Erschienen in: Journal of Phycology ; 50 (2014), 1. - S. 149-166 https://dx.doi.org/10.1111/jpy.12148

LIGULATE (, PHAEOPHYCEAE) REVISITED: D. JAPONICA SP. NOV. AND D. DUDRESNAYI DIFFER FROM D. LIGULATA1

Eun Chan Yang Culture Collection of Algae and Protozoa (CCAP), The Scottish Association for Marine Science Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland PA37 1QA, UK Marine Ecosystem Research Division, Korea Institute of Ocean Science & Technology, 787 Haeanro, Ansan 426 744, Korea Akira F. Peters Bezhin Rosko, 40 rue des pecheurs,^ 29250 Santec, Brittany, France Hiroshi Kawai Kobe University Research Center for Inland Seas, Rokkodai, Nadaku, Kobe 657 8501, Japan Rowena Stern SAHFOS, The Laboratory, Citadel Hill, The Hoe, Plymouth PL1 2PB, UK Takeaki Hanyuda Kobe University Research Center for Inland Seas, Rokkodai, Nadaku, Kobe 657 8501, Japan Ignacio Barbara Coastal Biology Research Group, Facultad de Ciencias, Universidad de A Coruna,~ Campus de la Zapateira, A Coruna~ 15071, Spain Dieter Gerhard Muller€ Fachbereich Biologie, Universit€at Konstanz, Konstanz D 78457, Germany Martina Strittmatter2 Culture Collection of Algae and Protozoa (CCAP), The Scottish Association for Marine Science Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland PA37 1QA, UK Willem F. Prud’Homme van Reine Netherlands Centre for Biodiversity Naturalis, Leiden University (section NHN), P.O. Box 9514, RA Leiden 2300, The Netherlands and Frithjof C. Kupper€ 3 Culture Collection of Algae and Protozoa (CCAP), The Scottish Association for Marine Science Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland PA37 1QA, UK Oceanlab, University of Aberdeen, Main Street, Newburgh, Scotland AB41 6AA, UK

The phylogeny of ligulate and sulfuric-acid taxa to four different species. (1) D. herbacea, containing species of Desmarestia, occurring containing broad-bladed and highly branched forms, worldwide from polar to temperate regions, was has dioecious . The three other species revised using a multigenic and polyphasic approach. have monoecious gametophytes: (2) D. ligulata which Sequence data, characteristics, and is profusely branched and, except for one subspecies, sporophyte morphology support reducing a total of 16 narrow-bladed, (3) Japanese ligulate Desmarestia, here described as D. japonica sp. nov., which is morphologically similar to D. ligulata but genetically 1Received 1 August 2012. Accepted 11 September 2013. 2 Present address: Station Biologique de Roscoff, UMR 7139 distant from all other ligulate taxa. This species may CNRS-Universite Pierre et Marie Curie-Paris VI, BP 74, Roscoff, have conserved the morphology of original ligulate F-29682, France. Desmarestia. (4) D. dudresnayi, including unbranched 3Author for correspondence: e-mail [email protected] or little branched broad-bladed taxa. A figure of the This article is dedicated to Aldo O. Asensi on the occasion of his holotype of D. dudresnayi, which was lost for decades, 80th birthday (September 28, 2012) for his contributions to brown algal and biogeography including Desmarestia. was relocated. The taxonomy is complemented by a Editorial Responsibility: H. Verbruggen (Associate Editor) comparison of internal transcribed spacer and

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Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-276196 150 cytochrome c oxidase subunit I (cox1) as potential 1977, 1981, 1989, Anderson 1985), dioecism versus barcode loci, with cox1 offering good resolution, monoecism of gametophytes (Anderson 1982, Peters reflecting species delimitations within the genus and Muller€ 1986, Ramirez et al. 1986, Ramirez and Desmarestia. Peters 1992), temperature tolerance of gameto- phytes (Peters and Breeman 1992, 1993), and Key index words: ; cox1; Desmarestia; DNA nuclear ribosomal ITS sequence data (van Oppen barcoding; multigene phylogeny; Phaeophyceae; et al. 1993, Peters et al. 1997, 2000) have been uti- psaA; rbcL; SSU-ITS; sulfuric acid lized to study the taxonomy, phylogeny, and bioge- Abbreviations: cox1, cytochrome c oxidase subunit I; ography of Desmarestia and the related monotypic ITS, internal transcribed spacer; ML, maximum genera Arthrocladia Duby, Himantothallus Skottsberg, likelihood and Phaeurus Skottsberg. Peters et al. (1997) hypoth- esized that Desmarestia originated in the Southern Hemisphere, possibly in high latitudes, and subse- The Desmarestiales is an order of large subtidal quently migrated to the Northern Hemisphere. marine brown algae with a heteromorphic life his- They suggested that the characteristic of strong acid- tory resembling that of the Laminariales or kelps. ity of the sporophytic cells evolved only once in the The macroscopic sporophytes are pseudoparenchy- desmarestialean lineage. matous, they may be bushy, feather-like, or consist The annual species of Desmarestia with of a single or several blades (Ramirez and Peters acid-containing thalli, which are in the focus of the 1992). The thalli are annual or perennial, can mea- present work, belong to a lineage of world-wide dis- sure up to 8 m in length, as observed in a Northeast tribution which is subdivided into a small clade of Pacific individual (Pease 1920), and these macro- taxa with terete thalli (e.g., D. viridis (O. F. Muller)€ scopic forms alternate with microscopic gameto- J.V. Lamouroux) and a larger clade of taxa with phytes that are either monoecious or dioecious bladed thalli (e.g., D. ligulata (Lightfoot) J.V. La- (Peters et al. 1997). A conspicuous character of mouroux). Although Peters et al. (1997) have most annual taxa of Desmarestia is a high concentra- shown the major evolutionary and biogeographic tion of sulfuric acid in the vacuoles (Schiff 1962, tendencies within the Desmarestiales, the systematic McClintock et al. 1982, Sasaki et al. 2004), which position, taxonomy, and nomenclature of several possibly serves to deter herbivores (Anderson and species, especially from the clade with bladed and Velimirov 1982, Pelletreau and Muller-Parker 2002). acid-containing thalli, have yet to be clarified. Opin- In molecular phylogenies, the Desmarestiales forms ions vary on how to treat this complex, ranging a well-supported clade within the brown algal crown from a single variable species (D. ligulata; Chapman radiation (Draisma et al. 2003, Kawai et al. 2007, 1972a) to a number of at least six genetically iso- Phillips et al. 2008, Silberfeld et al. 2010). With a lated taxa, potentially corresponding to species distribution from polar to warm-temperate climates, (Peters et al. 1997). The situation is complicated by Desmarestiales comprise dominant components of the fact that cases of significant morphological dif- the phytobenthos where other bed-forming brown ferences among co-occurring genetically similar algal taxa (i.e., Fucales, Laminariales and Phyllaria- forms exist (e.g., D. ligulata, D. gayana Montagne, ceae) are lacking (e.g., recolonization of barren and D. muelleri M.E. Ramırez et Peters in Chile), as grounds). This pattern is especially observed in the well as a case of similar morphologies in genetically Southern Hemishpere and, to a lesser extent, in the and geographically separated populations: isolates Northern Hemisphere (Peters et al. 1997). Desma- of D. ligulata from Japan differed genetically from restiales are also present in the understory of kelp D. ligulata isolates from Europe, South America, forests (e.g., Stegenga et al. 1997). Few records of New Zealand, or the northeast Pacific (Peters et al. Desmarestiales exist from tropical latitudes, how- 1997). In the present work, we have examined more ever, this may be due to the little studied deep-water specimens from Japan and more genetic markers to refugia (Taylor 1945, Graham et al. 2007). confirm the distinctness of the Japanese entity, The type genus Desmarestia J.V. Lamouroux con- which justifies its description as a different species. tains 30 species currently recognized (of 61 species Desmarestia dudresnayi J.V. Lamouroux ex Leman described in www.algaebase.org search on March 05, is a little-known ligulate taxon distributed in cold to 2012; Guiry and Guiry 2012) that are distributed warm-temperate regions of Europe, where it is rare worldwide from warm-temperate to polar regions. and confined to deep water. It is broad-bladed The type species of the genus, D. aculeata (Linna- (>25 mm width) and sparsely branched or eus) J.V. Lamouroux, is a perennial species which unbranched (Leman 1819, Drew and Robertson was described from Europe and occurs in the Arctic 1974, Anderson 1985) and opinions diverge whether and in cold-temperate regions of the Northern it should be regarded as an independent species, a Hemisphere (Lamouroux 1824, Luning€ 1990). Mor- subspecies or form of D. ligulata (Chapman 1972b), phology and ontogeny of sporophytes (Chapman or as conspecific with South African D. firma (C. 1972a,b, Anderson 1985, Stolpe et al. 1991, Wiencke Agardh) Skottsberg (Peters and Breeman 1992). et al. 1995, 1996), sporangial type (Moe and Silva The type locality of D. dudresnayi is St. Pol de Leon, 151 near Roscoff in northern Brittany (Sauvageau 1925). shi, Type): SAP109521; D. japonica (Muroran): SAP109522). So far there have been no culture or molecular They were transported back to the laboratory in sterilized sea studies of this entity, which is of nomenclatural water, cleaned, and sorted carefully under a dissecting micro scope. Epiphyte free parts of the thalli were rapidly frozen in importance because its description predates that of À75°C and freeze dried for subsequent DNA extraction. all other unbranched and of most branched species As Desmarestia dudresnayi is a rare species only a small num of ligulate Desmarestia. ber of sporophytes were collected in situ at the type locality DNA barcoding aims at providing a rapid and near St. Pol de Leon in Brittany (France; n = 4; L’Hardy unambiguous identification of biological materials, Halos 1972) and Galicia (Spain; n = 2; Barbara et al. 2004, based upon the rapid and cost-effective sequencing 2005). Morphological characters utilized by Chapman of a short strand of DNA typically of the five primer (1972b) were measured. The specimens of D. dudresnayi used for biometry were deposited in the herbarium of the Museum region of cox1 but now extends to other loci of Natural History, Paris (PC; unnumbered). Further speci (Hebert et al. 2003). In Phaeophyceae, DNA bar- mens from Galicia were deposited in the herbarium of the coding has been successful in identifying new and University of Santiago de Compostela (SANT), and an indi cryptic species. Mitochondrial cox1 and the nuclear vidual from Brittany was deposited in the herbarium of the rRNA ITS have been successful in identifying many University of California at Berkeley (UC; #UC 1746473). The brown algal species belonging to the Laminariales number of lateral blades was counted in previously collected (Lane et al. 2007, Macaya and Zuccarello 2010, specimens of D. dudresnayi housed at PC (Table 2). Isolation of gametophytes. Fragments a few mm2 in size were McDevit and Saunders 2010) and Fucales (Kucera cut out of fertile blades of freshly collected sporophytes of and Saunders 2008, McDevit and Saunders 2009). D. dudresnayi from Brittany and Galicia and of a sporophyte The cox1 locus reveals biogeographic patterns and of D. ligulata from Galicia and were inoculated in autoclaved cryptic diversity, but it is not uniformly useful in all Provasoli enriched seawater (Starr and Zeikus 1987) contain Á À1 Phaeophyceae, such as Macrocystis (Macaya and Zuc- ing GeO2 (6 mg L ) to prevent growth. They were cultivated at 10°C and 15°C in white light of 25 30 lmol carello 2010). The ITS has more variable sites and Á À2 Á À1 has proved useful in some genera but there have photons m s at a day length of 14:10 h LD. Clonal gametophye cultures were subisolated by pipetting single been difficulties interpreting results due to the pres- germlings. A gametophyte strain of D. dudresnayi from Brit ence of indels and genetic introgression (Kucera tany and gametophyte strains of D. dudresnayi and D. ligulata and Saunders 2008, McDevit and Saunders 2009, from Galicia were deposited in CCAP (Table 1). 2010). Sequencing and phylogenetic analyses. Genomic DNA was The primary objective of this study was a reassess- extracted from unialgal cultures or freeze dried field samples TM ment of ligulate, acid-producing Desmarestia phylog- using the DNeasy Plant Mini Kit (Qiagen, Hilden, Ger eny, based on the sequences of multiple and many) according to the manufacturer’s instructions. Polymer ase chain reactions (PCR) were performed using specific phylogenetically informative markers such as primers for each gene with a Taq PCR Master Mix KitTM (Qia nuclear small subunit (SSU) rDNA and ITS, mito- gen). The nuclear SSU and ITS regions were amplified using chondrial cox1, plastid psaA (photosystem I P700 the primers EAF3 and ITS055R, and sequenced using addi apoprotein A1), and ribulose-1,5-bisphosphate car- tional internal primers, such as 528F, 920F, EBR, 920R, 536R boxylase/oxygenase large subunit (rbcL). Including (Marin et al. 2003), a and b from Coleman et al. (1994). Plas D. dudresnayi was essential for the revision of this tidal psaA was amplified and sequenced using the primers psaA130F and psaA970R (Yoon et al. 2002). The mitochon species complex. Our results propose a practical drial cox1 was amplified using the primer pair GazF2 and nomenclature following Linnean classification crite- GazR2 (Saunders 2005). To amplify and sequence desmares ria. Finally, we compared the barcode loci of mito- tialean rbcL, we designed the specific primers rbcL77F chondrial cox1 and the nuclear rRNA ITS, to (5′ TGG GNT AYT GGG ATG CTG A 3′) and rbcL1471R (5′ evaluate their potential suitability as barcode mark- ATS AGG TGT ATC TGT TGA TGT 3′). PCR amplification was performed in a total volume of 50 lL, containing 0.5 ers for the genus Desmarestia. À units Á lL 1of Taq DNA Polymerase, 1 9 Qiagen PCR Buf l l fer, 1.5 mM MgCl2, and 200 M of each dNTP, 1 M of each MATERIALS AND METHODS primer (except for cox1, for which 300 nM of each primer were used), and 1 10 ng of template DNA. PCR of the SSU Taxon sampling. A total of 52 specimens and cultures were ITS region was carried out with an initial denaturation at investigated for this study (including four outgroup taxa; 95°C for 3 min, followed by 30 cycles of amplification (dena Table 1). Most of the Desmarestiales cultures and DNA turation at 95°C for 1 min, annealing at 50°C for 2 min and extracts used in the present study were the same as in previ extension at 68°C for 3 min) with a final extension step at ous studies (Peters and Breeman 1992, Ramirez and Peters 72°C for 5 min. PCR of cox1 was performed as follows: initial 1992, Peters et al. 1997) and they were deposited in the Cul denaturation at 94°C for 5 min followed by 35 cycles of dena ture Collection of Algae and Protozoa (CCAP; www.ccap.ac. turation at 94°C for 1 min, annealing at 50°C for 1 min, and uk). A specimen of ligulate Desmarestia was collected as drift extension at 72°C for 1 min with one final extension at 72°C material from the shore of Muroran (Western Hokkaido) on for 5 min. Amplified DNA was purified with the QIAquickTM July 14, 1989. A gametophyte isolate was made (CCAP 1306/ PCR Purification Kit (Qiagen) and sent to commercial 7), and a herbarium specimen was prepared (SAP109522). sequencing at the NERC Biomolecular Analytics Facility in More specimens were collected from Oshoro (Northwestern Edinburgh. Electropherogram outputs for each were edited Hokkaido) and Akkeshi (Eastern Hokkaido, Pacific Ocean) using the Chromas v.1.45 (http://www.technelysium.com.au/ in May 2009 and a part of their thalli were dried in silicagel chromas.html). Assembled sequences of nuclear SSU and ITS for DNA extractions, while the remainder of the thalli were were aligned using ClustalW implemented in SeaView v.4.3.3 pressed for herbarium specimens (Desmarestia japonica (Akke (Gouy et al. 2012; http://pbil.univ lyon1.fr/software/seaview. 152

TABLE 1. List of taxa and GenBank accession numbers used in the present study.

Nuclear Plastid Mitochondria Taxa Strain Locality SSU ITS cox1 psaA rbcL Desmarestia D. aculeata (Linnaeus) CCAP 1306/35 Helgoland, Germany HE866893 HE866855 HE866759 HE866785 HE866814 J.V. Lamouroux CCAP 1306/38 San Juan Island, HE866894 HE866856 HE866760 HE866815 Washington, USA EU681402 EU579882 AJ287847 D. anceps Montagne CCAP 1306/39 King George Island, HE866895 HE866857 HE866786 HE866816 Antarctica D. antarctica R.L. Moe CCAP 1306/41 King George Island, HE866896 HE866858 HE866817 et P.C. Silva Antarctica D. chordalis J.D. Cape Horn, Chile HE866897 HE866859 HE866818 Hooker & Harvey Sea Lion Island, HE866860 HE866829 Falkland Islands D. dudresnayi CCAP 1306/1 Ria de Arousa, HE866898 HE866861 HE866761 HE866787 HE866820 Lamouroux ex Galicia, Spain Leman CCAP 1306/2 Roscoff/St. Pol de HE866899 HE866862 HE866788 HE866821 Leon, Brittany, France (type locality) D. dudresnayi subsp. CCAP 1306/12 San Carlos, Valdivia, HE866900 HE866863 HE866762 HE866789 HE866822 patagonica Asensi Chile D. dudresnayi subsp. CCAP 1306/13 Montana de Oro HE866901 HE866864 HE866763 HE866790 HE866823 tabacoides Okamura State Park, California, USA Sageunjin 1, HE866902 HE866865 HE866791 HE866824 Gangreung, Korea D. herbacea (Turner) San Francisco, HE866903 HE866866 HE866764 HE866792 HE866825 Lamouroux California, USA Santa Barbara, HE866904 HE866867 HE866765 HE866793 HE866826 California, USA CCAP 1306/19 Playa Mendieta, HE866907 HE866870 HE866768 HE866796 HE866829 Paracas, Peru Horcon, Chile HE866910 HE866873 HE866771 HE866799 HE866832 (=D. latissima Setchell CCAP 1306/27 San Juan Island, HE866905 HE866868 HE866766 HE866794 HE866827 & Gardner) Washington, USA (=D. munda Setchell CCAP 1306/29 Bamfield, British HE866906 HE866869 HE866767 HE866795 HE866828 et Gardner) Columbia, Canada D. herbacea subsp. CCAP 1306/23 South Africa 1 HE866908 HE866871 HE866769 HE866797 HE866830 firma Skottsberg CCAP 1306/31 South Africa 10 HE866909 HE866872 HE866770 HE866798 HE866831 D. herbacea subsp. CCAP 1306/21 San Juan de Marcona, HE866911 HE866874 HE866772 HE866800 HE866833 peruviana Montagne Peru D. japonica sp. nov. CCAP 1306/7 Muroran, Hokkaido, HE866912 HE866875 HE866773 HE866801 HE866834 Japan Kawai Japan HE866913 HE866835 KU d5481, Oshoro, Hokkaido, HE866914 AB623009 HE866802 AB623010 KU d5486 Japan KU d5897, Akkeshi, Hokkaido, KU d5899 Japan Japan HE866915 HE866803 HE866836 D. latifrons (Ruprecht) CCAP 1306/33 Hearst Beach, CA, HE866916 HE866804 HE866837 Kutzing€ USA D. ligulata (Lightfoot) CCAP 1306/3 Faro de Larina, HE866917 HE866876 HE866838 Lamouroux Galicia, Spain CCAP 1306/5 Bamfield, Vancouver HE866918 HE866877 HE866774 HE866839 Island, British Columbia, Canada CCAP 1306/6 Cabo Raso, Chubut, HE866919 HE866878 HE866775 HE866805 HE866840 Argentina CCAP 1306/9 Kaikoura, New HE866920 HE866879 HE866776 HE866806 HE866841 Zealand CCAP 1306/10 Roscoff, Brittany, HE866921 HE866880 HE866807 HE866842 France CCAP 1306/11 San Carlos, Valdivia, HE866922 HE866881 HE866777 HE866808 HE866843 Chile L43060 EU681610 AJ287848 D. ligulata f. distans CCAP 1306/8 Ushuaia, Argentina HE866923 HE866882 HE866778 HE866844

(continued) 153

TABLE 1. Continued

Nuclear Plastid Mitochondria Taxa Strain Locality SSU ITS cox1 psaA rbcL D. ligulata subsp. CCAP 1306/15 Estaquilla, Los HE866883 HE866779 HE866845 gayana Montagne Muermos, X Region, Chile D. ligulata subsp. CCAP 1306/17 La Boca, Navidad, HE866924 HE866884 HE866780 HE866846 muelleri Ramirez et Chile Peters CCAP 1306/18 Isla Bridges, Ushuaia, HE866925 HE866885 HE866781 HE866809 HE866847 Argentina D. menziesii J. Agardh CCAP 1306/40 King George Island, HE866926 HE866886 HE866782 HE866810 HE866848 Antarctica Candlemas Island HE866927 HE866887 HE866849 GQ368260 GQ368333 GQ368318 D. viridis (O.F. CCAP 1306/14 Kiel, Germany HE866928 HE866888 HE866783 HE866811 HE866850 Muller)€ J.V. AJ295828 AY500367 EU681611 AJ287849 Lamouroux Arthrocladia villosa CCAP 1301/1 Topsail Island, North HE866929 HE866889 HE866851 (Hudson) Duby Carolina, USA Villefranche, France HE866930 HE866890 HE866852 EU681603 EU681589 Himantothallus CCAP 1313/1 King George Island, HE866931 HE866891 HE866784 HE866812 HE866853 grandifolius (A. Gepp Antarctica et E.S. Gepp) Zinova AJ229110 GQ368262 GQ368335 GQ368320 Phaeurus antarcticus King George Island, HE866932 HE866892 HE866813 HE866854 Skottsberg Antarctica Outgroup Fucus vesiculosus L. AY494079 AY372960 DQ307680 Laminaria digitata AJ344328 AY372964 AY372984 (Hudson) J.V. Lamouroux Saccorhiza polyschides L43059 EU681422 AY372965 AB045255 (Lightfoot) Batters Sporochnus EU681621 EU579937 pedunculatus (Hudson) C. Agardh

dent evolution model for each partition (five individual TABLE 2. Occurrence of laterals in specimens of Desmares genes) to minimize the effect on phylogeny of heterogeneity tia dudresnayi at the herbarium of the Museum of Natural among genes. The selected evolutionary models were general History, Paris (PC). time reversible substitution with the gamma distributed rate heterogeneity (GTR + G) model for DNA parts, the LG sub Number of thalli Number of stitution (Le et al. 2008) with empirical amino acid frequen laterals in cies and rate heterogeneity (LG + F + G) model for protein Without With branched parts. Herbarium laterals laterals individuals ML analyses were performed using the RAxML v.7.2.8 (Sta Thuret & Bornet 3 9 1 8 matakis 2006). We used “ f a” option for rapid bootstrap Sauvageau 17 2 1 analysis and the best likelihood tree searching using “ # 1000” Herbarium de 9713with default “ i” (automatically optimized SPR rearrange France ment) and “ c” (25 distinct rate categories) options of the Sum 29 18 program. The independent evolution model for all partition were unlinked by using “ m GTRGAMMA” and “ q” options. Bootstrap values (MLBS) were calculated using 1,000 replica html) then refined by eye with Se AlTM v2.0a11 (Sequencing tions under the same evolution model used for the best tree Alignment Editor Version 2.0 alpha 11; http://tree.bio.ed.ac. search. uk/software/seal/). The plastid and mitochondrial protein DNA barcoding analysis. For DNA barcoding analysis, cox1 coding genes were aligned manually with Se AlTM based on and ITS sequences were aligned with related phaeophycean inferred amino acid sequences. sequences using BioEditTM and MAFFTTM (Katoh et al. 1995). Two data sets were used for phylogenetic analyses. First, in Phylogenetic analyses were conducted in MEGA5 (Tamura the DNA data set (a total of 5,138 bp; c5dna data), we com et al. 2011). For pairwise distance calculations, both uncor bined all DNA alignments of psaA (675 bp), rbcL (1,257 bp), rected p distances and kimura 2 parameter (Kimura 1980) cox1 (655 bp), SSU (1,720 bp), and ITS (831 bp). Second, in models were calculated by MEGA5 and were found to be the protein + DNA mixed data set (3,413 characters; c5mix almost identical. The number of base differences per site was data), translated psaA (225 aa), rbcL (389 aa), and cox1 (218 calculated from averaging over all sequence pairs within each aa) were combined with SSU and ITS DNA sequences to species group. For cox1 and ITS, 556 and 447 positions were avoid possible artifacts of phylogenetic calculations such as analyzed, respectively, in the final data set. The analysis homoplasy at the third codon position. We used an indepen involved 324 and 253 sequences for cox1 and ITS respectively. 154

Codon positions included were lst, 2nd, 3rd, and noncoding. fresh water, similar to D. ligulata, D. viridis, etc. All position~ containing gaps and missing data were elimi (Sasaki et al. 2004). The thallus is composed of a nated. Species were defined based on clades obtained from large central axial cell surrounded by inner rhizoi­ phylogenetic analyses using all molecular markers in combi nation with nonmolecular characters (see Results and Discus dal filaments, large, colorless medullary cells, and sion). Within species and between species pairwise distances 1-2 layers of small, peripheral cells containing many were categorized into discrete bins and measured against discoid chloroplasts without pyrenoids. Unilocular their frequency. The barcoding cut off was determined as the zoidangia are conical, up to ~20 ).liD in height, smallest distance that encompassed all \\~thin species embedded in the peripheral layer of the entire thal­ distances. Minimum genus level distances were defined as the lus except for the basal part of the main axis and smallest pairwise distance observed between two species. This tips of the thalli U nizoids are -8 x 5 ~un in size, distance was applied to species to categorize them into bar code groups. The barcode groups were cross compared with containing a chloroplast with eyespot, and with the combined morphological and multigene phylogenies to longer anterior and shorter posterior flagella. Ga­ determine species and genus level boundaries for each metophytes are minute, uniseriate branched fila­ barcode marker. ments, monoecious, and oogamous (Nakahara 1984). Desmarestia dudresnayi: In Brittany, D. dudresnayi RESULTS was found on rock in the shade beneath an under­ Field collections, morphological and culture studies. water cliff (Le Paradis) and on a sublittoral reef (Ar Desmarestia j aponica sp. nov. Ligulate Desmarestia is Tourtu) at 20-25 m depth on three occasions in fairly common in northern J apan and an ecologi­ July and August 1999 and 2000. A total of four cally important component of seaweed communi­ specimens were available for measurement. The ties. It grows on rocks of more or less exposed holdfast was smooth and conical with a diameter of coasts in the shallow subtidal to 5-6 m (Fig. 1) and 1-3 mm, the stipe was terete, 1.5-3 em in length, is distributed around H okkaido and along the Paci­ and the blades had smooth margins. The phylloid fic coast of Northern Honshu. The sporophytic of the individual collected on J uly 18, 1999 (Fig. 2a) thalli are annual, growing from winter to late sum­ was 28 em in length and 6 em in width. The three mer, becoming fertile in late spring. The holdfast is other individuals had blades of 20 em length (apex cushion-shaped, bearing one to a few erect thalli. eroded) and 8 em width (Fig. 2b), 38 em length The erect thalli are light olive brown to brown in and 9 ern width, and 30 em length and 10.5 em color, 0.6-1 (-2) m in length, with a conspicuous width (not illustrated). The specimen with the main axis 2-6 (-20) mm in width, oppositely eroded apex had a pair of eroded laterals, the oth­ branched in 2-3 orders. The shape and width of ers were unbranched. The less eroded of the laterals the main axis as well as the branchlets are variable, was 12 em in length and 5 em in width. The con­ from linear to lanceolate. The main axis is com­ nections of the laterals to the main blade were not pressed cylindrical at the base, and flattened in terete like the stipe but flat and 4-5 m m wide. The other parts, with a conspicuous midrib. The branch­ central vein was distinct in the main blades of all lets are stipitate, narrower at the base, broadest at specimens, but lateral veins were obvious only in the middle portion, and becoming tapered at the one individual (Fig. 2b). They branched off at an distal end Young thalli have deciduous, trichothallic angle of less than 90° and were bifurcated toward filaments, and the thalli are pseudoparenchymatous. the margin. Cells of the sporophytes are strongly acidic, and I n Galicia, D. dudresnayi was growing on a substra­ turn bluish green when immersed or soaked in tum of maerl, pebbles, and broken shells, near the

F1c. 1. Dt!Smilrestia japtmica sp. nov. H. Kawai, T. Hanyuda, D.G. Muller, E.G. Yang, AF. Peters et F.G Kupper. Habit of grown up specimens (a and b) and juvenile specimen. (a) Akkeshi, Eastern Hokkaido; (b) Muroran, South western Hokkaido; (c) Oshoro, North western Hokkaido; scale bars 2 em. 155

Mhor (near Oban) on August 20, 2010. T he habitat a c (pebbles and small rocks on a mostly sandy seabed) A was different from the localities off Roscoff (under­ water cliff faces), but more resembling that where D. dudresnayi was encountered in Galicia - a seabed consisting mostly of gravel at ~1 5 m below low tide level, with D. dudresnayi thalli growing attached to small pebbles and sea shells. Despite two searches using SCUBA, no D. dudresnayi was found at the same locality in the summer of 2011. Gametophytes of D. dudresnayi from Brittany 0 developed only in the culture from the w1branched individual collected on J uly 18, 1999 (Fig. 2c). From Galicia, we obtained gametophytes both from the unbranched and the branched individual. All three cultures gave rise to monoecious gametophytes, indistinguishable from each other. T hey consisted d of branched creeping filaments 10-15 ~un in diame­ ter. Germlings became reproductive in l0°C and 15°C and bore antheridia and oogonia on the same thallus. Sporophytes developed from oogonia, with­ out release of eggs (Fig. 2c). Sporophytes of D. dud resnayi grown in ow- cultures to a lengt11 of several centimeters remained unbranched (Fig. 2d). Desmarestia ligulat«. The specimen of D. ligulata collected in Galicia was profusely branched. The maximum wid th of the main axis was 6 m m (Fig. 3). Gametophytes of D. ligulata from Galicia were monoecious (not shown as they were similar to previously studied isolates, e.g., Peters and Muller 1986, Ramirez and Peters 1992). The time required for gametogenesis in D. dudresnayi and D. ligulata Frc. 2. Desmarestia dwl:resnayi. (a and b) Branched and was compared. Vegetative gametophytes of both unbranched individuals collected at the type locality, St. Pol de species from Galicia were simultaneously inoculated Leon (Brittany). Herbarium specimens, scanned. In the lower specimen (b), bifurcation of lateral veins (white arrow) is visible. (c) Fertile monoecious gametophyte, laboratory culture, isolate from Brittany. Branched laterals with antheridia (A), oogonia (0), and young sporophytes (S) which have developed in situ from fertilized oogonia. (d) Jmoenile sporophytes from laboratory culture, after 2 months at I0°C. central channel of the Ria de Arousa (Barbara et al. 2004). Collections for the present work were made at 13-15 m depth in September 1997, with two spec­ imens measured T hey had narrow terete stipes of 1.5 em length, and in one a conical holdfast of 4 mm diameter was present. The blade of the first specimen was distally eroded, unbranched, 44 em in length and 17 em in width, the blade of the second individual was 61 em in length and 23 em in width. It had a single lateral of 9.5 em length and 5 em width whose connection to the main blade was 7 mm broad. The main axis and lateral veins were dearly visible. The lateral veins branched off at an angle of less than 90° and most of them were bifur­ cated. Like the individuals from Brittany, blades were devoid of marginal teeth or spines. In Scotland, D. dudresnayi was found in the nar­ row sea straits between Dunstaffnage and Eilean F1c. 3. Desman1stia ligul.ata from Rio de Arousa, Galicia. 156

at 10°C and the appearance of first young sporo- saturation signals found in all tests (coefficients of phytes (like those illustrated in Fig. 2c) was correlation were higher than 0.91, r2 = 0.999) recorded. D. dudresnayi gametogenesis took 14 d, except one; the third codon positions of psaA whereas in D. ligulata, it required only 10 d. showed the lowest coefficient of correlation 0.797 Molecular phylogeny. Sequence statistics obtained (r2 = 0.999). Rate heterogeneity of each gene was for the alignments of the five markers used in the evaluated by shape parameter (alpha) estimation. present study are summarized in Table 3. Nuclear ITS and cox1 showed relatively higher alpha values SSU rDNA and ITS required gaps for multiple (≥0.2) and SSU showed the lowest heterogeneity sequence alignment, however, no insertion and (0.02) among the five markers. deletion were found in the three protein markers. A total of 5,138 positions of five concatenated SSU was the most conserved gene (98.2% constant DNA sequences (c5dna; SSU rDNA + ITS + cox1 + positions) and mitochondrial cox1 was the most vari- psaA + rbcL) and 3,413 positions of mixed DNA/ able gene (27.2% variable positions) among the five protein sequences (c5mix; 862 aa from cox1, psaA genes used in the present study. The highest P-dis- and rbcL + 2,551 bp from SSU rDNA and ITS) were tance was found in cox1 (0.072 Æ 0.01), followed by used for phylogenetic analyses, respectively. ML psaA (0.047 Æ 0.005), ITS (0.026 Æ 0.004; ligulate trees of c5dna and c5mix were highly congruent Desmarestia species only), rbcL (0.02 Æ 0.002), and except for one different relationship. In the c5dna SSU (0.003). In each alignment, most variable posi- tree, Phaeurus antarcticus Skottsberg was grouped tions were identified as phylogenetically informative within a Desmarestia Himantothallus (DH) clade; in sites. For example, mitochondrial cox1 showed the the c5mix tree, P. antarcticus was a sister of the DH highest variable position content among genes clade (indicated by dotted arrow line in Fig. 4). (27.2%), and most variable positions (94%; 168/178 However, neither relationship had high statistical positions) were informative. However, in the ITS support. Since no saturation signals were found in alignment, more than half of the variable positions the saturation test, we used the c5dna phylogeny as were noninformative for phylogenetic analysis (52%; the best hypothesis. The type genus of the order 57/110 positions). The three protein-coding orga- Desmarestia was paraphyletic; i.e., D. anceps Montagne nelle genes (cox1, psaA, and rbcL) had similar pat- and D. antarctica R.L.Moe & P.C.Silva grouped with terns in variation, proportion of informative site, Himantothallus (MLBS 100% from c5dna and 91% base composition, and Ti/Tv ratio. The majority of from c5mix). substitutions occurred in the third codon position The sulfuric acid-containing Desmarestia species (e.g., 150 of 278 in cox1); AT bias was relatively were monophyletic with high bootstrap supports stronger (i.e., higher than 0.6); and transition (Ti) (MLBS, 100% from c5dna and 89% from c5mix). A was two times more abundant than transversion clade containing D. aculeata formed the sister group (i.e., Ti/Tv ratio higher than 2). To check for of the sulfuric acid-containing taxa (96% from potentially misleading phylogenetic signals of the c5dna and 77% from c5mix). The sulfuric acid-con- third codon position, we performed the saturation taining taxa were subdivided in five well-supported test for each gene. Uncorrected P distance and cor- clades: (1) D. viridis branched first, as the sister spe- rected distance with the Kimura 2-parameter evolu- cies to all ligulate taxa which form a monophyletic, tion model were used for determining the well supported group; (2) A Japanese species which coefficient of correlation. There were no significant will be described here as D. japonica sp. nov.; (3)

TABLE 3. Sequence statistics of Desmarestiales used in this study.

Nuclear Plastidial Mitochondrial SSU rDNAa ITSb cox1 psaA rbcL Number of sequences 41 26 30 34 47 Aligned positions (bp) 1720 730 655 675 1257 Indel positions 3 69 0 0 0 Variable positions, total 31 (1.8%) 110 (15.1%) 178 (27.2%) 135 (20%) 144 (11.5%) [1st/2nd/3rd codon] [23/5/150] [20/2/113] [28/7/109] Informative positions, total 24 (1.4%) 53 (7.3%) 168 (25.6%) 100 (14.8%) 110 (8.8%) [1st/2nd/3rd codon] [20/3/145] [11/0/89] [21/6/83] P distance: Mean Æ SD 0.003 Æ 0.0004 0.026 Æ 0.004 0.072 Æ 0.010 0.047 Æ 0.005 0.020 Æ 0.002 Base frequencies (A/C/G) 0.24/0.21/0.27 0.20/0.29/0.28 0.21/0.19/0.21 0.29/0.15/0.20 0.28/0.16/0.23 Ti/Tv ratio 1.77 2.10 2.43 3.76 2.29 Saturationc (r2 > 0.99) 0.994 0.942 0.952 0.797 0.915 Rate heterogeneity (alpha) 0.02 0.2 0.21 0.1 0.06 aHimantothallus grandifolius (GenBank AJ229110) was excluded because of missing data (only 503 bp in length). bSulfuric acidic Desmarestia taxa only. cCoefficient of correlation between uncorrected P distance and corrected distance with Kimura 2 parameter model. All positions were used for distance calculation in SSU rDNA and ITS; the third codon position result shown for cox1, psaA, and rbcL. 157

"D. munda" CCAP 1306/29 Canada e (5) D. herbacea San Francisco, USA e (5) "D. latissima" CCAP 1306/27 USA e (5) ssna D. herbacea Santa Barbara. USA e (5) D. herbacea subsp. peruviana CCAP 1306/21 Peru co 100ns D. herbacea CCAP 1306/19 Peru e (5} ...... , D. herbacea Hereon, Chile e (5} ·-(/) D. herbacea subsp. firma CCAP 1306/23 South Africa D. herbacea subsp. firma CCAP 1306/31 ~ D. dudresnayl CCAP 1306/1 Spain 0 (4 } ~"'!'lli"'-"~:::::::~=:=;:;::;::;;~=; 100/99 co D. dudresnayi CCAP 1306/2 France 0 D. dudresnayi subsp. patagonica CCAP 1306/12 Chile E: 96/- D. dudresnayi subsp. tabacoides Korea 0 (/) D. dudresnayi subsp. tabacoides CA, USAGG~:?:=~~!!~~[YI Q) D. ligutata CCAP 1306/3 Spain ® (36) - a 100/92 D. /igulata CCAP 1306/10 France D. ligulata D.ligulata "'0 D. Jigulata CCAP 1306/9 New Zealand ®(36) (.) 89197 D. ligulata subsp. gayana CCAP 1306/15 Chile (3B) D. ligula/a subsp. muel/eriCCAP 1306/17 Chile ® (3B) ro l::::::!+-t'"l' D.ligulata subsp. mue/leriCCAP 1306/18 Argentina®( ..., , ...... _. ,v. (.)

D.ligulata CCAP 1306/11 Chile® ( 3A) ~ D.ligulata f. distans CCAP 1306/8 Argentina e (3A) :::J D. ligulata CCAP 1306/6 Argentina e (3A) '+- 9 165 D. ligulata CCAP 1306/5 Canada e (3A) :::J D. japonica CCAP 1306/7 Japan (2) en D. japonica Akkeshi. Japan (2) D. japonica sp.nov. 100197 D. japonica Oshoro, Japan {2) D. Viridis CCAP 1306/14 Germany 0 (1) • • • 1001100 D. viridis 0 (1) D. vmd1s ------~

D. latifrons CCAP 1306/33 USA D. menziasii ® 921100 D. menziesii CCAP 1306/40 King George Island ® 1001100 D. menziesiiCandlemas Island 98188 D. chorda/is Chile D. chorda/is Felkland Island 56/- '------.,------Phaeurus antarcticus King George Island Himantothallus grandifolius Desmarestiales H. grandifolius CCAP 1313/1 King George Island e D. antarctica CCAP 1306/41 King George Island 100/91 D. anoeps CCAP 1306/39 King George Island 100/96 Arthroc/adia villose France ,____ ...;. 1 .;;..00;;../1;..;00~ A. villosa A. vi/losa CCAP 130111 USA

~9~8/~1~oot======~::;::;:~~~ Fucus vesiculosus r Saccorhiza polyschides '------Sporochnus pedunculatus '------Laminaria digitala 0.05

Flc. 4. Maximum likelihood phylogeny of the Desmarestiales based on combined small subunit + internal transcribed spacer (ITS) + cytochrome c oxidase subunit I (roxl) + psaA + rocL sequences (5,138 bp; c5dna data, 5 DNA sequences concatenated) under the indepen dent GTR + G model. The dotted arrow line indicates an alternative topology based on c5mLx (3,413 characters; 3 protein sequences [862 aa; exclude the last stop codon of rb.-.L] + 2 DNA sequences [2,551 bp] concatenated). The numbers near branches refer to the maximum likelihood bootstrap values derived from 1,000 bootstrap analyses based on the c5dna and c5mix data sets. Strain ID and locality are followed by ITS and caxl barcode labels (i.e. circle and number in parentheses, respectively). Numbers in parentheses (1 5) represent distinct coxl barcode species groups with $1.2% paitwise distance (PWD). Different patterned circles show distinct lTS barcode groups with g% PWD. For example, gray colored circles and number (5) in Desmarestia llerbacea indicate that aU samples grouped under the same species with ::>1.2% of cllXl and g% of ITS PWD, respectively. In D. ligulata, two coxl and ITS barcode groups were identified suggesting ongoing speciation. 158

D. ligulata isolates from Europe, Argentina, Chile, specimens. Genera- and species-level differences New Zealand, Canada, including strains originally overlapped considerably, mostly due to Alaria spp. identified as D. distans, D. gayana and D. muelleri; and only a modest genetic distance was found (4) D. dudresnayi (from France and Spain), D. pata between species and genera. However, significantly, gonica (Chile), and D. tabacoides (from Korea and the ITS-barcodes did maintain the same groups as USA); (5) D. herbacea from the Pacific Coast of the multigene phylogeny. Using 1.0% as a species- North and South America, D. latissima (USA) and level cut-off (see Fig. 5B, dashed line), ITS-barcode D. munda (Bristish Columbia), D. herbacea ssp. firma groups fell into two species-level groups and two (South Africa) and D. herbacea ssp. peruviana (Peru). single isolate groups in the sulfuric acid-containing DNA barcoding. We compared the DNA barcoding species. D. viridis was clearly confirmed as a separate utility of nuclear ITS and mitochondrial cox1. ITS species to other Desmarestia (2.8%–3.4%). D. japonica and cox1 showed larger rate heterogeneity values sp. nov. (Japan) was at the species boundary to its (≥0.2) than the other genes (Table 3). Cytochrome nearest neighbors, the D. dudresnayi specimen group c oxidase subunit I (cox1) sequence data were (0.8%–1.4%). The ITS sequences from the newly obtained from 30 Desmarestiales and three other defined D. ligulata formed two major, closely related phaeophycean specimens (Fucus vesiculosus Linna- and partially overlapping groups that showed eus, Laminaria digitata (Hudson) J.V. Lamouroux 1%–2.4% PWD difference to each other. D. ligulata and Saccorhiza polyschides (Lightfoot) Batters). To (Spain) was distinct from both these groups. All determine the utility of cox1 in delineating Desmares members of the newly defined D. dudresnayi group tia species, a comparison was made between genetic and a publicly available sequence, AJ439832, were distances of Desmarestia compared to those of six related at species-level. The D. herbacea group Phaeophyceae genera (Fig. 5A). Specimen identifi- (D. herbacea, D. herbacea subsp. firma, and D. herbacea cations of Desmarestia were based on the newly del- subsp. peruviana) were all related at species-level. imitated four species. Intraspecific PWDs were In summary cox1 shows better resolution with a ≤1.2% in 98% of cases of Phaeophyceae. Interspe- distinct separation between species and genera com- cies distances started at 2.4%. For barcode assign- pared to ITS. cox1 results confirm species limited by ments, identification of Desmarestia specimens were taxonomic and phylogenetic analysis. based on the newly delimitated four species. A cut-off value of 1.2% was used to define a species- barcode group. DISCUSSION Desmarestia cox1 species-level barcode groups con- Molecular phylogeny. Our new analyses employing formed to their respective phylogenetic clades, only nuclear, plastidial, and mitochondrial markers and D. ligulata contained two groups (3A,B). D. ligulata four outgroup taxa have confirmed the previous (Spain) showed only partial identity to D. ligulata phylogenetic tree of the Desmarestiales based on subspp. gayana and muelleri (Fig. 4). D. ligulata and ITS sequences (Peters et al. 1997). As in the previ- D. dudresnayi barcode groups showed more variation ous analysis, Antarctic and sub-Antarctic Desmarestia in genetic distance compared with D. herbacea. and Himantothallus as well as the monotypic genera Within the newly defined D. herbacea and D. dudres Arthrocladia and Phaeurus formed the early branches, nayi groups all members formed a species group although their hierarchy remained ambiguous. below the species-level cutoff of 1.2%. D. viridis Overall, our results confirm the monophyly of the formed its own separate species group that was at sulfuric acid-producing Desmarestia clade. It is the least 8.6% different to the ligulate specimens. sister group to the clade of the type species (Fig. 4). Within ligulate Desmarestia, D. japonica sp. nov. Furthermore, we confirmed that the sulfuric-acid (Japan; barcode group 2, Fig. 4) was clearly distinct clade is separated into D. viridis, branching off first, and showed the greatest distance to other Desmares and all ligulate forms, in which we distinguish four tia species, its nearest neighbor being D. ligulata major groups (Fig. 4): (1) Japanese D. japonica, (2) (New Zealand) at 3.0% PWD. D. ligulata sensu stricto (including forma distans, Evaluation of the ITS barcode locus was per- subsp. muelleri and subsp. gayana), (3) D. dudresnayi formed with 36 sequences of Desmarestia, one (including subsp. tabacoides and subsp. patagonica, sequence each from Himantothallus grandifolius, tentatively also subsp. sivertsenii [Tristan da Cunha] Phaeurus antarcticus, and Arthrocladia villosa, plus 214 and subsp. foliacea [NE Pacific]) and (4) D. herbacea phaeophycean sequences from six genera (five (incl. subsp. peruviana, subsp. firma, and the syn- being common with cox1 barcode analysis) available onyms D. latissima, D. munda, and D. mexicana). Our publically (Fig. 5B). Again, genetic distances were classification recognizes four instead of 16 species of compared with the newly delimited species defini- acid-containing ligulate Desmarestia (Table 4). The tions here. In our data set 18/23 species compari- criteria for recognizing subspecies are the following: sons showed equal or lower than 1.0% similarity (i) Genetic distance, but insufficient for declaring (see Fig. 5B, dashed line), although the frequency different species; (ii) Geographically disjunct of species between 1% and 1.14% is high because populations of the same species; (iii) Clear morpho- of greater representation from more divergent logical differences. 159

A cox1 Phaeophyceae

4000 . . 3500 . . 3000 J . >. . (..) c 2500 Jl . Q) . :J . 0" 2000 . Q),_ . lL . 1500 . : 1000 . 500 . • . I ['~ I . J: 0 I I . I I I I • I I •

PWD Category(%)

B ITS Phaeophyceae

600 . . 500 . . >. 400 . (..) . c . Q) . :J . 0" 300 . Q),_ . lL . 200 . . 100 +. . I I I 0 II II II I .I I . I • I I I . ON<::ri.OOOQNo::r\000 ON ciOOOO....i...i ...i~...t NN

PWD Category(%)

Fie. 5. Paitwise distance (PWD) distribution of Phaeophyceae for (A) roxl and (B) internal transoibed spacer (ITS) barcode markefli. Grey and black bars indicate intra and inteTlipecies distances, respectively, and the dashed line indicates the species level cut off applied to Desmarestiales in each case.

Desmarestia ligulata: Our isolates of D. ligulata The latter was not formally included in our analysis from Brittany (France) and from Galicia (Spain) because of incomplete data; rbCL sequences place it clustered together showing that European samples close to D. muelleri, as did the previous analysis of of this species are slightly genetically different from ITS data (Peters et al. 1997). To accommodate individuals from Argentina, Chile, New Zealand, these three taxa we propose to regard D. distans, and western Canada. Nevertheless, all together they which showed no genetic difference from a D. ligu form a highly supported clade, which represents lata sample from Argentina, as a narrow growth D. ligulata sensu stricto. However, samples belonging form of D. ligulata. This classification would agree to three other taxa fell within the same clade: D. di with the original treatment of this fotm (Sporochnus stans (C. Agardh) J. Agardh , D. muelleri M.E. ligulalus var. distans, Agardh 1824). For D. muelleri Ramirez et A F. Peters and D. gayana Montagne. and D. gayana, which show more significant mor- 160

TABLE 4. Recognized species of ligulate Desmarestiales with synonyms.

Species Infraspecific taxa Synonyms D. ligulata (Stackhouse) J.V. subsp. ligulata Lamouroux (see AlgaeBase why [subsp. ligulata]f.distans (C. Agardh) comb. nov. A.F. D. distans (C. Agardh) J. Stackhouse and not Lightfoot) Peters, E.C. Yang, F.C. Kupper€ & Prud’Homme van Agardh Reine subsp. muelleri (M.E.Ramırez et A.F.Peters) comb. nov. D. muelleri M.E. Ramırez et A.F. A.F. Peters, E.C. Yang, F.C. Kupper€ & Prud’Homme van Peters Reine subsp. gayana (Montagne) comb. nov. A.F. Peters, E.C. D. gayana Montagne Yang, & F.C. Kupper€ D. dudresnayi J.V. Lamouroux ex subsp. patagonica (Asensi) comb. nov. A.F. Peters, E.C. D. patagonica Asensi Leman Yang, F.C. Kupper€ & Prud’Homme van Reine subsp. tabacoides (Okamura) comb. nov. A.F. Peters, E.C. D. tabacoides Okamura Yang, F.C. Kupper€ & Prud’Homme van Reine subsp. foliacea (V.A. Pease) comb. nov. A.F. Peters, E.C. D. foliacea V.A. Pease Yang, F.C. Kupper€ & Prud’Homme van Reine subsp. sivertsenii (Baardseth) comb. nov. A.F. Peters, D. sivertsenii Baardseth E.C. Yang, F.C. Kupper€ & Prud’Homme van Reine D. herbacea (Turner) J.V. subsp. firma (C. Agardh) comb. nov. A.F. Peters, E.C. D. firma (C. Agardh) Skottsberg Lamouroux Yang, F.C. Kupper€ & Prud’Homme van Reine subsp. peruviana (Montagne) comb. nov. A.F. Peters, D. peruviana Montagne E.C. Yang, F.C. Kupper€ & Prud’Homme van Reine synonyms in subsp. herbacea: D. latissima Setchell & Gardner D. munda Setchell et N.L. Gardner D. mexicana E.Y. Dawson D. japonica H. Kawai, T. Hanyuda, D. ligulata (Lightfoot) J.V. D.G. Muller,€ E.C. Yang, A.F. Lamouroux Peters & F.C. Kupper€

phological differences from D. ligulata (and from (SSU, ITS, cox1, psaA, and rbcL) used in the present each other), we propose reduction to subspecific study suggested inclusion of Japanese ligulate rank (see Table 4 and New Combinations section). Desmarestia in the clade containing D. ligulata from Europe, i.e., the area of the type, as well as from all other regions of the area of distribution of this spe- NEW COMBINATIONS cies. Despite being morphologically similar to mate- [subsp. ligulata]f.distans (C. rial from outside Japan, Japanese ligulate Desmarestia Agardh) comb. nov. A.F. Peters, E.C. Yang, F.C. is also physiologically different: (i) gametogenesis in Kupper,€ & Prud’Homme van Reine Japanese specimens is under short-day photoperiod- Basionym and early description: Sporochnus ligula ic control (Nakahara 1984), whereas no effect of tus var. distans C. Agardh (1824) in Systema Algarum photoperiod was detected in gametogenesis of p. 261. strains of D. ligulata from western Canada (Peters Desmarestia ligulata subsp. muelleri (M.E. Ramırez and Muller€ 1986) and South America (Ramirez and et A.F. Peters) comb. nov. A.F. Peters, E.C. Yang, Peters 1992); (ii) gametophytes of two different iso- F.C. Kupper,€ & Prud’Homme van Reine lates from Hokkaido showed an upper survival tem- Basionym and early description: Desmarestia muel perature limit (USL) 1.5°C–2.9°C higher than leri M.E. Ramırez et A.F. Peters (1993) in Canadian D. ligulata gametophytes from Western Canada, Journal of Botany 70: p. 2442, figs. 12, 15, 34, 42, 43. Chile, New Zealand, Argentina, and Brittany (Peters Desmarestia ligulata subsp. gayana (Montagne) and Breeman 1992). This higher survival limit may comb. nov. A.F. Peters, E.C. Yang, F.C. Kupper,€ & help Japanese ligulate Desmarestia to occur in a Prud’Homme van Reine region with comparatively high summer tempera- Basionym and early description: Desmarestia gayana tures (up to ~25°C). , which is also Montagne (1852) in Flora Chileana, Plantas cellulares present in Japan (van Oppen et al. 1993), has a sim- 2, Algas in Gay, C. (ed.) Historia fisica y politica de ilar, high USL. D. aculeata, with a ~5°C lower USL, Chile 8: p. 243, pl. 14, fig. 1. does not occur in Japan and is only found further Japanese ligulate Desmarestia. The multigene North (Luning€ 1984, Peters and Breeman 1992). analyses of all isolates available for the clade of Furthermore, chromosome counts gave different bladed and sulfuric acid containing taxa confirmed results for Japanese ligulate Desmarestia (n = 52–56; the early branching of Japanese ligulate Desmarestia, Nakahara 1984) and western Canadian D. ligulata which had previously been referred to as D. ligulata (44 Æ 4; Peters and Muller€ 1986). Taken together, (e.g., Okamura 1907-9, 1936, Yoshida 1998), from the physiological and genetic separation of Japanese D. ligulata of other regions. None of the markers ligulate Desmarestia from D. ligulata sensu stricto sug- 161 gests that the Japanese entity must be recognized as occurring in western Europe from Scotland to Gali- a different species. The highly branched thallus cia, with isolated populations in Portugal (Barbara found both in the Japanese entity and in D. ligulata et al. 2006), the Mediterranean, particularly (Mes- sensu stricto, may represent the original morphol- sina, Italy; Drew and Robertson 1974) and Isle of Al- ogy of ligulate Desmarestia. boran (Rindi and Cinelli 1995). The specimens of Still, open questions remain regarding ligulate D. dudresnayi we collected from the type locality Desmarestia from the cold seas of the North-west were smaller than the individuals from Galicia (see Pacific. The case of Desmarestia kurilensis Yamada Barbara et al. 2004). Nevertheless, gametophytes (Yamada 1935) unfortunately has to remain unre- from both localities were monoecious and morpho- solved. The type specimen available at SAP did not logically similar. Their ITS sequences were similar yield DNA suitable for PCR, and the type locality indicating that the individuals from both localities (Urup, one of the central Kuril Islands) is practi- belong to the same species. Both populations of cally inaccessible for phycological studies. Ligulate D. dudresnayi sampled consisted of unbranched as Desmarestia from the east coast of Korea clustered well as sparsely branched individuals, consistent with within the D. dudresnayi clade, next to the entity pre- previous reports (Sauvageau 1925, Drew and Robert- viously called D. patagonica from Chile (Fig. 4). As son 1974). In the herbarium of the Natural History of now, we have no indication for the presence of Museum at Paris (PC), about one-third of the speci- D. japonica in Korea. mens of D. dudresnayi, that have been collected on Desmarestia japonica H. Kawai, T. Hanyuda, D.G. French coasts, are branched (Table 2), with up to Muller,€ E.C. Yang, A.F. Peters, & F.C. Kupper€ eight laterals (mode = 2). The laterals were con- sp. nov. nected to the main blade by a flattened stipe as in Thalli sporophytici annui 0.6–1 (-2) m alti, 2–6 the type of D. dudresnayi (Leman 1819, Fig. 4; see (-20) mm lati, pallide olivaceo-brunnei sed cum below). In other species of ligulate Desmarestia, sti- aciditate cellularum laesi viridescenti, ex haptero pes of branches are terete (see figures in Anderson conico vel complanato orientes; axe primario ple- 1985, Ramirez and Peters 1992). rumque prominente trichothallico pseudoparenchy- According to our multigene analyses, the clade mato, costa instructo, gignente ramos oppositos formed by D. dudresnayi and D. herbacea is phyloge- distichos determinati incrementi, bis vel ter ramifi- netically separated from D. ligulata (Fig. 4). Within catos; ramis ultimis brevibus et in juventute dentatis this clade, D. dudresnayi forms a subclade of taxa, propter filamenta terminalia incrementi trichothal- which have sparsely branched or unbranched thalli lici. Rami stipitati et ad basin apicemque attenuati; and usually broad blades. Gametophytes of D. dud thallus in sectione constans e cellula magna centrali resnayi are monoecious like those of D. ligulata. The axiali circumcincta filamentis rhizoideis interioribus, different timings required for gametogenesis in the cellulis medullariis magnis incoloribus, et 1 vel 2 same culture conditions provided additional evi- stratis cellularum peripheralium parvarum chloro- dence that there is a biological separation of D. dud plastos multos discoideos sine pyrenoidibus resnayi and D. ligulata, supporting their taxonomic continentium. Zoidangia unilocularia in strato separation based on sporophyte morphology peripherale immersa. Thalli gametophytici minuti (Leman 1819, Sauvageau 1925). A study about the filamentosi, ramis uniseriatis, monoecii oogami. recognition of oligoguluronates as defense elicitors Sporophytic thalli annual, 0.6–1 (-2) m tall, 2–6 in brown algae provided chemotaxonomic support (-20) mm wide, light olive brown in color, becoming for this notion: While sporophytes of D. dudresnayi greenish when damaged by cellular acidity, arising (strain CCAP 1306/1) recognized these cell wall from a conical or flattened holdfast; main axis usu- degradation products, reacting with an oxidative ally prominent, trichothallic, pseudoparenchyma- burst reminiscent of Laminaria species, sporophytes tous, with midrib, giving rise to opposite, distichous of D. aculeata and D. ligulata did not (Kupper€ et al. branches of limited growth branched to two to 2002). The hypothesis that D. dudresnayi represents three times; ultimate branches short and dentate a growth form of D. ligulata is thus rejected. with terminal filaments of trichothallic growth when Peters and Breeman (1992) hypothesized that young. Branches stipitate and attenuate at base and D. dudresnayi belongs to a group of taxa which are apex; in section thallus composed of a large central similar (possibly conspecific) to South African axial cell surrounded by inner rhizoidal filaments, D. firma, the latter in our molecular analyses being large, colorless medullary cells, and one to two lay- represented by a clade comprising two isolates of ers of small, peripheral cells containing many dis- D. firma from South Africa as well as D. herbacea, coid chloroplasts without pyrenoids. Unilocular D. latissima, D. munda, D. firma, and D. peruviana zoidangia embedded in peripheral layer. Gameto- from the Pacific coast of the Americas. This clade is phytic thalli minute, uniseriate branched filamen- genetically closer to D. dudresnayi than D. ligulata tous, monoecious, and oogamous. (Fig. 4) but all isolates had dioecious gametophytes D. dudresnayi and D. herbacea. A further focus while those of D. dudresnayi were monoecious. of the present work was the broad-bladed taxa and Although the genetic basis of the difference particularly D. dudresnayi which is a rare species between monoecism and dioecism in brown algae is 162 not known, we conclude that D. dudresnayi is a spe- and to reduce D. tabacoides and D. patagonica to sub- cies separate from the clade with dieocious gameto- species. The latter treatment may also be justified phytes. As all the dioecious taxa were genetically as for unbranched ligulate Desmarestia from Tristan da similar to each other as the different isolates of Cunha (South Atlantic; described as D. sivertsenii D. ligulata, we propose to merge them in a single Baardseth (Baardseth 1941) and from the northeast species, D. herbacea (Turner) Lamouroux, which is Pacific where it is described as D. foliacea (Pease the oldest valid name. Its type (BM 000562739; fig. 1917, 1920). Our isolate of unbranched Desmarestia 19 in Anderson 1985) is clearly from a broad-bladed from California, previously identified as D. tabacoides entity. However, the South African population (Peters et al. 1997) is slightly genetically different appears to be slightly separated genetically as well as from our new Korean sample and possibly repre- geographically, and we retain them as subspecies sents D. foliacea. Two specimens from Friday Harbor firma. The same reduction is proposed for the small (Washington, USA; type locality of D. foliacea), and narrow-bladed, i.e., morphologically different, kindly sent to us by Brian Wysor, were morphologi- D. peruviana. Based on our limited samples, the cally similar to unbranched D. dudresnayi. northeast Pacific taxa D. latissima and D. munda The literature knows two different spellings of the deserve no taxonomic separation from D. herbacea; specific epithet of D. dudresnayi, honoring the first in our opinion, D. latissima is a growth form from collector of the alga, Guy du Dresnay (1770–1837; the highly sheltered waters of Puget Sound (Wash- Dizerbo 1965). The longer spelling, dudresnayi,was ington, USA) and D. munda is another synonym of used in the protologue by Leman (1819). In fact, D. herbacea. Leman provided the name as D. dudresnay, contain- Due to its morphological similarity, D. mexicana ing an automatically correctable error; see Anderson Dawson (Dawson 1944) from Southern California, 1985, footnote on p. 438. The shorter epithet, dres of which we did not have any samples, is considered nayi, was employed by Lamouroux (1824) and many to belong to the same species (Pedroche et al. subsequent authors (Cotton 1912, Newton 1931, 2008). D. herbacea is possibly also the correct identifi- Dizerbo 1965, Chapman 1972b, Drew and Robert- cation for the deep-water ligulate Desmarestia son 1974, Fletcher 1987, Rindi and Cinelli 1995, reported as D. munda (Silva 1966) and as D. ligulata Barbara and Cremades 1996, Guiry and Nic Dhonn- (Graham et al. 2007) from Galapagos respectively. cha 2003). Others, such as Sauvageau (1925), Whether D. tropica, also described from Galapagos, Hamel (1931-39), Anderson (1985) and Barbara is another peculiar form or subspecies of D. herbacea et al. (2004, 2005, 2006) used the longer epithet. As remains to be examined. rule 60.1 of the Code of Botanical Nomenclature The closest relatives of our European isolates of imposes that the original spelling of the name is D. dudresnayi according to our sequence analyses are retained, dudresnayi is the correct epithet. samples originally identified as D. patagonica Asensi The fate of the holotype specimen of D. dud (Chile) and D. tabacoides (our new samples from resnayi, a branched individual with two small and Korea as well as the old isolate from California), two large laterals, is obscure (Chapman 1972b). which are all isolates with monoecious gameto- Anderson (1985) designated an unbranched speci- phytes. We had no cultures from our samples from men collected at the type locality by du Dresnay Korea but Japanese D. tabacoides was shown to be and now housed in Lamouroux’s collection in Caen monoecious (Nakahara and Nakamura 1971). In (CN) as lectotype. There is, however, a drawing of contrast to D. dudresnayi, where branched (Leman the holotype in the volumes of plates belonging to 1819) as well as unbranched (Montagne 1842, as Bory de Saint Vincent’s Dictionnaire des Sciences D. pinnatinervia Montagne; Crouan and Crouan Naturelles, which were published separately from 1852, as D. dudresnayi forma simplex; Sauvageau the protologue between 1816 and 1829. This draw- 1925) thalli have been reported (also see above), no ing featuring a branched individual was erroneously branched specimens are known from either D. pata referred to as plate number 43 by Sauvageau gonica (Asensi and Goncßalves 1972, Pinto 1989, (1925). In fact, plate number 43 contains either Ramirez and Peters 1992, Asensi and Kupper€ 2012) Alisma plantago or a set of figures of small fungi, or D. tabacoides. On the other hand, unbranched and later authors (e.g., Chapman 1972b) have sporophytes of D. dudresnayi and D. patagonica are apparently not seen the drawing. In the libraries of indistinguishable in size and morphology (compare Leiden University and the Natural History Museum, our Fig. 2 with the figures in Asensi and Goncßalves Paris, we located the figure, hand numbered as “40” 1972, Ramirez and Peters 1992). Due to monoecism and in “Volume II” of the plates, in the series on of D. dudresnayi, D. patagonica, and D. tabacoides we acotyledones. Below the figure, which corresponds have not attempted cross-fertility experiments. The exactly to the protologue, the name is provided in genetic distances among our samples of D. dud the short spelling, as “Desmarestia dresnayi (Lamx)” resnayi, D. patagonica, and D. tabacoides are compara- [or “dresnavi”]. A watercolor featuring the holotype ble to those among different samples of D. ligulata was found by Chantal Billard in the Lenormand and we thus propose to merge the unbranched to herbarium at Caen but the holotype itself is still little branched broad-bladed taxa in D. dudresnayi missing. To our opinion, the watercolor should be 163 regarded as iconotype (Fig. 6). As details of branch­ Basionym and early description: Sporochnus heWacea ing are important characteristics of D. dudresnayi it var. firma C. Agardh (1824) in Systema Algarum, p. 261. would still be useful to locate the holotype. Desmarestia herbacea subsp. pemviana (Montagne) AF. Peters, E.C. Yang, F.C. Kupper, & Prud'Homme van Reine comb. nov. NEW COMBINATIONS Basionym and early description: Desmarestia peruvi Desnun-estia dudt-esnayi subsp. patagonica (Asensi) ana Montagne (1839) in Plantes Cellulares, Algae, Flor AF. Peters, E.C. Yang, F.C. Kupper, & Prud'Homme ula Boliviensis stirpes novae et minus cognitae in: van Reine comb. nov. d'Orbigny, A (ed.): Voyage dans !'Amerique Meruidio Basionym and early description: Desmarestia patago nate Vol. 7, Botanique (2): p. 35, pl. 5, fig. 3. nica Asensi in Asensi, AO & Gon<;alves Carralves, M DNA barcoding. In this study, cox1 pairwise dis­ (1972) in Darwiniania 17: p. 378, fig. l. tance values for Desmarestiales within species and Desmarestia dud1-esnayi subsp. tabaroides (Okam­ between species, ranged from 0% to 1.2% and ura) AF. Peters, E.C. Yang, F.C. Kupper, & >2.4% respectively. These values were comparable to Prud'Homme van Reine comb. nov. 29 species from 20 genera of phaeophycean taxa Basionym and early description: Desmarestia tabaco reported by McDevit and Saunders (2009) at ides Okamura (1908) in leones ofjapanese algae 1: p. 0%-0.46% and >3% respectively. Desmarestiales 187, pl. 38, figs 1-4, pl. 39, figs 9-13. sequence diversity was similar to those of Laminaria Desmnrestia dudresnayi subsp. foliacea (V .A. Pease) (0%-0.5%, >2.9%) and Saccharina (Oo/o-1.2% and AF. Peters, E.C. Yang, F.C. Kupper, & Prud'Homme >2.1%). The only anomalous patterns in genetic van Reine comb. nov. cliversity were Macrocystis integrifolia and M pyrifera, Basionym and early description: Desmarestia folia whid1 had overlapping intra and interspecies ranges, cea V.A Pease (1920) in Puget Sound Marine Bi()logical compared to other l.aminariales. Recent results have Station Publication 2: p. 322, 342, pl. 58, figs ~10, pl. indicated these species should in fact be reduced to 61, figs 1-5. the one M. pyrifera (Demes et al. 2009, Macaya and Desmarestia dudresnayi subsp. sivertsenii (Baardy Zuccarello 2010). Our results indicate that cox1 is an eth) AF. Peters, E.C. Yang, F.C. Kupper, & excellent barcode marker for Desmarestiales, pre­ Prud'Homme van Reine comb. nov. clicting almost all of the species groups of the multi­ Basionym and early description: Desmarestia si gene phylogenetic analysis. Desmarestia japonica had vertsenii Baardseth (1941) in: Results of the Norwegian over four times larger sequence clivergence com­ Scientific Expedition to Tristan da Cunha 193 7-1939: 9: pared to all other Desmarestia species and therefore p. 28, figs 10, C-E, 12. warrants placement in a different species group and Desmarestia herbacea subsp . .finna (C. Agardb) AF. confirms results of systematic studies. ITS barcoding Peters, E.C. Yang, F.C. Kiipper, & Pmd'Homme van correctly identified species grouping, although with Reine comb. nov. much less resolution than cox1 as genetic clistances were smaller with greater than 1.0% PWD separating species. However, the ITS marker crucially lacks res­ olution and there is only 0.2% separating species and genus. The genetic distances for Desmccrestia ITS barcodes were similar to those of Saccharina latissima and Laminariales, whose species cut-off was greater than 1% (McDevit and Saunders 2010). The lack of species/genus separation was also observed for S. la tissima (Linnaeus) C.E. Lane, C. Mayes, DruehJ et G.W. Saunders, where the biogeographical bound­ aries established u~ing cox1-barcodes had collapsed using ITS-barcodes, with the authors speculating introgression as the cause (McDevit and Saunders 2010). It is possible that a lack of resolution in the ITS barcodes of Desmarestiales have occurred for similar reasons. For example D. japonica, a separate species, showed partial species level affinity with some but not all members of the unbranched to lit­ tle-branched Desmarestiales, a sister taxon to the monophyletic D. ligulata group. By cono·ast, the same Japanese specimen showed less similarity to the D. ligulata group. In summary, coxl performs well as a DNA barcoding marker in Desmarestiales,

Flc. 6. Iconotype of Desmo;reslia dudrestUL)'~ water color of the whilst ITS is more prone to error due to lack of holotype from Lenormand's herbarium in Caen, France. resolution. 164

Conclusions and outlook. Our data suggest reten- tion of four species of sulfuric-acid containing ligu- ACKNOWLEDGMENTS late Desmarestia. We recognized co-occurring and morphologically dissimilar forms, or geographically This work was supported by the BK21 Global Internship Pro separated populations, as subspecies, if they showed gram (Bio Brain Center for Daedock R&D Innopolis, Chung highly similar sequences. nam National University) funded by the Korea Research From a geographical point of view, all North Foundation to ECY, an access grant to the UK Natural Envi ronment Research Council (NERC, UK) Molecular Bioanalyt Atlantic taxa of Desmarestia have been reviewed, with ics Facility (MGF 154), core funding to the Culture the exception of the Moroccan D. tingitana Hamel, Collection of Algae and Protozoa (Oceans 2025 NF3) from which is well branched but has broader blades than the Natural Environment Research Council, and with fellow typical D. ligulata (Hamel 1931-39). The systematics ships from Studienstiftung des deutschen Volkes (German of the Southern Hemisphere taxa have also been Academic Merit Foundation) and the European Commission largely clarified (Moe and Silva 1977, 1981, 1989, to FCK (Marie Curie Fellowship, Program MAST III). This Anderson 1985, Ramirez and Peters 1992, Peters work also received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scot et al. 1997, 2000). Population genetic and ecological land) and their support is gratefully acknowledged. Further approaches are now desirable to find out what more we acknowledge the support of the European provokes the profound morphological differences Community research infrastructure action under the FP7 among South American D. ligulata, D. ligulata subsp. ‘capacities’ specific program ASSEMBLE (grant no. 227788). gayana and D. ligulata subsp. muelleri, and between MASTS is funded by the Scottish Funding Council (grant ref D. herbacea and D. herbacea subsp. peruviana. All erence HR09011) and contributing institutions. Thanks are due to the curator of CN, Chantal Billard, for searches in the materials studied so far originate from drift collec- – herbaria of Chauvin, Lamouroux and Lenormand, and to the tions where these taxa were found together yet it curator of PC, Bruno de Reviers, for providing access to speci is unclear whether they actually occur in the same mens and retrieval of old literature. We would also like to or different habitats. The North Pacific Ocean still thank Martin Sayer, Elaine Azzopardi and Hugh Brown of poses open taxonomic questions, and both terete the UK National Facility for Scientific Diving for supporting and ligulate taxa of Desmarestia are still in need of a our collections and surveys of Desmarestia dudresnayi at Dun comprehensive revision – the present work only pro- staffnage (Oban, Scotland). vided a start. Agardh, C. A. 1824. Systema Algarum. Berling, Lund 312 pp. Desmarestia remains a fascinating genus of brown Anderson, R. J. 1982. The life history of Desmarestia firma (C. Ag.) algae, from an ecological, physiological, develop- Skottsb. (Phaeophyceae, Desmarestiales). Phycologia 21:316 22. mental, and esthetic perspective. Even though a Anderson, R. J. 1985. Morphological and taxonomic relationship defense function of sulfuric acid accumulation (a among branched, ligulate members of the genus Desmarestia (Phaeophyceae, Desmarestiales), with particular reference to unique feature of Desmarestia species among all South African D. firma. Can. J. Bot. 63:437 47. brown algae) against grazers such as sea urchins Anderson, R. J. & Velimirov, B. 1982. An experimental investiga has been demonstrated (Pelletreau and Muller-Par- tion of the palatability of kelp bed algae to the sea urchin ker 2002), the underlying physiology and bio- Parechinus angulosus LESKE. P.S.Z.N.I: Mar Ecol. 3:357 73. ß chemistry of the process is only poorly Asensi, A. O. & Goncalves, M. 1972. Una nueva especie de alga parda: Desmarestia patagonica Asensi sp. nov. (Phaeophyta understood. So far, D. dudresnayi is the only mem- Desmarestiales). Darwiniana 17:378 83. ber of this genus for which oligoalginate recogni- Asensi, A. O. & Kupper, F. C. 2012. Seasonal periodicity and tion and an oxidative burst could be reproduction of brown algae (Phaeophyta) at Puerto Dese demonstrated (Kupper€ et al. 2002). Despite the ado (Patagonia). Bot. Mar. 55:217 28. Baardseth, E. 1941. The marine algae of Tristan da Cunha. Results observation of Saenko et al. (1978) of an iodine of the Norwegian Scientific Expedition to Tristan da Cunha 1937 content of 0.12% and a bromine content of 38 9:1 173. 0.13% dry weight in D. viridis from the sea of Barbara, I. & Cremades, J. 1996. Seaweeds of the Rıa de A Okhotsk, virtually nothing is known about the hal- Coruna (NW Iberian Peninsula, Spain). Bot. Mar. 39:371 88. ogen metabolism of Desmarestia species in general Barbara, I., Cremades, J., Calvo, S., Lopez Rodriguez, M. C. & Do – sil, J. 2005. Checklist of the benthic marine and brackish even though it is tempting to speculate that Galician algae (NW Spain). An. Jar. Bot. Madr. 62:69 100. they resemble other, morphologically complex Barbara, I., Cremades, J. & Veiga, A. J. 2004. A floristic study of a brown algae such as kelps (Kupper€ et al. 2008) in maerl and gravel subtidal bed in the Arousa rıa (Galicia, that respect. Also, gametophytes of D. viridis, Spain). Botanica Complutensis 28:35 46. Barbara, I., Diaz, P., Cremades, J., Pena, V., Lopez Rodriguez, M. D. ligulata, and D. herbacea as well as D. ligulata C., Berecibar, E. & Santos, R. 2006. Catalogo gallego de espe sporophytes turned out to be susceptible to the cies amenazadas y lista roja de las algas bentonicas marinas pathogen Eurychasma (Muller€ et al. de Galicia. Bol. Inf. Soc. Esp. Ficol. 35:9 19. 1999), but in general, the pathologies of Desma- Chapman, A. R. O. 1972a. Morphological variation and its taxo restiales remain poorly studied. Furthermore, Mo- nomic implications in the ligulate members of the genus Des marestia occurring on the west coast of North America. Syesis tomura and Sakai (1984) had found that both 5:1 20. iron and boron control gametogenesis in Desmares Chapman, A. R. O. 1972b. Species delimitation in the filiform, tia. Altogether, these are intriguing facets, warrant- oppositely branched members of the genus Desmarestia ing further, in-depth study of the life of this Lamour. (Phaeophyceae, Desmarestiales) in the northern peculiar brown algal genus. hemisphere. Phycologia 11:225 31. 165

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