular data, was required.
Studying ecophysiological aspects and the life history of
two monostromatic green algae from the Paraná coast,
Pellizzari et al. (2008) suggested that halotolerance could
be used as an additional character to distinguish between
the two monostromatic green algae species that had broad
morphological plasticity and overlapping characters. The
material was identified at that time as two different species
of Gayralia. The authors found that plantlets of G. brasil-
iensis (named as Gayralia sp. 1) had a broader tolerance
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to high salinities and irradiances, with correspondingly
high growth rates at a salinity of 30. In contrast, plant-
lets of G. oxysperma (named as Gayralia sp. 2) had higher
growth rates at salinities around 15. These results suggest
distinct physiological responses of both species that are
congruent with their distributions on the coast of Brazil.
Specifically, G. oxysperma is limited to the inner sectors of
estuaries or brackish waters, whereas G. brasiliensis grows
in mangroves and rocks from the outer sectors of estuaries
and on protected rocky shores, where salinities are > 25.
These data also suggest that G. brasiliensis, with its larger
thallus, higher growth rate, and wider tolerance to envi-
ronmental variations, has higher potential for aquacul-
ture than G. oxysperma.
During an ecophysiological study of monostromatic
green algae from New England (USA), Guo and Mathieson
(1992) reported distinct patterns of seasonal occurrence,
longevity, zonation, and horizontal distribution within
an estuary and in open coastal areas. These authors
concluded that Ulvaria obscura (Kützing) J. Agardh and
G. oxysperma are widely distributed in inner areas of the
estuaries and that Ulvaria is annual, whereas the occur-
rence of Gayralia is restricted to the summer in the North-
ern Hemisphere. In contrast, Monostroma grevillei and
P. undulatum (as M. pulchrum) are winter-spring annuals
and occur preferentially in higher salinities in sheltered
areas. These observations were useful for identifying
species populations. Bast et al. (2009b) reported the occur-
rence of M. latissimum in three distinct habitats of marine
and estuarine regions within Tosa Bay, Japan. Thallus
appearance, decay, and maturation of sporophytes were
influenced by salinity. Despite these differences, the
ontogeny and nucleotide sequences from natural popula-
tions and cultivated isolates were identical.
Gayralia brasiliensis had similarities and overlapping
of characters with G. oxysperma, M. grevillei, M. latis-
simum, and P. undulatum. However, neither saccate nor
tubular stages during thallus ontogeny were observed in
the material studied (G. brasiliensis). Moreover, we did
not observe fusion of biflagellate zooids, and neither a
Codiolum phase nor alternation of generations during
the life history. Risso et al. (2003) observed asexual repro-
duction and similar ontogenetic features in P. undulatum;
however, the zooids were quadriflagellate.
Bast et al. (2009a) mentioned the similarities in the
life history and thallus ontogeny between the asexual
strain of M. latissimum and G. brasiliensis (as Gayralia
sp. 1), and suggested that these species might be closely
related taxa. Comparing the ITS1 sequence available for
M. latissimum, the two species are closely related but still
have 5.5% divergence based on this region alone.
F. Pellizzari et al.: Gayralia brasiliensis sp. nov. 203
Gayralia brasiliensis seems to reproduce only asexu-
ally, as fusion of zooids was not observed during the cul-
turing period. However, other factors (e.g., presence of
certain bacteria) that could influence the process of zooid
fusion should be tested in further studies. The occurrence
of asexual lineages across geographic distribution ranges
has been observed for many species of foliaceous algae,
such as Ulva spinulosa Okamura et Segawa (Hiraoka et al.
2003), suggesting that the loss of the sexual phase is
common in many species.
The thallus ontogeny, morphology, and geographic
occurrence of G. brasiliensis partially agree with previ-
ous reports for a taxon identified as Monostroma sp. from
southeastern Brazil (Cordeiro-Marino et al. 1993, Braga
et al. 1997).
Of relevance is the fact that three current tropical
species of Monostroma, M. crepidinium var. pseudocrepi-
dinium V.J. Chapman, M. dactyliferum W.R. Taylor, and M.
ecuadoreanum W.R. Taylor (Taylor 1945), were last cited
∼70 years ago. Furthermore, Monostroma lindaueri V.J.
Chapman, M. antarcticum V.J. Chapman, and M. moorei
V.J. Chapman, described from Australia and New Zealand,
were cited by Chapman (1956) ∼60 years ago. Also con-
sidering that 1) citations for these taxa are limited and 2)
taxonomic identifications of these samples were unac-
companied by explicit indication of voucher specimens,
we presume that these references have little or no taxo-
nomic value, and their taxonomic status remains unclear
(Guiry and Guiry 2012).
The genus Monostroma was polyphyletic in our phylo-
genetic analysis, as also observed by Hayden and Waaland
(2002) and Bast et al. (2009b) on the basis of the com-
bined analysis of rbcL and SSU rDNA. The lectotype of the
genus is Monostroma bullosum (Roth) Thuret, a freshwa-
ter species described in France, and occurring in Europe,
Australia, and New Zealand. However, while 29 species
are currently accepted, molecular marker sequences are
available for only eight taxa. Therefore, analyses of other
species, especially material from the type location, are
necessary before establishing a new genus.
Considering our proposal of the new species G. brasil-
iensis and the actual species circumscription, the genus
Gayralia is paraphyletic. However, Gayralia sp. from Tan-
zania and Monostroma sp. “Hitoegusa” were not identified
to the species level. We propose that those specimens are
likely two other undescribed species of Gayralia. Further-
more, Monostroma nitidum from China is quite divergent
from M. nitidum AY026917, indicating misidentification of
one of these two specimens. We propose that the speci-
men from China represents probably another undescribed
species of Gayralia. However, the close proximity of
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204 F. Pellizzari et al.: Gayralia brasiliensis sp. nov.
M. latissimum to G. brasiliensis based on ITS1 sequences
suggests that the former should be transferred to the genus
Gayralia. Therefore, the genus Gayralia would encompass
other species, including those with alternation of hetero-
morphic generations and/or asexual life histories, and
thallus ontogenies with or without saccate stages, includ-
ing ontogenies with “filament-blade” development and
those with “filament-sac-blade” development only; this
expansion of Gayralia would include taxa with tolerance
of higher salinities, in addition to those that occur in only
estuarine ecosystems or brackish waters.
The formation of a Codiolum phase has traditionally
been considered as a ulotrichalean requisite. However,
the Codiolum phase was not observed in either monos-
tromatic species occurring in Brazil, and in this respect
they do not fit the older concept of the order. Monostroma
grevillei and M. nitidum are among the minority of species
that have a Codiolum phase, producing tubes for mature
zoospores. Golden and Garbary (1984) suggest that growth
and development of a thalloid and Codiolum phases for
Monostroma species varies considerably.
The incongruence in taxonomic information relating to
monostromatic green algae around the world, the absence
of recent revisions for the group, and a unique and ancient
identification key (Wittrock 1866) have resulted in taxonomic
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