Phylogenetic relationships of Polysiphonia - Algaebase.org

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Phylogenetic relationships of Polysiphonia (Rhodomelaceae, Rhodophyta) and its relatives based on anatomical and nuclear small-subunit rDNA sequence data Han-Gu Choi, Myung-Sook Kim, Michael D. Guiry, and Gary W. Saunders

Abstract: The aim of this study was to reassess monophyly of the genus Polysiphonia and determine the phylogenetic affinities of its component lineages among related red algae belonging to the Rhodomelaceae. Our “total evidence” approach, combining 28 anatomical characters and small-subunit ribosomal DNA sequence data for 25 ceramialean algae including 14 species of Polysiphonia sensu lato (including two species of the recently described genus Neosiphonia) and nine related Rhodomelaceae, indicates that Polysiphonia sensu lato consists of three strongly supported clades, Polysiphonia group, Neosiphonia group, and a “multipericentral” group, and a single taxon lineage consisting of Womersleyella setacea. The type species of the genus, Polysiphonia urceolata (= Polysiphonia stricta) from the north Atlantic, formed a distinct clade with Polysiphonia morrowii and Polysiphonia pacifica from the northwest and northeast Pacific, respectively. The Neosiphonia group included Neosiphonia japonica and Neosiphonia savatieri from the northwest Pacific, as originally proposed, Polysiphonia harveyi from the north Atlantic, which shares diagnostic features with this genus, and the anomalous Polysiphonia elongata and Polysiphonia virgata from the north Atlantic and South Africa, respectively. Boergeseniella and Vertebrata from the north Atlantic and Enelittosiphonia from the northwest Pacific associated solidly with the multipericentral Polysiphonia fucoides and Polysiphonia nigra from the north Atlantic. The implications for the taxonomy of Polysiphonia sensu lato and related genera within the Rhodomelaceae are discussed. Key words: Neosiphonia, nuclear small-subunit rDNA, phylogeny, Polysiphonia, Rhodomelaceae, Rhodophyta, systematics. Résumé : Le but de cette étude était de réévaluer la monophylie du genre Polysiphonia et de déterminer les affinités phylogénétiques de ses lignées constituantes parmi les algues rouges apparentées appartenant aux Rhodomelaceae. L’approche par preuve totale (“total evidence”), qui combine les données de 28 caractères anatomiques et séquences de petite sous-unité chez 25 algues céramialées comprenant 14 espèces de Polysiphonia sensu lato (incluant deux espèces du genre Neosiphonia récemment décrit) et neuf Rhodomelaceae apparentées, indique que les Polysiphonia sensu lato comportent trois clades fortement étayés, groupe Polysiphonia, groupe Neosiphonia et groupe “multipéricentrique”, ainsi qu’une lignée monotaxique constituée par le Womersleyella setacea. L’espèce type du genre, le Polysiphonia urceolata (=Polysiphonia stricta) de l’Atlantique nord, forme un clade distinct avec le Polysiphonia morrowi et le Polysiphonia pacifica du nord-ouest et du nord-est du Pacifique, respectivement. Le groupe Neosiphonia inclut le Neosiphonia japonica et le Neosiphonia savatieri du nord-ouest du Pacifique tel que proposé originalement, le Polysiphonia harveyi du nord de l’Atlantique, lequel partage ses caractéristiques diagnostiques avec ce genre, et les espèces irrégulières Polysiphonia elongata et Polysiphonia virgata de l’Atlantique nord et de l’Afrique du sud, respectivement. Les trois genres Boergeseniella et Vertebrata du nord de l’Atlantique et Enelittosiphonia du Pacifique nord-ouest montrent une solide association avec les espèces de Polysiphonia multipéricentriques, Polysiphonia fucoides et Polysiphonia nigra de l’Atlantique nord. Les auteurs discutent les implications taxonomiques pour les Polysiphonia sensu lato ainsi que pour les genres apparentés des Rhodomelaceae. Mots clés : Neosiphonia, petite sous-unité de l’ADNr nucléique, phylogénie, Polysiphonia, Rhodomelaceae, Rhodophyta, systématique. [Traduit par la Rédaction]

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Received July 31, 2001. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on December 18, 2001. H.-G. Choi1 and G.W. Saunders.2 Centre for Environmental and Molecular Algal Research, Department of Biology, University of New Brunswick, Fredericton, NB E3B 6E1, Canada. M.-S. Kim3 and M.D. Guiry. Department of Biology and The Martin Ryan Institute, University College Galway, The National University of Ireland, Galway, Ireland. 1

Present address: BK21, Department of Earth Systems & Environmental Sciences, Chonnam National University, Gwangju 500-757, Korea. 2 Corresponding author (e-mail: [email protected]). 3 Present address: Department of Biological Sciences, College of Natural Sciences, Pusan National University, Pusan 609-735, Korea. Can. J. Bot. 79: 1465–1476 (2001)

J:\cjb\cjb79\cjb-12\B01-128.vp Thursday, December 13, 2001 12:33:33 PM

DOI: 10.1139/cjb-79-12-1465

© 2001 NRC Canada


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Introduction Species of the largest red algal genus, Polysiphonia Greville (1824), are common and widely distributed on virtually all coasts of the world (Womersley 1979). The generic circumscription of Polysiphonia has been in an almost constant state of flux since C. Agardh’s (1817) original proposal (as Hutchinsia) (cf. Kim et al. 2000). Sprengel (1827) was the first to adopt the name Polysiphonia for the majority of the species placed by C. Agardh in Hutchinsia (Dixon and Irvine 1970) and included 27 species in the genus. J. Agardh (1863, p. 908) treated Polysiphonia as a diverse and speciose genus, which he divided primarily on the basis of thallus size into four subgenera: Herposiphonia, Oligosiphonia, Polysiphonia, and Ptilosiphonia. Kylin (1941) proposed the segregation of the genus Orcasia based on Polysiphonia senticulosa Harvey and characterized it as having young branches arising endogenously from the base of determinate branchlets. However, this genus has not gained general acceptance (cf. Kudo and Masuda 1988). Segi (1949) proposed the genus Enelittosiphonia based on Polysiphonia hakodatensis Yendo (= E. stimpsonii (Harvey) Kudo et Masuda), which has a prostrate ecorticate thallus with 8–12 pericentral cells. Hollenberg (1968a, 1968b) divided the Pacific species of Polysiphonia into two groups: Oligosiphonia for species with four pericentral cells and Polysiphonia for species with five or more pericentral cells. He also proposed several new genera, including Womersleyella Hollenberg (1967), based on Womersleyella pacifica Hollenberg, to which some species of Polysiphonia were later transferred (Norris 1992). Silva (1952) proposed the conservation of Polysiphonia over the earlier Vertebrata Gray (1821), Grammita Bonnemaison (1822), and Gratelopella Bory (1823). Kylin (1956) subsequently proposed that Vertebrata be reinstated based on Polysiphonia fastigiata (Roth) Greville to include only the type species of Gray’s genus (Vertebrata fastigiata (Roth) Gray 1821, p. 338), and also proposed four new genera including Boergeseniella (based on Polysiphonia fruticulosa (Wulfen) Sprengel), Diplocladia (based on Polysiphonia patersonis Sonder), Echinothamnion (based on Polysiphonia hystrix Hooker et Harvey), and Leptosiphonia (based on Polysiphonia schousoei Thuret in Bornet et Thuret) for species of the genus Polysiphonia sensu lato. Christensen (1967) accepted Kylin’s (1956) resurrection of Vertebrata because this species has several important diagnostic features such as all primordia developing into ecorticated polysiphonous branches without vegetative trichoblasts. He further synonymized V. fastigiata with Polysiphonia lanosa (Linnaeus) Tandy (1931, p. 226; based on Fucus lanosus Linnaeus 1767, p. 718) as Vertebrata lanosa (Linnaeus) T. Christensen. Subsequent authors have generally not accepted recognition of Vertebrata as distinct from Polysiphonia. For example, Maggs and Hommersand (1993) recognized Boergeseniella but not Vertebrata in Seaweeds of the British Isles. At present, therefore, Polysiphonia has a broad circumscription including at least 200 species (Stegenga et al. 1997). Polysiphonia, however, is quite variable in its anatomical features as noted by Hommersand (1963) and Maggs and

Can. J. Bot. Vol. 79, 2001

Hommersand (1993). Recently, Kim and Lee (1999) have proposed segregation of the genus Neosiphonia from Polysiphonia based on Neosiphonia flavimarina M.S. Kim et I.K. Lee from Korea. This species has lateral branch (including trichoblast) initials produced on successive segments, erect indeterminate branches developing from the main axes, rhizoids separated from pericentral cells by a wall, abundant vegetative trichoblasts, three-celled carpogonial branches, spermatangial branches arising from a branch of the trichoblasts, and tetrasporangia in a spiral series. To date, 11 species from Korea that were originally placed in the genus Polysiphonia have been transferred to Neosiphonia (Kim and Lee 1999). Previous molecular studies (Phillips 2000; Phillips et al. 2000) have indicated that a clade including Neosiphonia savatieri and V. lanosa (as P. lanosa) was moderately allied to Pleurostichidium falkenbergii Heydrich, or to Murrayella periclados (C. Agardh) Schmitz with weak support. McIvor et al. (1999), using the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) gene, concluded that the genus Polysiphonia is paraphyletic, consisting of four strongly supported lineages no more closely related to one another than they are to other genera such as Pterosiphonia. Considering the confusion surrounding traditional taxonomy in the genus Polysiphonia, we have generated a matrix of 28 anatomical characters for species representative of the main lineages of Polysiphonia and related genera to assess phylogenetic relatedness. In addition, we have generated small-subunit ribosomal DNA (SSU rDNA) sequence data for the same taxa to complement and test our anatomical analyses. The aims of this study were to reassess monophyly of the genus Polysiphonia and determine the phylogenetic affinities of its component lineages (including Neosiphonia) among related Rhodomelaceae by generating a more extensive phylogeny for this family.

Materials and methods SSU rDNA sequence data Collection information is provided in Table 1. To determine nuclear SSU rDNA sequences, samples were processed and DNA was extracted according to the protocol described by Saunders (1993). The SSU rDNA was PCR(polymerase chain reaction)-amplified from total genomic DNA using the primer combinations of Saunders and Kraft (1994, 1996). Agarose gel purification with the WizardTM PCR Preps DNA Purification System (Promega, Madison, Wis.) was used to clean PCR products. DNA cleaned by this method was sequenced with the dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosytems (ABI), Foster City, Calif.). Sequence data were collected with the ABI PRISM 310 Genetic Analyzer. Comparison and editing of sequence data were accomplished with the SeqEd DNA Sequence Editor (ABI) software package. The final alignment consisted of sequences representing 28 species, including 16 previously published red algal SSU sequences (Table 2). The 1857 aligned nucleotide positions of SSU data were edited to remove the 5′ and 3′ PCR primer regions (G01 & G07; Saunders and Kraft 1994), as well as ambiguously aligned regions, to yield 1709 base pairs for phylogenetic inference. All analyses were completed in PAUP 4.0b4a for the Macintosh (Swofford 1999). Maximum likelihood analyses used a model of equal rates at all sites, transversions weighted 2 to 1 © 2001 NRC Canada



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Table 1. Sample information for species of Rhodomelaceae included in our molecular investigations. Species collected

Location of samplea

Sample

GenBank

Boergeseniella fruticulosa (Wulfen) Kylin*

Intertidal, Spiddal, Co. Galway, Ireland, 14 May 1998. M-SK Intertidal, Akkeshi, Hokkaido, Japan, 9 May 1999. H.S. Yoon & S.M. Boo Intertidal on Grateloupia lanceolata (Okamura) Kawaguchi, Anin, Korea, 24 Oct. 1999. H-GC Intertidal, Spiddal, Co. Galway, Ireland, 13 Mar. 1998. M-SK Intertidal, Finavarra, Co. Clare, Ireland, 26 Feb. 1998. M-SK Intertidal, Spiddal, Co. Galway, Ireland, 17 Nov. 1997. M-SK Intertidal, Sachon, Korea, 8 Mar. 1997. M-SK Intertidal, Bradys Beach, Bamfield, B.C., Canada, 29 Apr. 1998. J. Warneboldt & J.T. Harper Intertidal, Finavarra, Co. Clare, Ireland, 26 Feb. 1998. M-SK Intertidal, Flambourough, England, 16 Jul. 1998. M-SK Intertidal, Olifantsbos Bay, Cape Province, South Africa, 21 Jan. 1998. MDG Isolate from Italy, NUIG Marine Algal Culture Collection. Fabio Rindi & MDG

CH049

AF427526

CH073

AF427527

CH092

AF427528

CH047

AF427529

CH044

AF427530

CH046

AF427531

CH043 GWS405

AF427532 AF427533

CH048

AF427534

CH052

AF427535

GWS360

AF427536

CH050

AF427537

Enelittosiphonia stimpsonii (Harvey) Kudo et Masuda* Neosiphonia japonica (Harvey) M.S. Kim et I.K. Lee Polysiphonia elongata (Hudson) Sprengel Polysiphonia fucoides (Hudson) Sprengel Polysiphonia harveyi Bailey Polysiphonia morrowii Harvey Polysiphonia pacifica Hollenberg Polysiphonia nigra (Hudson) Batters Polysiphonia stricta (Dillwyn) Greville* Polysiphonia virgata (C. Agardh) Sprengel Womersleyella setacea (Hollenberg) R.E. Norris a

M-SK, Myung-Sook Kim; H-GC, Han-Gu Choi; MDG, Michael D. Guiry. *The type species of the genus.

Table 2. Source of additional SSU sequence data included in the multiple alignment.

Ceramiales Dasyaceae Delesseriaceae Rhodomelaceae

Incertae sedis

Species and authority

Referencea

Dasya baillouviana (S.G. Gmelin) Montagne* Delesseria serrulata Harvey (outgroup) Bostrychia moritziana (Sonder ex Kützing) J. Agardh Heterocladia australis Decaisne* Laurencia filiformis (C. Agardh) Montagne Lenormandia muelleri Sonder Lenormandia prolifera (C. Agardh) J. Agardh Melanamansia mamillaris (Lamouroux ex C. Agardh) R.E. Norris Micropeuce strobiliferum J. Agardh* Murrayella periclados (C. Agardh) Schmitz* Neosiphonia savatieri (Hariot) M.S. Kim et I.K. Lee Pleurostichidium falkenbergii Heydrich* Rhodomela confervoides (Hudson) P.C. Silva* Sonderella linearis (Harvey) Schmitz* Vertebrata lanosa (Linneaeus) T. Christensen* Platysiphonia victoriae (Harvey ex J. Agardh) Womersley et Shepley

1 4 3 3 3 3 3 3 3 3 3 2 1 3 3b 4

a

1, Ragan et al. (1994); 2, Phillips (2000); 3, Phillips et al. (2000); 4, H.-G. Choi et al. (unpublished). As Polysiphonia lanosa Linnaeus. *The type species of the genus.

b

over transitions, and empirical base frequencies (random additions set to 10). Maximum likelihood analyses were subjected to 100 rounds of bootstrap resampling (random additions set to 3). Distance analyses used the Kimura (1980) 2-parameter correction to calculate evolutionary distances and trees were subsequently constructed with neighbor-joining (Saitou and Nei 1987). Distance analyses were subjected to 2000 rounds of bootstrap

resampling (Felsenstein 1985). For parsimony analyses there were 298 informative sites with gaps included as a fifth base. Parsimony was completed (50 random stepwise additions of taxa) using a heuristic search method with tree bisection–reconnection (TBR) branch swapping and all changes equally weighted. Parsimony analyses were subjected to 1000 rounds of bootstrap resampling (random additions set to 10). In all analyses, unrooted © 2001 NRC Canada



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Table 3. Characters used in cladistic analyses of anatomical data. Character No.

Character description

Vegetative structures 1 Shape of apical cell: pyramidal (0), cylindrical (1) 2 Division pattern of apical cell: transverse (0), oblique (1) 3 Cellulosympodial frond growth: absent (0), present (1) 4 Erect indeterminate branches are developed from: the main axis (0), an extensive creeping base (1) 5 Laterals from central-axial cells originating exogenously: absent (0), present (1) 6 Laterals from central-axial cells originating endogenously: present (0), absent (1) 7 Lateral branch initials including the trichoblast initials: separated by one or more naked internodal segments (0), formed on successive segments (1) 8 Adventitious laterals: present (0), absent (1) 9 Symmetry of primary phyllotaxy: bilateral/dorsiventral (0), radial (1) 10 Number of pericentral cells: four (0), five (1), exceeding five (2) 11 Uniseriate determinate branches: absent (0), present (1) 12 Vegetative unpigmented trichoblast: scarce or lacking (0), abundant (1) 13 Prostrate branches: absent (0), present (1) 14 Cortical cells: present (0), absent (1) 15 Connection between rhizoids and pericentral cells: separated by a cross wall (0), open (1) Female reproductive structures 16 Location of the fertile-axial cell of procarp: intercalary in the primary axial row (0), epibasal on pigmented laterals (1), or unpigmented trichoblasts (2) 17 Order of appearance of fertile pericentral cell: second or third (0), fourth to last to be cut off (1) 18 Carpogonial branch: four-celled (0), three-celled (1) 19 Number of sterile cell groups present before fertilization: two (0), one (1) 20 Auxiliary cells diploidized by connecting cell(s): present (0), absent (1) 21 Unconsolidated sterile tissue associated with the carposporophyte prior to fertilization: absent (0), present (1) Male reproductive structures 22 Spermatangial axes borne: on surface cortical cells (0), on pigmented determinate branches (1), at thefirst, and sometimes second, dichotomy of fertile trichoblasts (unpigmented) (2), on trichoblast initials (3) Tetrasporangial structures 23 Tetrasporangia per fertile-axial cell or axis tier: in pairs (0), in whorls (1), single (2) 24 Arrangement of tetrasporangia: in straight (0) and spiral (1) series 25 Distribution of tetrasporangia: aggregated in sori (0), packed into stichidia (1), scattered (2) 26 Post-sporangial cover cells borne on the sporangial mother cell: present (0), absent (1) 27 Presporangial cover cells borne on the sporangial mother cell: absent (0), present (1) 28 Cortical filaments covering tetrasporangium: absent (0), present (1) trees were calculated and the ingroup taxa subsequently rooted with Delesseria serrulata Harvey (Delesseriaceae, Ceramiales) designated as the outgroup (Choi et al. data not shown).

Anatomical data To assess phylogeny based on anatomical features and to investigate the evolution of these same characters in light of our molecular tree, 28 features were coded in unordered, multistate forms (Table 3) for 25 species (excluding three poorly known rhodomelacean algae, Lenormandia muelleri, Lenormandia prolifera, and Micropeuce strobiliferum; Table 4). For parsimony analyses of anatomical data only 15 rhodomelacean taxa were included (Enelittosiphonia, Murrayella, Polysiphonia elongata, Polysiphonia fucoides, Polysiphonia nigra, Polysiphonia pacifica, Polysiphonia virgata, and Womersleyella were excluded because their reproductive features are poorly known). Parsimony analyses were completed (random sequence addition, 50 replicates) using a heuristic search, steepest descent, and TBR branch swapping in PAUP with all changes equally weighted (unweighted parsimony) and with the 13 reproductive characters weighted 1.2 to 1 against the 15 vegetative characters (weighted par-

simony). Weighting of reproductive over anatomical features was considered warranted, as the former are recognized as more conservative in red algal lineages. A range of weighting strategies from 1.1:1 to 5:1 all resulted in the same tree, and the ratio of 1.2:1 was arbitrarily selected for presentation. Anatomical character states were mapped on the resulting tree(s) using PAUP (Swofford 1999). Parsimony analyses were subjected to 1000 rounds of bootstrap resampling (random additions set to 10). Unrooted trees were calculated and the ingroup taxa rooted with reference to Platysiphonia victoriae (H.-G. Choi, and G.W. Saunders data not shown) because Dasya and Delesseria differ substantially from the Rhodomelaceae for the characters in our matrix.

Cladistic analysis of combined SSU–anatomical data The anatomical data matrix was appended to the SSU data matrix. The combined matrix included 25 taxa (excluding Lenormandia spp. and Micropeuce) and 1737 characters, of which 326 were informative. Combined parsimony analyses were performed (random sequence addition, 50 replicates; gaps fifth state) using the heuristic search, steepest descent, and TBR branching swapping of PAUP with all changes equally weighted (unweighted © 2001 NRC Canada



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Table 4. Data matrix of anatomical characters and references for the species included in the cladistic analysis. Referencesa

Characters Taxon Dasyaceae Dasya baillouviana* Delesseriaceae Delesseria serrulata (outgroup) Rhodomelaceae Boergeseniella fruticulosa* Bostrychia moritziana Enelittosiphonia stimpsonii* Heterocladia australis* Laurencia filiformis Melanamansia mammilaris Murrayella periclados* Neosiphonia japonica Neosiphonia savatieri Pleurostichidium falkenbergii* Polysiphonia elongata Polysiphonia fucoides Polysiphonia harveyi Polysiphonia morrowii Polysiphonia nigra Polysiphonia pacifica Polysiphonia stricta* Polysiphonia virgata Rhodomela confervoides* Sonderella linearis* Vertebrata lanosa* Womersleyella setacea Incertae sedis Platysiphonia victoriae

1–10

11–20

21–28

1 1 1 0 1 1 1 0 1 1 1 0 0 0 0 0 0 0 0 0 0 1 1 ? 2 0 0 0

1b, 2b, 12

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0

11c, 16c

1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1

16 6d, 16d 3, 20 1, 4, 27 17, 22d 14d, 23d 4 19e, 24d 19f, 24d 6, 26 16g 16g 16 18 16h 13 25 21 16 7 10i, 16i 8, 15d, 23j

1 1 ? 0 1 0 ? 0 0 0 0 0 0 1 0 1 1 1 1 0 1 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 1 1 0 0 0 1 0 0 1 0 0 0 1 0 1 1 0 0 0 0 1

1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 0 1 1

0 0 0 1 0 0 1 0 0 0 1 1 0 1 1 1 1 ? 0 0 1 1

1 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 0 0 0 1

0 1 0 0 1 1 0 0 0 0 0 1 0 1 1 1 1 0 0 0 1 0

1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 ? 1 0 1 1

2 2 2 0 0 1 0 0 0 2 0 2 0 0 2 0 0 2 2 0 2 1

1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 1

1 0 1 1 1 1 0 1 1 1 0 0 1 0 0 0 0 0 1 0 0 1

1 1 1 0 1 0 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1

0 0 1 0 0 0 1 0 1 0 0 1 0 1 1 1 1 0 0 0 1 1

? 0 0 0 0 ? 0 0 0 0 0 0 0 1 0 1 1 ? ? ? 1 0

2 1 ? 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ? 2 1 2 ?

1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 ?

0 0 ? 0 0 0 ? 1 1 0 ? ? 1 0 ? ? 0 ? 0 0 0 ?

0 1 ? 1 0 0 ? 0 0 0 ? ? 0 0 ? ? 0 ? 0 0 0 ?

? 0 ? 1 1 ? ? 1 1 1 ? ? 1 1 ? ? 1 ? 0 1 1 ?

1 0 ? 1 1 1 ? 1 1 1 ? ? 1 1 ? ? 1 ? 1 1 1 ?

2 1 ? 2 2 3 ? 2 2 0 2 2 2 3 2 3 3 ? 1 1 3 3

2 0 ? 2 0 0 0 2 2 1 2 2 2 2 2 2 2 2 0 0 2 2

1 0 ? 1 0 0 0 1 1 ? 1 1 1 0 1 0 0 0 0 0 1 1

2 1 ? 1 1 1 1 2 2 1 2 2 2 2 2 2 2 2 2 1 2 2

1 0 ? 1 0 1 ? 1 1 1 1 1 1 1 ? 1 1 ? 1 1 1 1

1 1 ? 0 1 1 ? 1 1 1 1 1 1 1 ? 1 1 ? 1 1 1 1

0 1 ? 1 1 0 ? 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 1 1 0 0 1 0 0 0 0 1 0 0 0

5k, 9d

a

1, Falkenberg (1901); 2, Rosenberg (1933); 3, Segi (1949); 4, Kylin (1956); 5, Womersley and Shepley (1959); 6, Hommersand (1963); 7, Womersley (1965); 8, Hollenberg (1967); 9, Wynne (1969); 10, Rawlence and Taylor (1970); 11, Mikami (1972); 12, Parsons (1975); 13, Abbott and Hollenberg (1976); 14, Norris (1988); 15, Norris (1992); 16, Maggs and Hommersand (1993); 17, Nam et al. (1994); 18, Kim et al. (1994); 19, Kim (1995); 20, Masuda et al. (1995); 21, Stegenga et al. (1997); 22, Garbary and Harper (1998); 23, Abbott (1999); 24, Kim and Lee (1999); 25, Kim et al. (2000); 26, Phillips (2000); 27, Phillips et al. (2000). b As Dasya elegans. c As Delesseria violacea. d Information at generic level, not specific to the species used in our analyses. e As Carposiphonia japonica. f Carposiphonia savatieri. g Display both states 2 and 3 for character 22. h Character 12 is variable in P. nigra. i As Polysiphonia lanosa, character 15 is usually state 1 but rarely state 0. j As Polysiphonia setacea. k As Sarcomenia victoriae. *The type species of the genus.

parsimony), and were subjected to 2000 rounds of bootstrap resampling (random additions set to 10). In all analyses, unrooted trees were calculated and the ingroup taxa subsequently rooted, with Delesseria serrulata (Delesseriaceae, Ceramiales) designated as the outgroup (Choi et al. data not shown).

Results Phylogeny based on SSU rDNA sequence data The 12 SSU sequences newly completed for this study ranged from 1767 (Neosiphonia japonica) to 1795 (Polysiphonia stricta) base pairs in length. No ambiguities were observed in the SSU data except for Womersleyella

setacea (C or T at position 418). The sequences of N. japonica and Polysiphonia harveyi Bailey determined in this study were identical to that of N. savatieri (Phillips et al. 2000). Sequence data have been deposited in GenBank (Table 1). A maximum likelihood (–ln likelihood = 8182.9624) tree with bootstrap results from the maximum likelihood, distance, and parsimony analyses appended is presented (Fig. 1) for our SSU alignment (26 species with 1709 base pairs; excluding N. savatieri and P. harveyi because their SSU sequences are identical to that of N. japonica). The Rhodomelaceae (except Platysiphonia), form a solid monophyletic group (100% bootstrap replicates in all analyses). Sonderella, Bostrychia, and Heterocladia form the earliest divergences in the © 2001 NRC Canada



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Fig. 1. Tree constructed with maximum likelihood for the SSU alignment. Values at branches represent percentage of 100, 2000, and 1000 bootstrap replicates for maximum likelihood, distance, and parsimony analyses, respectively. Bold numbers are bootstrap values for the three major lineages within Polysiphonia sensu lato. Branches lacking values received less than 50% support. Scale bar = 0.01 substitutions per site. Delesseria serrulata Dasya baillouviana

DELESSERIACEAE DASYACEAE Platysiphonia victoriae Incertae sedis Bostrychia moritziana

Bostrychieae

Heterocladia australis

Heterocladieae Pleurostichid. falkenbergii Neos. japonica Polys. elongata

100/100 100

100/100 100

100/100 100

55/64 78

100/100 100

Polys. fucoides Polys. nigra

96/100 99

83/93 77

100/100 100

Polys. pacifica 87/96 Polys. morrowii 81

1

0>1

0>2

13

22

27

1>0

1>0

1>0

19

20

0>1

1>0

6

13

0>1

1>0

28 0>1

19

27

28

0>1

1>0

0>1

78

95

23 0>2

24

0>1

0>1

2>0

22 1>2

5

0>1 0>1

14

66

13

0>1

0>1 1>0

10 11

50

0>2 0>1

8 0>1

12 16 0>1 0>1

21 26 0>1 0>1

2

20

25

0>1

1>0

1>2

4 0>1

22 1>0

Polysiphonia group

“Multipericentral” group

Neosiphonia savatieri Neosiphonia japonica

Neosiphonia group

Polysiphonia harveyi

9 0>1

Heterocladieae

Vertebrata lanosa

0>1

1


Boerges. fruticulosa 7

18

4 1>0

0>2 0>1 0>1

55

Bostrychieae

Polysiphonia morrowii

0>1 1>0 2>3 1>0 0>1

1>2

Bostrychia moritziana

Polysiphonia stricta

25 10

Outgroup

1>0

6 8 11 12 14 22

2 4 10 15 0>1

10 24

2>0

Platysiphonia victoriae

23 0>1

monophyletic clade, the species of which shared one synapomorphic feature: tetrasporangia scattered in branches (character 25). The Neosiphonia group was weakly supported with an important synapomorphic feature: three-celled carpogonial branches (character 18). Boergeseniella and Vertebrata of the multipericentral group allied to the moderately supported Polysiphonia group. The taxa of these two groups share the vegetative features of oblique division of apical cells (character 2) and open connections between rhizoids and pericentral cells (character 15). The Polysiphonia group and the genus Vertebrata were allied by a shared absence of five vegetative features: laterals from central-axial cells originating endogenously (character 6), adventitious laterals (character 8), uniseriate determinate branches (character 11), vegetative trichoblasts (character 12) and cortical cells (character 14), as well as the shared presence of one male reproductive feature, spermatangial axes borne on trichoblast initials (character 22). The Polysiphonia group was defined by species having four pericentral cells (character 10) and tetrasporangia in straight series (character 24). With respect to Polysiphonia sensu lato, the weighted parsimony solution for anatomical data (Fig. 2) differed from the molecular tree (Fig. 1) in that the included representatives of

10

13

22

0>1

1>0

2>3

2

26

28

0>1

1>0

0>1

Melanam. mamillaris

Amansieae

Laurencia filiformis

Laurencieae

Rhod. confervoides

Rhodomeleae

Pleurost. falkenbergii

Pleurostichidieae

Sonderella linearis

Incertae sedis

the multipericentral group, the genera Boergeseniella and Vertebrata, were not specifically allied and failed to join the Neosiphonia group, but bootstrap support was absent for the former analysis (Fig. 2). Phylogeny based on combined SSU–anatomical data In a final analysis, combined SSU–anatomical data were subjected to parsimony in the absence of any weighting scheme and the single most parsimonious solution (length = 1106, consistency index = 0.590, retention index = 0.679) is presented (Fig. 3). Analysis of this data set generally echoed the molecular results (Fig. 1) in that Murrayella was moderately allied to a strongly supported clade of Pleurostichidium and Polysiphonia sensu lato. Within Polysiphonia sensu lato the Polysiphonia, Neosiphonia, and multipericentral groups were solidly resolved, the latter two groups strongly allied, whereas the affinities of Womersleyella relative to these three lineages were equivocal (Fig. 3). An association between the Neosiphonia and multipericentral groups was supported by reversals in three anatomical features; however, these characters changed a number of times on the combined tree, including within this lineage itself (Fig. 3). © 2001 NRC Canada



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Can. J. Bot. Vol. 79, 2001

Fig. 3. The single most parsimonious solution inferred from combined anatomical–SSU data with putative evolutionary pathway for the anatomical characters mapped. Bold numbers above the branches indicate bootstrap values (% of 2000 replicates). Branches lacking values received less than 50% support. Numbers above and below the solid and open bars correspond to anatomical characters and character-state changes (Tables 3 and 4), respectively. Solid bars, (syn)apomorphies; open bars, reversals. *Polysiphonia fucoides (as well as P. elongata, not indicated on figure) has spermatangial branches forming on both trichoblast initials and trichoblasts. 1

2

0>1 0>1

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6

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15

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DASYACEAE

Platysiphonia victoriae

Incertae sedis

Bostrychia moritziana

Bostrychieae


Heterocladieae

Pleurost. falkenbergii

Pleurostichidieae

Womers. setacea Polys. pacifica

7 13

6 12

DELESSERIACEAE

Dasya baillouviana

Polys. stricta

80

0>1 1>0

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8 12 9 11 12 16 22

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23

Delesseria serrulata

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7 22*

Polys. morrowii

Incertae sedis

Polysiphonia group

Neos. savatieri Neos. japonica Polys. harveyi Polys. elongata

Neosiphonia group

1>0

Polys. virgata Enelitt. stimps. Boerg. fruticul.

Vert. lanosa Polys. nigra

“Multipericentral” group

Polys. fucoides

0>1 2>2,3

20

22

25

1>0

3>1

1>2

26 1>0

All members of the Polysiphonia group share three diagnostic features: the absence of uniseriate determinate branches (character 11); open connections between rhizoids and pericentral cells (character 15); and the formation of tetrasporangia in a straight series (character 24). These features, however, have evolved in parallel in Polysiphonia virgata (characters 11 and 24) of the Neosiphonia group and Vertebrata (character 15) of the multipericentral group (Fig. 3). Species of the Neosiphonia group share the presence of adventitious laterals (character 8) and cortication (character 14) as well as three-celled carpogonial branches (character 18), except for N. savatieri having an alternative state for character 14. Characters 8 and 14 have evolved in parallel to the Neosiphonia group in the ancestor to Boergeseniella–Enelittosiphonia and Boergeseniella, respectively, within the multipericentral group. All taxa of the multipericentral group share a key diagnostic feature: more than five pericentral cells (character 10). This feature has also evolved in parallel in Polysiphonia virgata, which unequivocally joined the Neosiphonia group in our molecular and combined analyses. The alliance of Womersleyella and the three groups mentioned previously was supported by two tetrasporangial fea-

Polysiphonia sensu lato

16 17 20 25

22

Murrayella periclados

Lophothalieae

Rhodom. confervoides

Rhodomeleae

Melanam. mamillaris

Amansieae

Laurencia filiformis

Laurencieae

Sonderella linearis

Incertae sedis

tures: a single tetrasporangium per axial tier (character 23) and tetrasporangia scattered in the frond (character 25). The relationships of Pleurostichidium and Womersleyella relative to one another and the three groups, however, were unresolved with the combined analysis (Fig. 3), similar to the molecular results (Fig. 1).

Discussion Polysiphonia sensu lato is currently placed in the tribe Polysiphonieae Schmitz (1889) of the subfamily Rhodomeloideae Hommersand (Maggs and Hommersand 1993). Similar to previous molecular studies (Phillips 2000; Phillips et al. 2000), our molecular and combined data indicate that Pleurostichidium, Pleurostichidieae, is allied to Polysiphonia sensu lato with strong support, and Murrayella, Lophothalieae, diverges at the base of this lineage with moderate support. Relationships among the component species and genera of Polysiphonia sensu lato are complex in light of our analyses and we recognize three groups that are discussed below. © 2001 NRC Canada



Choi et al.

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Fig. 4. Comparison of six selected diagnostic characters between the (i) Polysiphonia, (ii) multipericentral, and (iii) Neosiphonia groups. A, number of pericentral cells (numbered circles)(character 10) and presence or absence of cortication (smaller circles peripheral to pericentral cells)(character 14); B, connection between rhizoids and pericentral cells open or with a crosswall (character 15); C, carpogonial branch three or four-celled (character 18); D, spermatangial axes development (character 22); and E, straight or spiral arrangement of tetrasporangia (tetrahedrally divided circles)(character 24). a, Boergeseniella is corticated; b, open or rarely separated by a crosswall in Vertebrata lanosa; c, on both the trichoblasts and trichoblast initials in P. fucoides, and on trichoblast initials in Vertebrata lanosa; d, cortication absent in N. savatieri, P. virgata has more than four pericentral cells; e, not known for P. elongata and P. virgata of this group; f, except P. virgata (straight series).

i

B

A

D

C

2 4

3

1

4

su

2 3

ii

1

2

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1

6

4 3

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E

7

5

a

b

3

su

2

c

1

iii 2 4

3

1 2

3

d Type Polysiphonia urceolata (= P. stricta) and additional species of the genus Polysiphonia In our analyses, the Polysiphonia group includes the type species of the genus, Polysiphonia urceolata (= P. stricta) from the north Atlantic, P. morrowii from the northwest Pacific, and P. pacifica from the northeast Pacific. These species share the important diagnostic features outlined in Fig. 4, as well as an absence of uniseriate determinate branches (character 11). In addition to these three species, the Polysiphonia group may include: Polysiphonia atlantica Kapraun et J.N. Norris (cf. Maggs and Hommersand 1993); P. atlantica sensu M.S. Kim and I.K. Lee (1996); Polysiphonia carettia Hollenberg and Polysiphonia decussata Hollenberg (cf. Abbott and Hollenberg 1976); Polysiphonia namibiensis Stegenga et Engledow (cf. Stegenga et al. 1997); Polysiphonia pungens Hollenberg (cf. Womersley 1979); Polysiphonia scopulorum Harvey (cf. Abbott and Hollenberg 1976; Stegenga et al. 1997; Abbott 1999); Polysiphonia senticulosa Harvey (cf. Kim et al. 2000); Polysiphonia shepherdii

su 1

e

f

Womersley and Polysiphonia subtilissima Montagne (cf. Womersley 1979; Abbott 1999); and Polysiphonia tuberosa Hollenberg (cf. Abbott 1999). Multipericentral group including the genus Vertebrata The multipericentral group allied to the Neosiphonia group in our molecular and combined analyses. This group includes species of four recognized genera from the north Atlantic and northwest Pacific: Boergeseniella, Enelittosiphonia, Polysiphonia, and Vertebrata. They share the important synapomorphic feature of more than five pericentral cells (character 10), and the included species variously share other key anatomical features with the Neosiphonia and Polysiphonia group (Fig. 4). Polysiphonia fucoides and P. nigra from the north Atlantic group together with strong support in our molecular and combined analyses, and share, in addition to the several important diagnostic features of the group (Fig. 4), the development of erect indeterminate branches from the main axes © 2001 NRC Canada



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(character 4), although none of these specifically ally these species (Fig. 3). The former has spermatangial branches on both trichoblast initials and trichoblasts (character 22), whereas the latter, as well as Boergeseniella and other members of the multipericentral group in Britain and Ireland except for Vertebrata, have spermatangial branches that form only on the trichoblasts (Fig. 4). These species are clearly distinct both anatomically and in our molecular analyses from Polysiphonia sensu stricto and may require transfer to Boergeseniella (Fig. 1) or may be referred to a separate genus pending further investigation (Fig. 3). In contrast to other members of the multipericentral group, V. lanosa has a unique combination of features: vegetative trichoblasts are absent (character 12; also true for P. fucoides, and variable for P. nigra); the connections between rhizoids and pericentral cells are open (character 15; rarely separated in Vertebrata); spermatangial branches are formed only on trichoblast initials (character 22); and the species is exclusively epiphytic on the brown algae Ascophyllum nodosum (Linnaeus) Le Jolis and Fucus vesiculosus Linnaeus. Owing to this combination of features in addition to our molecular analyses, which do not ally this species with Polysiphonia sensu stricto, we support Kylin (1956) and Christensen (1967) in their recognition of the genus Vertebrata. One approach to dealing with taxonomy in the multipericentral group might be to sink all species into the single genus Vertebrata, but a thorough systematic investigation of the genera and species of this group is necessary prior to formal taxonomic proposals. Extending Neosiphonia to include P. harveyi In our combined analyses, a strongly supported Neosiphonia group included N. japonica and N. savatieri from the northwest Pacific, P. elongata and P. harveyi from the north Atlantic, and P. virgata from South Africa. These taxa are joined by the diagnostic features outlined in Fig. 4, although exceptions occur, and in some cases important character states are unknown, particularly for P. elongata and P. virgata (Table 4, Fig. 4 caption). In addition, these taxa share the presence of adventitious laterals (character 8). Polysiphonia elongata grows on bedrock, stones, shells, various algae, and occasionally on stipes of Laminaria hyperborea (Gunnerus) Foslie (Maggs and Hommersand 1993), and shares some key diagnostic features (four pericentral cells and a spiral series of tetrasporangia; Fig. 4) with Neosiphonia, as well as erect indeterminate branches developing from the main axis (character 4) and the presence of adventitious laterals (character 8). However, P. elongata clearly allied to P. virgata (an alliance lacking support from anatomical features used in the current study) not to the clade including Neosiphonia spp. and P. harveyi in our molecular and combined analyses (Figs. 1 and 3). The South African endemic species P. virgata, an epiphyte of Ecklonia maxima (Osbeck) Papenfuss and occasionally Laminaria pallida Greville (Stegenga et al. 1997), has been variously assigned to the monotypic genus Carradoriella (Kylin 1956, as Carradoria; Silva et al. 1996) or included in Polysiphonia (Wynne 1986). It shares some key diagnostic features with the multipericentral (12–16 pericentral cells)

Can. J. Bot. Vol. 79, 2001

and Polysiphonia groups (tetrasporangia in straight series; Fig. 4), but it unequivocally joined the Neosiphonia group, in particular P. elongata, in our molecular and combined analyses (Figs. 1 and 3). If the indications of our molecular data are correct, the newly established Neosiphonia could be subsumed into Carradoriella, but the two may ultimately resolve as sister genera. It would be premature to make formal proposals until additional molecular data are investigated and the female and male reproductive features of P. elongata and P. virgata are clarified. Polysiphonia harveyi shares the many diagnostic features of this group with Neosiphonia (Fig. 4), in addition to forming laterals endogenously from central axial cells (character 6) and the presence of abundant vegetative unpigmented trichoblasts (character 12), and fits comfortably within this genus based on anatomical and combined SSU–anatomical analyses. Bailey (1848) first described P. harveyi from Connecticut. This species occupies a wide range of inter- and sub-tidal habitats in both the northwest Pacific and north Atlantic, but it is regarded as an alien in European waters (Maggs and Hommersand 1993; Maggs and Stegenga 1999; McIvor et al. 2001). McIvor et al. (2001) recently concluded that Japan is the centre of diversity and origin for P. harveyi based on rbcL sequences from isolates of P. harveyi and various congeners from the Pacific and north Atlantic. Taxonomic proposal As a result of our molecular results and the anatomical features shared by P. harveyi with Neosiphonia, we propose the following new combination: Neosiphonia harveyi (Bailey) M.-S. Kim, H.-G. Choi, Guiry et G.W. Saunders, comb.nov. Basionym: Polysiphonia harveyi Bailey, Am. J. Sci. Arts, Ser. 2, 6: 38. 1848. Type locality: Stonington, Connecticut, U.S.A. Lectotype: TCD.

Acknowledgements We are grateful to Sylva Donaldson and Nancy MacAfee for technical assistance. We thank S.M. Boo, H.S. Yoon, J. Warneboldt, and J. Todd Harper for providing some of the samples used in this study. This work was supported by a Natural Sciences and Engineering Research Council of Canada Grant and Canada Research Chair Program funds to GWS, and Korean Science and Engineering Foundation postdoctoral fellowships to H-GC and M-SK.

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