Morphology, ontogeny, and phylogenetic position of
Transcrição
Morphology, ontogeny, and phylogenetic position of
DOI 10.1515/bot-2012-0197 Botanica Marina 2013; aop 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, Chlorophyta), is described from Brazil on the basis of ontogenetic, morphological, and molecular phylogenetic data. Liberation of zooids from the monostromatically bladed thalli occurred by disintegration of the zooidangium wall, releasing four biflagellate cells with prominent eyespots. Fusion of zooids was not observed. After zooid attachment, cell divisions gave rise to uniseriate filaments that developed into fan-shaped blades. Only asexual reproduction was observed; this took place through recycling of blades by germination of zooids. Phylogenetic analysis based on rDNA internal transcribed spacer (ITS) sequences showed that G. brasiliensis specimens formed a monophyletic group closely related to Monostroma nitidum and two unidentified monostromatic species from Japan and Tanzania. The genus Monostroma was polyphyletic in our analysis. ITS sequences also confirmed the presence of a second monostromatic species of Ulotrichales in the Brazilian coast, Gayralia oxysperma. There were overlapping morphological and life history traits between G. brasiliensis and G. oxysperma. However, these two species were clearly divergent based on ITS sequences and ontogeny. Keywords: Gayralia brasiliensis sp. nov.; Gayralia oxysperma; internal transcribed spacer; ontogeny; Ulotrichales. *Corresponding author: Franciane Pellizzari, Laboratório de Ficologia e Qualidade de Água do Mar, Universidade Estadual do Paraná (UNESPAR), Campus FAFIPAR, Comendador Correa Júnior 117, 82203-280 Paranaguá, PR, Brazil, E-mail: [email protected]; [email protected] Mariana C. Oliveira, Amanda da Silva Medeiros and Eurico C. Oliveira: Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, CEP 05508900 São Paulo, SP, Brazil Nair S. Yokoya: Núcleo de Pesquisa em Ficologia, Instituto de Botânica, Secretaria do Meio Ambiente do Estado de São Paulo, Av. Miguel Estefano 3687, CEP 04301-012 São Paulo, SP, Brazil Eurico C. Oliveira: Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-970, Brazil Introduction Monostromatic members of the green algal order Ulotrichales are widely distributed from temperate to tropical seas (Leliaert et al. 2012). Some species have economic importance, mainly in the food and cosmetic industries (Pellizzari et al. 2007, Pise et al. 2012). Chemicals with antiviral and anticoagulant properties were recently isolated from some species in the genera Monostroma Thuret and Gayralia Vinogradova (Cassolato et al. 2008, Zhang et al. 2008). However, the taxonomy, biology, phylogeny, and biogeography of this group remain largely understudied. The taxonomy has undergone several nomenclatural changes since 1960 (e.g., Gayral 1965, Bliding 1968, Vinogradova 1969, Tatewaki 1972). Tentative revisions resulted in taxon transfers, and current species are mainly assigned to the genera Monostroma, Gayralia, and Protomonostroma Vinogradova. The genus Monostroma is cosmopolitan, comprising 60 species, of which only 29 are currently accepted taxonomically (Guiry and Guiry 2012). Gayralia and Protomonostroma are monospecific, comprising G. oxysperma (Kützing) K.L. Vinogradova ex Scagel, P.W. Gabrielson, D.J. Garbary, L. Golden, S.C. Lindstrom, J.C. Oliveira et T.B. Widdowson and P. undulatum (Wittrock) K.L. Vinogradova, respectively. Protomonostroma undulatum is found in southern Argentina and Antarctica (Boraso de Zaixso et al. 2003, Quartino et al. 2005, Oliveira et al. 2009). Monostromatic members of the Ulotrichales vary little in external morphology; however, the genera differ in the numbers of flagella on reproductive cells, ontogeny, and life history (Bast et al. 2009a). Gayralia oxysperma has saccate stages before developing laminar thalli, while Monostroma may or may not have “Codiolum” and tubular stages (Gayral 1965). Protomonostroma undulatum has a long blade with dentate margins, asexual reproduction, and quadriflagellate zooids (Boraso de Zaixso et al. 2003). In Brazil, monostromatic members of the Ulotrichales have been reported as Ulvaria oxysperma (Kützing) Authenticated | [email protected] author's copy Download Date | 2/13/13 1:28 PM 2 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 different 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 ontogeny 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 overlapping features that were insufficient to establish species identities. Hayden and Waaland (2002) used a molecular approach to provide a phylogenetic framework for the Ulotrichales and Ulvales; however, in addition to G. oxysperma and P. undulatum, DNA sequences are available 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 identification 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 Gayralia 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 microscope (Axioskop 2; Zeiss, Göttingen, Germany): thallus margins; position, size, and shape of holdfast; dimension and shape of cells; and shape and number of chloroplasts 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 photomicroscope (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 Authenticated | [email protected] author's copy Download Date | 2/13/13 1:28 PM F. Pellizzari et al.: Gayralia brasiliensis sp. nov. 3 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 Accession No. Voucher specimen Gayralia oxysperma (Kützing) K.L. Vinogradova ex Scagel, P.W. Gabrielson, D.J. Garbary, L. Golden, M.W. Hawkes, S.C. Lindstrom, J.C. Oliveira et T.B. Widdowson PR: Antonina Bay, PR, 09 Jul 2006, F. Pellizzari SP: Lagoinha Beach, Ubatuba, SP, 07 Aug 2005, M.T. Fujii SC: Laguna, 23 Nov 2010, E.C. Oliveira KC143758 KC143759 SPF56197 KC143760 SPF57337 Gayralia brasiliensis Pellizzari, M.C. Oliveira et N.S. Yokoya sp. nov. SC: Sambaqui Point, Florianópolis, SC, 01 Jun 2006, E.C. Oliveira PR-1: Guaratuba Bay, PR, 11 Aug 2006, F. Pellizzari PR-2: Maciel (cultivation net/pneumatophore), Paranaguá Bay, PR, 01 Jan 2005, F. Pellizzari SP: Dura Beach, Ubatuba, SP, 14 Oct 2008, E.C. Oliveira KC143761 SPF56250 KC143762 KC143763 KC143764 RJ: Marambaia Island (Cadim), RJ, 15 Aug 2006, F. Pellizzari ES: Piúna Beach, Piúna, ES, 12 Sep 2007, D. Barata BA-1: Tucuruipe Beach, Trancoso, Caraíva, BA, 07 May 2007, D. Barata BA-2: Caravelas, BA, 26 Jan 2008, E.C. Oliveira PE-1: Itapissuma, PE, 09 May 2007, E.C. Oliveira PE-2: Itapissuma, PE, 09 May 2007, E.C. Oliveira Zanzibar, Tanzania, 05 Dec 2009, E.C. Oliveira KC143765 SPF56198 SPF56157 SPF56199 SP427739 SPF56199 SPF57054 SPF56285 KC143766 KC143767 SPF57026 SPF57024 KC143768 KC143769 KC143770 KC143771 SPF57027 SPF57025 SPF57025 SPF57055 Gayralia sp. 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 grinding in liquid nitrogen) using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The ITS region was amplified by PCR using the following primers: 5′-TAGGTGAACCTGCGGAAGGAT-3′ (Milstein and Oliveira 2005) and 5′-ATATGCTTAAGTTCAGCGGGT-3′ (Bellorin et al. 2002), with the following 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, Piscataway, 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 heuristic 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. Authenticated | [email protected] author's copy Download Date | 2/13/13 1:28 PM 4 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 macroscopic 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 marginal zone discoloration, disintegration of the zooidangium A wall, and the release of four biflagellate zooids. Zooid fusions and a Codiolum phase were not observed; therefore, 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 settling. 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 monostromatic 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 projections. 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 C 1 cm D B 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. Authenticated | [email protected] author's copy Download Date | 2/13/13 1:28 PM F. Pellizzari et al.: Gayralia brasiliensis sp. nov. 5 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 pyrenoids. 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 flagellum 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 conditions, reproduction occurred only by asexual recycling of the gametophytic blades through germination of biflagellate zooids, followed by several mitotic divisions. As there was no fusion of biflagellated zooids, the Codiolum 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 developed 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 identical 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 Monostroma 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 circumscriptions, the monostromatic genus Protomonostroma is monophyletic, Gayralia is paraphyletic, and Monostroma is polyphyletic. 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). Authenticated | [email protected] author's copy Download Date | 2/13/13 1:28 PM 6 F. Pellizzari et al.: Gayralia brasiliensis sp. nov. Discussion PE BA ES SP RJ 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 relationships of the new species Gayralia brasiliensis (boldface) and other available species. Bootstrap support values for MP (normal type) and NJ (italics) analyses are shown on the branches. Bayesian posterior probabilities are represented by branch thicknesses (as indicated in the figure). 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 existence 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 different classification tools and lack of agreement on the biological features considered as relevant for the separation of taxa. Phenotypic variation in this group is well documented. Kida (1990) demonstrated different morphologies in Monostroma latissimum related to distinct habitats, which is commercially recognized in Japan as “Hirohanohitoegusa”. Also, a taxonomic key proposed for only the Northern Hemisphere (Wittrock 1866) has generated disagreement 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. latissimum and quadriflagellate zooids in P. undulatum. The thallus ontogeny of G. oxysperma has a saccate intermediate 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. brasiliensis (named as Gayralia sp. 1) had a broader tolerance Authenticated | [email protected] author's copy Download Date | 2/13/13 1:28 PM F. Pellizzari et al.: Gayralia brasiliensis sp. nov. 7 to high salinities and irradiances, with correspondingly high growth rates at a salinity of 30. In contrast, plantlets 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 environmental variations, has higher potential for aquaculture 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 occurrence of Gayralia is restricted to the summer in the Northern 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 occurrence 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 populations and cultivated isolates were identical. Gayralia brasiliensis had similarities and overlapping of characters with G. oxysperma, M. grevillei, M. latissimum, 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 reproduction 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. Gayralia brasiliensis seems to reproduce only asexually, as fusion of zooids was not observed during the culturing 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 previous 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. pseudocrepidinium 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 considering that 1) citations for these taxa are limited and 2) taxonomic identifications of these samples were unaccompanied by explicit indication of voucher specimens, we presume that these references have little or no taxonomic value, and their taxonomic status remains unclear (Guiry and Guiry 2012). The genus Monostroma was polyphyletic in our phylogenetic analysis, as also observed by Hayden and Waaland (2002) and Bast et al. (2009b) on the basis of the combined analysis of rbcL and SSU rDNA. The lectotype of the genus is Monostroma bullosum (Roth) Thuret, a freshwater 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. brasiliensis and the actual species circumscription, the genus Gayralia is paraphyletic. However, Gayralia sp. from Tanzania and Monostroma sp. “Hitoegusa” were not identified to the species level. We propose that those specimens are likely two other undescribed species of Gayralia. Furthermore, Monostroma nitidum from China is quite divergent from M. nitidum AY026917, indicating misidentification of one of these two specimens. We propose that the specimen from China represents probably another undescribed species of Gayralia. However, the close proximity of Authenticated | [email protected] author's copy Download Date | 2/13/13 1:28 PM 8 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 heteromorphic generations and/or asexual life histories, and thallus ontogenies with or without saccate stages, including 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 monostromatic 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 instability in this group. The use of molecular data, as a tool complementary to morphology and life history studies, is essential for better understanding the diversity and taxonomy of monostromatic green algae. Some recent and relevant studies show that molecular phylogenetic data are indispensable in assessing taxon boundaries in the Ulotrichales and Ulvales (Ichihara et al. 2009, Rinkel et al. 2012). The addition of ITS sequences was necessary to establish the identification of the monostromatic green alga occurring on the Brazilian coast. 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