Recent Advances in the Phylogeny and Taxonomy of Laminariales, with Special Reference to the Newly Discovered Basal Member Aureophycus

Perspectives in Phycology, Vol. 1 (2014), Issue 1, p. 27–40 Article Published online June 2014

Recent advances in the phylogeny and taxonomy of Laminariales, with special reference to the newly discovered basal member Aureophycus

Hiroshi Kawai

Kobe University Research Center for Inland Seas, Rokkodai, Nadaku, Kobe 657-8501, Japan; [email protected]

With 10 figures

Abstract: Laminarialean species are the largest photosynthetic organisms in aquatic environments and constitute a significant ecological element of coastal ecosystems in temperate and colder seas. Laminariales has been believed to be clearly delimited in the ordinal taxonomic rank by the morphology and life history patterns, however, their phylogeny and taxonomy has been considerably revised in the past two to three decades largely based on fine structure as revealed by transmission electron microscopy, life history studies employing unialgal cultures, and molecular phylogenetic analyses using diverse genetic markers. The families Akkesiphycaceae and Pseudochordaceae were described as basic members, whereas Phyllariaceae and Halosiphon were transferred to Tilopteridales. In the derived members, familial taxonomy was considerably revised by the molecular studies, and the finding ofAureophycus .

Keywords: Aureophycus, kelp, Laminariales, molecular phylogeny, Phaeophyceae, taxonomy

Introduction basal and derived groups, partly due to the lack of any significant fossil record and incomplete molecular phylogeny. Laminarialean species (so-called kelps) are the largest pho- A majority of kelp taxa are only distributed in the tosynthetic organisms in aquatic environments, sometimes Northern Hemisphere, and only four genera (i.e., Ecklonia, exceeding 50 m in length, and constitute a significant eco- Eisenia, Lessonia and Macrocystis) are reported from the logical element of coastal ecosystems in temperate and Southern Hemisphere, except for Undaria pinnatifida colder seas, providing habitat for diverse plants and animals which spread by anthropogenic introductions by such as (Bold & Wynne 1985; Dayton 1985; Graham & Wilcox fisheries and ship operations (Voisin et al. 2005; Uwai et al. 2000). They are the most structurally complex seaweeds, 2006). Therefore, Laminariales are considered to have with two distinctive generations, although they are sister originated in the North Eastern Pacific and spread to the to a group consisting of small filamentous members (i.e., other distributional ranges including the Atlantic and the Ectocarpales s.l. including the brown algal ‘model’ species Southern Hemisphere (Lüning & tom Dieck 1990; Bolton Ectocarpus siliculosus) (Kawai et al. 2007; Phillips et al. 2010). The divergence time of Laminariales has been sug- 2008; Silberfeld et al. 2010; Cock et al. 2010). The largest gested to be relatively recent (< 100 Ma), but the phyloge- kelps such as Macrocystis pyrifera exhibit differentiation ography is still poorly understood (Silberfeld et al. 2010). between stipe and blade and have buoyancy to maintain the This review aims to summarize the advances in the stud- distal portion at the water’s surface for photosynthesis, while ies of taxonomy and phylogeny of Laminariales in the past bearing reproductive structures basically only near the base two to three decades, with special reference to the morphol- on special blades (sporophylls). ogy and life history of the basal members, including the Currently more than 30 genera in 8 families are recog- recently described Aureophycus. nized in the order (Guiry & Guiry 2013), generally grouped into basal (‘primitive’) and derived (‘advanced’) families (Kawai et al. 2013). The taxonomy and phylogeny of Ordinal taxonomy of Laminariales Laminariales have been considerably revised in the past two to three decades largely based on fine structure as revealed The order Laminariales Migula (1909) has been believed by transmission electron microscopy (TEM), life history to be clearly delimited by the heteromorphic (haplodiplon- studies employing unialgal cultures, and molecular phylo- tic) life history alternating between a large parenchymatous genetic analyses using diverse genetic markers. However, (polystichous) sporophyte with intercalary growth zone, and a there still remains a considerable apparent gap between the filamentous, oogamous gametophyte (Kylin 1916; Sauvageau

Fig. 1. Chorda species. a, C. filum; b, C. asiatica; c, C. rigida; d, C. kikonaiensis; e, cross section of fertile sporophyte with unicellular paraphyses; f, female gametophyte and oogonia of C. asiatica; g, male gametophyte and mature and emptied antheridia; h, TEM micrograph of emptied antheridia; i, tip of young sporophyte with phaeophycean hairs (embryo). In addition to the type species C. filum (a), three new species distributed in the Pacific Ocean (b-d) have been described.

1916; Oltmanns 1922; Setchell & Gardner 1925; Fritsch 1945; the presence of protruded-type pyrenoids without invaginations Bold & Wynne 1985; South & Whittick 1987; van den Hoek in the chloroplasts (Evans 1966; Hori 1971, 1972; Kawai 1992; et al. 1995). Later, Kawai & Sasaki (2000) extended the defi- de Reviers & Rousseau 1999; Nagasato et al. 2003; Tanaka et al. nition of the order to include a taxon with plano-anisogamy 2007; Silberfeld et al. 2011). Because of the high divergence in (Kawai 1986) by the establishment of Akkesiphycaceae the morphology and life history patterns within Ectocarpales s.l. Kawai et Sasaki and its inclusion in the order. In contrast, and by the inclusion of Akkesiphycaceae in Laminariales, other Phyllariaceae Tilden, including Saccorhiza and Phyllariopsis morphological features traditionally used in distinguishing the (= Phyllaria), which used to be placed in the order, was trans- two orders have much less taxonomic value (i.e., development ferred to Tilopteridales Bessey mainly because of their geneti- of parenchymatous thalli, heteromorphic life history, oogamy, cally distant phylogenetic positions (Sasaki et al. 2001). number of chloroplasts in each cell, presence of trumpet-shaped On the other hand, a relatively close systematic relation- hyphae and localized growth zone). ship of Desmarestiales Setchell et Gardner and Sporochnales C. Sauvageau with Laminariales has been proposed based on their resemblance in sperm morphology (Fig. 7), life his- Evolution and taxonomy of basal tory, sexual reproduction patterns (Clayton 1984; Müller et Laminariales al. 1985a, 1985b; Kawai 1992), and early molecular phylogenetic studies (Tan & Druehl 1996; Peters 1998; Boo et Within Laminariales, traditionally two taxa distinguished by al. 1999; de Reviers & Rousseau 1999). However, by more gross morphology have been recognized: Chorda, with terete recent molecular phylogenetic studies covering broader thalli (Fig. 1), and all other genera, with more morphologi- taxa and using multiple genetic markers, their monophyly cally differentiated thalli (i.e., stipe and blade). Therefore, was not supported, and therefore those phenotypic similari- the order Laminariales has been divided into two groups, ties are understood as evolutionary convergence (Kawai & basal (‘primitive’) members and derived (‘advanced’ or Sasaki 2000; Phillips et al. 2008; Silberfeld et al. 2010). ‘evolved’) members, and the latter were sometimes treated The currently generally accepted sister taxon of Laminariales as Laminariales s.s. (de Reviers & Rousseau 1999; Yoon is Ectocarpales s.l. Bessey (Kawai et al. 2007; Phillips et al. 2008; et al. 2001). Silberfeld et al. 2010). Ectocarpales s.l. (including Chordariales, Before 1980s, only Chorda was recognized as a basal mem- Dictyosiphonales, Ectocarpales s.s. and Scytosiphonales) is at ber of Laminariales. Later Kawai and Kurogi (1985) described present principally characterized by the cytological feature of another basal member of the Laminariales, Pseudochorda Phylogeny and taxonomy of Laminariales 29

Fig. 2. Pseudochorda species. a, b, P. nagaii; c, d, P. gracilis; e, cross section of fertile sporophyte of P. gracilis indicating the multicellular paraphyses and unilocular zoidangium; f. male gametophyte bearing antheridia in group; g. TEM micrograph of fertile antheridia; h, female gametophyte with fertile and emptied oogonia; i, terminal portion of young sporophyte in culture (f-i, P. nagaii). Note that P. nagaii has characteristic antheridia in cluster resembling those in Akkesiphycus. nagaii which showed a heteromorphic life history alternat- Subsequently, Kawai (1986) suggested a close taxo- ing between terete, parenchymatous sporophytes and monoe- nomic relationship between Akkesiphycus lubricus Yamada cious, oogamous gametophytes in Pseudochorda nagaii et Tanaka and Pseudochorda based on morphology and life (Tokida) Inagaki, which used to be classified in Chordariales history (Fig. 3). Originally, A. lubricus had been placed in Setchell & Gardner (Tokida 1938; Inagaki 1958). Thereby, Scytosiphonales Feldmann or Dictyosiphonales Setchell & they suggested the transfer of P. nagaii to Laminariales and Gardner, being characterized by relatively large foliose thalli proposed a new family Pseudochordaceae Kawai et Kurogi with a stipe, resembling kelps in gross morphology, but with (Fig. 2). Later a second species of Pseudochorda, P. gracilis a more fragile blade with simpler anatomy lacking trumpet- Kawai et Nabata (Kawai & Nabata 1990; Fig. 2) was added shaped hyphae and with multicellular paraphyses (Yamada to the genus, and the authors emended the genus to recognize & Tanaka 1944; Kurogi & Yamada 1970). The sporophyte that the gametophytes of P. nagaii are dioecious but sexually anatomy as well as the heteromorphic life history with dioe- monomorphic, whereas P. gracilis gametophytes are dioecious but sexually monomorphic gametophytes in A. lubricus cious and sexually dimorphic which is a more common state bear close similarity to P. nagaii (and hence to Laminariales), in derived Laminariales. although the gametes of Akkesiphycus show anisogamy (both 30 H. Kawai

Fig. 3. Akkesiphycus lubricus. a, juvenile sporophytes; b, young sporophyte; c, mature sporophyte; d, longitudinal section of sporophyte with multicellular paraphyses; e, female gametophyte with fertile and emptied (arrowheads) female gametangia; f, male gametophyte bearing antheridia in group (arrowhead); g, TEM micrograph of fertile male gametangia in group; h, whole mount TEM micrograph of male and female plano-gametes. Arrowheads show anterior flagella. Note that Akkesiphycus and Pseudochorda lack localized growth zone as well as the trumpet-shaped hyphae, characteristic of derived Laminariales. male and female gametes have an eyespot and female gam- rence of monoecious (Saccorhiza dermatodea (Bachelot de etes have flagella; Figs 3, 7). However, Motomura & Sakai la Pylaie) J. Agardh) or dioecious but monomorphic game- (1988) showed the occurrence of a residual flagella in the eggs tophytes (Phyllariopsis brevipes (C. Agardh) Henry et South of Laminaria angustata Kjellman (= Saccharina angustata and P. purpurascens (C. Agardh) Henry et South). However, (Kjellman) Lane, Mayes, Druehl et Saunders). Therefore, at Phyllariaceae is unique in having a vascular system made of least some of the ‘oogamy’ of Laminariales is, in the strict solenocysts and allelocysts, which appear functionally compa- sense, aplano-anisogamy, and the gap between the plano- rable (Emerson et al. 1982) to the trumpet-shaped hyphae in anisogamy of Akkesiphycus and the oogamy of Laminariales Chordaceae Dumortier and derived Laminarilaes, but whose has less taxonomic importance (Kawai 1986). cells are multinucleate, unlike any other brown algal cells Henry & South (1987) suggested the reappraisal of the (Fritsch 1945; Henry & South 1987). family Phyllariaceae Tilden based on some primitive charac- Later, molecular phylogenetic data have provided clues teristics of the members (Henry 1987) such as lack of meris- for elucidating the cryptic diversity of Chorda at the species tematic rhizoidal holdfast, annual nature of sporophytes, lack level. Only one Chorda species, C. filum (L.) Stackhouse, of mucilaginous organs and mucilage caps on paraphyses, was recognized after the transfer of Chorda tomentosa presence of eyespots in zoospores (meiospores), and occur- Lyngbye to Halosiphon (as H. tomentosus (Lyngbye) Phylogeny and taxonomy of Laminariales 31

Fig. 4. Aureophycus aleuticus. a, juvenile sporophyte; b, mature sporophyte; c, cross section of basal part of blade; d, longitudinal section of basal part of blade; e, cortex of blade showing trumpet-shaped hyphae (arrows); f, epidermal cells of blade; g, cross section of stipe; h, longitudinal section of stipe. Note that mucilaginous organs are absent in the Aureophycus sporophyte.

Jaasund; Peters 1998). This transfer was first suggested Evolution and taxonomy of derived based on morphological, life history and biochemical data Laminariales (Maier 1984; Kogame & Kawai 1996), and was later confirmed by molecular data (Peters 1998; Sasaki et al. 2001). Furthermore, Halosiphon was placed in an independent fam- Early evolution of derived Laminariales ily Halosiphonaceae Jaasund ex Kawai et Sasaki (Sasaki et al. 2001) in Tilopteridales. Derived laminarialean species are distinctive in their elab- Within Chorda, later a second species, Chorda rigida orate sporophyte morphology: 1) Differentiation among Kawai et Arai was described based on morphological and blade, stipe and meristematic rhizoidal holdfast (haptera); molecular phylogenetic data (Kawai et al. 2001). Later, 2) Presence of a localized growth zone between blade and largely based on molecular phylogeny, further taxonomic stipe; 3) Perennial erect thalli in some taxa; 4) Presence of divergence of the genus became evident: A third species, characteristic trumpet-shaped hyphae, comparable to sieve Chorda kikonaiensis Sasaki et Kawai was described, and tubes in vascular plants, which are essential for the devel- Asian C. filum was shown to be independent from Atlantic opment of the intercalary growth zone located at the base C. filum and was therefore designated as C. asiatica Sasaki et of the blade, as well as the development of a meristematic Kawai (Sasaki & Kawai 2007). It is interesting that Atlantic rhizoidal holdfast (Reinke 1892; Fritsch 1945; Parker 1965; C. filum, which is considered to have diverged more recently Schmitz & Srivastava 1974, 1975) under the low light inten- compared to Asian taxa, based on molecular phylogenetic sity conditions of the bottom of the sea where those tissues data (Sasaki & Kawai 2007), demonstrates considerably of large sporophytes are located. greater morphological diversity in thallus anatomy (South & The order Laminariales has traditionally included four Burrows 1967) than that observed within each Asian species, families: Chordaceae, Alariaceae Setchell et Gardner, and this has hindered discovery of the species divergences in Laminariaceae Bory de Saint-Vincent, and Lessoniaceae the Pacific Ocean (Sasaki & Kawai 2007). Setchell et Gardner (Setchell & Gardner 1925; Bold & Wynne 32 H. Kawai

Fig. 5. Aureophycus aleuticus. a, Sporophytes growing on intertidal rocks. Arrowhead shows well-developed disc-shaped holdfast; b, Sporophytes of different developmental stages. Arrows show flattened stipes and arrowheads show basal systems (discoidal holdfast forming sorus). Note that the basal systems show unilateral development in the early stages, and the iridescent color of the basal systems including those of rather young thalli showing signs of sorus formation; c, developed basal systems overlapping each other, with iridescent color on the surface (arrow) showing sorus formation; d, erect part of sporophyte with characteristic marginal thickening at the transitional zone between blade and stipe; e, basal part of stipe and disc-shaped holdfast. Arrow shows sporangial sorus. Asterisks show secondarily developed holdfast; f, bottom of holdfast; g, h, sorus under transmitted (g) and strong epi-illumination showing iridescence of sorus (h). Note the difference between vegetative portion (asterisk) and sorus (arrow). i, fertile sorus showing partly detached cuticle (arrow). Asterisk shows premature sorus; j, cuticles of sorus.

1985). There has been some controversy in the familial tax- Genetic markers used in the phylogenetic onomy of the derived members, however, the designation of studies in Laminariales and Phaeophyceae basal (‘primitive’) and derived (‘advanced’) members was quite clear. In spite of the sporophyte gross morphology being considerably different from derived Laminariales, Chordaceae Molecular phylogenetic studies of brown algae using have been recognized as a member of the order because of the nucleotide sequences began with RNA sequencing. The similarity in the life history pattern (i.e., heteromorphic life principal target was 5S ribosomal RNA, and although the history alternating between sexually dimorphic, oogamous divergence time of the brown algae from other strameno- gametophyte and large sporophyte) and occurrence of the pile classes (heterokonts) was roughly estimated (e.g., trumpet-shaped hyphae (Kylin 1918; Fritsch 1945). < 200 Ma from Bacillariophyceae), there was not sufficient Phylogeny and taxonomy of Laminariales 33

Fig. 6. Aureophycus aleuticus . a, female gametophyte with Fig. 7. Schematic presentation of laminarialean flagellated fertile oogonium; b, female gametophyte (left) and fertile male cells. a, zoospore of Akkesiphycaceae, Pseudochordaceae and gametophyte with antheridia (right); c, d, female gametophyte Chordaceae, with longer anterior and shorter posterior flagella with embryos. provided with eyespot and flagellar swelling/green flagellar autofluorescence of the posterior flagellum; b, zoospore of derived families (Aureophycaceae, Costariceae, Alariaceae, resolution for discussing the phylogenetic relationships Laminariaceae, Lessoniaceae) lacking eyespot and flagellar within Phaeophyceae (Lim et al. 1983, 1986). Then the tar- swelling/green flagellar autofluorescence of the posterior flagellum; c, female gamete of Akkesiphycaceae, including get sequences shifted to DNA by the general application several chloroplast, eyespot and associate structures; d, male of the Maxam-Gilbert method (Maxam & Gilbert 1977) gamete of Akkesiphycus similar to a, but a smaller cell; e, and the Sanger (chain-termination) method (Sanger et al. sperm of Pseudochordaceae, Chordaceae and derived families provided with longer posterior flagellum. Note that chloroplasts 1975), followed by the improvement of sequencing meth- are reduced, and lack eyespot and associated structures. Note ods and the invention of the PCR (polymerase chain reac- that the type-e sperm resemble those in Desmarestiales and tion) method and automated sequencers. Sporochnales in the flagellation pattern, and considered to show Early studies of brown algal DNA phylogeny largely tar- their phylogenetic affinity before molecular studies. geted nuclear 18S rDNA (Saunders & Druehl 1992; Kawai et al. 1995; Boo et al. 1999), rDNA ITS (internal transcribed spacer) (Serrão et al. 1999; Yotsukura et al. 1999; Peters The monophyly of Lessoniaceae, defined by morphological et al. 2000; Coyer et al. 2001), chloroplast rbcL sequences characters of the stipe branched from the base or above, was (Valentin & Zetsche 1990; Siemer et al. 1998; Draisma & questioned by Fain et al. (1988) based on the results of RFLD Prud’homme van Reine 2001; Draisma et al. 2002), and mito- (restriction fragment length difference) analyses, suggest- chondrial cox3 (Kogame et al. 2005; Uwai et al. 2007, 2009; ing the necessity of taxonomic reassessment of the familial Ni-Ni-Win et al. 2008, 2011). Apart from the above diverse assignments of Nereocystis, Macrocystis and Lessoniopsis. regions have also been tested to find sufficient resolution for Saunders & Druehl (1993) supported the use of rDNA ITS solving evolutionary and taxonomic problems: rDNA IGS sequence analyses and suggested the transfer of Lessoniopsis to (intrageneric spacer) (Kim et al. 2002, 2003; Yotsukura et al. Alariaceae. Later, by 18S rDNA molecular phylogeny, Boo et 2002, 2006; Draisma et al. 2012), 26S rDNA (Rousseau & de al. (1999) confirmed the monophyly of derived Laminiarilaes Reviers 1999; Erting et al. 2004), and rbcS (Kogame et al. including members of Laminariaceae, Lessoniaceae and 1999). Later, multiple gene regions were used for testing the Alariaceae, and suggested that the Pseudochordaceae might reliability of the genetic markers for elucidating more precise have branched off first from the laminarialean lineage that phylogenies, and to improve statistical reliability (Stache- leads, through the Chordaceae, to the derived Laminariales. Crain et al. 1997). Still later, concatenated sequences came They further supported the distant phylogenetic relation- into use for molecular phylogenetic analyses (Lane et al. ship of Halosiphon tomentosus and Saccorhiza polyschides 2006; Silberfeld et al. 2010; Kawai et al. 2013). (Lightfoot) Batters from Laminariales. Yoon et al. (2001) examined the phylogeny of derived Laminariales using rbc spacer and rDNA ITS sequences, and Molecular phylogenetic studies in derived obtained 8 clades (i.e., Hedophyllum-, Macrocystis-, Alaria-, Laminariales Agarum-, Ecklonia-, Lessonia-, Laminaria- and Egregia- clades). They considered that some of the clades corre- Familial level taxonomy of derived Laminariales has been sponded to the Tribes suggested by Kützing, Bory de Saint examined by many authors using different genetic markers. Vincent, Setchell, and Setchell & Gardner (i.e., Alarieae, 34 H. Kawai

Fig. 8. Molecular phylogenetic tree (MP) based on concatenated DNA sequences of 8 genes (chloroplast rbcL, atpB, psaA, psaB, psbA, psbC and mitochondrial cox1 and cox3 genes). Modified from Kawai et al. (2013).

Ecklonieae, Egregieae, Laminarieae, Hedophylleae, in derived laminarialean species, but it had trumpet-shaped Agareae, Cymathaereae, Lessoniaeae, Lessoniopseae and hyphae characteristic for Chorda and derived Laminariales. Macrocysteae; Setchell & Gardner 1925) and suggested Although no reproductive structures were found, a new subdivision of the order into 8 families as one of the pos- genus Aureophycus in Laminariales was proposed, and the sible scenarios, although they did not propose any actual species was described as A. aleuticus because of the char- taxonomic treatment. Later, Lane et al. (2006) reexamined acteristic vegetative morphology of the sporophyte. By a the phylogeny of derived Laminariales using ITS and 26S molecular phylogenetic study using rbcL, ITS1-5.8S-ITS2 rDNA, rbcL-rbcS and nad6 gene sequences, and suggested rDNA and nad6 sequences, A. aleuticus was shown to be phy- that they consist of three major clades roughly correspond- logenetically independent from any ‘derived’ Laminariales ing to Alariaceae (‘Group-1’), newly proposed Costariaceae and may be basal to Alariaceae (Kawai et al. 2008). (‘Group-2’), and Laminariaceae/Lessoniaceae (‘Group-3). Later a second locality of this species was discovered Yoon et al. (2001) suggested that Egregia was the most basal at St. George Island in the Bering Sea. By seasonal collec- taxon in the derived Laminariales, but Lane et al. (2006) tions at this site, unique reproductive structures formed on concluded that this was an artifact of biased taxon sampling. the basal system (holdfast) were found (Fig. 5, Kawai et al. 2013). No sori were found on the blades, and no separate sporophylls have been found on the stipes, even on thalli with zoidangia on the basal system, and the authors concluded Discovery of Aureophycus aleuticus, and its evolutionary implications that the sori on the basal system were the sole reproductive structures in the Aureophycus sporophyte. The anatomy of As mentioned above, there has been a large gap between the sori was similar to those found in ‘derived’ Laminariales basal and derived Laminariales in regard to various features. on the blades or on sporophylls, and the developmental pat- The discovery of Aureophycus aleuticus Kawai, Hanyuda, terns of zoospores released from the unilocular zoidangia Lindeberg et Lindstrom (Kawai et al. 2008) and its familial and the morphology of gametophytes were essentially the assignment (Kawai et al. 2013) provides a clue for elucidat- same as those of derived Laminariales (Fig. 6), but the locus ing the relationship between the two. of sorus formation is unique in Laminariales and any other An unknown laminarialean species of distinctive brown algae. morphology, exceeding 2 m in height, was discovered at The phylogenetic position of Aureophycus, based on the Kagamil Island in the Aleutian Islands during 2006 and concatenated sequences of 8 genes (chloroplast rbcL, atpB, 2007 (Kawai et al. 2008). It had a unique sporophyte with psaA, psaB, psbA, psbC and mitochondrial cox1 and cox3), large discoidal holdfast, considerably flattened stipe, and a was found to be either basal to all derived Laminariales, characteristic fragile blade with obvious V-shaped thicken- or to the clade of Alariaceae including Alaria, Undaria, ings at the transition zone to the stipe (Fig. 4). The sporo- Pterygophora and Pleurophycus. However, based on the phyte had no mucilaginous structures such as are common results of character mapping of representative taxonomic fea- Phylogeny and taxonomy of Laminariales 35

Fig. 9. Schematic presentation of an evolutionary scenario in Laminaliales. tures onto the two topology options, the authors concluded tougher thalli supported by denser inner cortical filaments that Aureophycus was basal to all ‘derived’ Laminariales as in Laminaria (Costariaceae and Laminariaceae or ‘Group (Figs. 8, 9, Kawai et al. 2013). 2’ + ‘Group 3’). This may further support a hypothesis that Among the characteristic morphological features of derived the ancestral laminarialean species connecting ‘basal’ and Laminariales (absent in basal taxa), the evolution of the fol- ‘derived’ Laminariales had prostrate thalli similar to the lowing characters was especially important for achieving a prostrate system of Aureophycus (Fig. 10). This is because large thallus size: 1) a tough sporophytic thallus composed it seems unlikely that the location of reproductive sori on of dense subcortical filaments associated with mucilaginous the blades or sporophylls secondarily moved to the basal organs (e.g. mucilaginous gland cells, mucilaginous ducts); holdfast, and were also lost from the original location on the 2) an intercalary growth zone allowing the development of blade or sporophylls. perennial sporophytes; 3) pneumatocysts (bladder) or inflation of stipes to providing buoyancy of the sporophytes; 4) a meristematic rhizoidal holdfast to increase the number of rhizoids in Taxonomic updates in selected genera accordance with the increase of the pulling force of waves and of derived Laminariales buoyancy due to the enlargement of the sporophyte. Aureophycus lacks most of these features. The growth Agarum Dumortier pattern of the Aureophycus sporophyte is still not fully clear Traditionally 4–5 species have been recognized in Agarum: but the blade is annual, and the meristematic activity of the i.e., A. clathratum Dumortier (= C. cribrosum Bory de Saint- intercalary growth zone appears to be rather limited. Based Vincent), A. fimbriatum, A. oharaense Yamada (Y. Yamada on morphological comparisons and molecular phylogeny, 1961), A. yakishiriense (Y. Yamada 1962, but as a form of A. Kawai et al. (2013) suggested that Aureophycus exhibits clathratum f. yakishiriense in I. Yamada (1974)) and A. turn- some of the most ancestral morphological features of derived eri (Klochkova 1998). Miyata & Yotsukura (2005) reported Laminariales: The finding that the basal system shows uni- the genetically distant relationship between A. oharaense lateral development may suggest the possibility that the and A. cribrosm (= A. clathratum) by rbc spacer and ITS-2 ancestral kelps connecting Aureophycus and Chordaceae rDNA sequences. By molecular phylogenetic study using originally had a prostrate type of sporophyte, and have sec- ITS rDNA, cox1, cox3 and rbc spacer sequences, Boo et al. ondarily evolved foliose erect thalli as seen in Aureophycus (2011) showed closer genetic relationship between A. clath- (Fig. 10). ratum and A. yakishiriense (= C. cribrosum f. yakisiriense), Then the erect thalli might have further developed elabo- and suggested treating them as subspecies of A. clathratum. rate thalli in two different manners, one by supporting the Furthermore, because the monophyly of Thalassiophyllum membranous blade by a midrib as in Alaria (Alariaceae or clathrus (S.G. Gmelin) Postels et Ruprecht was not sup- ‘Group 1’ in Lane et al. (2006)), and the other by developing ported by the sequence data, they suggested synonymizing 36 H. Kawai

Fig. 10. Schematic presentation of an evolutionary scenario of sporophyte morphology from basal to derived families in Laminariales.

Thallasiophyllum to Agarum, although no discussions of confirmed the polyphyly of Laminaria in the analyses using the morphological features were given for characterizing ITS and 26S rDNA, rbcL-rbcS, and nad6 gene sequences, the genus. Thereby, they recognized 5 species in the genus and proposed the resurrection of the genus Saccharina Agarum: i.e., A. clathratum subsp. clathratum, A. clath- Stackhouse for one of the clades. They further suggested ratum subsp. yakishiriense, A. clathrus, A. fimbriatum, A. that Kjellmaniella Miyabe in Okamura is synonymous to oharaense and A. turneri. Saccharina (lectotype: S. plana Stackhouse).

Alaria Greville Lessonia Bory de Saint-Vincent Alaria is widely distributed in the cold temperate regions The species of Lessonia are distinguished by the massive of the Northern Hemisphere, and includes one of the larg- branched to fused holdfasts, the stipe branched from the est buoyant species A. fistulosa, comparable to Macrocystis base or above, and the blade with basal splits and lacking and Nereocystis. However, because of the remarkable mor- pneumatocysts. Lessonia laminarioides Postels et Ruprecht, phological plasticity, the taxonomy has been somewhat con- reported from the Sea of Ochotsk, has been removed from fused. More than a hundred species have been described Lessonia based on molecular phylogenetic study using psaA in the genus, and currently around a dozen species are rec- and rbcL gene sequences, and transferred to a new genus ognized (Widdowson 1971; Lüning & tom Dieck 1990). Pseudolessonia Cho, Klochkova, Krupnova et Boo (Cho Lüning & tom Dieck (1990) suggested that A. grandifolia et al. 2006). Cho et al. (2006) suggested close phyloge- and A. pylaii were conspecific to A. esculenta. Later, Kraan netic relationship among Pseudolessonia with Nereocystis, et al. (2000, 2001) examined the taxonomy of the genus by Macrocytis, Pelagophycus and Postelsia, and pointed out hybridization experiments and rbc spacer sequences, and that all of them share splits in the base of the blade. By these supported the notion that A. grandifolia was conspecific to taxonomic revisions, the known distributions of Lessonia, A. esculenta. Lane et al. (2007) suggested that A. filstulosa the only genus in Lessoniaceae, are solely in the Southern was genetically distant from other Alaria species by cox1 Hemisphere. sequence analyses, and proposed placing it in a new genus Druehlia Lane et Saunders, although their species level tax- Macrocystis C. Agardh onomy has not been clarified. The species level taxonomy of Macrocystis, the largest marine macrophyte, has been controversial. Historically Laminaria Lamouroux more than 10 species have been described in the genus and Yoon et al. (2001) suggested that members of Laminaria 17 taxa (species and varieties) were combined into a sin- were divided into two distant clades based on rbc spacer gle species M. pyrifera by Hooker (1847). However, Bory and ITS rDNA sequence analyses. Later, Lane et al. (2006) de Saint-Vincent recognized M. integrifolia and M. angus- Phylogeny and taxonomy of Laminariales 37 tifolia by the holdfast morphology (Bory de Saint-Vincent Acknowledgements: I am grateful to Drs Eric Henry and Taizo 1826), and Hay (1986) added M. laevis based on its smooth Motomura for their helpful comments on the manuscript. blade morphology. Since then those four (M. angustifolia, M. integrifolia, M. laevis and M. pyrifera) species have been generally recognized. However, this taxonomy was contra- dicted by crossing experiments (Lewis et al. 1986; Lewis References & Neushul 1994; Westermeier et al. 2007), and based on molecular phylogenetic study using ITS rDNA region DNA Bold, H.C. & Wynne, M.J. (1985): Introduction to the Algae: sequences, Coyer et al. (2001) suggested that Macrocystis is Structure and reproduction. 2nd ed. − Prentice-Hall, Englewood monospecific and recognized onlyM. pyrifera. Furthermore, Cliffs, NJ, USA/ pp. 720. the conspecificity of those four species was confirmed by Bolton, J. (2010): The biogeography of kelps (Laminariales, morphological re-assessment (Demes et al. 2009) and Phaeophyceae): a global analysis with new insights from recent genetic study using the cox1 gene (Macaya & Zuccarelo 2010). advances in molecular phylogenies. − Helgol. Mar. Res. 64: 263–279. Boo, S.M., Lee, W.J., Yoon, H.S., Kato, A. & Kawai, H. (1999): Molecular phylogeny of Laminariales (Phaeophyceae) inferred Geographical origin of Laminariales from small subunit ribosomal DNA sequences. − Phycol. Res. 47: 109–114. Lim et al. (1986) estimated the divergence of Phaeophyceae Boo, G.H., Lindstrom, S.C., Klochkova, N.G., Yotsukura, N., from Bacillariophyceae to be around 200 Ma. Later Yang, E.C., Kim, H.G., Waaland, J. R., Cho, G.Y., Miller, K.A. Silberfeld et al. (2010) published a time-calibrated molec- & Boo, S.M. (2011): Taxonomy and biogeography of Agarum ular phylogenetic tree based on 10 mitochondrial, chlo- and Thallasiophyllum (Laminariales, Phaeophyceae) based on roplast and nuclear gene sequences, and suggested the sequences of nuclear, mitochondrial, and plastid markers. − branching time of Laminariales from Ectocarpales to be Taxon 60: 831–840. Bory de Saint-Vincent, J.B.G.M. (1826): Macrocyste. − In: around 100 MA, and that of Chordaceae and derived fami- Audouin, I. (Eds.), Dictionnaire Classique d’Histoire Naturelle. lies to be around 85 Ma. Paris. Vol. 10, pp. 8–10. The fossil evidence of the evolution of the Phaeophyceae Cho, G.Y., Klochkova, N.G., Krupnova, T.N. & Boo, S.M. (2006): is scanty, because of their generally soft tissues composed The reclassification of Lessonia laminarioides (Laminariales, of polysaccharides such as alginates, fucoidans and cel- Phaeophyceae): Pseudolessonia gen. nov. − J. Phycol. 42: lulose, very limited occurrence of calcified taxa (i.e., only 1289–1299. Padina spp. and Newhousia imbricate Kraft, Saunders, Clayton, M.N. (1984): Evolution of the Phaeophyta with particular Abbott et Haroun), preference for exposed habitats where reference to the Fucales. − Prog. Phycol. Res. 2: 11–46. sedimentation is not common, and relatively recent evolu- Cock, J.M. (2010): The Ectocarpus genome and the independent evolution of multicellularity in the brown algae. − Nature 465: tion compared with red and green algae. The oldest generally 617–621. accepted phaeophycean fossil is Julescranea grandicornis Coyer, J.A., Smith, G.J. & Andersen, R.A. (2001): Evolution of from the Tertiary, described from the upper Miocene, which Macrocystis spp. (Phaeophyceae) as determined by ITS1 and is intermediate in appearance between laminarialean species ITS2 sequences. − J. Phycol. 37: 574–585. Pelagophycus and Nereocystis (Parker & Dawson 1965). Dayton, P.K. (1985): Ecology of kelp communities. − Ann. Rev. Because of the abundance of laminarialean taxa and the Ecol. Syst. 16: 215–245. occurrence of basal taxa such as Pseudochordaceae in the Demes, K.W., Graham, M.H. & Suskievicz, T.S. (2009): Pacific, Laminariales has been considered to have origi- Phenotypic plasticity reconciles incongruous molecular nated in the Pacific (Lüning & tom Dieck 1990). The occur- and morphological taxomies: The giant kelp, Macrocystis (Laminariales, Phaeophyceae), is a monospecific genus. − rence of Phyllariaceae and Halosiphon only in the Atlantic J. Phycol. 45: 1266–1269. Ocean (Lüning 1990; Lüning & tom Dieck 1990) posed a Draisma, S.G.A., Eurlings, M.C.M & Lim, P.-E. (2012): High intra- dilemma for this notion. Later they were shown to be dis- individual sequence variation in the nuclear rDNA LSU-5S inter- tant from Laminariales by molecular phylogeny, and fur- genic spacer in the Sargassaceae (Fucales, Phaeophyceae). − thermore the addition of another basal laminarialean taxon J. Appl. Phycol. 24: 1372–1379. Akkesiphycaceae gave further support for the notion that Draisma, S.G.A., Olsen, J., Stam, W. & Prud’homme van Reine, Laminariales originated in the Northwestern Pacific (Kawai W.F. (2002): Phylogenetic relationship within the Sphacelariales & Sasaki 2000; Sasaki et al. 2001). More recently, Bolton (Phaeophyceae): rbcL, RUBISCO spacer and morphology. − (2010) reviewed the biogeography of Laminariales, and sup- Europ. J. Phycol. 37: 385–401. Draisma, S.G.A. & Prud’homme van Reine, W.F. (2001): ported the notion that laminarialean taxa originated in the Onslowiaceae fam. nov. (Phaeophyceae). − J. Phycol. 37: cool-water region of the Northern Hemisphere (Northwestern 647–649. North Pacific), and further proposed that at least four differ- Emerson, C.J., Buggeln, R.G. & Bal, A.K. (1982): Translocation ent genera (Laminaria, Macrocystis, Eisenia and Ecklonia) in Saccorhiza dermatodea (Laminariales, Phaeophyceae): crossed the tropical region to the Southern Hemisphere. anatomy and physiology. − Can. J. Bot. 60: 2164–2184. 38 H. Kawai

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