Chinese Journal of Oceanology and Limnology Vol. 31 No. 3, P. 552-559, 2013 http://dx.doi.org/10.1007/s00343-013-2216-x

Culture observation and molecular phylogenetic analysis on the blooming green alga valida (, ) from China*

DENG Yunyan (邓蕴彦) 1, 2 , TANG Xiaorong (汤晓荣)3 , ZHAN Zifeng (詹子锋)1 , TENG Linhong (滕林宏) 1, 2 , DING Lanping (丁兰平) 1, 4 , HUANG Bingxin (黄冰心) 1, 4 , ** 1 Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 2 Graduate University of Chinese Academy of Sciences, Beijing 100049, China 3 Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao 266003, China 4 Shantou University, Shantou 515063, China

Received Sep. 6, 2012; accepted in principle Oct. 13, 2012; accepted for publication Dec. 17, 2012 © Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2013

Abstract The marine green alga Chaetomorpha valida fouls aquaculture ponds along the coastal cities of Dalian and Rongcheng, China. Unialgal cultures were observed under a microscope to determine the developmental morphological characters of C. valida . Results reveal that gametophytic fi laments often produce lateral branches under laboratory culture conditions, suggesting an atypical heteromorphic life cycle of C. valida between unbranched sporophytes and branched gametophytes, which differs from typical isomorphic alternation of Chaetomorpha species. The shape of the basal attachment cell, an important taxonomic character within the genus, was found variable depending on environmental conditions. The 18S rDNA and 28S rDNA regions were used to explore the phylogenetic affi nity of the taxa. Inferred trees from 18S rDNA sequences revealed a close relationship between C. valida and Chaetomorpha moniligera . These results would enrich information in general biology and morphological plasticity of C. valida and provided a basis for future identifi cation of green tide forming algae.

Keyword: blooming green algae; Chaetomorpha valida; branch; culture observation; heteromorphic life cycle; molecular phylogeny

1 INTRODUCTION and Rongcheng in China (Chi et al., 2009; Deng et al., 2011a, 2011b, 2012a). Its potential for southward The green algal genus Chaetomorpha belongs to the expansion along the Chinese coast was predicted in family (Cladophorales, Chlorophyta) Deng et al., 2011b. Astonishing quantities of algal and is characterized by unbranched fi laments. biomass drifted into aquaculture ponds, which resulted of the genus has been based primarily on in a deleterious physic-chemical environment and morphological attributes and about 60 species are caused large economic losses to local aquaculture. currently recognized (Guiry and Guiry, 2012). Chaetomorpha valida (Hooker et Harvey) Kützing,

1849, a marine species originally described from * Supported by General and Major Projects of the National Natural Tasmania, Australia, has been recorded in Australia Science Foundation of China (Nos. 31070185, 31093440), the Science (Womersley, 1984), New Zealand (Womersley, 1984), and Technology Project of Guangdong Province (Nos. 2011B031100010, 2012A020200007), the Talent Introduction Projects of Guangdong Fiji (South and Skelton, 2003) and China (Chi et al., Province Universities and Colleges, the Team Project of Natural Science 2009; Deng et al., 2011a, 2011b, 2012a; Ding and Foundation of Guangdong Province (No. S2011030005257), the Grant Luan, 2013). It typically grows on intertidal to supra- of the Key Laboratory of Integrated Marine Monitoring and Applied littoral rocks that are subject to strong surge. In the past Technologies for Harmful Algal Blooms, SOA (MATHAB) (No. MATHAB 20100301), and the Science and Technology Project of Shantou few years, seasonal accumulations of C . valida have City, China (No. 2011-162) constituted a major nuisance to the coasts of Dalian ** Corresponding author: [email protected] No.3 DENG et al.: Culture observation and phylogenetic analysis on C . valida 553

Only mechanical eradication has been used to al., 1990) results. The newly generated sequences ameliorate the periodic green algal blooms, which has were aligned using Clustal X (Thompson et al., 1997) not proved very effective. and ambiguously aligned positions were excluded Since its original description in 1849, C. valida has using BIOEDIT (Hall, 1999). The fi nal alignment for been poorly documented. The morphology and the 18S region comprised 18 taxa and 1 417 nucleotide taxonomy of the species has been discussed by positions; the 28S region included 16 taxa and 505 Womersley (1984), South and Skelton (2003), Chi et nucleotide positions. Because of a large numbers of al. (2009), Ding and Luan (2013), and the life cycle indels, the ITS sequences could not be aligned, and has been studied by Deng et al. (2011a). Gaps remain therefore the ITS gene data was excluded from the in our knowledge about its phylogenetic relationships. phylogenetic analysis. Phylogenetic trees were To further reveal the morphology and the phylogeny constructed using the maximum likelihood (ML) of C. valida , we examined its morphological method with the PHYML V. 2.4.4 (Guindon and development based on culture experiment, and Gascuel, 2003) and Bayesian inference (BI) approach sequenced 18S rDNA, ITS and 28S rDNA gene data as implemented in MRBAYES V. 3.1.2 (Ronquist and for a phylogenetic analysis. Huelsenbeck, 2003), using the GTR+I+G model selected by MODELTEST (Posada and Crandall, 2 MATERIAL AND METHOD 1998). Sequences of the genus Boodlea (Siphonocladales, Chlorophyta) were used as the 2.1 Specimen collection outgroup. Chaetomorpha valida samples were collected from aquaculture ponds in Rongcheng, Shandong, China 3 RESULT (37°16′N, 122°41′E) on 24 August 2008. Healthy thalli were selected, thoroughly cleaned and incubated 3.1 Morphological observations in sterilized seawater at 22°C. The morphology of the specimens from Rongcheng, 2.2 Unialgal culture experiment China matched previous descriptions of Chaetomorpha valida from China in terms of shape Unialgal culture were developed to investigate the and size (Table 1; Chi et al., 2009; Deng et al., 2011a; reproductive processes, morphogenesis and growth Ding and Luan, 2013). Thalli were deep green in characteristics of C. valida. Thalli were maintained in color, rigid in texture, and form straight, uniseriate, an incubator under 22°C in a 10 h: 14 h L: D cycle at unbranched fi laments (Fig.1a), nearly 1 m long. a light intensity of 72 μmol photons/(m2 ·s). Filaments Filaments without marked constrictions at septa developed sporangia within 7–14 days and unialgal (Fig.1b) were unattached and entangled to form cultures were established by isolation of zoospores. spongy tufts. Cells were barrel-shaped, 300–450 μm Enriched seawater (Deng et al., 2011a, 2011b) used in diameter, 300–1 000 μm long and 1.8–2.8 times as for unialgal cultures was renewed every 7 days. long as wide. Cells of unattached fi laments were Growth and development were observed regularly equal diameter throughout. Apical cells were with a light microscope. Observation and cylindrical with a rounded or obtuse tip (Fig.1c). Cell microphotography was conducted with a Nikon E400 walls were 10–20 μm thick, striated and lamellated. (Tokyo, Japan), Zeiss Stemi 2000-c (Wetzlar, Chloroplasts were parietal and reticulate with Germany) microscope and Nikon Coolpix 4500 numerous pyrenoids (Fig.1d). (Tokyo, Japan) camera. 3.2 Culture experiment 2.3 Molecular phylogenetic analysis Unialgal culture studies were started from 18S rDNA, ITS and 28S rDNA regions were zoospores, which were produced by transformation of sequenced for phylogenetic analysis. DNA extraction, vegetative cells into zoosporangia (Fig.2a). Matured PCR amplifi cation and sequencing were performed as zoospores were discharged from papilla-shaped described (Leliaert et al., 2007; Teng, 2011). The liberation pores (Fig.2b). Zoospores were ovoid, related sequences (18S rDNA, max identities ≥95%; measuring (6–9)×(10–13) μm (Table 2), and each of 28S rDNA, max identities ≥86%) were downloaded them had a red eyespot and a chloroplast with many from GenBank according to the BLAST (Altschul et pyrenoids. Four equal fl agella were present on the 554 CHIN. J. OCEANOL. LIMNOL., 31(3), 2013 Vol.31

Table 1 Comparison of selected features of Chaetomorpha valida populations recorded from China

Present isolation Chi et al. (2009) Deng et al. (2011a) Ding and Luan (2013) Color Deep green Deep green Light green Deep green Texture Rigid Rigid and crisp Firm and crisp Firm Thalli shape Nearly uniform diameter Nearly uniform diameter Nearly uniform diameter Nearly uniform diameter Cell shape Barrel-shaped Barrel-shaped Cylindrical Cylindrical Cell length(μm) 300–1 000 200–460 150–700 300–1 000 Cell width (μm) 300–450 200–300 265–475 280–400 Length-width ratio 1.8–2.8 1–2 0.5–2.1 1–3.3 Unattached or attached Holdfast shape Attached with basal rhizoidal Unattached Unattached with basal holdfast discs Upper intertidal Upper intertida or Upper intertidal Upper intertidal to supralittoral Habitats aquaculture ponds aquaculture ponds aquaculture ponds rocks or aquaculture ponds

ab

1 mm 500 μm

cd

500 μm 100 μm

Fig.1 Chaetomorpha valida : morphology in culture a. General morphology; b. A fi lament without marked constrictions at septa; c. An apical cell with an obtuse tip (arrow); d. A cell with numerous pyrenoids. Arrows note pyrenoids. anterior of the zoospores (Fig.2c). Zoospores lost obvious apico-basal polarity at that time. Initial cells motility in several minutes and became sub-spherical divided into two differentiated cells: an apical cell after settling (Fig.2d). Zoospore germination was the and a basal cell (Fig.2f). The apical cell divided bipolar erect type. It began with upward and continuously to form erect uniseriate fi laments, while downward cell elongation, leading to apical and the proximal ends of basal cells elongated to produce rhizoidal portions (Fig.2e). Cells appeared to have rhizoidal holdfasts, which were aseptate and extended No.3 DENG et al.: Culture observation and phylogenetic analysis on C . valida 555

ab c

de i

f g hj

kmn

Fig.2 Chaetomorpha valida : events of reproduction and development observed in culture a. Early stage of the formation of zoosporangia. Arrows note zoosporangia. Scale bar=500 μm. b. Emptied zoosporangium. Arrows note liberation pores. Scale bar=300 μm. c. A quadrifl agellate zoospore. Scale bar=10 μm. d. A settled zoospore. Scale bar=10 μm. e. A zoospore elongating to form apical and basal portions. Scale bar=20 μm. f. A germling with an apical cell and a basal cell. Scale bar=40 μm. g, h. Early stage of the branches formation. Scale bar=500 μm. i, j, k. Gametophytic fi laments with lateral branches. Scale bar=500 μm. m. A bifl agellated gamete. Scale bar=10 μm. n. Two gametes fusing at their sides. Scale bar=10 μm. by apical growth. Cells produced lateral protuberances, which Zoospore germination gave rise to gametophytes functioned as apical cells for lateral branches and gametophytic fi laments with lateral branches (Fig.2g–h). Acute angles formed between lateral were frequently observed in our study (Fig.2g–k). branches and primary fi laments. The lateral branch Branching always commenced in cells within healthy could be composed of dozens of cells and no longer fi laments, usually in the apical or intercalary position. produced a secondary branch (Fig.2i–k). 556 CHIN. J. OCEANOL. LIMNOL., 31(3), 2013 Vol.31

Cladophora ohkuboana 39/0.65 AB062708 Cladophora sakaii AB062709

Chaetomorpha linum AB062702

Chaetomorpha antennina AB062700 35/* 61/0.88 98/1.00 Chaetomorpha crassa AB062701

47/0.53 62/0.83 Rhizoclonium grande AB062714

100/1.00 Chaetomorpha valida JQ308276 95/1.00 Chaetomorpha moniligera AB062703

Rhizoclonium riparium AB202077 98/* 100/1.00 Rhizoclonium hieroglyphicum AB256042 97/1.00 Cladophora albida Z35317

93/1.00 Cladophora sericea Z35320

78/0.98 Cladophora glomerata AB062706

65/0.96 Cladophora vagabunda Z35316 Chaetomorpha okamurae AB062704

Chaetomorpha catenata Z35418

Cladophora socialis AM498753

Boodlea composita AF510156

0.01 Fig.3 Nuclear 18S rDNA topology of Chaetomorpha The most likely ML tree with branch lengths. The Bayesian tree is inferred using MrBayes and the ML tree are similar in topology. Node support is as follow: ML bootstrap/BI (MrBayes) posterior probability. “*” refl ects minor differences in topology that could not be represented on the consensus tree. The new sequence submitted in this study is in bold.

After maturation, dioecious gametophytes yielded Table 2 Chaetomorpha valida : swarmer dimensions in isogametes. The shape and behavior of gametes culture resembled those of zoospores except that gametes had Swarmer Length (μm) Width (μm) two fl agella (Fig.2m) and their sizes were smaller Zoospore 11.5±0.74 7.1±0.69 than those of zoospores (Table 2). When heterosexual Gamete 8.4±0.81 5.3±0.63 gametes met, gamete fusions took place (Fig.2n) and resulted in zygotes. Zygotes were (5–10)×(9–16) μm Zygote 12.7±1.51 7.4±1.06 in size (Table 2) with two-eyespots. Germination of Values are Means±SD; n =30. zygotes was similar to that of zoospores except that no branch formation was observed. 0.1% dissimilarity), and they branched closely in all 3.3 Phylogenetic analysis the 18S rDNA trees (Fig.3). In all 28S rDNA topologies, C . valida formed a single clade nesting within the The newly generated sequences of the partial 18S Chaetomorpha branch with low support (Fig.4). rDNA, ITS and 28S rDNA regions of Chaetomorpha valida were deposited into GenBank under accession 4 DISCUSSION numbers JQ308276, JQ308262 and JX995108, 4.1 Morphology respectively. The BLAST search results showed that the ITS sequence was 91% identical to the closest Chaetomorpha is characterized by uniseriate, sequence, Chaetomorpha sp. (GenBank No. straight, unbranched fi laments attached with an FR694876), and 88% identical to the next closest elongate, thick-walled, basal cell or loose-lying sequence, Chaetomorpha norvegica (GenBank No. without basal cell. C. valida was recognized by fi rm FR694877). The 18S sequences of C . valida and and crisp textured tufts, composed of nearly uniform Chaetomorpha moniligera were nearly identical (only diameter fi laments without marked constrictions at No.3 DENG et al.: Culture observation and phylogenetic analysis on C . valida 557

Chaetomorpha norvegica 77/0.68 FN257509 regional pools vs. usually epiphytic on Sargassum Chaetomorpha sp. FR694875 20/* Chaetomorpha melagonium FN257511 spp. or culture rafts in subtidal zones; Table 3). 100/1.00 Chaetomorpha gracilis FN687242 Compared with Chaetomorpha moniligera (Fig.3), 26/* 83/0.86Chaetomorpha brachygona AJ544759 C. valida was nearly equal in diameter throughout, Chaetomorpha sp. FN687243 96/0.92 Chaetomorpha aerea FM205025 while C. moniligera had gradually increasing diameter 42/* Chaetomorpha valida JX995108 from base to apex. Some other important diagnostic Chaetomorpha antennina FN687237 38/* characters, such as thalli height and shape, cell shape 96/1.00 Chaetomorpha sp. FN687244 74/95 Chaetomorpha crassa AJ544767 and diameter, were also markedly distinct between 100 /1.00 Chaetomorpha spiralis AJ544766 the two taxa (Table 3), and that warranted the Rhizoclonium africanum Cladophora albida FN687245 recognition of separate species. 24/* AM503433 100/1.00 Cladophora vagabunda AJ544760 Boodlea composite AJ544731 4.2 Culture observation

0.1 We investigated the developmental morphogenesis Fig.4 Nuclear 28S rDNA topology of Chaetomorpha of C. valida in culture. In our study, male and female The most likely ML tree with branch lengths. The Bayesian tree inferred gametes discharged from gametophytes fused into using MrBayes and the ML tree are similar in topology. Node support is as zygotes. Zygotes settled and developed into follow: ML bootstrap/BI (MrBayes) posterior probability. “*” refl ects minor unbranched sporophytes, which then released differences in topology that could not be represented on the consensus tree. The new sequence submitted in this study is in bold. zoospores. Zoospores germinated and developed into newly branched gametophytes to complete the life cycle (Fig.5). The developmental morphologies of C. valida here were similar as in Deng et al. (2011a), Zoospores except for the branch formation, which contrasts with the apparently constant morphology of Chaetomorpha species in their natural habitat. Although the formation Branched of lateral branches in cultured Chaetomorpha has been Unbranched gametophytes sporophytes documented in Leliaert et al. (2011), of note here were that gametophytes have several branches, which were not seen in the sporophytes. An atypical heteromorphic Sporophytic stage Gametophytic stage life cycle was suggested for C. valida , alternating between unbranched sporophytes and branched gametophytes (Fig.5), which was in confl ict with the ♂ Gametes typical isomorphic alternation of Chaetomorpha Zygotes ♀ species (Deng et al., 2011a). However, it is unknown metes Ga whether the atypical phenomenon was genetically constrained or environmentally plastic. Molecular phylogenetic data illustrated that unbranched Fig.5 The heteromorphic life cycle of Chaetomorpha valida fi lamentous thalli (Chaetomorpha or Rhizoclonium ) in culture independently evolved from branched forms several Male and female gametes discharged from gametophytes fused into times within the Cladophorales (Hanyuda et al., 2002). zygotes. Zygotes settled and developed into unbranched sprophytes, which This suggested that the unbranched fi lamentous then released zoospores. Zoospores germinated and developed into new architecture evolved early from branched condition branched gametophytes to complete the life cycle. and branching genetic potential might still present in Chaetomorpha. Besides, Environmental cues were septa (Chi et al., 2009; Ding and Luan, 2013). inferred at least partly account for the branch induction Compared with a morphologically closely related of Chaetomorpha (Leliaert et al., 2011). Further species, Chaetomorpha linum , C. valida usually had culture studies of unbranched and branched algae wider cell length-width ratio (1.8–2.8 vs. 0.5–1, under various culture conditions are warranted. In occasionally more than 1; Table 3). C. valida could be addition, future comparative transcriptome studies separated from the morphologically closely related would facilitate understanding of the genetic factors species Chaetomorpha vieillardii (as Chaetomorpha controlling morphogenesis. crassa ) by habitat (often drifting in the upper intertidal Chaetomorpha are usually unattached without 558 CHIN. J. OCEANOL. LIMNOL., 31(3), 2013 Vol.31 basal cells, or attached with discoid, conical, hapteroid 4.4 Complementary approaches for Cladopho- or rhizoidal holdfast. The characters of basal raceae taxonomy attachment cells are important taxonomic criteria within the genus. However, the trait seemed to be Traditional method was inadequate for variable in C. valida , since loose-lying fi laments were Chaetomorpha taxonomy because of the limited found without basal cells in nature (Chi et al., 2009; number of characteristics that could be used for reliable Teng, 2011; Ding and Luan, 2013), discoid holdfasts morphological identifi cation. Most Chaetomorpha were described by Deng et al. (2011a) and rhizoidal were purely delineated using morphological traits, basal cells were found in this study. Basal attachment which led to taxonomic confusion. Diffi culties were cell formation has been previously recorded as encountered especially when transitional stages susceptible to environmental conditions in unbranched specimens were collected (Patel, 1971). Cladophorales (Nienhuis, 1975; Price, 1976). For years, numerous attempts were aimed to Therefore, specimens under different cultural embrace other approaches, such as cytological, conditions should be examined to test intraspecifi c biochemical and ultrastructural methods, were used variation within Chaetomorpha . to acquire supplementary information for green algal taxonomy (Mathieson et al., 1981). Such additional 4.3 Phylogenetic analysis diagnostic evidence was particularly benefi cial to inherently polymorphic groups. Several studies have 18S rDNA and 28S rDNA, the two molecular verifi ed the usefulness of culture observations on markers that have been widely applied to species morphological-taxonomic problems in the classifi cation and phylogenetic analysis (Blomster et Cladophorales (van den Hoek, 1963; Price, 1967; al., 1999; Hayden et al., 2002, 2003; Leliaert et al., Patel, 1971; Kornmann, 1972; Nienhuis, 1975); and 2007, 2009, 2011; Teng, 2011; Deng et al., 2012b), molecular tools were invaluable at offering insights were employed to investigate the phylogenetic affi nity into phylogenetic relationships. Our study revealed of C. valida . Phylogenetic trees (Fig.3–4) clearly that the combination of culture observations with revealed that our material clustered with other molecular tools was helpful to differentiate Chaetomorpha species and belonged to the genus morphologically simple taxa like Chaetomorpha . Chaetomorpha . The 18S sequences of C . valida and Since morphology is not a good indicator for species Chaetomorpha moniligera were nearly identical, and boundaries and relationships in Cladophorales they clustered with each other closely in all the 18S (Bakker et al., 1995a, 1995b; Leliaert et al., 2009; rDNA analyses (Fig.3). Although relationships 2011), by virtue of such supplementary methods between the two species were uncertain in our ITS associated with morphology, efforts to clarify and 28S rDNA analysis because of the lack of confusion and catalogue species need to be prioritized sequences from C . moniligera , the high similarity and in Cladophoraceae taxonomy. topology of the 18S gene sequences strongly supported their close phylogenetic affi nity. 5 ACKNOWLEDGMENT The systematics of many unbranched fi lamentous green algal taxa, especially Chaetomorpha species, Sincere thanks to Mr. LIAN Shaoxing for has remained unresolved. 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