A NEW BROWN ALGAL ORDER, ISHIGEALES (PHAEOPHYCEAE ...

J. Phycol. 40, 921–936 (2004) 

r 2004 Phycological Society of America 

DOI: 10.1111/j.1529-8817.2004.03160.x 

A NEW BROWN ALGAL ORDER, ISHIGEALES (PHAEOPHYCEAE), ESTABLISHED ON 

THE BASIS OF PLASTID PROTEIN-CODING rbcL, psaA, AND psbA REGION 

COMPARISONS 1 

Ga Youn Cho, Sang Hee Lee, and Sung Min Boo 2 

Department of Biology, Chungnam National University, Daejon 305-764, Korea 

The brown algal family Ishigeaceae currently 

includes a single genus, Ishige Yendo, with two species. 

The relationship of the family to other brown 

algal lineages is less studied in terms of their plastid 

ultrastructure and molecular phylogeny. We determined 

the sequences of rbcL from four samples of 

the two Ishige species and nine putative relatives 

and the psaA andpsbA sequences from 37 representatives 

of the brown algae. Analyses of individual 

and combined data sets resulted in similar trees; 

however, the concatenated data gave greater resolution 

and clade support than each individual gene. 

In all the phylogenies, the Phaeophyceae was well 

resolved, the Ectocarpales being placed in a terminal 

position and the Ishigeaceae ending up in a 

basal position. From our ultrastructural study, 

we concluded that the pyrenoid is absent in the 

Ishigeaceae, despite the presence of a rudimentary 

pyrenoid in I. okamurae. These results suggest that 

the Ishigeaceae is an early diverging brown lineage. 

Our molecular and morphological data, therefore, 

lead us to exclude the Ishigeaceae from the Ectocarpales 

s.l., which have an elaborate pyrenoid, and 

to propose its own order Ishigeales ord. nov. The 

Ishigeales is distinguished by oligostichous structure 

of thalli, phaeophycean hairs formed within 

cryptostomata, unilocular sporangia transformed 

from terminal cortical cells, and plurilocular sporangia 

lacking sterile terminal cells. This study is the 

first to document the utility of the psaA and psbA 

sequences for brown algae and also the first report 

on the multigene phylogeny of the Phaeophyceae 

based on three protein-coding plastid genes. 

Key index words: Ishige; Ishigeaceae; Ishigeales ord. 

nov.; Phaeophyceae; phylogeny; psaA; psbA; pyrenoid; 

rbcL; taxonomy; ultrastructure 

Abbreviations: BS, bootstrap value; LSU, large subunit 

of rDNA; ML, maximum likelihood; MP, maximum 

parsimony; psaA, PSI P700 chl a apoprotein 

A1 gene; psbA, PSII thylakoid protein D1 gene; 

rbcL, large subunit of RUBISCO gene; SH, 

Shimodaira-Hasegawa; SSU, small subunit of rDNA 

1 Received 4 September 2003. Accepted 9 June 2004. 

2 Author for correspondence: e-mail smboo@cnu.ac.kr. 

921 

Ishige is a genus of brown algae that contains two 

species. Both are summer annuals and occur exclusively 

in the warm waters of the Pacific Ocean (Yendo 

1907, Setchell and Gardner 1924, Tseng 1983, Lee 

et al. 2003). The genus is characterized by cylindrical 

to foliose thalli, hairs in cryptostomata, uniseriate 

plurilocular sporangia lacking sterile terminal cells, 

and an isomorphic life history (Yendo 1907, Ajisaka 

1989, Tanaka in Hori 1993, Lee et al. 2003). The 

genus was based on I. okamurae, which was described 

from two different forms (filiform and foliose forms) 

collected in Shimoda on the Pacific coast of Japan. The 

foliose thalli are considered abnormal branches of the 

filiform type, because the former is commonly epiphytic 

on the latter. Ishige okamurae occurs in the upper 

intertidal zone along the coasts of Korea (Lee et al. 

2003), Japan (Yendo 1907, Yoshida 1998), and China 

(Tseng 1983). Okamura (in Segawa 1935) described 

I. foliacea, the second member of the genus, based on 

the foliose type, which has a cortical layer much thinner 

than that of I. okamurae. However, Chihara (1969) 

showed that I. foliacea is a later homonym of Polyopes 

sinicola Setchell et Gardner (1924), revising the name to 

I. sinicola (Setchell et Gardner) Chihara. Ishige sinicola 

occurs as an epiphyte on I. okamurae in Korea, 

Japan, and China (Tseng 1983, Yoshida 1998, Lee 

et al. 2003), whereas the former occurs alone along 

the northern coast of the Gulf of California (Setchell 

and Gardner 1924). 

On establishing the genus Ishige, Yendo (1907) 

putatively put it within the family Fucaceae on the 

grounds of cryptostomata in the foliose form ( 5 I. sinicola). 

However, Okamura (in Segawa 1935) established 

the monotypic family Ishigeaceae based on the zoospores 

in the unilocular sporangia of I. sinicola, and 

then Okamura (1936) placed the Ishigeaceae within 

the Punctariales, because the apical cells of the assimilatory 

filaments change into unilocular sporangia 

where zoospores with two lateral flagella are produced. 

Finally, Arasaki (1943) concluded that the Ishigeaceae 

belonged to the order Chordariales on the basis of its 

heteromorphic life history, motile gametes in microscopic 

gametophytes, and plurilocular sporangia similar 

to those of some chordarialean species. In contrast, 

Ajisaka (1989) proposed that the family Ishigeaceae 

may not belong to the Chordariales, because I. okamurae 

has uniseriate plurilocular sporangia lacking 

sterile terminal cells, whereas the Chordariales have

922 

unilocular sporangia formed on the basal or middle 

part of the assimilatory filaments. Nevertheless, the 

family Ishigeaceae is still classified in the Chordariales 

in textbooks of algae (Yoshida 1998). 

The presence of a pyrenoid is a diagnostic character 

for the Phaeophyceae at the ordinal level (Evans 1966, 

Hori and Ueda 1975, Kawai 1992). This is supported 

by previous studies using small subunit (SSU), large 

subunit (LSU), and rbcL DNAs (Tan and Druehl 1994, 

de Reviers and Rousseau 1999, Peters and Ramírez 

2001). The Ectocarpales have a large pedunculate 

pyrenoid with a cap layer in the discoid plastids of 

vegetative cells (Evans 1966, Hori and Ueda 1975). 

The Scytothamnales have a pyrenoid in the center of 

stellate plastids (Peters and Clayton 1998), whereas 

Asterocladon, Asteronema, and Bachelotia, which are insertae 

sedis, have several terminal pyrenoids in the ribbon-shaped 

plastids (Müller et al. 1998). In contrast, 

the pyrenoid is absent in other brown algae (Evans 

1966, 1968, Chi 1971, Hori 1971, Hori and Ueda 

1975, Henry 1984). The absence/presence of the pyrenoid 

in the Ishigeaceae has been contentious. Hori 

(1971) observed a small pyrenoid in the plastids of the 

vegetative cells of I. okamurae from Japan but found no 

pyrenoids in the plastids of I. sinicola. He therefore 

questioned the absence of the pyrenoid in the latter 

species because of the congeneric relationship of the 

two species (Hori 1971, Hori and Ueda 1975). 

Rousseau and de Reviers (1999) proposed the 

broad concept of the order Ectocarpales, including 

the Chordariales, Dictyosiphonales, Punctariales, and 

Scytosiphonales and excluding the Ralfsiales and taxa 

with stellate plastids, based on combined SSU þ LSU 

rDNA sequences. This is corroborated by other DNA 

phylogenies (de Reviers and Rousseau 1999, Rousseau 

et al. 2000, 2001, Draisma et al. 2001, 2003, Peters and 

Ramírez 2001, Cho et al. 2003). de Reviers and Rousseau 

(1999) placed the Ishigeaceae and 22 other families, 

formerly assigned to the aforementioned different 

orders, within the Ectocarpales s.l. Then, Peters 

and Ramírez (2001) reclassified all 23 families into 

just 5 families, viz. Acinetosporaceae, Adenocystaceae, 

Chordariaceae, Ectocarpaceae, and Scytosiphonaceae, 

using DNA data, life history, and plastid structure data. 

In this classification, the previous chordarialean and 

dictyosiphonalean families are synonymized with the 

Chordariaceae (Peters and Ramírez 2001). This forces 

Ishige, the only member of the family Ishigeaceae, to be 

included in the Chordariaceae s.l. Based on partial sequences 

of SSU rDNA, Lee et al. (2003) suggested that 

the Ishigeaceae form an independent lineage with 

some affinity with the early lineage of the Ectocarpales 

s.l. However, the relationship of the family Ishigeaceae 

to the other brown algal lineages has not been addressed 

because taxon sampling was limited. 

The present study investigated the phylogenetic relationships 

of the family Ishigeaceae in the class 

Phaeophyceae by analyses of sequence data derived 

from three independent protein-coding plastid genes. 

First, we analyzed the rbcL gene from Ishige and puta- 

GA YOUN CHO ET AL. 

tive relatives and subsequently compiled an rbcL data 

set that included previously published sequences from 

GenBank. The rbcL gene has frequently been used to 

study various brown algal groups at a variety of taxonomic 

levels (Siemer et al. 1998, Draisma et al. 2001, 

Peters and Ramírez 2001, Cho et al. 2003). The second 

coding gene we chose was the psaA gene, which encodes 

the PSI P700 chl a apoprotein A1 (Morden and 

Sherwood 2002). To date, psaA has been analyzed for 

only two species, Fucus vesiculosus (Pearson et al. 2001) 

and Pylaiella littoralis (Yoon et al. 2002a), in the Phaeophyceae. 

The third protein-coding gene we used is the 

psbA gene, which encodes the PSII thylakoid protein 

D1 (Morden and Sherwood 2002). The psbA has been 

analyzed for two species, Ectocarpus siliculosus (Winhauer 

et al. 1991) and Pylaiella littoralis (Yoon et al. 

2002a), in the Phaeophyceae. Samples of 37 algae including 

two outgroup taxa, selected from the taxa used 

in forming the rbcL data set described above, were 

available for the psaA and psbA analyses. Because psaA 

and psbA gene sequences provide better resolution for 

the deep branches of the red and dinophycean algal 

phylogenies (Morden and Sherwood 2002, Yoon et al. 

2002a, b, Yang and Boo 2004), both genes should be 

useful for reconstructing the phaeophycean phylogeny 

at higher ranks. 

When multiple independent data sets are available 

for a phylogenetic study, the investigator uses combined 

data sets as well as individual data sets for phylogenetic 

reconstructions. For combing individual data 

sets, Bull et al. (1993) suggested that congruent data 

sets only can be combined for phylogenetic analyses, 

whereas Gatesy et al. (1999a), who advocated a ‘‘total 

evidence’’ methodology, showed that the combination 

of incongruent data can increase the resolution and the 

support within phylogenetic trees, revealing if a ‘‘hidden 

signal’’ is present in the different data sets. However, 

advocates of these two approaches agree that use 

of multiple data sets improve phylogeny estimation 

(Lavoué et al. 2003, Shimabukuro-Dias et al. 2004). In 

this study, we follow an empirical strategy, conducting 

both separate and simultaneous analyses. Together 

with molecular analyses of the Ishigeaceae, we studied 

the ultrastructure of plastids in the vegetative cells of 

I. okamurae, the type species of the genus. 

MATERIALS AND METHODS 

Samples. The starting point for this work was our collection 

of previously published rbcL sequences of the brown 

algaefromGenBank.WecompiledrbcL sequences from 57 

taxa, which consisted of the type genus or representatives of 

13 brown algal orders, 4 ‘‘ordinal-level taxa’’ (Choristocarpaceae, 

Onslowiaceae, Phyllariaceae, and Asterocladon/Asteronema) 

(see Table 2 in Draisma et al. 2003), and 3 outgroup 

taxa (Schizocladia ischiensis, Tribonema aequale, andPhaeothamnion 

confervicola). Thirteen rbcL sequences we determined 

here were added to this data set: two samples from each of 

Ishige okamurae and I. sinicola and nine other brown algae. 

Then, psaA andpsbA regions from 37 taxa, including Tribonema 

aequale and Schizocladia ischiensis as outgroup taxa, were 

analyzed. Both psaA and psbA sequences from Pylaiella littoralis

and the psbA sequence from Ectocarpus siliculosus were downloaded 

from GenBank (Winhauer et al. 1991, Yoon et al. 

2002a). The psaA sequence of Fucus vesiculosus was not used 

because of its insufficient length (330 nt, Pearson et al. 2001). 

The specimens and their corresponding GenBank accession 

numbers are listed in Table 1. 

Analyses of rbcL, psaA, and psbA regions. Total DNA was 

extracted from approximately 0.01 g of dried thalli ground 

in liquid nitrogen using a DNeasy Plant Mini Kit (Qiagen 

Gmbh, Hilden, Germany), according to the manufacturers’ 

instructions, and then dissolved in 150 mL of distilled water. 

Extracted DNA was stored at 201 Candusedtoamplifythe 

rbcL, psaA, and psbA regions. 

The rbcL region was amplified and sequenced using the 

method of Kogame et al. (1999) and Yoon and Boo (1999). 

Primers PRB-FO, F2, F3, R1A, R2, R3A, RS1, and RS2 were 

used for most brown algae. For Cutleria, Ishige, Sargassum, and 

Sphacelaria, primer RbcL68F (Draisma et al. 2001), instead of 

PRB-FO, was used. The same DNA aliquot was used for amplifying 

the psaA andpsbA regions, and the amplification and 

sequencing reactions for these regions were the same as those 

used for rbcL. The psaA region was amplified and sequenced 

using primers psaA130F, psaA870F, psaA970R, and 

psaA1760R (Yoon et al. 2002a). The psbA region was amplified 

using primers psbA-F and psbA-R2 and sequenced using 

primers psbA-F, psbA-600R, psbA-500F, and psbA-R2 (Yoon 

et al. 2002a). The PCR products were purified using a High 

Pure TM PCR Product Purification Kit (Roche Diagnostics 

GmbH, Mannheim, Germany) according to the manufacturers’ 

instructions. The sequences of the forward and reverse 

strands were determined for all taxa using an ABI PRISM TM 

377 DNA Sequencer (Applied Biosystems, Foster City, CA, 

USA). The electropherogram output for each sample was edited 

using the program Sequence Navigator v. 1.0.1 (Applied 

Biosystems). 

All sequences of the rbcL gene from 70 taxa (67 brown algae 

and 3 outgroups) were collated using the multisequence editing 

program, SeqPup (Gilbert 1995), and aligned by eye to 

compare our sequences with those published previously (Draisma 

et al. 2001, Cho et al. 2003). The undefined sequences at 

the 5 and 3 0 ends of the rbcL data set were coded as missing 

data. The psaA sequences from 38 taxa (36 brown algae, including 

previously published sequence of Pylaiella littoralis, and 

2 outgroups) were also aligned by eye (Table 1). The psbA sequences 

from 39 taxa (37 brown algae, including previously 

published sequences of Ectocarpus siliculosus and Pylaiella littoralis, 

and 2 outgroups) were aligned by eye, as for rbcL gene 

(Table 1). All alignments posed no problems because there 

were no gaps in each data set of these three protein-coding 

genes. The alignments are available at Treebase (study accession 

S1095) under accession numbers M1870 (psbA), M1871 

(rbcL), M1872 (psaA), and M1873 (rbcL þ psaA þ psbA). 

Phylogenetic analyses. Four data sets were used for the 

phylogenetic analyses: 70 taxa for rbcL, 39 taxa for psbA, and 

38 taxa for both the psaA andcombinedrbcL þ psaA þ psbA 

data sets. To infer the level of nucleotide saturation of the 

rbcL, psaA, and psbA sequences, uncorrected p-distances were 

plotted against corrected pairwise distances using the Hasegawa-Kishino-Yano 

85 model (Hasegawa et al. 1985) for the 

first, second, and third codon positions and all positions. We 

also conducted the partition homogeneity test (incongruence 

length difference [ILD] test of Farris et al. 1994), implemented 

in PAUP* 4.0b8 (Swofford 2002). The partition homogeneity 

test used 1000 replicates, each with 100 random 

sequence-addition replicates using tree bisection-reconnection 

(TBR) branch swapping. 

Maximum parsimony (MP) trees were constructed for each 

data set with PAUP* using a heuristic search algorithm with the 

following settings: 100 random sequence-addition replicates, 

ISHIGEALES ORD. NOV. 923 

TBR branch swapping, MulTrees, all characters unordered 

and unweighted, and branches with a maximum length of zero 

collapsed to yield polytomies. The bootstrap values (BS) for the 

resulting nodes were assessed using bootstrapping with 1000 

replicates. 

For maximum likelihood (ML) and Bayesian analyses, we 

performed a likelihood ratio test using Modeltest 3.06 (Posada 

and Crandall 1998) to determine the best available model for 

individual and combined data sets. For all data sets, the best 

model was a general time reversible (GTR) model with a gamma 

correction for among-site variation (G) and invariant sites 

(I). ML analyses (heuristic search with 10 random sequenceaddition 

replicates, TBR branch swapping, and MulTrees on) 

were performed using the GTR þ G þ I model. BS analysis was 

conducted by performing replicate ML searches, with two random 

sequence-addition replicates, using the same search conditions 

as described above. We succeeded in performing 100 

bootstrap replicates for each of individual and combined data 

sets using two processors. To run simultaneous bootstrap replicates 

in different processors, we prepared two batch files that 

differed in their starting random seed number (seed number, 

0), each specified to run 50 bootstrap replicates and to save the 

resulting bootstrap trees into files. A 50% majority rule consensus 

bootstrap tree was estimated by aggregating and 

weighting trees accordingly to the number of trees found in 

each bootstrap replicate, so that the bootstrap replicates had 

equal weight. 

Bayesian phylogenetic analyses were performed using 

MrBayes 3.0 (Huelsenbeck and Ronquist 2001). Each analysis 

was initiated from a random starting tree, and the program 

was set to run four chains of Markov chain Monte Carlo iterations 

simultaneously for 2,000,000 generations with trees 

sampled every 100th generation. The likelihood scores stabilized 

at approximately 50,000 generations, and thus the first 

500 trees were burned. For the purpose of comparison with 

bootstrapping, we chose to consider nodes with Bayesian posterior 

probabilities (PP) greater than 0.9 (e.g. the node appears 

in greater than 90% of sampled trees) as being well supported. 

The SH test (Shimodaira and Hasegawa 1999) was used to 

compare statistically alternative phylogenetic hypotheses, focusing 

on the inclusion of the Ishigeaceae within the Ectocarpales 

or other putative relatives. The SH test was conducted 

using PAUP*, with resampling estimated log-likelihood optimization 

and 10,000 bootstrap replicates. 

Morphology of plastids in Ishige okamurae. Apical parts of 

field-collected thalli were prepared for EM according to the 

following protocol. Specimens were fixed with 3% glutaraldehyde 

in 0.1 M cacodylate buffer (pH 7.2) containing 2% 

NaCl and 0.1% CaCl2 for 3 h at 41 C and then postfixed in 2% 

osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 h. 

The apical parts were rinsed with cold distilled water, cut, 

and transferred into test tubes at 41 C. They were dehydrated 

in a graded series of ethanol and propylene oxide at 41 Cand 

infiltrated gradually with Spurr’s epoxy resin (Spurr 1969). 

After polymerization of the resin at 701 C for 24 h, serial sections 

(120–200 sections per sample) were cut with a diamond 

knife on an Ultracut E ultramicrotome (Reichert-Jung, Germany) 

and mounted on Formvar-coated slot grids. Thin sections 

were stained with Reynold’s lead citrate (Reynolds 

1963) and uranyl acetate and examined with a JEM-1010 

transmission electron microscope (JEOL, Ltd., Tokyo, Japan) 

at the Center for Research Facilities, Chungnam National 

University. 

RESULTS 

The rbcL alignment. The sequences determined in 

the present study were 1467 nt long, except for 

the two Ishige species (1325 and 1368 nt), Cutleria

924 

TABLE 1. Taxa, collection site or data source, and GenBank accession number of the rbcL, psaA, and psbA sequences. 

GenBank accession no. 

rbcL/psaA/psbA 

Taxa Collection site, date, voucher, or reference of rbcL/psaA/psbA 


Phaeophyceae 

Ectocarpales 

Acinetosporaceae 

Geminocarpus austro-georgiae Skottsberg Peters and Ramírez (2001) AJ295830/ — / — 

Pylaiella littoralis (Linnaeus) Kjellman Assali et al. (1990)/Yoon et al. (2002a)/ibid X55372/AY119724/AY119760 

Adenocystaceae 

Adenocystis utricularis (Bory) Skottsberg Peters and Ramírez (2001)/Barton, Maxwell Bay, Antarctica, 25 January 2000, PE001/ibid AJ295823/AY372939/AY528824 

Caepidium antarcticum J. Agardh Peters and Ramírez (2001) AJ295826/ — / — 

Utriculidium durvillei Skottsberg Peters and Ramírez (2001) AJ295835/ — / — 

Chordariaceae 

Asperococcus fistulosus (Hudson) Hooker Cho et al. (2003)/Port Erin Bay, Isle of Man, UK, 9 July 2002, PE002/ibid AY095321/AY372940/AY528825 

Chordaria flagelliformis (O.F.Müller) J. Agardh Cho et al. (2003)/Avacha Bay, Kamchatka, Russia, 24 July 1998, PE003/ibid AY095324/AY372941/AY528826 

Coelocladia arctica Rosenvinge Siemer et al. (1998) AF055395/ — / — 

Delamarea attenuata (Kjellman) Rosenvinge Siemer et al. (1998)/Nakhodka, Russia, 22 May 2002, PE004/ibid AF055396/AY372942/AY528827 

Dictyosiphon foeniculaceus (Hudson) Greville Avacha Bay, Kamchatka, Russia, 28 July 1998, PE005 AY372973/AY372943/AY528828 

Elachista fucicola (Velley) Areschoug Siemer et al. (1998) AF055398/ — / — 

Giraudia sphacelarioides Derbès et Solier Siemer et al. (1998) AF055399/ — / — 

Hummia onusta (Kützing) Fiore Siemer et al. (1998) AF055402/ — / — 

Isthmoplea sphaerophora (Harvery) Kjellman Siemer et al. (1998) AF055403/ — / — 

Myriotrichia clavaeformis Harvey Siemer et al. (1998) AF055408/ — / — 

Punctaria latifolia Greville Cho et al. (2003)/Hoedong, Jindo, Korea, 9 March 2001, PE010/ibid AY095322/AY372948/AY528833 

Sphaerotrichia divaricata (C. Agardh) Kylin Siemer et al. (1998) AF055412/ — / — 

Streblonema maculans (Hamel) South et Tittley Rousseau and de Reviers (1999) AY157694/ — / — 

Striaria attenuata (Greville) Greville Siemer et al. (1998) AF055415/ — / — 

Ectocarpaceae 

Ectocarpus sp. Hoedong, Jindo, Korea, 9 March 2001, PE011 AY372978/AY372949/AY528834 

E. siliculosus (Dillwyn) Lyngbye Winhauer et al. (1991) —/ —/ X56695 

Scytosiphonaceae 

Chnoospora implexa J. Agardh Kogame et al. (1999) AB022231/ — / — 

Colpomenia sinuosa (Mertens ex Roth) Derbès etSolier Kogame et al. (1999)/Guryongpo, Pohang, Korea, 6 November 2000, PE012/ibid AB022234/AY372950/AY528835 

in Castagne 

Hydroclathrus clathratus (C. Agardh) Howe Kogame et al. (1999)/Tsuyazaki, Fukuoka, Japan, 7 March 1999, PE013/ibid AB022233/AY372951/AY528836 

Myelophycus simplex (Harvey) Papenfuss Cho et al. (2003)/Daesado, Wando, Korea, 13 June 1999, PE014/ibid AY095320/AY372952/AY528837 

Petalonia fascia (O.F.Müller) Kuntze Kogame et al. (1999)/Ile de Batz, Roscoff, France, 5 April 2000, PE015/ibid AB022243/AY372953/AY528838 

Rosenvingea intricate (J. Agardh) Boergesen Kogame et al. (1999) AB022232/ — / — 

Scytosiphon lomentaria (Lyngbye) Link Kogame et al. (1999)/Seongsan, Jeju, Korea, 22 March 2000, PE016/ ibid AB022238/AY372954/AY528839 

Choristocarpaceae 

Choristocarpus tenellus (Kützing) Zanardini Draisma et al. (2001) AJ287862/ — / — 

Cutleriales 

Cutleria cylindrica Okamura Hoedong, Jindo, Korea, 9 March 2001, PC001 AY372979/AY372955/AY528840 

C. multifida (J. E. Smith) Greville Burrowes et al. (2003) AY157692/ — / — 

Zanardinia prototypes (Nardo) Nardo Burrowes et al. (2003) AY157693/ — / — 

Desmarestiales 

Desmarestia ligulata (Stackhouse) Lamouroux Draisma et al. (2001) AJ287848/ — / — 

D. viridis (Müller) Lamouroux Siemer and Pedersen (unpublished data) AF207799/ — / — 

Desmarestia sp. Penguin Rookery, Maxwell Bay, Antarctica, 11 January 2000, PD001 AY372980/AY372956/AY528841 

Himantothallus grandifolius (A. et E. Gepp) Zinova Draisma et al. (2001) AJ287850/ — / — 

Dictyotales 

Dictyota dichotoma (Hudson) Lamouroux Draisma et al. (2001) AJ287852/ — / — 

Dictyota sp. Guryongpo, Pohang, Korea, 15 March 2001, PDI001 AY422654/AY372957/AY528842 

Zonaria diesingiana J. Agardh Ishigaki Island, Okinawa, Japan, 19 January 1998, PDI002 AJ295823/AY372958/AY528843 

Fucales 

Ascophyllum nodosum (Linnaeus) Le Jolis Draisma et al. (2001)/Neeltjejans, Netherlands, 13 August 1997, PF001/ibid AJ287853/AY372959/AY528844

Table 1. (Contd.) 

GenBank accession no. 

rbcL/psaA/psbA 

Taxa Collection site, date, voucher, or reference of rbcL/psaA/psbA 

Fucus vesiculosus Linnaeus Burrowes et al. (2003)/Coos Bay, Oregon, USA, 16 May 2001, PF002/ibid AY157695/AY372960/AY528845 

Sargassum horneri (Turner) C. Agardh Seosang, Namhaedo, Korea, 3 November 2002, PF003 AY372981/AY372961/AY528846 

Ishigaceae 

Ishige okamurae Yendo Hanrim, Jeju, Korea, 4 December 2002, PE006 AY372974/AY372944/AY528829 

I. okamurae Kominato, Chiba, Japan, 28 July 2002, PE007 AY372975/AY372945/AY528830 

I. sinicola (Setchell et Gardner) Chihara Hanrim, Jeju, Korea, 4 December 2002, PE008 AY372976/AY372946/AY528831 

I. sinicola Kominato, Chiba, Japan, 28 July 2002, PE009 AY372977/AY372947/AY528832 

Laminariales 

Alaria crassifolia Kjellman in Kjellman et Petersen Hakodate, Hokkaido, Japan, 21 August 1999, PL001 AY372982/AY372962/AY528847 

Chorda filum (Linnaeus) Stackhouse Oshoro, Hokkaido, Japan, 25 April 2002, PL002 AY372983/AY372963/AY528848 

Laminaria digitata (Linnaeus) Lamouroux Port Erin Bay, Isle of Man, UK, 9 July 2000, PL003 AY372984/AY372964/AY528849 

Onslowiaceae 

Onslowia endophytica Searles Draisma et al. (2001) AJ287864/ — / — 

Verosphacela ebrachia Henry Draisma et al. (2001) AJ287867/ — / — 


Phyllariaceae 

Phyllariopsis brevipes ssp. brevipes (C. Agardh) 

Sasaki et al. (2001) AB045244/ — / — 

Henry et South 

Saccorhiza polyschides (Lightfoot) Batters Sasaki et al. (2001)/Ile Callot, Roscoff, France, September 1999, PP001/ibid AB045256/AY372965/AY528850 

Ralfsiales 

Analipus japonicus (Harvey) Wynne Cho et al. (2003)/Boiler Bay, Oregon, USA, 16 May 2001, PR001/ibid AY095323/AY372966/AY528851 

Scytothamnales 

Scytothamnus australis (J. Agardh) Hooker et Harvey Peters and Ramírez (2001)/Scorching Bay, Wellington, New Zealand, 3 August 2001, AJ295833/AY372967/AY528852 

PS001/ibid 

Splachnidium rugosum (Linnaeus) Greville Peters and Ramírez (2001)/Scorching Bay, Wellington, New Zealand, 3 August 2001, AJ295834/AY372968/AY528853 

PS002/ibid 

Sphacelariales 

Alethocladus corymbosus (Dickie) Sauvageau Draisma et al. (2001) AJ287860/ — / — 

Halopteris filicina (Grateloup) Kützing Draisma et al. (2001)/Seongsan, Jeju, Korea, DNA from Y. S. Keum/ibid AJ287894/AY372969/AY528854 

Sphacelaria divaricata Montagne Draisma et al. (2001) AJ287889/ — / — 

S. divaricata Seongsan, Jeju, Korea, DNA from Y. S. Keum AY372985/AY372970/AY528855 

Sporochnales 

Carpomitra costata (Stackhouse) Batters Sasaki et al. (2001)/Munseom, Jeju, Korea, 8 April 2003, PSP001/ibid AB045257/AY372971/AY528856 

Sporochnus radiciformis (B. Brown ex Turner) C. Agardh Kawai and Sasaki (2000) as S. scoparuis Harvey /Cheonbu, Ulreungdo, Korea, 

AB037142/AY528861/AY528857 

26 August 2003, PSP002/ibid 

Syringodermatales 

Microzonia velutina (Harvey) J. Agardh Burrowes et al. (2003) AY157697/ — / — 

Syringoderma phinneyi Henry et Müller Draisma et al. (2001)/Moe 2, Culture of Prof. D. G. Müller (Konstanz, Germany)/ibid AJ287868/AY528862/AY528858 

Tilopteridales 

Haplospora globosa Kjellman Kawai and Sasaki (2000) AB037138/ — / — 

Tilopteris mertensii (Turner in Smith) Kützing Sasaki et al. (2001) AB045260/ — / — 

Incertae sedis 

Asterocladon lobatum Müller et al. Peters and Ramírez (2001) AJ295824/ — / — 

Asteronema rhodochortonoides (Boergesen) Müller et Parodi Peters and Ramírez (2001) AJ295825/ — / — 

Phaeothamniophyceae 

Phaeothamnion confervicola G. Lagerheim Bailey et al. (1998) AF064746/ — / — 

Schizocladiophyceae 

Schizocladia ischiensis Henry, Okuda et Kawai Kawai et al. (2003)/CCMP2287/ibid AB085615/AY528863/AY528859 

Tribophyceae 

Tribonema aequale Pascher Bailey and Andersen (1998)/UTEX 50/ibid AF084611/AY372972/AY528860 

CCMP, Provasoli-Guillard National Center for Culture of Marine Phytoplankton; UTEX, Culture Collection of Algae at the University of Texas at Austin. Bold numbers 

indicate new sequences published in the present study.

926 

TABLE 2. Nucleotide composition of the rbcL, psaA, and psbA and statistics from MP analyses of the individual and combined 

data sets including outgroups. 

cylindrica (1372 nt), Sargassum horneri (1292 nt), and 

Sphacelaria divaricata (1383 nt), which were amplified 

with different primers. The 70 aligned rbcL sequences 

had 685 (46.7%) variable bases and 558 (38.0%) 

parsimoniously informative sites. There were excesses 

of adenine (29.49%) and thymine (32.21%) at all 

codon positions. Transversions were more common 

than transitions for all codon positions (Ti/Tv 5 0.95) 

(Table 2). 

The uncorrected sequence divergence (p-distance) 

values for the rbcL region within Ishige ranged from 

6.77% between I. okamurae and I. sinicola from Korea to 

7.10% between I. okamurae and I. sinicola from Japan. 

The samples of I. okamurae from Korea and Japan differed 

by seven nucleotides (0.51% sequence divergence), 

and there was a difference of two nucleotides 

(0.15% sequence divergence) between I. sinicola from 

Korea and Japan. Ishige differed from other ectocarpalean 

algae between 14.01% (between I. okamurae from 

Japan and Dictyosiphon foeniculaceus) and 16.61% 

(between I. sinicola from Japan and Pylaiella littoralis) 

sequence divergence. 

The psaAalignment. The sequences determined for 

the psaA region totaled 1488 nt. For the 38 aligned 

sequences of the psaA gene, 661 (44.4%) bases were 

variable and 578 (38.8%) were parsimoniously informative. 

There were excesses of adenine and thymine 

at all codon positions (29.52% and 35.62%, 

respectively). Transversions and transitions occurred 

at approximately similar frequencies for all codon 

positions (Ti/Tv 5 1.02) (Table 2). 

The sequence divergence for the psaA gene within 

Ishige ranged from 8.27% (between I. okamurae from 

Japan and I. sinicola from Korea) to 8.74% (between 

I. okamurae Korea and I. sinicola from Japan). Ishige 

okamurae from Korea and Japan differed by 11 nt 

(0.74% sequence divergence), and I. sinicola from Korea 

and Japan differed by 5 nt (0.34% sequence divergence). 

Ishige diverged from other ectocarpalean algae 

between 16.4% (between I. sinicola from Korea and 

Dictyosiphon foeniculaceus) and 18.82% (between 

I. okamurae from Japan and Adenocystis utricularis). 

The psbA alignment. The sequences determined 

for the psbA region totaled 885 bp. For the 39 aligned 

rbcL psaA psbA Combined 

Number of taxa 70 38 39 38 

Nucleotides (bp) 1467 1488 885 3840 

Base frequency (A/C/G/T) 0.2949/0.1627/0.2203/0.3221 0.2952/0.1564/0.1922/0.3562 0.2467/0.1825/0.2077/0.3632 

Number of transitions/ 191379/200661 (0.95) 74550/73082 (1.02) 29095/23450 (1.24) 

transversions (Ti/Tv ratio) 


Variable sites (%) 685 (46.7) 661 (44.4) 306 (34.6) 1583 (41.2) 

Informative sites (%) 558 (38.0) 578 (38.8) 226 (25.5) 1296 (33.8) 

Number of MP trees 2 2 4 1 

MP tree length 4062 3387 1021 7243 

Consistency index 0.278 0.333 0.378 0.344 

Retention index 0.561 0.457 0.554 0.477 

sequences of the psbA gene, 306 (34.6%) bases were 

variable and 226 (25.5%) were parsimoniously informative. 

There were excesses of adenine and thymine 

at all codon positions (24.67% and 36.32%, 

respectively). Transitions were more common than 

transversion for all codon positions (Ti/Tv 5 1.24) 

(Table 2). 

The sequence divergence for the psbA gene within 

Ishige ranged from 6.44% (between I. okamurae and I. 

sinicola from Japan) to 6.89% (between I. okamurae and 

I. sinicola from Korea). Ishige okamurae from Korea and 

Japan differed by 6 nt (0.68% sequence divergence), 

and I. sinicola from Korea and Japan differed by 2 nt 

(0.23% sequence divergence). Ishige diverged from 

other ectocarpalean algae between 9.49% (between 

I. okamurae from Korea and Petalonia fascia) and 

11.86% (between I. sinicola from Korea and Adenocystis 

utricularis). 

Saturation tests. Saturation tests showed that the 

first and second codon positions for all three genes 

were conserved and the third codon positions were 

the most variable. The scatter plots (not shown) for 

the rbcL, psaA, and psbA sequences were linear and 

showed no evidence of multiple hit problems for all 

three codon positions. Therefore, we included all 

events for the three codon positions in the phylogenetic 

analysis. 

Partition homogeneity tests. Partition homogeneity 

tests between rbcL and psaA were significant 

(P 5 0.002, mean of three tests); however, with 

Desmarestia or the kelp genera (Chorda, Alaria, and 

Laminaria) excluded, the results were not significant 

(P 5 0.102 and 0.150, respectively), indicating congruence. 

The psbA data was incongruent with the 

psaA andrbcL data sets (at the P 0.01 level) even 

with Desmarestia and/or the kelp genera excluded. 

The P value was not changed when outgroup taxa or 

uninformative sites were excluded. So, we provided 

all the phylogenetic trees reconstructed from individual 

and combined data sets. 

Phylogenetic relationships. The independent analyses 

of rbcL, psaA, psbA, and rbcL þ psaA þ psbA data 

sets resulted in congruent, though not identical, 

phylogenetic reconstructions. The statistics for the

MP analyses are compared among individual and 

combined data sets (Table 2), and the ML trees for 

all four data sets are shown in Figures 1 through 4. 

The rbcL tree (Fig. 1) showed that all the phaeophycean 

algae investigated here were strongly monophyletic 

(100% BS for ML, 92% BS for MP, and 

PP 5 1), having the Schizocladiophyceae as the sister 

taxon. The basal-most taxon of the Phaeophyceae was 

the Choristocarpaceae. Ishige okamurae and I. sinicola 

from Korea and Japan were strongly monophyletic 

(100% BS for ML and MP and PP 5 1), and the Ishigeaceae 

was the sister taxon to all other remaining 

taxa (100% BS for ML, 79% BS for MP, and PP 5 1). 

The Dictyotales, Onslowiaceae, Sphacelariales, and 

Syringodermatales formed a clade, although the clade 

was only supported by Bayesian PP. The remaining 11 

higher taxa, including the Phyllariaceae and Asterocladon/Asteronema 

group, formed a monophyletic clade 

(56% BS for ML, 51% BS for MP, and PP 5 1). The 

Ectocarpales, comprising the Acinetosporaceae, Adenocystaceae, 

Chordariaceae, Ectocarpaceae, and Scytosiphonaceae, 

was strongly monophyletic (94% BS for 

ML, 90% BS for MP, and PP 5 1) and was placed in a 

terminal position. The Laminariales and Asterocladon/ 

Asteronema group clustered with the Ectocarpales (88% 

BS for ML, 85% BS for MP, and PP 5 1). However, 

interrelationships of most of other orders and families 

were not well resolved. 

The psaA tree (Fig. 2) is similar to the rbcL tree in 

having the monophyletic Phaeophyceae with the Ishigeaceae 

in a basal position and the Ectocarpales in a 

terminal position. The clade of the Sphacelariales, Dictyotales, 

and Syringodermatales was not supported 

with bootstrap values or Bayesian probabilities. The 

remaining taxa formed a monophyletic clade (61% BS 

for ML, 59% BS for MP, and PP 5 1). However, the 

psaA tree is different from the rbcL tree in having the 

Desmarestiales as the sister taxon to the Laminariales 

(66% BS for ML, 75% BS for MP, and PP 5 1), and the 

Laminariales is not monophyletic. 

The psbA tree (Fig. 3) had a similar topology to the 

rbcL and psaA trees in the positions of the Ishigeaceae 

and the Ectocarpales within the Phaeophyceae. However, 

the psbA tree is different from the rbcL and psaA 

trees in having the Syringodermatales separated from 

the clade of the Dictyotales and Sphacelariales. The 

latter two orders were not supported with ML and MP 

bootstrap values but were supported with 0.91 and 

0.97 PP, respectively. 

Although the tree of the concatenated data set was 

similar to the rbcL tree than the psaA andpsbA trees, all 

these trees were congruent in having the monophyletic 

Phaeophyceae with the Ishigeaceae in a basal position 

and the Ectocarpales in a terminal position (Fig. 4). 

Most of orders and families included in the present 

study received higher bootstrap values and Bayesian 

probabilities than each individual data set. The Dictyotales, 

Sphacelariales, and Syringodermatales formed a 

clade supported with 65% BS for ML and 1 PP. The 

other remaining taxa produced a monophyletic clade 


(93% BS for ML, 94% BS for MP, and PP 5 1). The 

Laminariales was monophyletic (97% BS for ML, 90% 

BS for MP, and PP 5 1) and was shown to be the sister 

taxon to the Ectocarpales, but the sister relationship 

was not supported with bootstrap values. The interrelationships 

between most of orders and families were 

not well resolved. 

The SH tests using the combined data set showed 

that the best position for the Ishigeaceae was on 

the basal branch of the Phaeophyceae. The alternative 

topologies that force the genus to be placed into a clade 

within the Chordariaceae, Ectocarpales, Fucales, 

Sphacelariales, Dictyotales, or Syringodermatales 

were significantly worse than the best tree (Po0.05, 

Table 3). 

Morphology of plastids and ultrastructure of Ishige okamurae. 

Thalli of I. okamurae (Fig. 5a) contain plastids 

in the vegetative cells of the assimilatory 

filaments. They are discoid, and the number is three 

to four per cell (Fig. 5b). The ultrastructure is typical 

of other brown algal plastids. No pyrenoid was found 

in most of the plastids in our samples, but a small 

exserted pyrenoid was very rarely observed (Fig. 5, c 

and d). 

DISCUSSION 

This study presents the first psaA and psbA phylogenies 

of the Phaeophyceae based on 38–39 representatives 

from 13 orders or ‘‘ordinal-level families,’’ 

including two outgroup taxa. The psaA and psbA phylogenies 

are compared with the rbcL tree from 70 taxa 

from 17 orders, including 4 ordinal-level taxa and 3 

outgroup species. To our knowledge, such a large data 

set containing three protein-coding plastid genes has 

not been used for the Phaeophyceae. Although the 

rbcL region is more variable (46.7%) than the psaA 

(44.4%) and psbA (34.6%) regions, the psaA region contains 

more informative sites (38.8%) than the psbA 

(25.5%) and rbcL (38%) regions. Lower consistency index 

of the rbcL region (0.278) among three genes is 

probably due to the number of taxa investigated. The 

result that the consistency index values of the psaA 

(0.333) and psbA (0.378) in the present study are lower 

than those (0.493 in psaA and 0.383 in psbA) in previous 

study of red algae (Yang and Boo 2004) may be 

assigned to taxon sampling of brown algae at the ordinal 

level than that of red algae at the genus level. A 

comparison of other phylogenetic signals in individual 

and combined data sets is given in Table 2. 

Recently, several studies pointed out that the results 

of congruence tests should not be used as criteria for 

combining individual data sets. The reliability of the 

congruence tests such as the ILD test has been questioned 

(Lavoué et al. 2003, Shimabukuro-Dias et al. 

2004), and some different data sets, being in conflict 

over some parts of a tree, may be congruent over other 

parts (Gatesy et al. 1999b). Although there are no signs 

of saturation of each gene sequence, the ILD test detected 

a significant incongruence (at the Po0.05 level)

928 


FIG. 1. ML tree for the Ishigeaceae and other phaeophyceaen algae estimated from the rbcL sequence data (GTR þ G þ I model, 

–ln likelihood 5 20839.33098; G 5 0.798767; I 5 0.463896; A–C 5 1.223060, A–G 5 4.104599, A–T 5 1.183624, C–G 5 2.299422, 

C–T 5 7.838568, G–T 5 1). The bootstrap values shown above the branches are from ML/MP methods and dashes indicate o50% 

support of bootstrap. Thick branches indicate Bayesian posterior probabilities 0.9.

among the rbcL, psaA, and psbA data sets in the present 

study. This is a contrast to congruence among the same 

three genes in the ceramiaceous red algal tribe Griffithsieae 

(Yang and Boo 2004). However, the combined 

sequences of the three genes have more resolving 

power and clade support for all ordinal taxa including 

the three orders, the Laminariales not resolved in the 

psaA and psbA trees and the Dictyotales and Sphacelariales 

not supported in the psbA tree, than the sequences 

of individual gene. The individual and combined data 

sets are congruent in phylogenetic positions of the Ishigeaceae 

and the Ectocarpales within the Phaeophyceae 

investigated. It appears that incongruence among 

the three genes is probably attributed to unstable relationships 

among orders, which are a feature well 

known in phylogenetic reconstructions of the Phaeophyceae 

(Draisma et al. 2001, 2003, Rousseau et al. 

2001). Our studies show that statistically incongruent 

data can be combined for a better understanding of 

brown algal phylogeny, although the rbcL, psaA, and 


FIG. 2. ML tree for the Ishigeaceae 

and other phaeophyceaen algae 

estimated from the psaA sequence 

data (GTR þ G þ I model, –ln likelihood 

5 16551.60179; G 5 0.568987; 

I 5 0.492528; A–C 5 2.626896, A–G 5 

4.898248, A–T 5 0.072912, C–G 5 

3.757704, C–T 5 12.922112, G–T 5 1). 

The bootstrap values shown above the 

branches are from ML/MP methods 

and dashes indicate o50% support of 

bootstrap. Thick branches indicate 

Bayesian posterior probabilities 0.9. 

psbA genes possess different phylogenetic information. 

Our results indicate that the psaA and psbA genes, both 

being independent to each other (Morden and Sherwood 

2002), are useful for other brown algal phylogeny 

and have more resolution when combining with 

other molecular data such as the rbcL gene. 

In the present study, the Phaeophyceae are well resolved, 

having the Schizocladiophyceae as sister taxon 

in the rbcL and combined data sets, although the 

Tribophyceae was the sister to the Phaeophyceae in 

the psaA and psbA data sets. Based on trees of individual 

and combined data sets, the Phaeophyceae 

consist of two large groups. The ‘‘basal group’’ included 

the Choristocarpaceae, Ishigeaceae, Dictyotales, 

Sphacelariales, and Syringodermatales and the ‘‘crown’’ 

contained the Ectocarpales, Fucales, Laminariales, and 

other many lineages. These two groups are also reflected 

in the trees of previous rbcL (Draisma et al. 2001, 

2003) and LSU þ partial SSU data sets (Rousseau et al. 

2001). However, because the interrelationships of the

930 

orders or families within each of the two groups were 

not resolved, we focus on the phylogenetic and taxonomic 

implications of the Ishigeaceae compared with 

other relative orders. 

In all phylogenies reconstructed from three protein-coding 

genes, the Ectocarpales was placed in a 

terminal position, whereas the Ishigeaceae ended up 

in a basal position. The monophyly of the Ectocarpales, 

which includes the largest number of taxa in our study, 

is strongly supported and, within the order, the five 

families established by Peters and Ramírez (2001) are 

well resolved, although the Chordariaceae appear paraphyletic. 

The long-held concept of an ancestral Ectocarpales 

and derived Fucales (van den Hoek et al. 

1995) is not supported by our psaA andpsbA gene 

phylogenies as well as rbcL gene, as is seen in previous 

rbcL (Draisma et al. 2001, 2003) and LSU þ partial 

SSU trees (Rousseau et al. 2001). 

The main finding of our study is that the family Ishigeaceae 

does not cluster with members of the Chord- 



and other phaeophyceaen algae estimated 

from the psbA sequence data 

(GTR þ G þ I model, –ln likelihood 5 

5953.44945; G 5 0.472723; I 5 0.402634; 

A–C 5 1.245033, A–G 5 9.15766, A–T 5 

8.589725, C–G 5 1.088511, C–T 5 

23.338746, G–T 5 1]. The bootstrap 

values shown above the branches are 

from ML/MP methods and dashes indicate 

o50% support of bootstrap. 

Thick branches indicate Bayesian posterior 

probabilities 0.9. 

ariaceae such as Chordaria flagelliformis or other taxa of 

the Ectocarpales. These results contradict the current 

classifications of Yoshida (1998) that assign the family 

to the Chordariales (or the Chordariaceae s.s.). Our 

trees confirm that the Ectocarpales s.l. (Peters and 

Ramírez 2001), which is circumscribed by a pedunculated 

pyrenoid in discoid plastids and mostly a heteromorphic 

life history (Peters and Ramírez 2001, Draisma 

et al. 2003), cannot contain the genus Ishige. Instead, 

all analyses of our rbcL, psaA, psbA, and combined data 

sets indicate that the family Ishigeaceae consistently 

forms a basal group in the Phaeophyceae. The Ishigeaceae 

was the basal most in the psaA, psbA, combined 

data sets, and SH tests because Choristocarpus is 

not included; however, the Ishigeaceae was the penultimate 

basal taxon, after the Choristocarpaceae, in the 

rbcL tree. These results suggest that the Ishigeaceae 

might have diverged early in brown algal evolution. 

This is the first time that such a hypothesis has 

been proposed on either morphological or molecular

evidence. A closer look on morphological features of 

the Ishigeaceae that support their probable early divergence 

is described below. 

From the ultrastructural details of the vegetative 

cells of I. okamurae from Korea, it is clear that most cells 

do not have a pyrenoid or vary rarely have a pyrenoid. 

The pyrenoid is very small, exserted (Fig. 5, c and d), 

and similar to that of I. okamurae from Japan (Fig. 12 in 

Hori 1971). The small exserted pyrenoid of the species 

is considered a relatively rudimentary type when compared 

with the elaborate pedunculate pyrenoid of the 

Ectocarpales (Evans 1968). Ishige sinicola also lacks a 

pyrenoid (Hori 1971, Hori and Ueda 1975). Therefore, 

we conclude that the Ishigeaceae lack a pyrenoid 

in plastid. We suspect that Hori (1971) might wonder 

at the absence of a pyrenoid in Ishige, believing it to 

belong to the Chordariales, which have pyrenoids. The 

vegetative cells of the Dictyotales, Fucales, Laminariales, 

and Sphacelariales also lack pyrenoids, although 

a rudimentary form occurs in their gametes or zoo- 



and other phaeophyceaen algae 

estimated from combined rbcL þ 

psaA þ psbA sequence data (GTR þ G þ 

I model, –ln likelihood 5 37313.27150; 

G 5 0.756446; I 5 0.516176; A–C 5 

2.052989, A–G 5 5.063074, A–T 5 

1.442179, C–G 5 2.684154, C–T 5 

12.351116, G–T 5 1). The bootstrap 

values shown above the branches are 

from ML/MP methods and dashes indicate 

o50% support of bootstrap. 

Thick branches indicate Bayesian posterior 

probabilities 0.9. 

spores (Evans 1966, Chi 1971). Therefore, the presence 

of the ‘‘true’’ pyrenoid (not a ‘‘rudimentary’’ one 

in which the exact nature is not demonstrated and 

perhaps not homologous) is apomorphic in the Phaeophyceae. 

Our results do not accord with the view of 

Evans (1966, 1968) that the presence of the pyrenoid is 

plesiomorphic. However, because the occurrence and 

development of the pyrenoid varies with the life history 

stage and physiological conditions (Bourne and 

Cole 1968), further ultrastructural studies of the 

plastids of various brown algae are required before 

the phylogenetic significance of the pyrenoid can be 

substantiated. 

The two species of Ishigeaceae grow via small apical 

cells (Fig. 1 in Lee et al. 2003). Apical growth occurs in 

all other members of the basal group, such as the 

Choristocarpaceae, Dictyotales, Sphacelariales, Onslowiaceae, 

and Syringodermatales, which have an apical 

cell, a group of apical cells, or marginal cells cutting 

off segments proximally (Prud’homme van Reine

932 

TABLE 3. Results of the Shimodaira-Hasegawa tests used to 

evaluate alternative hypotheses of the Ishigeaceae position 

in the phylogeny of the psaA þ psbA þ rbcL data set (see 

Fig. 4). 

Hypotheses tested –ln L Difference –ln L P 

Basal position 37313.27150 best 

Chordariaceae 37415.16034 101.88883 0.0000 a 

Ectocarpales 37366.85999 53.58849 0.0020 a 

Fucales 37395.40131 82.12981 0.0000 a 

Sphacelariales 37368.18768 54.91617 0.0118 a 

Dictyotales 37366.71234 53.44084 0.0143 a 

Syringodermatales 37359.43507 46.16357 0.0193 a 

a Significant at 0.05 level. 

1982, Henry 1984, Bold and Wynne 1985). We agree 

with Draisma et al. (2001, 2003) and Rousseau et al. 

(2001) that apical growth is plesiomorphic. In this context, 

it is interesting to speculate whether apical growth 

in spermatochnacean ectocarpoids, such as Chordariopsis, 

Spermatochnus, and Stilophora (Fritsch 1945), is 

plesiomorphic or convergent. However, the apical 

growth in the Fucales may be derived in that the apical 

meristem is composed of one or more cells that can 

divide in several directions, generating three-dimensional 

thalli (Graham and Wilcox 2000). In other 

crown group algae, thalli grow for trichothallic, intercalary, 

or marginal meristems (Bold and Wynne 1985, 

Graham and Wilcox 2000). 

The internal tissues of the thallus of the Ishige 

species consist of cortex and medulla; the cortex 

is composed of assimilatory filaments and pseudoparenchymatous 

tissue (Ajisaka 1989, Figs. 5 and 11 

in Lee et al. 2003), and the medulla contains colorless 

hypheal cells connected to adjacent hypheal cells or 

directly to cortical cells (Ajisaka 1989, Tanaka in Hori 

1993, Lee et al. 2003). This oligostichous organization 

is similar to the haplostichous structure of uniseriate 

filament of the Choristocarpaceae (Fritsch 1945). 

Both haplostichous and polystichous structures are 

found in the Sphacelariales (Fritsch 1945, Prud’homme 

van Reine 1982). However, the Dictyotales and 

Syringodermatales have polystichous organization 

alone (Fritsch 1945, Henry 1984, Draisma et al. 

2003). Therefore, a series of evolutionary changes 

may have led from haplostichous structures to polystichous 

structures in the internal organization of the 

thalli of the basal brown algae. However, it appears that 

the internal organization of the thallus might have 

evolved independently several times among the crown 

group of the brown algae (Draisma et al. 2003). 

One of the most distinctive characters of the Ishigeaceae 

is the presence of the cryptostomata on the 

surface of thalli, which are well illustrated in previous 

studies (Fig. 8 in Yendo 1907, Figs. 2 and 10 in Lee 

et al. 2003). Phaeophycean hairs originate from cells 

in the medulla. In the presence of cryptostomata, 

the Ishigeaceae is similar to crown groups, such as 

the Fucales, Scytothamnales, and Adenocystis of the 

Ectocarpales (Clayton 1984, 1985). However, the 


scytothamnalean cryptostomata mature into conceptacles 

containing unilocular sporangia, whereas the 

fucalean cryptostomata do not form gametangial 

branches. There are a variety of other genera of the 

Ectocarpales, such as Colpomenia, Chnoospora, and Leathesia, 

in which there are groups of phaeophycean 

hairs growing from shallow epithermal pits (Fritsch 

1945, Clayton 1984). Our molecular phylogenies 

based on the rbcL, psaA, and psbA genes place taxa 

with cryptostomata, viz. Ishigeaceae, Scytothamnales, 

and Fucales, in different clades. Adenocystis formed a 

clade independent from the scytosiphonacean genera, 

despite their belonging to the Ectocarpales. These results 

demonstrate that the cryptostomata in the family 

Ishigeaceae is a homoplastic character that misled 

Yendo (1907) into classifying the genus in the family 

Fucaceae. At the present state of knowledge, the 

occurrence of cryptostomata in the Phaeophyceae is 

evolutionarily enigmatic. On the other hand, the 

phaeophycean hairs are present on branches in the 

Sphacelariales (Prud’homme van Reine 1982) or are 

associated with sporangial sori in the Dictyotales (Bold 

and Wynne 1985), whereas they are absent in the 

Choristocarpaceae, Onslowiaceae, and Syringodermatales 

(Henry 1984, 1987, Womersley 1987, Draisma 

and Prud’homme van Reine 2001). Our multigene 

trees do not provide phylogenetic correlation between 

the cryptostomata and the phaeophycean hairs. 

The unilocular sporangia of I. okamurae are produced 

from the outermost cortical cells (Tanaka in 

Hori 1993, Lee et al. 2003). The plurilocular sporangia 

of the species originate from the assimilatory filaments, 

are uniseriate, and lack sterile terminal cells 

(Ajisaka 1989, Tanaka in Hori 1993, Lee et al. 2003). 

Both unilocular and plurilocular sporangia occur in 

the thalli of I. sinicola from the south coast of Korea 

(Lee et al. 2003). The observations of Ajisaka (1989) 

and Lee et al. (2003) contradict those of Arasaki (1943) 

that the plurilocular sporangia of I. sinicola in culture 

were gametangia and similar to those of certain species 

of the Chordariales, in which the unilocular sporangia 

are always formed in the basal or middle part of the 

assimilatory filaments, and both types of sporangia are 

present on the sporophytes of some species (Inagaki 

1958, Ajisaka 1989, Yoshida 1998). However, the 

Dictyotales have meiosporic tetrasporangia (Bold and 

Wynne 1985), and the Choristocarpaceae, Onslowiaceae, 

and Sphacelariales have unilocular zoidangia, 

plurilocular zoidangia, or multicellular propagules 

(Prud’homme van Reine 1982). 

Ajisaka (1989) reported that plurispores of I. okamurae, 

released from the plurilocular sporangia, develop 

directly into typical filamentous thalli through a 

pseudoparenchymatous prostrate disc, which is sterile 

and functions as a perennial holdfast system. A similar 

perennial basal system is found in the Sphacelariales, 

including the Choristocarpaceae (Fritsch 1945). Perennial 

systems are found in Ralfsia verrucosa (Areschoug) 

Areschoug, Desmarestia aculeata (Linnaeus) 

Lamouroux, and the Aglaozonia-stage of Cutleria monoica

Ollivier (Fritsch 1945). Because the latter three taxa 

were not included in the present study, they are beyond 

the scope of our discussion. Tanaka (in Hori 

1993) demonstrated that I. okamurae has an isomorphic 

life cycle, consisting of sporophytes that form plurilocular 

sporangia and gametophytes bearing unilocular 

sporangia. In the same chapter, Tanaka doubted the 

heteromorphic life history of I. sinicola observed by 

Arasaki (1943). Both plurilocular and unilocular sporangia 

occurred in erect thalli of I. okamurae (Figs. 7 and 

8 in Lee et al. 2003) and I. sinicola (Figs. 15 and 16 in 

Lee et al. 2003) during a year-round collection from 

Namhaedo on the south coast of Korea. All these studies 

led us to conclude that the family Ishigeaceae has 

an isomorphic life history, with macroscopic sporophytes 

and gametophytes. Isomorphic life cycles have 

been reported for the Dictyotales, Sphacelariales, and 

Onslowiaceae (Prud’homme van Reine 1982, Henry 


FIG. 5. Thallus and plastid of Ishige okamurae. (a) A herbarium specimen collected on 28 June 2001 in Geojedo, Korea. Scale bar, 

2 cm. (b) Discoid plastids (arrows) in assimilatory cell. Scale bar, 10 mm. (c) Ultrastructure of a plastid, showing thylakoids, without 

pyrenoid. Scale bar, 1 mm. (d) A small exserted pyrenoid (arrow) in a plastid. Scale bar, 0.4 mm. 

1987, van den Hoek et al. 1995, Draisma and Prud’homme 

van Reine 2001). In the Choristocarpaceae, thalli 

with either uni- or plurilocular sporangia look similar 

and could also indicate an isomorphic life history 

(Fritsch 1945, Burrowes et al. 2003). However, the 

Syringodermatales have a heteromorphic life history 

(Henry 1984). Although some members of the Scytosiphonaceae 

have an isomorphic type (Cho et al. 

2003), the Ectocarpales and other brown algae have 

heteromorphic life histories or single diploid generations 

(Graham and Wilcox 2000). 

Interestingly, both species of Ishige are distributed in 

the warmer waters of the Pacific Ocean (Yendo 1907, 

Setchell and Gardner 1924, Tseng 1983, Lee et al. 

2003). Choristocarpus tenellus of the Choristocarpaceae 

inhabits the Mediterranean Sea (Fritsch 1945). The 

Dicytotales are very diverse in tropical and subtropical 

waters (Bold and Wynne 1985), and the Sphacelariales

934 

TABLE 4. A taxonomic comparison of the Ishigeaceae with other basal brown algae that have symplesiomorphic characters such as apical growth and discoid pyrenoidless 

plastids. 

Ishigeaceae Choristocarpaceae Onslowiaceae Sphacelariales Dictyotales Syringodermatales 

Thallus construction Oligostichous Haplostichous Oligostichous Haplostichous or Polystichous Oligostichous 

of sporophytes 

polystichous 

Perennial disc Present Present Present? Present Rarely present Absent 

Cryptostomata Present Absent Absent Absent Absent Absent 

Phaeophycean hairs Within cryptostomata Absent Absent On branches Associated with 

Absent 

reproductive organs 

Propagules Absent With a large apical cell Without a large Without a large 

Absent Absent 

apical cell 

apical cell 

Plurilocular From cortical layer of On branches of On branches of On branches of From the surface From the surface cells of 

sporangia 

gametophytes gametophytes 

gametophytes 

gametophytes cells of gametophytes gametophytes 

Unilocular Many spores, from the Many spores, on Many spores, on Many spores, on Tetraspores, from the Bi-, tetraspores, from the 

sporangia 

cortical layer of 

branches of 

branches of branches of sporophytes surface cells of surface cells of 

sporophytes 

sporophytes 

sporophytes 

sporophytes 

sporophytes 

Gametes Isogametes Isogametes Anisogametes Mostly iso- and Egg and spermatozoids Isogametes 

anisogametes 

Life cycle Isomorphic Probably isomorphic Isomorphic Mostly isomorphic Isomorphic Heteromorphic 

Pheromone Not detected Not detected Not detected Ectocarpene/ Dictyotene, multifidene Viridene 

hormosirene, 

desmarestene 


Data from Fritsch (1945), Prud’homme van Reine (1982), Henry (1984, 1987), Womersley (1987), Ajisaka (1989), Tanaka (in Hori 1993), Draisma and Prud’homme van Reine 

(2001), Pohnert and Boland (2002), and Lee at al (2003). 

and Syringodermatales are distributed from tropical to 

Antarctic waters (Prud’homme van Reine 1982, Henry 

1984). The distributions of living taxa of these basal 

groups speculate that the brown algae may have originated 

in tropical waters during a geological period 

with warm climatic conditions, whereas most brown 

algae in the crown group appear to exhibit the greatest 

diversity in terms of species and morphology in cold 

waters (Bold and Wynne 1985). The Paleozoic may 

have been the period of the earliest brown algae, as 

suggested by Clayton (1984), or the Mesozoic, which 

roughly corresponds to 155 million years ago based on 

rDNA SSU sequence divergence (Medlin et al. 1997) 

or approximately 200 million years ago based on 5S 

rRNA sequences (Lim et al. 1986). 

Our molecular study shows conclusively that the 

family Ishigeaceae produces a basal brown algal group 

with the Choristocarpaceae, Dictyotales, Sphacelariales, 

Onslowiaceae, and Syringodermatales. These early 

diverging brown algae have apical growth and 

pyrenoidless discoid plastids as morphological 

symplesiomorphy. In addition, the Ishigeaceae is similar 

to the Dictyotales, Sphacelariales, Onslowiaceae, 

and probably Choristocarpaceae in its isomorphic life 

history. However, our rbcL, psaA, and psbA phylogenies 

indicate that the family Ishigeaceae makes an independent 

lineage from the early diverging brown algae 

in having oligostichous organization of thallus, cryptostomata, 

and uni- and plurilocular sporangia within 

cortical layer (Table 4). Our studies on the three protein 

coding plastid genes and ultrastructure of plastids 

do not confirm the current classification of the family 

Ishigeaceae within the Ectocarpales s.l. or Chordariaceae 

s. str. Although we did not examine the Ascoseirales, 

which is only order of the Phaeophyceae not 

included in the present study because of no collection, 

it does not appear that the inclusion of the taxon would 

change our finding that the Ishigeaceae is a distinct 

basal group clearly separated from other brown algal 

orders or families. The unique molecular and ultrastructural 

evidence that defines the Ishigeaceae as an 

early diverging but independent brown algal group is 

best expressed by placement of the family in a separate 

order, Ishigeales ord. nov. 

Ishigeales ord. nov. G. Y. Cho et Boo 

Diagnosis: Novus ordo Phaeophycearum. Plantae 

isomorphicae, ad 20 cm altae, ex haptero parvo aut basi 

crustosa extenta crescentes, epiphyticae vel epilithicae. 

Augmen per cellulas apicales. Frondes ramosae teretes vel 

foliosae, medulla corticeque instructae. Cortex pseudoparenchymatus, 

filamentis assimilantibus. Cellulae plastides 

aliquot discoideos sine pyrenoide continentes. Medulla cum 

hyphis. Pili phaeophycei caespitosi, e cryptostomatibus 

orientes. Sporangia unilocularia e cellulis corticalibus 

transformatis facta, terminalia. Sporangia plurilocularia 

e filamentis assimilantibus transformatis facta, uniseriata, 

sine cellula terminali. 

Type family: Ishigeaceae Okamura in Segawa with 

the same characteristics as the order.

Type genus: Ishige Yendo 

New order of Phaeophyceae. Plants isomorphic, to 

20 cm high, growing from a small holdfast, or an 

extended crustose base, epiphytic or epilithic. 

Growth from apical cells. Fronds branched, terete, 

or foliose, with medulla and cortex. Cortex pseudoparenchymatous, 

with assimilatory filaments. Cells 

containing several discoid plastids without pyrenoids. 

Medulla with hyphae. Phaeophycean hairs clustered, 

growing from cryptostomata. Unilocular sporangia 

transformed from cortical cells, terminal. Plurilocular 

sporangia transformed from assimilatory filaments, 

uniseriate, lacking a terminal cell. 

We appreciate Drs. Joana Kain, Nina Klotchkova, Wendy Nelson, 

and Nathalie Simon for help with the collection of samples 

and their identification; Drs. Christos Kataros, Tae Jun 

Han, and Wook Jae Lee for sharing their samples; Drs. 

Suzanne Fredericq, Giovanni Furnari, and Mark A. Garland 

for Latin translation; and Dr. Willem F. Prud’homme van 

Reine for valuable comments. We thank W. J. Lee for providing 

his unpublished sequences and Dr. Yeun Sim Keum for 

sharing DNA stocks. We sincerely thank Drs. Debashish 

Bhattacharya and Hwan Su Yoon for providing information 

on the psaA and psbA regions. The director of the Marine Station 

in Chiba, Japan arranged accommodation of the collection. 

This study was supported by KRF grant 2002-070- 

C00083 to S. M. B. 

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