Tracing of Systematic Characters of Bangiales (Rhodophyta) Based on Molecular Phylogeny Inferred from Sequences of Nuclear Small Subunit Rrna Genes

Suisanzoshoku 52(2), 185-198 (2004)

Tracing of Systematic Characters of Bangiales (Rhodophyta) Based on Molecular Phylogeny Inferred from Sequences of Nuclear Small Subunit rRNA Genes

Kazuyo MATSUYAMASERISAWA1'4, Satoru YAMAZAIU2,Yuklhlro KITADE3,6, Yukihiko SERISAWA4,6,Kazuyoshi KUWANO5and Naotsune SAGA3*

Abstract: Tracing of 15 systematic characters of Bangiales in which the genus Bangia and Porphyra are included was conducted based on a molecular phylogenetic tree constructed from small subunit (SSU)rRNA gene sequence data of 57 strains using ACCTRAN optimization. The SSU rRNA gene sequence data of Porphyra were determined in seven species including three unregistered species. In the phylogenetic tree, most strains diverged largely into three clades, one Bangia clade and two Bangia-Porphyra complex clades. This indicates that the genus Bangia and Porphyra are not monophyletic. In tracing, the evolution of gametophyte shape (systematic character of the genus) from cylindrical to leafy or leafy to cylindrical is considered to have occurred at least once or twice, respectively. The evolution of the cell layer in gametophytes (systematic character of the subgenus Diploderma) from monostromatic to distromatic is considered to have occurred once, but monostromatic strains appeared in two lineages. The evolution of chloroplast number per cell in gametophytes (systematic character of the subgenus Diplastidia) from monoplastid to diplastid is suggested to have occurred twice in different lineages. Although several assemblages were confirmed, strains with the same character status appeared in various lineages in terms of other systematic characters. Therefore, the systematic characters used in the present study were concluded not to reflect the SSU rRNA phylogeny. This is the first report on tracing systematic characters in Bangiales.

Key words: Bangiales; Phylogeny; SSU rRNA gene; Systematic character

Bangiophyceae (Rhodophyta) is comprised of at several billion US dollars. The following are five orders including Bangiales (van den Hoek principal factors for a successful aquaculture of et al. 1995) and Bangiales contains only one Porphyra, securing high-quality strains, mass- family (Bangiaceae) with two genera, Bangia production of seedlings, and managing a culture and Porphyra (Chihara 1997). Some of them ground. To understand diversity in Bangiales are edible, and they are called layer or•enori•fin plants in which some aqua-industrially valuable Japanese. Layer is the most important marine species are included and to clarify phylogeny crop and has been used widely in Japan, and and evolution various characters of them are the annual sales of their products are estimated a profitable subject for improving high-quality Received May 12, 2004: Accepted May 31, 2004. 1Marine Ecosystems Research Department , Japan Agency for Marine-Earth Science and Technology, Natsushima 2-15, Yokosuka, Kanagawa 237-0061, Japan. 2 Graduate School of Marine Science and Technology , Tokai University, Orido 3-20-1, Shimizu, Shizuoka 424-8610, Japan. 3 Graduate School of Fisheries Sciences , Hokkaido University, Minato 3-1-1, Hakodate, Hokkaido 041-8611, Japan. 4 Marine Bcosystems Research Center , Chiba University, Uchiura 1, Amatsu-kominato, Chiba 299-5502, Japan. 5 Graduate School of Science and Technology , Nagasaki University, Nagasaki, Nagasaki 852-8521, Japan. 6JSPS Research Fellow , Japan Society for the Promotion of Science, Ichiban 6, Chiyoda, Tokyo 102-8471, Japan. * Corresponding author , e-mail: [email protected] 186 K. Matsuyama-Serisawa, S. Yamazaki, Y. Kitade, Y. Serisawa, K. Kuwano and N. Saga

strains. Because of the economic importance Although Bangia had been considered to of Porphyra, extensive taxonomic studies be a sister group to Porphyra (Garbary et al. have been performed in this genus (Kurogi 1980), recent molecular phylogenetic trees 1972; Conway et al. 1975; Lindstrom and Cole have contradicted the traditional placement of 1992a, 1992b) and over 130 species have been Bangia (Stiller and Waaland 1993; Oliveira et described worldwide (Yoshida et al. 1997). al. 1995; Brodie et al. 1997; Woolcott and King The taxonomy of Bangia is complex and 1998) . Based on sequences of nuclear small- many species are unified, but at least several subunit (SSU) rRNA genes, Oliveira et al. species have remained (Cole and Sheath 1990). (1995) showed that the distromatic species, Although some species of Bangia occur in fresh Porphyra amplissima, is more closely related water, most species of Bangia and Porphyra to the monostromatic P. leucosticta than to the grow in intertidal rocky substrata in seawater distromatic P miniata, despite the fact that from subarctic to subtropical regions (Sheath these species were previously placed in the and Cole 1984; Cole and Sheath 1990). subgenus Diploderma. Stiller and Waaland Species of both Bangia and Porphyra have a (1993) also suggested that the distromatic dimorphic life cycle with a macroscopic thallus species P. cuneiformis appeared more closely of gametophyte generation and a microscopic related to the monostromatic P. nereocystis and filament of sporophyte generation (conchocelis P. thuretii than to the distromatic P. scizophylla phase) (Honi 1993) . Generic concepts divided on the basis of restriction fragment-length into Bangia and Porphyra are based on the polymorphism analysis. However, no studies morphological characters of their macroscopic have been conducted to determine the thalli; the thallus of Bangia represents a molecular phylogeny of diplastidic species cylindrical form, while that of Porphyra is a of Porphyra. Thus molecular information on leafy form. Kurogi (1972) classified Porphyra Bangiales is insufficient. Furthermore, no into three subgenera, Diplastidia, Diploderma, tracing of systematic characters based on and Porphyra, based on the number of cell molecular phylogeny has yet been performed layers (monostroma or distroma) and the in Bangiales. plastid number included per cell (monoplastid The aim of the present study was to trace or diplastid) . In general, species of Porphyra the systematic characters of Bangiales based have been delineated by morphological and on molecular phylogeny using sequences of cytological features of the gametophytes nuclear SSU rRNA genes. (Chang and Zheng 1960; Miura 1961, 1968; Kurogi 1972; Oliveira and Cole 1975; Abbott Materials and Methods and Hollenberg 1976; Cole and Sheath 1990; Lindstrom and Cole 1992a; Lindstrom and Cole Seven species of Porphyra were used for 1993; Notoya et al. 1993; Matsuo et al. 1994; determination of SSU rRNA sequences. Their Miyata and Kikuchi 1997; Nelson et al. 1998; original collection sites and dates are presented Yoshida 1998). in Table 1. Except for P amplissima and

Table 1. List of species in which the SSU rRNA sequence was determined in the present study and their original collection sites and dates Systematic Character Tracing in Bangiales 187

P. variegata, they were grown in ESS2 medium software (Strimmer and von Haeseler 1996).

(Kitade et al. 1996) at 15•Ž, 50ƒÊmol m-2s-1, with To confirm the confidence intervals of each cool white fluorescent lamps and a photoperiod branch, the bootstrap method was employed of 10-h light : 14-h dark in our laboratory. (Felsenstein 1985) or the quartet puzzling Leafy gametophytes of P. amplissima and P. supported value was calculated (Strimmer and variegata were collected in the field, stored at von Haeseler 1996) with 1000 replicates. Other 4•Ž until use and used for DNA extraction after Bangiophyceae algae, Erythrocladia sp. and cleaning of epiphytes. Genomic DNA from the Erythrotrichia carnea (Erythropeltidales), were species of Porphyra used in the present study selected as an outgroup. was extracted applying the method reported by The following systematic characters Yamazaki et al. (2003) except for P. amplissima were used for cladistic analysis: shape of and P. variegata (Yamazaki et al. 1996) and the gametophytic thallus, number of cell layers, SSU rRNA gene was amplified from genomic number of chloroplasts per cell, thickness, DNA using the polymerase chain reaction color, marginal undulation, marginal dentation,

(PCR) with the primers (PY1 and PYR1) and number of chromosomes per cell, season of reaction conditions described by Yamazaki appearance, sexuality, distribution of male et al. (1996). When the amplification of DNA and female cells, division form of sexually was difficult or could not be detected, SSU reproductive cells (Hus formula), and the rRNA was obtained using nested PCR (White presence of asexual reproduction, all of which et al. 1990) with PY1 (Yamazaki et al. 1996) referred to gametophytes; and presence of and PYNESTRI (5'-TTGTTACGACTTCTC asexual reproduction of sporophytes (Table 3). CTTCC-3') . The amplified fragments were The statuses of these characters were coded purified by isopropanol precipitation. The based on previous studies (Chang and Zheng purified double-stranded DNA fragments were 1960; Miura 1961, 1968; Kurogi 1972; Oliveira directly sequenced using a Genetic Analyzer and Cole 1975; Abbott and Hollenberg 1976; 310 (Applied Biosystems Japan, Tokyo). Whole Cole and Sheath 1990; Lindstrom and Cole regions used in this study were confirmed by 1992a; Lindstrom and Cole 1993; Notoya et al. sequencing in both directions. 1993; Matsuo et al. 1994; Miyata and Kikuchi Alignment of SSU rRNA gene sequences 1997; Nelson et al. 1998; Yoshida 1998) (Table of Bangiales registered in the DDBJ/EMBL/ 4) . Tracing of these characters was conducted GenBank (Muller et al. 1998; Kunimoto et al. based on the SSU rRNA gene tree constructed 1999; Broom et al. 1999) was performed using the NJ method (showing the highest using the CLUSTAL W computer program bootstrap value) using the MacClade 3.05

(Thompson et al. 1994; Higgins et al. 1996) program (Maddison and Maddison 1992) with and then refined by eye (Table 2) . Some ACCTRAN optimization. In case two or more sequence data (e.g., AF123051, AF133792, strains formed one cluster on the SSU rRNA AF136419, and AF136425) of Bangiales, gene tree, e. g., P. haitanensis, P. pseudolinearis, which were also registered in the database P suborbiculata, P. tenera, P. umbilicalis, and of GenBank, were not used because their P yezoensis, they were reduced to one strain alignment was impossible. Neighbor-joining (Table 2).

(NJ) analyses were performed using the DNADIST, NEIGHBOR, SEQBOOT, and Results CONSENSE (1000 bootstrapped replicates) programs from the PHYLIP package The SSU rRNA gene sequences of seven

(Felsenstein 1993) . Parsimony trees were species including three unregistered species constructed using PAUP version 3.1.1 software of Porphyra were determined and registered

(Swofford 1993). Maximum likelihood (ML) with the database of DDBJ/EMBL/GenBank analyses were performed using PUZZLE 4.0.2 (Table 2) . The lengths of the SSU rRNA gene 188 K. Matsuyama-Serisawa, S. Yamazaki, Y. Kitade, Y. Serisawa, K. Kuwano and N. Saga

Table 2. List of strains used for molecular phylogenetic analysis

* Strains used for tracing of systematic characters . Systematic Character Tracing in Bangiales 189

Table 3. Systematic characters of Bangiales and their status codes used in the present study

The characters are composed of morphological, cytological, ecological, and reproductive features of gametophytes (1-14) and reproductive features of sporophytes (15).

Table 4. Status of systematic characters corresponding to Table 3

Code•e?' indicates no information. sequences of the seven species were from 1800 Three phylogenetic trees, the NJ tree, by to 2300 bp. The SSU rRNA gene sequences most parsimonious (MP) tree, and ML tree, of 50 strains of Bangiales and two strains of were constructed based on the SSU rRNA Erythropeltidales (outgroup) were obtained sequences of the 59 strains. MP analysis from the GenBank database and a total of produced 54 parsimonious trees with 1969 steps 59 strains including seven newly registered (consistency index: 0.53, retention index: 0.73). species was used for construction of the Among the three trees, the highest bootstrap phylogenetic trees (Table 2). values were confirmed in the NJ tree (Fig. 1). 190 K. Matsuyama-Serisawa, S. Yamazaki, Y. Kitade, Y. Serisawa, K. Kuwano and N. Saga

Fig. 1. Phylogenetic tree of Bangiales inferred from sequences of nuclear SSU rRNA genes constructed using the neighbor-joining method. Scale bar indicates an evolutionary distance of 0.1 nucleotide substitution per position in the sequence. The numbers under the branches indicate bootstrap values greater than 50.

In all trees, Porphyra sp. (AF117239) branched clade A, strains of Bangia branched first and first, then Bangia sp. (AF043364) branched, then strains of Porphyra branched. In the and other Bangiales were largely divided into Bangia Glade, only strains of Bangia branched. three clades. All strains of Porphyra except one In the Bangia-Porphyra complex clade B, all (AF117239) were contained in the two clades distal branches (Fig. 1, clades a-i) were divided with some strains of Bangia. Among the three similarly in all trees, but the ML tree formed clades, the Bangia-Porphyra complex clade A a polytomy, and the order of branching in diverged first, then the Bangia Glade diverged, the MP tree was partially different from that and finally the Bangia-Porphyra complex clade in the NJ tree. The strains of P. haitanensis, B diverged. In the Bangia-Porphyra complex P. suborbiculata, P. tenera, P. umbilicalis, and Systematic Character Tracing in Bangiales 191

Fig. 2. Cladistic trees of systematic characters 1-3 of Bangiales based on the neighbor-joining tree inferred from sequences of nuclear SSU rRNA genes using ACCTRAN optimization. The statuses of systematic characters used in the analysis are given in Tables 3 and 4.

P. yezoensis were gathered within the same genus or subgenus were as follows (Fig. 2). branches in each species, while strains of P. Character 1 (shape of gametophytes): Both amplissima and P. pseudolinearis were divided cylindrical and leafy strains were included into different branches. in Bangia-Porphyra complex clades A and Tracing of three systematic characters of a B. In the Bangia-Porphyra complex clade B, 192 K. Matsuyama-Serisawa, S. Yamazaki, Y. Kitade, Y. Serisawa, K. Kuwano and N. Saga

Fig. 3a

Fig. 3a-c. Cladistic trees of systematic characters 4-15 of Bangiales based on the neighbor-joining tree inferred from sequences of nuclear SSU rRNA genes using ACCTRAN optimization. The statuses of systematic characters used in the analysis are given in Tables 3 and 4.

cylindrical strains were gathered in clade c and 2 (number of cell layers in gametophytes): all other distal branches were composed of Monostromatic strains appeared in Bangia- leafy strains. The Bangia clade was composed Porphyra complex clades A and B. Distromatic only of cylindrical strains. Thus cylindrical strains appeared only in the Bangia-Porphyra strains separated the three clades. Character complex clade B and gathered in clade d. Systematic Character Tracing in Bangiales 193

Fig. 3b

Character 3 (number of chloroplasts per appeared in various lineages as follows (Fig. cell in gametophytes): Most strains were 3a-c) . Character 4: The thin form of thallus judged to have one chloroplast per cell. Only appeared in Bangia-Porphyra complex clades two strains of diplastid appeared in clade e and A and B. The thick form of thallus appeared in clade i of the Bangia-Porphyra complex clade B. clades d-h and clade i of the Bangia-Porphyra Similarly, in each systematic character 4-15, complex clade B. Character 5: Most strains which separate the species of Bangia and were judged to be pinkish-red to deep rose- Porphyra, strains with the same character status pink. Nine strains with a reddish-brown to 194 K. Matsuyama-Serisawa, S. Yamazaki, Y. Kitade, Y. Serisawa, K. Kuwano and N. Saga

Fig. 3c

brown-red color appeared in 7 positions of strains with microscopic marginal dentation Bangia-Porphyra complex clades A and B. appeared in clade a, clade b, and clade e of the Character 6: Porphyra strains with marginal Bangia-Porphyra complex clade B. Character undulation appeared in clades a-e and clade i 8: Strains with three chromosomes appeared of the Bangia-Porphyra complex clade B (clade in clades a-d, clade h, and clade i of the c is presumptive). Character 7: Porphyra Bangia-Porphyra complex clade B (clade c and Systematic Character Tracing in Bangiales 195

clade h are presumptive). Strains with four in all three trees (NJ, MP, and ML trees) (Fig. chromosomes appeared in the Bangia-Porphyra 1) . As strains of Bangia separated into the complex Glade A and in clade b, clades e-g, three clades, it is considered that the genus and clade i of the Bangia-Porphyra complex Bangia and Porphyra are not monophyletic and clade B. Strains with five chromosomes Bangia is not a sister group of Porphyra. The appeared in the Bangia-Porphyra complex same context was also confirmed by Oliveira clade A and clade e and clade i of the Bangia- et al. (1995). It is interesting that strains of P. Porphyra complex clade B. Character 9: All amplissima and P. pseudolinearis did not gather strains appearing in winter were contained in in one branch in each species. In those strains, the Bangia-Porphyra complex clade B. Some further taxonomic investigations are thought strains appearing in summer were included to be necessary. Also, further studies will be in the Bangia-Porphyra complex clade A and required in the strains of unidentified species clade d and clade i of the Bangia-Porphyra (B. sp. and P, sp.) of Bangia and Porphyra dealt complex clade B. Character 10: Most strains with in the present study. were monoecious. Dioecious strains appeared Species of Bangiales have mainly been in clades e-h of the Bangia-Porphyra complex classified based on 15 systematic characters clade B. Character 11: Strains with separated (Chang and Zheng 1960; Miura 1961, 1968; distribution of sexual cells by a vertical line Kurogi 1972; Oliveira and Coll 1975; Abbott were included in the Bangia-Porphyra complex and Hollenberg 1976; Cole and Sheath 1990; clade A and clade d and clade i of the Bangia- Lindstrom and Cole 1992a; Lindstrom and Porphyra complex clade B. Strains with mixed Cole 1993; Notoya et al. 1993; Matsuo et al. distribution of sexual cells were contained 1994; Miyata and Kikuchi 1997; Nelson et al. in the Bangia-Porphyra complex clade B. 1998; Yoshida 1998), and the genus Porphyra Character 12: Strains with 128 of the Hus was divided into three subgenera based on formula of spermatangia were included in the number of cell layers and chloroplasts per Bangia-Porphyra complex clade A, and strains cell (Kurogi 1972). Among the 15 systematic with the three types of the Hus formula were characters, the number of cell layers was mixed in the Bangia-Porphyra complex clade reported not to reflect the molecular phylogeny B. Character 13: Strains with 16 and 32 of the of Porphyra (Stiller and Waaland 1993; Oliveira Hus formula of carposporangia were included et al. 1995). In the present study, from the in the Bangia-Porphyra complex clade A. Strains cladistic tree of systematic character 1 (shape with three types of the Hus formula were of gametophytes) which separates the genera mixed in the Bangia-Porphyra complex clade B. of Bangia and Porphyra, it is considered that Character 14: Some strains in which asexual an evolution of gametophyte shape from reproduction was confirmed appeared in the cylindrical to leafy occurred at least twice Bangia-Porphyra complex clade B. Character and an evolution of the shape from leafy to 15: Most strains formed conchospores. cylindrical once (Fig. 2) . From the cladistic Strains forming unseparated conchospores or tree of systematic character 2 (number of cell conchospores and monospores appeared in layers in gametophytes), which separates the clade i or clade d, respectively. subgenus Diploderma from the genus Porphyra, it is considered that an evolution of cell layers Discussion from monostromatic to distromatic occurred once, but monostromatic strains appeared in In phylogenetic trees inferred from SSU two lineages. The cladistic tree of systematic rRNA sequences, it is clear that the 57 strains character 3 (number of chloroplasts per cell in of Bangiales diverged largely into three clades, gametophytes), which separates the subgenus the Bangia-Porphyra complex clade A, Bangia Diplastidia from the genus Porphyra, suggests clade, and Bangia-Porphyra complex clade B that an evolution of chloroplast number per cell 196 K. Matsuyama-Serisawa, S. Yamazaki, Y. Kitade, Y. Serisawa, K. Kuwano and N. 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SSU rRNA遺 伝 子 の配 列 よ り推 定 した分子系 統 に基 づ く ウシケ ノ リ目植物(紅 色 植物 門)の 分 類形 質 の復元

芹 澤(松 山)和 世 ・山﨑 悟 ・北 出幸 広 ・芹 澤 如 比 古 ・桑 野 和 可 ・嵯峨 直 恆

ウ シケ ノ リ 目植 物 の15分 類 形 質 を57株 のSSU rRNAに よる分 子系 統 樹 を基 にACCTRAN最 適化 配 置 に よって復 元 した。系 統樹 で は,多 くの株 が3つ の ク レー ド,す な わ ち,1つ の ウシケ ノ リク レー ドと2つ の ウ シケ ノ リーアマ ノ リ複 合 ク レー ドに分 岐 した 。 これ は ウシケ ノ リ属 や アマ ノ リ属 が各 々 単 系統 で は ない こ とを示 して い る。復 元 の結 果,配 偶 体 の形態(属 の分 類形 質)の 進 化 は少 な くと も 円柱状 か ら葉 状へ2回,葉 状 か ら円柱 状へ1回 起 き;配 偶 体 の細胞 層(フ タエ アマ ノ リ亜 属 の分類 形 質)の 進化 は一層 か ら二層 へ1回 起 き;配 偶体 の葉 緑体 数(フ タツボ シア マ ノ リ亜属 の分 類形 質)の 進 化 は,1個 か ら2個 へ2回,異 なる系 統 で起 こった と示 唆 され た。 他 の分類 形 質 につ い て も,い く つか の ま とま りは確 認 されたが,同 じ形 質状 態 を持つ株 が 様 々 な系 統 で見 られ上 記 の分類 形 質 は どれ

もSSU rRNAの 系統 を反映 してい ない と結論 づ け られた。 なお,本 報 は ウシケ ノ リ 目植物 の分 類形 質 の復元 に関す る初 め ての報 告 であ る。