Phylogenetic relationships of Polysiphonia (Rhodomelaceae, Rhodophyta) and its relatives based on...

Phylogenetic relationships of Polysiphonia(Rhodomelaceae, Rhodophyta) and its relativesbased on anatomical and nuclear small-subunitrDNA 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 af-finities of its component lineages among related red algae belonging to the Rhodomelaceae. Our “total evidence” ap-proach, combining 28 anatomical characters and small-subunit ribosomal DNA sequence data for 25 ceramialean algaeincluding 14 species of Polysiphonia sensu lato (including two species of the recently described genus Neosiphonia) andnine 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 ofWomersleyella setacea. The type species of the genus, Polysiphonia urceolata (= Polysiphonia stricta) from the north At-lantic, formed a distinct clade with Polysiphonia morrowii and Polysiphonia pacifica from the northwest and northeast Pa-cific, respectively. The Neosiphonia group included Neosiphonia japonica and Neosiphonia savatieri from the northwestPacific, as originally proposed, Polysiphonia harveyi from the north Atlantic, which shares diagnostic features with thisgenus, and the anomalous Polysiphonia elongata and Polysiphonia virgata from the north Atlantic and South Africa, re-spectively. Boergeseniella and Vertebrata from the north Atlantic and Enelittosiphonia from the northwest Pacific associ-ated solidly with the multipericentral Polysiphonia fucoides and Polysiphonia nigra from the north Atlantic. Theimplications 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 affinitésphylogénétiques 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 depetite sous-unité chez 25 algues céramialées comprenant 14 espèces de Polysiphonia sensu lato (incluant deux espècesdu genre Neosiphonia récemment décrit) et neuf Rhodomelaceae apparentées, indique que les Polysiphonia sensu latocomportent 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 ur-ceolata (=Polysiphonia stricta) de l’Atlantique nord, forme un clade distinct avec le Polysiphonia morrowi et le Polysi-phonia pacifica du nord-ouest et du nord-est du Pacifique, respectivement. Le groupe Neosiphonia inclut leNeosiphonia japonica et le Neosiphonia savatieri du nord-ouest du Pacifique tel que proposé originalement, le Polysi-phonia harveyi du nord de l’Atlantique, lequel partage ses caractéristiques diagnostiques avec ce genre, et les espècesirrégulières 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 mon-trent une solide association avec les espèces de Polysiphonia multipéricentriques, Polysiphonia fucoides et Polysiphonianigra de l’Atlantique nord. Les auteurs discutent les implications taxonomiques pour les Polysiphonia sensu lato ainsique 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] Choi et al. 1476

Can. J. Bot. 79: 1465–1476 (2001) © 2001 NRC Canada

1465

DOI: 10.1139/cjb-79-12-1465

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 ofNew 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 NationalUniversity of Ireland, Galway, Ireland.

1Present address: BK21, Department of Earth Systems & Environmental Sciences, Chonnam National University, Gwangju 500-757,Korea.

2Corresponding author (e-mail: [email protected]).3Present address: Department of Biological Sciences, College of Natural Sciences, Pusan National University, Pusan 609-735, Korea.

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

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Introduction

Species of the largest red algal genus, PolysiphoniaGreville (1824), are common and widely distributed on vir-tually all coasts of the world (Womersley 1979). The genericcircumscription of Polysiphonia has been in an almost con-stant state of flux since C. Agardh’s (1817) original proposal(as Hutchinsia) (cf. Kim et al. 2000). Sprengel (1827) wasthe first to adopt the name Polysiphonia for the majority ofthe species placed by C. Agardh in Hutchinsia (Dixon andIrvine 1970) and included 27 species in the genus. J. Agardh(1863, p. 908) treated Polysiphonia as a diverse and speciosegenus, which he divided primarily on the basis of thallussize into four subgenera: Herposiphonia, Oligosiphonia,Polysiphonia, and Ptilosiphonia.

Kylin (1941) proposed the segregation of the genusOrcasia based on Polysiphonia senticulosa Harvey and char-acterized it as having young branches arising endogenouslyfrom the base of determinate branchlets. However, this ge-nus has not gained general acceptance (cf. Kudo and Masuda1988). Segi (1949) proposed the genus Enelittosiphoniabased on Polysiphonia hakodatensis Yendo (= E. stimpsonii(Harvey) Kudo et Masuda), which has a prostrate ecorticatethallus with 8–12 pericentral cells. Hollenberg (1968a,1968b) divided the Pacific species of Polysiphonia into twogroups: Oligosiphonia for species with four pericentral cellsand Polysiphonia for species with five or more pericentralcells. He also proposed several new genera, includingWomersleyella Hollenberg (1967), based on Womersleyellapacifica Hollenberg, to which some species of Polysiphoniawere later transferred (Norris 1992).

Silva (1952) proposed the conservation of Polysiphoniaover the earlier Vertebrata Gray (1821), GrammitaBonnemaison (1822), and Gratelopella Bory (1823). Kylin(1956) subsequently proposed that Vertebrata be reinstatedbased on Polysiphonia fastigiata (Roth) Greville to includeonly the type species of Gray’s genus (Vertebrata fastigiata(Roth) Gray 1821, p. 338), and also proposed four new gen-era including Boergeseniella (based on Polysiphoniafruticulosa (Wulfen) Sprengel), Diplocladia (based onPolysiphonia patersonis Sonder), Echinothamnion (based onPolysiphonia hystrix Hooker et Harvey), and Leptosiphonia(based on Polysiphonia schousoei Thuret in Bornet etThuret) for species of the genus Polysiphonia sensu lato.Christensen (1967) accepted Kylin’s (1956) resurrection ofVertebrata because this species has several important diag-nostic features such as all primordia developing intoecorticated polysiphonous branches without vegetativetrichoblasts. He further synonymized V. fastigiata withPolysiphonia lanosa (Linnaeus) Tandy (1931, p. 226; basedon Fucus lanosus Linnaeus 1767, p. 718) as Vertebratalanosa (Linnaeus) T. Christensen. Subsequent authors havegenerally not accepted recognition of Vertebrata as distinctfrom Polysiphonia. For example, Maggs and Hommersand(1993) recognized Boergeseniella but not Vertebrata in Sea-weeds of the British Isles.

At present, therefore, Polysiphonia has a broad circum-scription including at least 200 species (Stegenga et al. 1997).Polysiphonia, however, is quite variable in its anatomical fea-tures as noted by Hommersand (1963) and Maggs and

Hommersand (1993). Recently, Kim and Lee (1999) haveproposed segregation of the genus Neosiphonia fromPolysiphonia based on Neosiphonia flavimarina M.S. Kim etI.K. Lee from Korea. This species has lateral branch (includ-ing trichoblast) initials produced on successive segments,erect indeterminate branches developing from the main axes,rhizoids separated from pericentral cells by a wall, abundantvegetative trichoblasts, three-celled carpogonial branches,spermatangial branches arising from a branch of thetrichoblasts, and tetrasporangia in a spiral series. To date, 11species from Korea that were originally placed in the genusPolysiphonia have been transferred to Neosiphonia (Kimand Lee 1999).

Previous molecular studies (Phillips 2000; Phillips et al.2000) have indicated that a clade including Neosiphoniasavatieri and V. lanosa (as P. lanosa) was moderately allied toPleurostichidium falkenbergii Heydrich, or to Murrayellapericlados (C. Agardh) Schmitz with weak support. McIvor etal. (1999), using the large subunit of ribulose-1,5-bisphosphatecarboxylase/oxygenase (rbcL) gene, concluded that the genusPolysiphonia is paraphyletic, consisting of four strongly sup-ported lineages no more closely related to one another thanthey are to other genera such as Pterosiphonia.

Considering the confusion surrounding traditional taxon-omy in the genus Polysiphonia, we have generated a matrixof 28 anatomical characters for species representative of themain lineages of Polysiphonia and related genera to assessphylogenetic relatedness. In addition, we have generatedsmall-subunit ribosomal DNA (SSU rDNA) sequence datafor the same taxa to complement and test our anatomicalanalyses. The aims of this study were to reassess monophylyof the genus Polysiphonia and determine the phylogeneticaffinities of its component lineages (including Neosiphonia)among related Rhodomelaceae by generating a more exten-sive phylogeny for this family.

Materials and methods

SSU rDNA sequence dataCollection information is provided in Table 1. To determine nu-

clear SSU rDNA sequences, samples were processed and DNA wasextracted according to the protocol described by Saunders (1993).The SSU rDNA was PCR(polymerase chain reaction)-amplifiedfrom total genomic DNA using the primer combinations ofSaunders and Kraft (1994, 1996). Agarose gel purification with theWizardTM PCR Preps DNA Purification System (Promega, Madi-son, Wis.) was used to clean PCR products. DNA cleaned by thismethod was sequenced with the dRhodamine Terminator Cycle Se-quencing Ready Reaction Kit (PE Applied Biosytems (ABI), Fos-ter City, Calif.). Sequence data were collected with the ABIPRISM 310 Genetic Analyzer. Comparison and editing of se-quence data were accomplished with the SeqEd DNA SequenceEditor (ABI) software package.

The final alignment consisted of sequences representing 28species, including 16 previously published red algal SSU se-quences (Table 2). The 1857 aligned nucleotide positions of SSUdata were edited to remove the 5′ and 3′ PCR primer regions (G01& G07; Saunders and Kraft 1994), as well as ambiguouslyaligned regions, to yield 1709 base pairs for phylogenetic infer-ence. All analyses were completed in PAUP 4.0b4a for theMacintosh (Swofford 1999). Maximum likelihood analyses used amodel of equal rates at all sites, transversions weighted 2 to 1

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over transitions, and empirical base frequencies (random addi-tions set to 10). Maximum likelihood analyses were subjected to100 rounds of bootstrap resampling (random additions set to 3).Distance analyses used the Kimura (1980) 2-parameter correctionto calculate evolutionary distances and trees were subsequentlyconstructed with neighbor-joining (Saitou and Nei 1987). Dis-tance analyses were subjected to 2000 rounds of bootstrap

resampling (Felsenstein 1985). For parsimony analyses therewere 298 informative sites with gaps included as a fifth base. Par-simony was completed (50 random stepwise additions of taxa) us-ing a heuristic search method with tree bisection–reconnection(TBR) branch swapping and all changes equally weighted. Parsi-mony analyses were subjected to 1000 rounds of bootstrapresampling (random additions set to 10). In all analyses, unrooted

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Species and authority Referencea

CeramialesDasyaceae Dasya baillouviana (S.G. Gmelin) Montagne* 1Delesseriaceae Delesseria serrulata Harvey (outgroup) 4Rhodomelaceae Bostrychia moritziana (Sonder ex Kützing) J. Agardh 3

Heterocladia australis Decaisne* 3Laurencia filiformis (C. Agardh) Montagne 3Lenormandia muelleri Sonder 3Lenormandia prolifera (C. Agardh) J. Agardh 3Melanamansia mamillaris (Lamouroux ex C. Agardh) R.E. Norris 3Micropeuce strobiliferum J. Agardh* 3Murrayella periclados (C. Agardh) Schmitz* 3Neosiphonia savatieri (Hariot) M.S. Kim et I.K. Lee 3Pleurostichidium falkenbergii Heydrich* 2Rhodomela confervoides (Hudson) P.C. Silva* 1Sonderella linearis (Harvey) Schmitz* 3Vertebrata lanosa (Linneaeus) T. Christensen* 3b

Incertae sedis Platysiphonia victoriae (Harvey ex J. Agardh) Womersley etShepley

4

a1, Ragan et al. (1994); 2, Phillips (2000); 3, Phillips et al. (2000); 4, H.-G. Choi et al. (unpublished).bAs Polysiphonia lanosa Linnaeus.*The type species of the genus.

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

Species collected Location of samplea Sample GenBank

Boergeseniella fruticulosa (Wulfen) Kylin* Intertidal, Spiddal, Co. Galway, Ireland,14 May 1998. M-SK

CH049 AF427526

Enelittosiphonia stimpsonii (Harvey) Kudo et Masuda* Intertidal, Akkeshi, Hokkaido, Japan,9 May 1999. H.S. Yoon & S.M. Boo

CH073 AF427527

Neosiphonia japonica (Harvey) M.S. Kim et I.K. Lee Intertidal on Grateloupia lanceolata (Okamura)Kawaguchi, Anin, Korea, 24 Oct. 1999. H-GC

CH092 AF427528

Polysiphonia elongata (Hudson) Sprengel Intertidal, Spiddal, Co. Galway, Ireland,13 Mar. 1998. M-SK

CH047 AF427529

Polysiphonia fucoides (Hudson) Sprengel Intertidal, Finavarra, Co. Clare, Ireland,26 Feb. 1998. M-SK

CH044 AF427530

Polysiphonia harveyi Bailey Intertidal, Spiddal, Co. Galway, Ireland,17 Nov. 1997. M-SK

CH046 AF427531

Polysiphonia morrowii Harvey Intertidal, Sachon, Korea, 8 Mar. 1997. M-SK CH043 AF427532Polysiphonia pacifica Hollenberg Intertidal, Bradys Beach, Bamfield, B.C., Canada,

29 Apr. 1998. J. Warneboldt & J.T. HarperGWS405 AF427533

Polysiphonia nigra (Hudson) Batters Intertidal, Finavarra, Co. Clare, Ireland,26 Feb. 1998. M-SK

CH048 AF427534

Polysiphonia stricta (Dillwyn) Greville* Intertidal, Flambourough, England,16 Jul. 1998. M-SK

CH052 AF427535

Polysiphonia virgata (C. Agardh) Sprengel Intertidal, Olifantsbos Bay, Cape Province,South Africa, 21 Jan. 1998. MDG

GWS360 AF427536

Womersleyella setacea (Hollenberg) R.E. Norris Isolate from Italy, NUIG Marine Algal CultureCollection. Fabio Rindi & MDG

CH050 AF427537

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

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



trees were calculated and the ingroup taxa subsequently rootedwith Delesseria serrulata Harvey (Delesseriaceae, Ceramiales)designated as the outgroup (Choi et al. data not shown).

Anatomical dataTo assess phylogeny based on anatomical features and to investi-

gate the evolution of these same characters in light of our moleculartree, 28 features were coded in unordered, multistate forms (Table 3)for 25 species (excluding three poorly known rhodomelacean algae,Lenormandia muelleri, Lenormandia prolifera, and Micropeucestrobiliferum; Table 4).

For parsimony analyses of anatomical data only 15rhodomelacean taxa were included (Enelittosiphonia, Murrayella,Polysiphonia elongata, Polysiphonia fucoides, Polysiphonia nigra,Polysiphonia pacifica, Polysiphonia virgata, and Womersleyella wereexcluded because their reproductive features are poorly known). Par-simony analyses were completed (random sequence addition, 50replicates) using a heuristic search, steepest descent, and TBRbranch swapping in PAUP with all changes equally weighted (un-weighted parsimony) and with the 13 reproductive charactersweighted 1.2 to 1 against the 15 vegetative characters (weighted par-

simony). Weighting of reproductive over anatomical features wasconsidered warranted, as the former are recognized as more conser-vative in red algal lineages. A range of weighting strategies from1.1:1 to 5:1 all resulted in the same tree, and the ratio of 1.2:1 wasarbitrarily selected for presentation. Anatomical character stateswere mapped on the resulting tree(s) using PAUP (Swofford 1999).Parsimony analyses were subjected to 1000 rounds of bootstrapresampling (random additions set to 10). Unrooted trees were calcu-lated and the ingroup taxa rooted with reference to Platysiphoniavictoriae (H.-G. Choi, and G.W. Saunders data not shown) becauseDasya and Delesseria differ substantially from the Rhodomelaceaefor the characters in our matrix.

Cladistic analysis of combined SSU–anatomical dataThe anatomical data matrix was appended to the SSU data ma-

trix. The combined matrix included 25 taxa (excludingLenormandia spp. and Micropeuce) and 1737 characters, of which326 were informative. Combined parsimony analyses were per-formed (random sequence addition, 50 replicates; gaps fifth state)using the heuristic search, steepest descent, and TBR branchingswapping of PAUP with all changes equally weighted (unweighted

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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 pigmentedlaterals (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) series25 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)

Table 3. Characters used in cladistic analyses of anatomical data.



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

Results

Phylogeny based on SSU rDNA sequence dataThe 12 SSU sequences newly completed for this study

ranged from 1767 (Neosiphonia japonica) to 1795(Polysiphonia stricta) base pairs in length. No ambiguitieswere observed in the SSU data except for Womersleyella

setacea (C or T at position 418). The sequences of N. japon-ica and Polysiphonia harveyi Bailey determined in this studywere 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) treewith bootstrap results from the maximum likelihood, distance,and parsimony analyses appended is presented (Fig. 1) for ourSSU alignment (26 species with 1709 base pairs; excludingN. savatieri and P. harveyi because their SSU sequences areidentical to that of N. japonica). The Rhodomelaceae (exceptPlatysiphonia), form a solid monophyletic group (100% boot-strap replicates in all analyses). Sonderella, Bostrychia, andHeterocladia form the earliest divergences in the

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Choi et al. 1469

Characters Referencesa

Taxon 1–10 11–20 21–28DasyaceaeDasya baillouviana* 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, 12DelesseriaceaeDelesseria serrulata (outgroup) 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

RhodomelaceaeBoergeseniella fruticulosa* 1 1 0 1 1 0 1 0 1 2 1 1 1 0 ? 2 1 0 0 ? 1 2 2 1 2 1 1 0 16Bostrychia moritziana 1 1 0 1 1 0 0 1 0 2 0 0 1 0 0 1 1 0 1 0 0 1 0 0 1 0 1 1 6d, 16d

Enelittosiphonia stimpsonii* 1 ? 0 1 1 0 0 0 1 2 1 1 1 1 0 ? ? ? ? ? ? ? ? ? ? ? ? ? 3, 20Heterocladia australis* 1 0 0 0 1 1 0 0 1 0 1 1 0 0 0 2 1 0 1 1 1 2 2 1 1 1 0 1 1, 4, 27Laurencia filiformis 1 1 0 0 1 0 0 1 1 0 1 1 1 0 0 2 1 0 0 1 1 2 0 0 1 0 1 1 17, 22d

Melanamansia mammilaris 1 0 0 0 1 0 0 1 1 1 1 1 0 0 ? 2 1 0 0 ? 1 3 0 0 1 1 1 0 14d, 23d

Murrayella periclados* 1 ? 0 1 1 1 0 0 1 0 1 0 1 1 0 2 1 ? ? ? ? ? 0 0 1 ? ? ? 4Neosiphonia japonica 1 0 0 0 1 0 1 0 1 0 1 1 0 0 0 2 1 1 0 1 1 2 2 1 2 1 1 0 19e, 24d

Neosiphonia savatieri 1 0 0 0 1 0 1 0 1 0 1 1 0 1 0 2 1 1 0 1 1 2 2 1 2 1 1 0 19f, 24d

Pleurostichidium falkenbergii* 0 0 0 1 0 0 0 0 0 2 1 1 1 0 0 2 1 0 0 1 1 0 1 ? 1 1 1 0 6, 26Polysiphonia elongata 1 0 0 0 1 1 0 0 1 0 1 0 1 0 0 2 1 ? ? ? ? 2 2 1 2 1 1 0 16g

Polysiphonia fucoides 1 0 0 0 1 1 1 1 1 2 1 0 1 1 0 2 1 ? ? ? ? 2 2 1 2 1 1 0 16g

Polysiphonia harveyi 1 0 0 0 1 0 0 0 1 0 1 1 1 0 0 2 1 1 0 1 1 2 2 1 2 1 1 0 16Polysiphonia morrowii 1 1 0 1 1 1 0 1 1 0 0 0 1 1 1 2 1 0 0 1 1 3 2 0 2 1 1 0 18Polysiphonia nigra 1 0 0 0 1 1 0 1 1 2 1 0 1 1 0 2 1 ? ? ? ? 2 2 1 2 ? ? ? 16h

Polysiphonia pacifica 1 1 0 1 1 1 0 1 1 0 0 0 1 1 1 2 1 ? ? ? ? 3 2 0 2 1 1 0 13Polysiphonia stricta* 1 1 0 1 1 1 0 1 1 0 0 0 1 1 1 2 1 0 0 1 1 3 2 0 2 1 1 0 25Polysiphonia virgata 1 1 0 0 1 ? 0 0 ? 2 0 0 1 0 ? ? ? ? ? ? ? ? 2 0 2 ? ? 0 21Rhodomela confervoides* 1 1 0 0 1 0 0 0 1 2 1 1 1 0 ? 2 1 0 0 0 1 1 0 0 2 1 1 0 16Sonderella linearis* 0 0 0 0 0 0 0 0 0 0 0 0 1 0 ? 1 1 0 0 1 1 1 0 0 1 1 1 0 7Vertebrata lanosa* 1 1 0 0 1 1 0 1 1 2 0 0 1 1 1 2 1 0 0 1 1 3 2 1 2 1 1 0 10i, 16i

Womersleyella setacea 1 1 0 1 1 1 1 0 1 1 1 1 1 1 0 ? ? ? ? ? ? 3 2 1 2 1 1 0 8, 15d, 23j

Incertae sedisPlatysiphonia victoriae 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

a1, 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).

bAs Dasya elegans.cAs Delesseria violacea.dInformation at generic level, not specific to the species used in our analyses.eAs Carposiphonia japonica.fCarposiphonia savatieri.gDisplay both states 2 and 3 for character 22.hCharacter 12 is variable in P. nigra.iAs Polysiphonia lanosa, character 15 is usually state 1 but rarely state 0.jAs Polysiphonia setacea.kAs Sarcomenia victoriae.*The type species of the genus.

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



Rhodomelaceae, but relationships among these taxa are equivo-cal. The remainder of the ingroup taxa are allied with moderateto strong support. Among these remaining taxa, Laurencia di-verges early, and two lineages of species are recognized: theAmansieae–Micropeuce–Rhodomela lineage and theMurrayella–Pleurostichidium–Polysiphonia sensu lato lineage.In the latter, Murrayella has diverged at the base of a stronglysupported lineage (100% bootstrap replicates in all analyses)including Enelittosiphonia, Neosiphonia, Pleurostichidium,Polysiphonia, Vertebrata, and Womersleyella. Three strongly(100% replicates) supported groups and two single taxon lin-eages were resolved within this complex: (i) Polysiphoniagroup, consisting of P. stricta, the type species of the genusfrom the north Atlantic, Polysiphonia morrowii from the north-west Pacific and P. pacifica from the northeast Pacific;(ii) Neosiphonia group, including N. japonica from the north-west Pacific (representing also N. savatieri from the northwestPacific and P. harveyi from the north Atlantic with identicalSSU sequences), P. elongata from the north Atlantic, and P.virgata from South Africa; (iii) a “multipericentral” group in-cluding the three genera Boergeseniella, Enelittosiphonia andVertebrata, as well as the multipericentral Polysiphonia spp.,P. fucoides and P. nigra from the north Atlantic;(iv) Pleurostichidium; and (v) Womersleyella. The Neosiphonia

group was strongly (100% replicates in all analyses) allied tothe multipericentral group. The relationships ofPleurostichidium and Womersleyella relative to one anotherand the three groups outlined previously were unresolved.Maximum likelihood, distance, and parsimony trees differed inthe relative positioning of Enelittosiphonia and Vertebratawithin the multipericentral group, and Pleurostichidium andWomersleyella within Polysiphonia sensu lato, but bootstrapsupport was absent in all cases (Fig. 1).

Phylogeny based on anatomical dataUnweighted parsimony of the anatomical characters gener-

ated 47 trees (length = 62, consistency index = 0.484, retentionindex = 0.619), whereas weighted parsimony (13 reproductivecharacters weighted 1.2 to 1 against 15 vegetative characters)resolved a single most parsimonious solution (length = 67, con-sistency index = 0.493, retention index = 0.626). A strict con-sensus tree of the equally parsimonious solutions underunweighted parsimony failed to resolve relationships amongmost of the included taxa (not shown). The single most parsi-monious solution for weighted parsimony was selected for pre-sentation with the distribution of character–state changesappended (Fig. 2). In this tree (Fig. 2) the Neosiphonia group,Polysiphonia group and multipericentral group formed a

© 2001 NRC Canada

1470 Can. J. Bot. Vol. 79, 2001

Delesseria serrulata

Dasya baillouviana

Platysiphonia victoriae

Bostrychia moritziana

Heterocladia australis

Pleurostichid. falkenbergii

Neos. japonica

Polys. elongata

Polys. virgata

Boerg. fruticulosa

Polys. fucoides

Polys. nigra

Vert. lanosa

Enelitt. stimpsonii

Womersleyella setacea

Polys. stricta

Polys. pacifica

Polys. morrowii

Murrayella periclados

Rhodomela confervoides

Micropeuce strobiliferum

Melanamansia mamillaris

Lenormandia prolifera

Lenormandia muelleri

Laurencia filiformis

Sonderella linearis

100/100

99/98

90/88

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<50

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77

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99

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81

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Neosiphonia

group

“Multipericentral”

group

Polysiphonia

group

DELESSERIACEAE

DASYACEAE

Incertae sedis

Bostrychieae

Heterocladieae

Pleurostichidieae

Lophothalieae

Rhodomeleae

Brongniartelleae

Amansieae

Laurencieae

Incertae sedis

Incertae sedis

52/62

<50

Po

lysip

ho

nia

sen

su

lato

Fig. 1. Tree constructed with maximum likelihood for the SSU alignment. Values at branches represent percentage of 100, 2000, and1000 bootstrap replicates for maximum likelihood, distance, and parsimony analyses, respectively. Bold numbers are bootstrap valuesfor the three major lineages within Polysiphonia sensu lato. Branches lacking values received less than 50% support. Scale bar = 0.01substitutions per site.



monophyletic clade, the species of which shared onesynapomorphic feature: tetrasporangia scattered in branches(character 25). The Neosiphonia group was weakly supportedwith an important synapomorphic feature: three-celledcarpogonial branches (character 18). Boergeseniella andVertebrata of the multipericentral group allied to the moder-ately supported Polysiphonia group. The taxa of these twogroups share the vegetative features of oblique division of api-cal cells (character 2) and open connections between rhizoidsand pericentral cells (character 15). The Polysiphonia groupand the genus Vertebrata were allied by a shared absence offive vegetative features: laterals from central-axial cells origi-nating endogenously (character 6), adventitious laterals (char-acter 8), uniseriate determinate branches (character 11),vegetative trichoblasts (character 12) and cortical cells (charac-ter 14), as well as the shared presence of one male reproductivefeature, spermatangial axes borne on trichoblast initials (char-acter 22). The Polysiphonia group was defined by species hav-ing four pericentral cells (character 10) and tetrasporangia instraight series (character 24).

With respect to Polysiphonia sensu lato, the weighted parsi-mony solution for anatomical data (Fig. 2) differed from themolecular tree (Fig. 1) in that the included representatives of

the multipericentral group, the genera Boergeseniella andVertebrata, were not specifically allied and failed to join theNeosiphonia group, but bootstrap support was absent for theformer analysis (Fig. 2).

Phylogeny based on combined SSU–anatomical dataIn a final analysis, combined SSU–anatomical data were

subjected to parsimony in the absence of any weightingscheme 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 generallyechoed the molecular results (Fig. 1) in that Murrayellawas moderately allied to a strongly supported clade ofPleurostichidium and Polysiphonia sensu lato. WithinPolysiphonia sensu lato the Polysiphonia, Neosiphonia, andmultipericentral groups were solidly resolved, the lattertwo groups strongly allied, whereas the affinities ofWomersleyella relative to these three lineages were equivo-cal (Fig. 3). An association between the Neosiphonia andmultipericentral groups was supported by reversals in threeanatomical features; however, these characters changed anumber of times on the combined tree, including withinthis lineage itself (Fig. 3).

© 2001 NRC Canada

Choi et al. 1471




Polysiphonia stricta

Polysiphonia morrowii

Vertebrata lanosa

Boerges. fruticulosa

Neosiphonia savatieri

Neosiphonia japonica

Polysiphonia harveyi

Melanam. mamillaris


Rhod. confervoides

Pleurost. falkenbergii

Sonderella linearis

Outgroup

Bostrychieae


group

Polysiphonia

group

Neosiphonia

group

Heterocladieae

Amansieae

Laurencieae

Rhodomeleae

Pleurostichidieae

Incertae sedis

22

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6614

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1>0

28

0>1

2

0>1

20

1>0

25

1>2

4

0>1

22

1>0

23

0>1

Fig. 2. The single most parsimonious solution for anatomical data with the 13 reproductive characters weighted 1.2 to 1 against 15vegetative characters (weighted parsimony). Bold numbers above the branches indicate bootstrap values (% of 1000 replicates).Branches lacking values received less than 50% support. Numbers above and below the solid and open bars correspond to anatomicalcharacters and character-state changes (Tables 3 and 4), respectively. Solid bars, (syn)apomorphies; open bars, reversals.



All members of the Polysiphonia group share three diag-nostic features: the absence of uniseriate determinatebranches (character 11); open connections between rhizoidsand pericentral cells (character 15); and the formation oftetrasporangia in a straight series (character 24). These fea-tures, however, have evolved in parallel in Polysiphoniavirgata (characters 11 and 24) of the Neosiphonia group andVertebrata (character 15) of the multipericentral group(Fig. 3). Species of the Neosiphonia group share the pres-ence of adventitious laterals (character 8) and cortication(character 14) as well as three-celled carpogonial branches(character 18), except for N. savatieri having an alternativestate for character 14. Characters 8 and 14 have evolved inparallel to the Neosiphonia group in the ancestor toBoergeseniella–Enelittosiphonia and Boergeseniella, respec-tively, within the multipericentral group. All taxa of themultipericentral group share a key diagnostic feature: morethan five pericentral cells (character 10). This feature hasalso evolved in parallel in Polysiphonia virgata, which un-equivocally joined the Neosiphonia group in our molecularand combined analyses.

The alliance of Womersleyella and the three groups men-tioned previously was supported by two tetrasporangial fea-

tures: a single tetrasporangium per axial tier (character 23)and tetrasporangia scattered in the frond (character 25). Therelationships of Pleurostichidium and Womersleyella relativeto one another and the three groups, however, were unre-solved with the combined analysis (Fig. 3), similar to themolecular results (Fig. 1).

Discussion

Polysiphonia sensu lato is currently placed in the tribePolysiphonieae Schmitz (1889) of the subfamilyRhodomeloideae Hommersand (Maggs and Hommersand1993). Similar to previous molecular studies (Phillips 2000;Phillips et al. 2000), our molecular and combined data indi-cate that Pleurostichidium, Pleurostichidieae, is allied toPolysiphonia sensu lato with strong support, andMurrayella, Lophothalieae, diverges at the base of this lin-eage with moderate support. Relationships among the com-ponent species and genera of Polysiphonia sensu lato arecomplex in light of our analyses and we recognize threegroups that are discussed below.

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1472 Can. J. Bot. Vol. 79, 2001

78

9

0>1

11

0>1

12

0>1

16

1>2

22

1>2

DELESSERIACEAE

DASYACEAE

Incertae sedis

Bostrychieae

Heterocladieae

Pleurostichidieae

Incertae sedis

Lophothalieae

Rhodomeleae

Amansieae

Laurencieae

Incertae sedis

Delesseria serrulata

Dasya baillouviana




Pleurost. falkenbergii

Womers. setacea

Polys. stricta

Polys. pacifica

Polys. morrowii

Neos. savatieri

Neos. japonica

Polys. harveyi

Polys. elongata

Polys. virgata

Enelitt. stimps.

Boerg. fruticul.

Vert. lanosa

Polys. nigra

Polys. fucoides

Murrayella periclados

Rhodom. confervoides

Melanam. mamillaris


Sonderella linearis

Polysiphonia

group

Neosiphonia

group


group

1

0>1

3

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2

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6

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5

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10011

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894

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8019

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5622

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8

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12

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0>22

1>026

1>0

20

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22

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25

1>210

0>1

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994

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2

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10014

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97

0>1

11, 24

1>0210

0>2

7

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14

1>0

2

0>110

0>2

4, 12

0>1

6, 8

1>0

15

0>1

22

2>3

99

99

Po

lysip

ho

nia

sen

su

lato

1>0 1>0 1>0 1>0 1>0

7

0>1

22*

2>2,3

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



Type Polysiphonia urceolata (= P. stricta) and additionalspecies of the genus Polysiphonia

In our analyses, the Polysiphonia group includes the typespecies of the genus, Polysiphonia urceolata (= P. stricta)from the north Atlantic, P. morrowii from the northwest Pa-cific, and P. pacifica from the northeast Pacific. These speciesshare the important diagnostic features outlined in Fig. 4, aswell as an absence of uniseriate determinate branches (charac-ter 11). In addition to these three species, the Polysiphoniagroup may include: Polysiphonia atlantica Kapraun et J.N.Norris (cf. Maggs and Hommersand 1993); P. atlantica sensuM.S. Kim and I.K. Lee (1996); Polysiphonia carettiaHollenberg and Polysiphonia decussata Hollenberg (cf.Abbott and Hollenberg 1976); Polysiphonia namibiensisStegenga et Engledow (cf. Stegenga et al. 1997); Polysiphoniapungens Hollenberg (cf. Womersley 1979); Polysiphoniascopulorum Harvey (cf. Abbott and Hollenberg 1976;Stegenga et al. 1997; Abbott 1999); Polysiphonia senticulosaHarvey (cf. Kim et al. 2000); Polysiphonia shepherdii

Womersley and Polysiphonia subtilissima Montagne (cf.Womersley 1979; Abbott 1999); and Polysiphonia tuberosaHollenberg (cf. Abbott 1999).

Multipericentral group including the genus VertebrataThe multipericentral group allied to the Neosiphonia

group in our molecular and combined analyses. This groupincludes species of four recognized genera from the northAtlantic and northwest Pacific: Boergeseniella,Enelittosiphonia, Polysiphonia, and Vertebrata. They sharethe important synapomorphic feature of more than fivepericentral cells (character 10), and the included species var-iously share other key anatomical features with theNeosiphonia and Polysiphonia group (Fig. 4).

Polysiphonia fucoides and P. nigra from the north Atlanticgroup together with strong support in our molecular andcombined analyses, and share, in addition to the several im-portant diagnostic features of the group (Fig. 4), the devel-opment of erect indeterminate branches from the main axes

© 2001 NRC Canada

Choi et al. 1473

i

2

3

14

A D EB

su

1

2

3

4

C

ii4

2

1

36

87

5su

1

2

3

4

ab c

iii2

3

14

su1

2

3

d e f

Fig. 4. Comparison of six selected diagnostic characters between the (i) Polysiphonia, (ii) multipericentral, and (iii) Neosiphoniagroups. A, number of pericentral cells (numbered circles)(character 10) and presence or absence of cortication (smaller circles periph-eral 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 ar-rangement of tetrasporangia (tetrahedrally divided circles)(character 24). a, Boergeseniella is corticated; b, open or rarely separated bya crosswall in Vertebrata lanosa; c, on both the trichoblasts and trichoblast initials in P. fucoides, and on trichoblast initials inVertebrata lanosa; d, cortication absent in N. savatieri, P. virgata has more than four pericentral cells; e, not known for P. elongataand P. virgata of this group; f, except P. virgata (straight series).



(character 4), although none of these specifically ally thesespecies (Fig. 3). The former has spermatangial branches onboth trichoblast initials and trichoblasts (character 22),whereas the latter, as well as Boergeseniella and other mem-bers of the multipericentral group in Britain and Ireland ex-cept for Vertebrata, have spermatangial branches that formonly on the trichoblasts (Fig. 4). These species are clearlydistinct both anatomically and in our molecular analysesfrom Polysiphonia sensu stricto and may require transfer toBoergeseniella (Fig. 1) or may be referred to a separate ge-nus pending further investigation (Fig. 3).

In contrast to other members of the multipericentralgroup, V. lanosa has a unique combination of features: vege-tative trichoblasts are absent (character 12; also true forP. fucoides, and variable for P. nigra); the connections be-tween rhizoids and pericentral cells are open (character 15;rarely separated in Vertebrata); spermatangial branches areformed only on trichoblast initials (character 22); and thespecies is exclusively epiphytic on the brown algaeAscophyllum nodosum (Linnaeus) Le Jolis and Fucusvesiculosus Linnaeus. Owing to this combination of featuresin addition to our molecular analyses, which do not ally thisspecies with Polysiphonia sensu stricto, we support Kylin(1956) and Christensen (1967) in their recognition of the ge-nus Vertebrata.

One approach to dealing with taxonomy in themultipericentral group might be to sink all species into thesingle genus Vertebrata, but a thorough systematic investiga-tion of the genera and species of this group is necessaryprior to formal taxonomic proposals.

Extending Neosiphonia to include P. harveyiIn our combined analyses, a strongly supported

Neosiphonia group included N. japonica and N. savatierifrom the northwest Pacific, P. elongata and P. harveyi fromthe north Atlantic, and P. virgata from South Africa. Thesetaxa are joined by the diagnostic features outlined in Fig. 4,although exceptions occur, and in some cases importantcharacter states are unknown, particularly for P. elongataand P. virgata (Table 4, Fig. 4 caption). In addition, thesetaxa share the presence of adventitious laterals (character 8).

Polysiphonia elongata grows on bedrock, stones, shells,various algae, and occasionally on stipes of Laminariahyperborea (Gunnerus) Foslie (Maggs and Hommersand1993), and shares some key diagnostic features (fourpericentral cells and a spiral series of tetrasporangia; Fig. 4)with Neosiphonia, as well as erect indeterminate branchesdeveloping from the main axis (character 4) and the pres-ence of adventitious laterals (character 8). However,P. elongata clearly allied to P. virgata (an alliance lackingsupport from anatomical features used in the current study)not to the clade including Neosiphonia spp. and P. harveyi inour molecular and combined analyses (Figs. 1 and 3).

The South African endemic species P. virgata, an epiphyteof Ecklonia maxima (Osbeck) Papenfuss and occasionallyLaminaria pallida Greville (Stegenga et al. 1997), has beenvariously assigned to the monotypic genus Carradoriella(Kylin 1956, as Carradoria; Silva et al. 1996) or included inPolysiphonia (Wynne 1986). It shares some key diagnosticfeatures with the multipericentral (12–16 pericentral cells)

and Polysiphonia groups (tetrasporangia in straight series;Fig. 4), but it unequivocally joined the Neosiphonia group,in particular P. elongata, in our molecular and combinedanalyses (Figs. 1 and 3). If the indications of our moleculardata are correct, the newly established Neosiphonia could besubsumed into Carradoriella, but the two may ultimately re-solve as sister genera. It would be premature to make formalproposals until additional molecular data are investigatedand the female and male reproductive features ofP. elongata and P. virgata are clarified.

Polysiphonia harveyi shares the many diagnostic featuresof this group with Neosiphonia (Fig. 4), in addition to form-ing laterals endogenously from central axial cells (character6) and the presence of abundant vegetative unpigmentedtrichoblasts (character 12), and fits comfortably within thisgenus based on anatomical and combined SSU–anatomicalanalyses. Bailey (1848) first described P. harveyi from Con-necticut. This species occupies a wide range of inter- andsub-tidal habitats in both the northwest Pacific and north At-lantic, 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 concludedthat Japan is the centre of diversity and origin for P. harveyibased on rbcL sequences from isolates of P. harveyi and var-ious congeners from the Pacific and north Atlantic.

Taxonomic proposalAs a result of our molecular results and the anatomical

features shared by P. harveyi with Neosiphonia, we proposethe 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 MacAfeefor technical assistance. We thank S.M. Boo, H.S. Yoon, J.Warneboldt, and J. Todd Harper for providing some of thesamples used in this study. This work was supported by aNatural Sciences and Engineering Research Council of Can-ada Grant and Canada Research Chair Program funds toGWS, and Korean Science and Engineering Foundationpostdoctoral fellowships to H-GC and M-SK.

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Phylogenetic relationships of Polysiphonia (Rhodomelaceae, Rhodophyta) and its relatives based on...

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