Molecular phylogeny of two unusual brown algae,Phaeostrophion irregulare and Platysiphon glacialis,proposal of the Stschapoviales ord. nov. and Platysiphonaceae fam. nov., and a re‐examination of divergence times for brown algal orders

The molecular phylogeny of brown algae was examined using concatenated DNA sequences of seven chloroplast and mitochondrial genes (atpB, psaA, psaB, psbA, psbC, rbcL, and cox1). The study was carried out mostly from unialgal cultures; we included Phaeostrophion irregulare and Platysiphon glacialis because their ordinal taxonomic positions were unclear. Overall, the molecular phylogeny agreed with previously published studies, however, Platysiphon clustered with Halosiphon and Stschapovia and was paraphyletic with the Tilopteridales. Platysiphon resembled Stschapovia in showing remarkable morphological changes between young and mature thalli. Platysiphon, Halosiphon and Stschapovia also shared parenchymatous, terete, erect thalli with assimilatory filaments in whorls or on the distal end. Based on these results, we proposed a new order Stschapoviales and a new family Platysiphonaceae. We proposed to include Phaeostrophion in the Sphacelariales, and we emended the order to include this foliose member. Finally, using basal taxa not included in earlier studies, the origin and divergence times for brown algae were re‐investigated. Results showed that the Phaeophyceae branched from Schizocladiophyceae ~260 Ma during the Permian Period. The early diverging brown algae had isomorphic life histories, whereas the derived taxa with heteromorphic life histories evolved 155–110 Ma when they branched from the basal taxa. Based on these results, we propose that the development of heteromorphic life histories and their success in the temperate and cold‐water regions was induced by the development of the remarkable seasonality caused by the breakup of Pangaea. Most brown algal orders had diverged by roughly 60 Ma, around the last mass extinction event during the Cretaceous Period, and therefore a drastic climate change might have triggered the divergence of brown algae.     

Molecular phylogeny of Punctaria mageshimensis reveals evidence for its transfer to Spatoglossum as S. mageshimense (Dictyotales,Phaeophyceae)

Taxonomy of the little‐studied brown algal species Punctaria mageshimensis (Ectocarpales s.l.) was reexamined by molecular phylogeny and morphology. In the genetic analyses of newly collected specimens using plastid rbcL and psaA gene sequences, the specimens morphologically referable to P. mageshimensis were phylogenetically distant from Ectocarpales s.l. and were included in the clade of Spatoglossum (Dictyotales). Morphological reexamination of the type specimen and newly collected specimens confirmed its systematic position in Dictyotales: Branched thallus; cushion‐shaped rhizoidal holdfast occasionally forming secondary holdfast at the bottom of the thallus; many discoidal plastids without pyrenoid per cell; tetrasporangium‐like reproductive structures with dark, homogeneous cell content; occurrence of hair tufts. Genetically P. mageshimensis was most related to a reported sequence of Spatoglossum asperum, but P. mageshimensis was considerably different from S. asperum as well as other known Spatoglossum species in the deep habitat and in having scarcely‐branched lanceolate and considerably thickened thallus. In conclusion, we propose the transfer of P. mageshimensis to Spatoglossum as S. mageshimense comb. nov.     

Molecular phylogeny of Zeacarpa (Ralfsiales,Phaeophyceae) proposing a new family Zeacarpaceae and its transfer to Nemodermatales

Zeacarpa leiomorpha is a crustose brown alga endemic to South Africa. The species has been tentatively placed in Ralfsiaceae, but its ordinal assignment has been uncertain. The molecular phylogeny of brown algae based on concatenated DNA sequences of seven chloroplast and mitochondrial gene sequences (atpB, psaA, psaB, psbA, psbC, rbcL, and cox1) of taxa covering most of the orders revealed the most related phylogenetic relationship of Z. leiomorpha to Nemoderma tingitanum (Nemodermatales) rather than Ralfsiaceae (Ralfsiales). Morphologically, Zeacarpa and Nemoderma share crustose thallus structure and multiple discoidal chloroplasts without pyrenoids in each cell, however, the formation of lateral unilocular zoidangia in tufts in loose upright filaments in Zeacarpa is distinctive in brown algae. Considering the relatively distant genetic divergence between the two taxa, comparable to that among families or orders in representative brown algae, in addition to the above‐mentioned unique morphological features, we propose the classification of Zeacarpa in a new family Zeacarpaceae in the order Nemodermatales.     

Plastid Genome of <Emphasis Type="Italic">Dictyopteris divaricata</Emphasis> (Dictyotales,Phaeophyceae): Understanding the Evolution of Plastid Genomes in Brown Algae

Dictyotophycidae is a subclass of brown algae containing 395 species that are distributed worldwide. A complete plastid (chloroplast) genome (ptDNA or cpDNA) had not previously been sequenced from this group. In this study, the complete plastid genome of Dictyopteris divaricata (Okamura) Okamura (Dictyotales, Phaeophyceae) was characterized and compared to other brown algal ptDNAs. This plastid genome was 126,099 bp in size with two inverted repeats (IRs) of 6026 bp. The D. divaricata IRs contained rpl21, making its IRs larger than representatives from the orders Fucales and Laminariales, but was smaller than that from Ectocarpales. The G + C content of D. divaricata (31.19%) was the highest of the known ptDNAs of brown algae (28.94–31.05%). Two protein-coding genes, rbcR and rpl32, were present in ptDNAs of Laminariales, Ectocarpales (Ectocarpus siliculosus), and Fucales (LEF) but were absent in D. divaricata. Reduced intergenic space (13.11%) and eight pairs of overlapping genes in D. divaricata ptDNA made it the most compact plastid genome in brown algae so far. The architecture of D. divaricata ptDNA showed higher similarity to that of Laminariales compared with Fucales and Ectocarpales. The difference in general features, gene content, and architecture among the ptDNAs of D. divaricata and LEF clade revealed the diversity and evolutionary trends of plastid genomes in brown algae.     

Phylogeny of the Northeast Pacific brown algal (Phaeophycean) orders as inferred from 18S rRNA gene sequences

Partial 18S rRNA gene sequences have been determined for thirteen brown algae representing nine Northeast Pacific brown algal orders: Chordariales, Desmarestiales, Dictyosiphonales, Dictyotales, Ectocarpales, Fucales, Scytosiphonales, Sphacelariales and Syringodermatales. These sequences were compared with published sequences from a kelp (Laminariales), a xanthophyte and a bacillariophyte. A preliminary phylogeny generated by the neighbor-joining phylogeny inference method indicated that the class Phaeophyceae is a monophyletic group in relation to the xanthophyte and the bacillariophyte. Further, bootstrap analysis of the phylogeny consistently grouped together all the representatives belonging to the orders Ectocarpales, Chordariales, Dictyosiphonales and Scytosiphonales and separated them from the representatives belonging to the other brown algal orders. These results offer valuable insights into the controversial brown algal orders' phylogeny and provide additional data to the phylogenetic relationship study among the chromophyte classes.     

Further investigations on the PHAEOTHAMNIOPHYCEAE using a multigene phylogeny,with descriptions of five new species

We examined 12 strains representing eight species classified in the algal class Phaeothamniophyceae (Heterokontophyta). Based upon a five-gene molecular phylogeny (nuclear-encoded SSU rRNA and plastid-encoded psaA, psbA, psbC, and rbcL) and light microscopic observations, we describe five new species: Phaeoschizochlamys santosii sp. nov., Phaeoschizochlamys siveri sp. nov., Phaeothamnion wetherbeei sp. nov., Stichogloea dopii sp. nov. and Stichogloea fawleyi sp. nov. The Phaeothamniophyceae, as delimited here, form a natural group that is sister to the Aurearenophyceae. Molecular phylogenetic analyses proved more reliable than morphological characters for distinguishing species. Evolutionary trends with the SI clade of the heterokont algae are discussed.     

Circumscription of the order Ectocarpales (Phaeophyceae): bibliographical synthesis and molecular evidence

Phylogenetic analyses of 27 brown algae including the type genera of the orders Chordariales, Dictyosiphonales, Ectocarpales sensu stricto, and Scytosiphonales, using partial SSU + LSU combined rDNA sequence data, supports a broadly circumscribed order Ectocarpales. This order is redefined to include taxa possessing an exserted, pedunculated pyrenoid. Previous then taxa were placed in the Ectocarpales sensu stricto, the Chordariales, the Dictyosiphonales, or the Scytosiphonales. Algae either lacking a pyrenoid, and sometimes included in the Ectocarpales (Tilopteridales, Ralfsiales sensu Nakamura), or which possess a non-pedunculated pyrenoid (such as those placed in the recently proposed order Scytothamnales, as well as Asteronema, Asterocladon and Bachelotia), are excluded from the Ectocarpales.     

Tinocladia sanrikuensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

The new species Tinocladia sanrikuensis sp. nov. H.Kawai, K.Takeuchi & T.Hanyuda (Ectocarpales s.l., Phaeophyceae) is described from the Pacific coast of the Tohoku region, northern Japan based on morphology and DNA sequences. The species is a spring–summer annual growing on lower intertidal to upper subtidal rocks and cobbles on relatively protected sites. T. sanrikuensis has a slimy, cylindrical, multiaxial erect thallus, slightly hollow when fully developed, branching once to twice, and resembles T. crassa in gross morphology. The erect thalli are composed of a dense medullary layer, long subcortical filaments, and assimilatory filaments of 11–35 cells, up to 425 μm long and curved in the upper portion. Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most closely related to T. crassa and has the same basic thallus structures but differs in having thinner and longer assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes support the distinctness of this species.     

A MOLECULAR ANALYSIS OF ANALIPUS AND RALFSIA (PHAEOPHYCEAE) SUGGESTS THE ORDER ECTOCARPALES IS POLYPHYLETIC1

We report partial 18S ribosomal DNA sequences of Analipus japonicus (Harvey) Wynne and Ralfsia fungiformis (Grunnerus) Setchell et Gardner. These sequences were compared with the corresponding sequences of 13 brown algae representing six phaeophycean orders: Dictyotales, Ectocarpales, Fucales, Laminariales, Sphacelariales, and Syringodermatales. These 15 brown algae included 10 ectocarpoids representing nine ectocarpacean (sensu Gabrielson et al. 1989) families: Chordariaceae, Dictyosiphonaceae, Ectocarpaceae, Elachistaceae, Heterochordariaceae, Leathesiaceae, Punctariaceae, Ralfsiaceae, and Scytosiphonaceae. We addressed the controversial taxonomic placement of A. japonicus and R. fungiformis in the Ectocarpales by analysis of DNA sequences. Neighbor-joining and maximum parsimony-inferred phylogenies provided evidence that A. japonicus and R. fungiformis are not closely associated with the other representatives of the Ectocarpales. Bootstrap analyses suggest polyphyly of the order Ectocarpales (sensu Gabrielson et al.). However, our analysis failed to resolve the phylogenetic relationship between A. japonicus and R. fungiformis. Our results suggest that the ectocarpoids are just as distantly related to A. japonicus and R. fungiformis as they are to members of the advanced orders Desmarestiales, Dictyotales, Fucales, Laminariales, Sphacelariales, and Syringodermatales.     

Utility of rbcS gene as a novel target DNA region for brown algal molecular systematics

The usefulness of molecular phylogenetic studies has increased remarkably as the quantity and quality of available DNA sequences has increased. When compared with the progress that has occurred in angiosperms and animals, there have been relatively few target DNA regions identified for use in taxonomic studies of brown algae. Therefore, in this study, we developed a new set of primers to amplify Rubisco small subunit (rbcS) gene sequences and determined the rbcS gene sequences of various species of brown algae including those belonging to Dictyotales, Ectocarpales, Fucales and Sphacelariales. The level of sequence variations in the rbcS gene varied according to the brown algal lineages. When focusing on the relationship of species within the genus Sargassum, the rbcS gene sequences provided useful information regarding the phylogenetic relationship among sections of the subgenus Bactrophycus. Based on the broad applicability and phylogenetic utility of the rbcS gene, we suggest that the sequence be used as a new target region for the molecular systematics of brown algae.     

GENOMIC INSIGHTS INTO EVOLUTIONARY RELATIONSHIPS AMONG HETEROKONT LINEAGES EMPHASIZING THE PHAEOPHYCEAE1

Heterokonts comprise a large and diverse group of organisms unified by the heterokont biflagellate condition. Monophyly of many of these lineages is well established, but evolutionary relationships among the various lineages remain elusive. Among these lineages, the brown algae (Phaeophyceae) are a monophyletic, taxonomically diverse, and ecologically critical group common to marine environments. Despite their biological and scientific importance, consensus regarding brown algal phylogeny and taxonomic relationships is missing. Our long‐term research goal is to produce a well‐resolved taxon‐rich phylogeny of the class to assess evolutionary patterns and taxonomic relationships among brown algal lineages and their relationship to other closely related heterokont groups. To accomplish this goal and augment existing loci for phaeophycean‐wide systematic studies, we generated expressed sequence tags (ESTs) from several major brown algal lineages and from the heterokont lineage representing the closest sister group to brown algae. To date, we have successfully constructed cDNA libraries for two lineages (Choristocarpus tenellus Zanardini and Schizocladia ischiensis E. C. Henry, Okuda et H. Kawai) and in the library test phase obtained up to 1,600 ESTs per organism. Annotation results showed a gene discovery rate of 45%–50% for each library revealing 500–700 unique genes from each organism. We have identified several potential genes for phylogenetic inference and used these loci for preliminary molecular clock analyses. Our molecular clock analysis suggests that the basal divergence in brown algae occurred around the time of the pennate‐centric diatom divergence. Here we report this analysis and other uses of ESTs in brown algal phylogenomics and the utility of these data for resolving the phylogeny of this group.     

A molecular-assisted floristic survey of crustose brown algae (Phaeophyceae) from Malaysia and Lombok Island,Indonesia based on rbcL and partial cox1 genes

Studies on the crustose brown algae are relatively few despite a long history of studies conducted since the 1800s, with temperate species forming the bulk of these studies. There is a need for more focus on crustose brown algae particularly in the tropics as they are generally different from those in the temperate regions. Taxonomic confusion arising from morphological simplicity largely dependent on the reproductive structures and overlap in morpho-anatomical features among species necessitates the use of molecular techniques. This study is dedicated to a better understanding of the diversity of these understudied algae in the Indo–Malay region. Specimens collected from Peninsular Malaysia, Sabah (Borneo) and Lombok Island in Indonesia were identified using molecular markers from the plastid rubisco large subunit (rbcL) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes in tandem with morphology and anatomy. Three Mesospora spp., two putative Diplura spp. and the cosmopolitan Neoralfsia expansa were identified in this study, including a new record of Mesospora negrosensis for Malaysia. Despite their morpho-anatomical similarities, Mesospora and Diplura occur in widely divergent clades within the brown algae, the former in the Mesosporaceae in the Ralfsiales, the latter in an unclassified clade sister to the Ishigeales. All six species occurred both in Malaysia and Lombok Island except for M. elongata and M. negrosensis, respectively. The rbcL marker performed better in the elucidation of phylogeny among the brown algal orders, whereas cox1-5′ is more suited as a barcoding marker for species level identification.     

PHYLOGENETIC RELATIONSHIPS OF THE BROWN ALGAL ORDERS ECTOCARPALES, CHORDARIALES, DICTYOSIPHONALES, AND TILOPTERIDALES (PHAEOPHYCEAE) BASED ON RUBISCO LARGE SUBUNIT AND SPACER SEQUENCES

Phylogenetic relationships among 23 species of morphologically simple brown algae belonging to the Ectocarpales sensu stricto , Chordariales, Dictyosiphonales, and Tilopteridales sensu stricto , Phaeophyceae (Fucophyceae), were analyzed using chloroplast-encoded RUBISCO large subunit gene sequences ( rbc L) and the associated RUBISCO spacer sequences. Comparison of the observed and expected sequence divergence at the three codon positions of rbc L showed that the level of mutational saturation within the brown algae is minor. Thus, rbc L is well suited for phylogenetic studies in this group. Unweighted parsimony analyses and a neighbor-joining distance analysis were performed using unambiguously aligned rbc L sequences from the above four orders, one marine raphidophyte and two Tribophyceae (Xantophyceae). Polyphyly of Tilopteridales sensu lato (i.e. including Dictyosiphonales) is verified; we therefore recommend the use of Tilopteridales in the strict sense. The Ectocarpales, Chordariales, and Dictyosiphonales are paraphyletic with respect to each other, forming a highly interwoven clade. A separate parsimony analysis of the RUBISCO spacer as well as a combined rbc L and spacer analysis supported the close relationship among the latter three orders, adding to the evidence that they should be subsumed into the Ectocarpales sensu lato.     

A RIBOSOMAL DNA PHYLOGENY SUPPORTS THE CLOSE EVOLUTIONARY RELATIONSHIPS AMONG THE SPOROCHNALES,DESMARESTIALES, AND LAMINARIALES (PHAEOPHYCEAE)1

We report six complete 18S ribosomal DNA (rDNA) sequences representing five brown algal orders: Sporochnus comosus C. A. Agardh (Sporochnales), Chorda tomentosa Lyngbye (Chordaceae, Laminariales), Saccorhiza polyschides (Lightfoot) Batters (Phyllariaceae, Laminariales), Desmarestia ligulata (Lightfoot) Lamouroux (Desmarestiales), Ectocarpus siliculosus (Dillwyn) Lyngbye (Ectocarpales), and Scytosiphon lomentaria (Lyngbye) J. G. Agardh (Scytosiphonales). These sequences were compared with published laminarialean (Alaria marginata Postel et Ruprecht [Alariaceae] and Macrocystis integrifolia Bory [Lessoniaceae]) and fucalean (Fucus gardneri Silva) rDNA sequences for phylogeny inference using both the distance-matrix and parsimony methods. The inferred 18S phylogenies clustered Sporochnus, Desmarestia, Chorda, Saccorhiza, Alaria, and Macrocystis in an assemblage. This Sporochnales–Desmarestiales–Laminariales (S-D-L) complex was consistently separated from the Ectocarpales, Scytosiphonales, and Fucales by bootstrap analyses. The inferred phylogenies are consistent with several possible evolutionary processes leading to this S-D-L complex. Members in this assemblage lack eyespots in their sperm, and their sperm have the atypical brown algal flagellation: shorter anterior and longer posterior flagella. In addition, they are oogamous with a heteromorphic alternation of generations between a microscopic gametophyte and a macroscopic sporophyte. Members of the S-D-L complex can be separated into different phylogenetic lines based on the presence/absence of eyespots in their meiospores. Our findings support the contention that the Sporochnales, Desmarestiales, and Laminariales are closely related. In addition, our rDNA tree suggests that the Laminariales is paraphyletic.     

Molecular phylogeny of small brown algae,with special reference to the systematic position of Caepidium antarcticum (adenocystaceae,ectocarpales)

Nuclear ribosomal DNA (3′-SSU, ITS, 5′-LSU) and plastid-encoded (rbcL and Rubisco spacer) sequences were determined in Caepidium antarcticum and compared to homologous sequences of relatives from Ectocarpales, Scytothamnales, and other brown algae. Plastidial sequences confirmed a previous conclusions from nuclear ribosomal sequences that some taxa with stellate plastids (Asterocladon and relatives) form the closest outgroup to the Ectocarpales as yet identified. To reconcile nomenclature with the clades resolved in recent molecular studies, we propose a subdivision of the Ectocarpales in five families. Plastidial sequences support the recent proposal of Adenocystaceae, and all sequences suggest that Caepidium should be included in this family. As a further result, Geminocarpus was shown to belong to the same clade as Pylaiella and a number of other brown algae with an isomorphic life history and discoid plastids. We recognise this clade, whose correct name is Acinetosporaceae, as another family in the Ectocarpales. We also propose to unite a number of genetically related taxa, which were formely classified in different families, in an extended Chordariaceae. The remaining species of the Ectocarpales belong to Scytosiphonaceae and to Ectocarpaceae, the latter containing only Ectocarpus and Kuckuckia.     

Undaria mitochondrial large subunit ribosomal DNA (Phaeophyceae, Alariaceae): Structural properties and use for phylogenetic analysis

We sequenced mitochondrial 23S ribosomal DNA (rDNA) from two Undaria species and compared them with homologous sequences from related brown algae. The 23S rDNA sequences from Undaria pinnatifida (Harvey) Suringar were 2707 bp in length, of which 1290–1292 sites were assigned to stem regions and the rest to loop regions in the rRNA secondary structure model. In Undaria undarioides (Yendo) Okamura, the sequences were 2708–2709 bp in length and 1290 sites were assigned to stems. There were three sequence types among the seven U. pinnatifida individuals examined and two among the four U. undarioides individuals. A maximum parsimony analysis of these sequence data with those from related brown algal species resolved a robust monphyly of each of the two Undaria species, suggesting the utilities of 23S rDNA for phylogeny at species or intraspecific level in brown algae. Accounting for variations among sequences parsimoniously, sequence changes were sorted into 423 events, of which 165 were mapped to stems of the secondary structure.     

Morphological and Taxonomic Interpretation of <Emphasis Type="Italic">Orestovia</Emphasis> and <Emphasis Type="Italic">Schuguria</Emphasis>, Enigmatic Devonian Plants from Russia

A detailed study of the external and internal surfaces of the cuticle of Orestovia and Schuguria using scanning electron microscopy and confocal laser microscopy has shown that the structures formerly interpreted as stomata or reproductive organs are most likely to be gland cells producing mucilage. Each structure represents a single large cell with a complex aperture on the external wall. In addition, a reproductive organ similar to multilocular sporangia known in brown algae of the order Ectocarpales has been found in attachment to a compression fossil of Orestovia. These data suggest similarity of the genera studied with brown algae.     

MOLECULAR EVIDENCE FROM nrDNA ITS SEQUENCES THAT LAMINARIOCOLAX (PHAEOPHYCEAE, ECTOCARPALES SENSU LATO) IS A WORLDWIDE CLADE OF CLOSELY RELATED KELP ENDOPHYTES

Marine brown algae living as endophytes in macroalgae are morphologically simple and their taxonomy is particularly difficult. A molecular phylogeny for endophytic taxa isolated from kelps and red algae, and for putative epiphytic and free-living relatives, was inferred from partial small subunit and complete internal transcribed spacer nuclear ribosomal DNA sequences. It has revealed the following results. (1) Three species of endophytes isolated from members of the Laminariales are closely related. They form a clade together with the epi-endophytic species Laminariocolax tomentosoides (Farlow) Kylin. Members of the clade possess uniseriate plurilocular sporangia, and they may form erect filaments. Laminariocolax eckloniae sp. nov., occurring in the South African host Ecklonia maxima (Osbeck) Papenfuss, is described. The new combinations, Laminariocolax aecidioides (Rosenvinge) comb. nov. and L. macrocystis (Peters) comb. nov., are proposed for two taxa previously classified in Gononema and Streblonema , respectively. (2) The genus Laminariocolax occurs worldwide in temperate areas, and the phylogeny of the taxa studied is in agreement with biogeographic distribution. (3) Laminariocolax belongs to the Ectocarpales sensu lato. The genus is more closely related to Chordaria than to Dictyosiphon, Ectocarpus, or Scytosiphon . (4) Two brown endophytes ( Streblonema spp.), isolated from red algae, are closely related to each other and may form a sister clade to Laminariocolax .