RESOLVING EVOLUTIONARY RELATIONSHIPS AMONG THE BROWN ALGAE USING CHLOROPLAST AND NUCLEAR GENES

RESOLVING EVOLUTIONARY RELATIONSHIPS AMONG THE BROWN ALGAE USINGCHLOROPLAST AND NUCLEAR GENES1

Naomi Phillips2,3

Centre for Environmental and Molecular Algal Research, University of New Brunswick, Fredericton, New Brunswick E3B 4E1, Canada

Renaud Burrowes, Florence Rousseau, Bruno de Reviers

Departement Systematique et Evolution, UMR 7138 UPMC-MNHN-CNRS-IRD Systematique, adaptation, evolution, Museum national

d’histoire naturelle, 57, Rue Cuvier, CP 39, 75231 Paris cedex 05, France

and Gary W. Saunders

Centre for Environmental and Molecular Algal Research, University of New Brunswick, Fredericton, New Brunswick E3B 4E1, Canada

The brown algae are one of the largest and mostimportant groups of primary producers in benthiccoastal marine environments. Despite their biologi-cal importance, consensus regarding their taxo-nomic or evolutionary relationships remains elusive.Our goal was to produce a taxon-rich two-gene (rbcLand LSU rDNA) phylogeny. Key species weresequenced to represent each order and family in theanalyses across all 19 orders and �40 families,including selected outgroups Schizocladiophyceaeand Xanthophyceae. Our results are in sharp con-trast to traditional phylogenetic concepts; the Ecto-carpales are not an early diverging clade, nor do theFucales diverge early from other brown algae.Rather, Choristocarpus is sister to the remainingbrown algae. Other groups traditionally consideredto have primitive features are actually recentlydiverged lineages, turning traditional phylogeneticconcepts upside down. Additionally, our resultsallow for the assessment, in the broadest context, ofmany of the historical and more recent taxonomicchanges, resulting in several emended groups alongwith proposals for two new orders (Onslowiales,Nemodermatales) and one new family (Phaeosipho-niellaceae).

Key index words: 28S rDNA; brown algae; Fucales;LSU rDNA; marine algae; Phaeophyceae; phylo-genetics; rbcL

Abbreviations: BA, Bayesian analysis; G, gamma;GTR, general-time-reversible model; I, invariablesites; ME, minimum evolution; MP, maximumparsimony; rbcL, large subunit of RUBISCO gene

Brown algae are among one of the most impor-tant groups of primary producers in coastal marine

environments (Mann 1982, Andersen 1992). Accord-ing to de Reviers and Rousseau (1999), there are�2,000 known species in 270 genera and 13–19families. Brown algae exhibit a wide range of mor-phological forms, from simple microscopic filaments(e.g., Streblonema, Ectocarpus) to large and complexparenchymatous plants (e.g., Macrocystis). Economic-ally, brown algae are major sources of food, naturalproducts (e.g., alginic acid), and biomedical sup-plies (Andersen 1992). Despite the fact that brownalgae are critical marine resources (biologically, eco-logically, and economically) and the progress madewith recent molecular work (cf. Draisma et al.2003), consensus in terms of a natural classificationsystem that reflects overall phylogenetic relation-ships remains elusive.

Traditional classification systems for brown algaeare based on four basic features: (i) type of lifecycle, (ii) type of gamy, (iii) mode of growth, and(iv) morphological construction of the thallus (Boldand Wynne 1985, de Reviers and Rousseau 1999).These features have been arbitrarily assigned ances-tral and derived evolutionary states and used to con-struct classification systems that hypotheticallyreflect phylogenetic relationships. In most treat-ments, the ‘‘simple brown algae,’’ or Ectocarpales,represent the ancestral stock from which all otherlineages have evolved (Kylin 1933, Papenfuss 1953,Scagel 1966, Wynne and Loiseaux 1976), with theexception of the Fucales. Most authors consideredthe Fucales a separate lineage that diverged early inthe evolutionary history or were sister to the brownalgae due to their gametic life cycle, which is atypi-cal for the group. However, some authors considerthe Fucales to be a divergent lineage within thebrowns (van den Hoek et al. 1995).

Molecular data offer an alternate means to evalu-ate taxonomic and phylogenetic concepts such asthose of the brown algae. Among the first workers toapply molecular data (18S rDNA) to testsystematic concepts in brown algae were Tan andDruehl (1993, 1994). Although limited in scope,

1Received 14 January 2007. Accepted 25 July 2007.2Author for correspondence: e-mail [email protected] address: Biology Department, Arcadia University,

450 S. Easton Rd., Glenside, PA 19038, USA.

J. Phycol. 44, 394–405 (2008)� 2008 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2008.00473.x

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their work challenged the ancestral placement of the‘‘simple’’ species, indicating that brown algae mayactually fall into two groups: one in which cells have astalked pyrenoid, and the other for taxa lacking orwith only a reduced pyrenoid (Tan and Druehl 1993,1994).

The next publications to evaluate phaeophycean-wide systematic concepts with molecular data werethose of de Reviers and Rousseau (1999) and Rous-seau et al. (2001). These were more comprehensivestudies in terms of taxon sampling, as well as thegenes used (based on partial SSU and LSU rDNAdata), but produced poorly supported phylogeniesfor the majority of included orders owing to the con-servative nature and limited size of the ribosomalregions used. These studies did, however, suggestthat the Dictyotales, along with the Sphacelarialesand Syringodermatales, represent early diverginglineages of brown algae with the remaining ordersclustered into a ‘‘crown radiation’’ suggestive of arapid radiation (Rousseau et al. 2001).

A subsequent investigation used the previouslypublished rDNA (partial SSU and LSU rDNA) andrbcL data, as well as new rbcL and partial LSU rDNAsequences, to investigate these issues further(Draisma et al. 2001). This study resolved the samebasic brown algal tree as earlier studies (Rousseauet al. 2001) with early diverging lineages (i.e., Dic-tyotales) sister to a poorly resolved ‘‘crown radia-tion,’’ while also providing the first evidence thatthe earliest diverging brown alga was not Ectocarpus(or an ectocarpalean member) but instead wasChoristocarpus tenellus (Choristocarpaceae), a speciesvariously placed within the Sphacelariales (Fritsch1945) or considered as distinct from this order(Prud’homme van Reine 1982). The early diver-gence of Choristocarpus has been further verified bya study using chloroplast genes to evaluate the posi-tion of the enigmatic genus Ishige (Cho et al. 2004).This later work established the order Ishigeales andplaced it as another early diverging lineage ofbrown algae. Thus, molecular data, although limitedby resolution and ⁄ or taxon representation, indicatethat many of the orders traditionally consideredancestral (Ectocarpales) are actually derived,whereas some orders traditionally considered to bederived (e.g., Dictyotales) are actually early diver-ging groups.

The aim of this study was to produce a compre-hensive phylogeny based on chloroplast (rbcL) andnuclear (LSU ⁄ 28S) gene regions to evaluate sys-tematic relationships among the brown algae. Ourstudy extends previous work (Tan and Druehl 1993,1994, de Reviers and Rousseau 1999, Draisma et al.2001, 2003, Rousseau et al. 2001, Cho et al. 2004)by using a more comprehensive taxon samplingacross all brown algal orders and families (for rbcLwith 16 taxa added, representing five additionalorders and seven families) and a greater proportionof the LSU rDNA gene (�3,000 bp, as well as 46

new LSU rDNA sequences) to further resolve brownalgal relationships. The resulting topologies wereused to test traditional systematic concepts of theclass, as well as assess current and historical ordinal-and familial-level concepts. Key species wereselected to represent each brown algal order andfamily in the analyses (including types where possi-ble) across all orders, including outgroups from theSchizocladiophyceae and Xanthophyceae.

MATERIALS AND METHODS

Taxa selection and DNA extraction. Taxa were selected torepresent each order and family based on available materialwith type species ⁄ genera used where possible (Table S1, seethe supplementary material). An exemplar was used torepresent Halopteris in the combined analyses (global andlocal) formed by H. filicina for rbcL and H. pseudospicata forLSU. DNA was extracted from either fresh or silica-preservedmaterial as in Phillips et al. (2001) or Lane et al. (2006).

PCR and sequencing. PCR reactions for both the chloroplastencoded rbcL and nuclear LSU (28S rDNA) gene regions wereaccomplished in either a 2400 GeneAmp PCR System (AppliedBiosystems, Foster City, CA, USA) or an Icycler (Bio-RadLaboratories, Hercules, CA, USA) using the high fidelityTakara Ex-Taq DNA polymerase kit (Pan Vera, Madison, WI,USA) according to the manufacturer’s specifications. PCRconditions and primers for LSU rDNA were as described inHarper and Saunders (2001) with slight modifications (Laneet al. 2006). For rbcL, primers and PCR conditions followedLane et al. (2006) or Phillips et al. (2005).

Global and local gene alignments. Sequences were assembledusing the program Sequencher (Gene Codes Corp., AnnArbor, MI, USA) and aligned by eye and compiled into thevarious alignments with the program SeqPup (Gilbert 1995). Inaddition to the data determined here, sequences were acquiredfrom GenBank (Table S1). Representatives from the Schizo-cladiophyceae (the closest heterokont lineage to the brownalgae) and the Xanthophyceae (see Bailey et al. 1998, Kawaiet al. 2003) were employed as outgroups in global alignments.However, these groups are divergent in relationship to brownalgae, potentially causing saturation (rbcL) and alignmentissues (LSU rDNA). Thus, the global alignments were used toestablish deep relationships among the brown lineages, withsubsequent removal of the outgroups to generate localalignments to facilitate phylogenetic analyses within the class.Seven different alignments were constructed—five single geneand two combined: (i) global rbcL alignment, including allthree codon positions, 1,467 bp, 73 brown algae, plus theoutgroup Schizocladiophyceae; (ii) global rbcL alignment,including only the first and second codon positions, 984 bp,73 brown algae, and the outgroup Schizocladiophyceae;(iii) local rbcL alignment, including all three codon positions,1,368 bp (alignment truncated owing to missing data in somegroups), and 64 taxa (excluding the Dictyotales, Discospor-angiales, and Schizocladiophyceae); (iv) global LSU rDNAalignment, including 2,517 bp (excluding ambiguously alignedregions totaling �182 bp), 58 brown algae, and two Xantho-phyceae; (v) local LSU rDNA alignment, including 2,621 bp(excluding ambiguously aligned regions totaling �134 bp), 49taxa (excluding the Dictyotales, Discosporangiales, andXanthophyceae); (vi) global rbcL ⁄ LSU combined alignmentcomprising 3,419 bp, 54 brown algae, and two Xanthophyceae;(vii) local rbcL ⁄ LSU combined alignment comprising 3,989 bp,47 taxa (excluding the Dictyotales, Discosporangiales, andXanthophyceae).

Gene saturation and congruency. The chloroplast-encodedrbcL was partitioned by codon, and saturation levels were

PHYLOGENY OF THE PHAEOPHYCEAE 395

investigated by comparing uncorrected distances (p) toKimura-2-parameter and Modeltest corrected values (Kimura1980, Posada and Crandall 1998).

Congruency between the rbcL and LSU rDNA gene regionsin our two concatenated alignments was evaluated with thePartition Homogeneity Test (or Incongruence Length Differ-ential Test, ILD, Farris et al. 1995) using the program PAUP*version 4.0 (Swofford 2002). Invariant sites were first removedas suggested by Cunningham (1997), and 1,000 runs (10replicate sequence additions) were completed using theheuristic search option.

Phylogenetic analyses. Three methods of phylogenetic recon-struction were employed to explore evolutionary relationshipsamong the various lineages of brown algae: maximumparsimony (MP), minimum evolution (ME), and Bayesiananalysis (BA). MP and ME were accomplished using PAUP*; BAwas carried out using MrBayes 3.1 (Huelsenbeck and Ronquist2003). Hierarchical likelihood ratio tests were performed usingModeltest version 3.0 (Posada and Crandall 1998) to determineappropriate likelihood model parameters for the ME analyses.MP and ME analyses were completed using 100 heuristic searchreplicates, gaps treated as missing data, and tree-bisection-reconnection (TBR). Bootstrap values (Felsenstein 1985) forboth MP and ME were obtained using 2,000 replicates (10random sequence additions) under the heuristic searchoption. Bayesian analyses were completed using theGTR + I + G model. Alignments were partitioned by codonand by gene with model parameters estimated independentlyfor each partition using the unlink command. Analyses wererun in 106 generation (four chains, with trees sampled every100 generations) increments until a consensus was reachedbetween the two parallel runs (i.e., when the standard deviationof split frequencies reaches �0, Huelsenbeck and Ronquist2003). The runs were then extended for another 106

generations. Consensus trees and posterior probabilities werecalculated excluding the ‘‘burn in’’ trees as determined by theconsensus point of the two parallel runs.

RESULTS

Global analyses. Global analysis with the full rbcLalignment showed significant divergences (5- to 10-fold) between uncorrected distances (p) and Kimura-2-parameter and Modeltest values for the third codonposition indicating saturation, whereas divergenceswere minimal for the first and second codon posi-tions. Consequently, the third codon position wasexcluded in the second rbcL alignment, leaving 984characters with 348 variable characters, of which 153were parsimony informative. According to Modeltest,a GTR+ I + G model was the best fit for the data(note, GTR+ I + G was the best fit for all the align-ments). All three methods of phylogenetic inference(MP, ME, BA) resulted in nearly identical trees for allwell-supported nodes; thus, the Bayesian tree isdepicted with support (Fig. 1).

The first brown algal lineage to diverge wasC. tenellus (for taxonomic authors, see Table S1;Choristocarpaceae, Discosporangiales), followed bythe Ishigeales, and, finally, the remaining brownalgae clustered into two poorly supported clades: theSphacelariales, Syringodermatales, Dictyotales, Ons-lowiaceae, and Phaeostrophionaceae ⁄Bodanella (S ⁄ S ⁄D ⁄ O ⁄ P) lineage; and the crown group clade (CGC),containing the remaining brown algal orders. There

was support for monophyly of some lineages withinthe CGC and S ⁄ S ⁄ D ⁄ O ⁄ P groups, but little resolu-tion of relationships among them (Fig. 1).

Analyses of the global LSU rDNA alignmentincluded 720 variable positions, of which 532 wereparsimony informative. The LSU rDNA dataresolved the same basic topology as the rbcL tree forthe included groups (no data were available for theIshigeales, Onslowiaceae, Phaeostrophionaceae, andBodanella) and will be discussed in more detail inthe combined analyses below (Fig. S1, see the sup-plementary material).

The Partition Homogeneity Test for the com-bined global rbcL and LSU rDNA suggested that thedata could be combined (P = 0.0012). The com-bined rbcL ⁄ LSU alignment had 1,002 variable sites,of which 623 were parsimony informative. Modeltestindicated that the GTR + I + G model of evolutionbest fit the data. Consistent with the topologies ofthe rbcL and LSU rDNA alignments, the combinedanalyses resolved C. tenellus (Choristocarpaceae, Dis-cosporangiales) as sister to the rest of the brownalgae (Fig. 2). The remaining brown algae (exclu-sive of the Ishigeales) clustered into the two groupsresolved in the single gene analyses. The S ⁄ S ⁄ D hadstrong support for the individual orders, but not forthe branching pattern among them (Fig. 2). Also,note the long branches in the Dictyotales. Likewise,there was a strong support for the CGC cladeand some of the established orders within it (e.g.,Ectocarpales, Fucales), but little resolution of rela-tionships among the orders (Fig. 2).

Local analyses. All three global analyses placedC. tenellus (Choristocarpaceae, Discosporangiales) assister to the other lineages of brown algae (Figs. 1and 2), while the global rbcL analyses additionallyestablished the Ishigeales (although not supported)as the next lineage to diverge. Overall, the branchingorder within the S ⁄ S ⁄ D ⁄ O ⁄ P clade was not resolved,and the monophyly of this group was at best weak inall analyses; however, these taxa were always sister tothe well-supported CGC (Fig. 2), making them anexcellent outgroup for the latter. Thus, to reducesaturation and alignment issues in order to resolverelationships within the CGC, the S ⁄ S ⁄ D clade(exclusive of the Dictyotales, which were removedowing to their long branches) was used to root theCGC clade in a series of ‘‘local’’ analyses.

The local rbcL alignment had 697 variable sites,of which 512 were parsimony informative. The CGCresolved as two groups, with the Desmarestiales awell-supported lineage sister (moderately sup-ported) to a weakly supported clade including theremainder of the CGC taxa (Fig. 3). This lattergroup further divided into two assemblages: a rea-sonably supported Laminariales and Ectocarpalesgroup, with the constituent families well resolved,and a well-supported Sporochnales clade; and a sec-ond grouping of the remaining CGC orders, whichwere poorly resolved except for relationships among

396 NAOMI PHILLIPS ET AL.

the included members of the Fucales. There werealso two notable taxonomic incongruities: inclusionof Phaeosiphoniella cryophila (Tilopteridales) withinthe Laminariales and an association, although withlimited support, of Nemoderma tingitanum (Ralfsiales)as sister to the Fucales.

The local LSU rDNA alignment had 527 variablepositions, of which 360 were parsimony informative.The local LSU rDNA data resolved the same generaltopology as the rbcL tree for the included groups(note: no data were available for the Onslowiaceae,

Phaeostrophionaceae, or Bodanella; Fig. S2 in thesupplementary material).

Similar to the results for the global analyses, thePartition Homogeneity Test for the combined localrbcL and LSU rDNA supported their combination(P = 0.001). The combined alignment had 1,167variable sites, of which 813 were parsimony informa-tive. Overall, the combined analyses resulted inincreased support for the branching order amongmany of the lineages of the CGC (Fig. 4). The CGCgroup clustered into two strongly supported clades:

Fig. 1. Bayesian phylogeny based on the global alignment of rbcL sequences including first and second codons. Numbers abovebranches indicate support from MP, ME, and BA, respectively. Support below 50% is represented by -, while support ‡90% is representedby * for one or more of the values. BA, Bayesian analysis; ME, minimum evolution; MP, maximum parsimony.


the Desmarestiales and a lineage containing theremaining taxa (Fig. 4). The latter clustered intotwo clades: one for the Ectocarpales and Laminar-iales; and a second for the Sporochnales, Scytotham-nales, Tilopteridales, Cutleriales, and the Fucales.There was little resolution among these latterorders, but all were monophyletic, and strong sup-port was recovered for some families of the Fucales.The taxonomic incongruities noted in the single-gene analyses were also evident: the clusteringof P. cryophila (Tilopteridales) with the Laminariales,

and of N. tingitanum (Ralfsiales) as sister to theFucales.

DISCUSSION

Through comprehensive taxon sampling (acrossall orders and most families) and sequencing oftwo genic regions, this study has made significantprogress in addressing systematic issues within oneof the largest classes of benthic primary produ-cers, the brown algae. We were able to minimize

Fig. 2. Bayesian phylogeny based on the global combined rbcL ⁄ LSU alignment. Numbers above branches indicate statistical supportfrom MP, ME, and BA, respectively. Support below 50% is represented by -, while support ‡90% is represented by * for one or more ofthe values. Number sign (#) highlights branch support for nested analyses. D, Dictyotales. BA, Bayesian analysis; ME, minimum evolution;MP, maximum parsimony.


alignment and saturation issues by using globalanalyses to resolve the deeper relationships andusing that knowledge to complete local analysesto assess family- and ordinal-level relationships.Using this approach resulted in the resolutionof several ordinal relationships not recoveredin earlier work (e.g., Rousseau et al. 2001, see

Figs. 3 and 4). Both rbcL and LSU rDNA analysesgave nearly identical topologies for the well-sup-ported clades, and these were further supportedby the combined analyses. Thus, in the broadestcontext, this work allows not only a comprehen-sive evaluation of traditional systematic concepts,but also assessment of many recently proposed

Fig. 3. Bayesian phylogeny based on the local rbcL alignment including all codon positions. Numbers above branches indicate statisti-cal support from MP, ME, and BA, respectively. Support below 50% is represented by -, while support ‡90% is represented by * for one ormore of the values. Boxes around support values highlight nodes with increased statistical support over previous studies (Rousseau et al.2001). A, Ascoseirales; D, Desmarestiales; Du, Durvillaeaceae; E, Ectocarpales; F, Fucales; i, incertae sedis; H, Himanthaliaceae; L, Laminar-iales; N, Notheiaceae; Ne, Nemodermatales; O, Onslowiales; R, Ralfsiales; S, Sargassaceae; Sc, Scytothamnales; Se, Seirococcaceae; Sp,Sporochnales; Spc, Spacelariales; Sy, Syringodermatales; T, Tilopteridales. BA, Bayesian analysis; ME, minimum evolution; MP, maximumparsimony.


taxonomic changes among and within the variouslineages.

Consistent with previous results (Draisma et al.2001, 2003, Burrowes et al. 2003, Cho et al. 2004),our global analyses (separate and combined) estab-lished that Choristocarpus (Choristocarpaceae, Discos-

porangiales) is a distant sister to the remainingbrown algal taxa. This result stands in sharp contrastto prevailing traditional hypotheses where the Ecto-carpales are proposed as the basal stock, and theFucales a distant sister lineage (Kylin 1933, Papen-fuss 1953, Scagel 1966, Wynne and Loiseaux 1976).

Fig. 4. Bayesian phylogeny based on the local combined rbcL ⁄ LSU alignment. Numbers above branches indicate statistical supportfrom MP, ME, and BA, respectively. Support below 50% is represented by -, while support ‡90% is represented by *. Boxes around supportvalues highlight nodes with increased statistical support over previous studies (Rousseau et al. 2001). A, Ascoseirales; D, Desmarestiales; E,Ectocarpales; H, Hormosiraceae; i, incertae sedis; L, Laminariales; Ne, Nemodermatales; S, Sargassaceae; Sc, Scytothamnales; Sp, Sporoch-nales; Spc, Spacelariales; Sy, Syringodermatales; T, Tilopteridales; X, Xiphophoraceae. BA, Bayesian analysis; ME, minimum evolution; MP,maximum parsimony.


However, with the exception of apical growth, Chor-istocarpus does fit the traditional concept of plesio-morphic characters attributed to the brown algalancestral stock because of its filamentous habit andlikely isomorphic life history (Kylin 1933, Papenfuss1953, Scagel 1966, Wynne and Loiseaux 1976).

This work clearly establishes that Choristocarpus(Choristocarpaceae, Discosporangiales) represents aseparate lineage only distantly related to members ofthe Sphacelariales and supports its removal from thisorder. Fritsch (1945) included Choristocarpus in theSphacelariales on the basis of its apical growth, simi-larity with the genus Sphacella (a uniseriate exceptionamong Sphacelariales), and the presence of vegeta-tive propagules resembling early stages of these struc-tures for Sphacelaria tribuloides. Later, Prud’hommevan Reine (1982) argued against the inclusion ofChoristocarpus within the Sphacelariales based onmorphological incongruities and the negative bleachresponse. However, Draisma et al. (2002) determinedthat the simple structure of Sphacella was the result ofa secondary reduction and not homologous to Choris-tocarpus. Consequently, on the basis of morphologicaland molecular grounds, Draisma et al. (2002) sug-gested Choristocarpus should be placed in a new order.Recent rbcL and partial 18S rDNA sequence dataplace Choristocarpus in a clade with Discosporangiummesarthrocarpum at the base of the brown algal tree inthe recently amended Discosporangiales (Kawai et al.2007). This placement is supported by our data.

The next lineage resolved in our phylogenies(rbcL) was the recently described Ishigeales (Choet al. 2004), which supports recognition of thisorder as a monophyletic entity and its placementamong the early lineages of brown algae. Althoughconsistent with earlier results (de Reviers and Rous-seau 1999, Draisma et al. 2001, Cho et al. 2004),little more can be inferred about the relationshipsamong these deeply divergent orders due to poorresolution.

The remaining brown algae resolve into twogroups: the S ⁄ S ⁄ D ⁄ O ⁄ P group (Syringodermatales,Sphacelariales, Dictyotales, Onslowiaceae [LSUrDNA data lacking], Phaeostrophionaceae ⁄ Bodanella[LSU rDNA data lacking]) and the crown group(remaining browns). As mentioned previously, littlemore can be stated about the branching orderamong the S ⁄ S ⁄ D ⁄ O ⁄ P group because data have yetto resolve relationships among these lineages. How-ever, the orders of the S ⁄ S ⁄ D ⁄ O ⁄ P cluster are them-selves monophyletic. In support of this evolutionaryalliance, representatives of all four orders(S ⁄ S ⁄ D ⁄ O) share apical growth, have cells withnumerous plastids lacking pyrenoids, and are char-acterized by polystichous construction. Members ofthe Syringodermatales were included in the Dictyo-tales prior to Henry (1984) establishing a separateorder.

In terms of taxonomy, inclusion of Onslowiaand Verosphacela in the Sphacelariales results in a

paraphyletic assemblage (Fig. 3). This incongruitywas first noted by Draisma et al. (2001) and waslater confirmed by Cho et al. (2004). Draisma andPrud’homme van Reine (2001) removed Onslowiaand Verosphacela from the Choristocarpaceae andestablished the Onslowiaceae to accommodate thesetwo genera, but left the family incertae sedis, sug-gesting that a new order was necessary (Draismaet al. 2003). The molecular results presented here,along with the works of Draisma et al. (2001, 2003)and Cho et al. (2004), clearly establish that theOnslowiaceae warrants ordinal status. A diagnosisfor the new order Onslowiales is provided below.

Earlier studies were unsuccessful in resolvingordinal relationships among the crown grouplineages and concluded that this result was probablydue to a rapid radiation event leading to theselineages (Draisma et al. 2001, Rousseau et al. 2001).Our research suggests that at least some of this poorresolution was probably due to a combination ofoutgroup artifacts and limitations of the generegions used. Consequently, by rooting the treeinternally in our local analyses, we were able toincrease resolution of the branching order amongseveral lineages of the crown group. In our analyses,the Desmarestiales resolved as a monophyletic line-age sister to the remaining taxa of the CGC. Tradi-tional classifications have considered theDesmarestiales and Sporochnales as being closeallies and have even included these lineages in a sin-gle order (Russell and Fletcher 1975, Clayton 1981)owing to their similar mode of construction. Ourdata do not support this close association. Membersof the Desmarestiales have uniaxial filamentous con-struction with a bidirectional intercalary meristem(see Fritsch 1945). This mode of growth may wellbe homologous to the more complex multifacialmeristems of the Laminariales, supporting the Des-marestiales as a potential evolutionary intermediaryconsistent with its position in the molecular trees.

The remaining taxa group either with the Lami-nariales and Ectocarpales, or with the Sporochnalesto Fucales clade (Fig. 4). From a molecular stand-point, there has been an established associationbetween the former two orders (Tan and Druehl1993, 1994). Our analyses are consistent with theseearlier data. In contrast, traditional classificationschemes place the Ectocarpales as an ‘‘ancestral’’ or‘‘primitive’’ group because of their simple construc-tion and isomorphic life histories, whereas thekelps, with their complex thalli and heteromorphiclife histories, were considered derived. The twoorders are nonetheless sister lineages in the molecu-lar trees, indicating that ‘‘simple’’ construction andlife histories in the Ectocarpales are possibly derivedrather than ancestral states.

In terms of the circumscription of the Ectocar-pales, our analyses support removal of taxa with stel-late plastids with immersed pyrenoids and their(excluding Asterocladon and Bachelotia) inclusion in


the order Scytothamnales (Peters and Clayton1998). Our analyses also support a new definitionfor the Ectocarpales, to include only taxa with anexerted pedunculated pyrenoid and isogamy or ani-sogamy (Siemer et al. 1998, Rousseau and deReviers 1999a). Other studies have also supportedthis new definition (excluding taxa such as Astero-nema [Scytothamnales, Muller et al. 1998], Ishige[Ishigeales, Cho et al. 2004], Stschapovia [Tilopteri-dales, Kawai and Sasaki 2004], Phaeostrophion[Phaeostrophionaceae, Kawai et al. 2005], andAsterocladon [Incertae sedis, Uwai et al. 2005]).Currently, five lineages are resolved within theEctocarpales (Figs. 1 and 3), consistent with thesuggestion of Peters and Ramırez (2001) to collapsethe numerous ectocarpalean families to five: theAcinetosporaceae, Adenocystaceae, Chordariaceae,Ectocarpaceae, and Scytosiphonaceae.

In terms of the Laminariales, our data confirmthe suggestion of Sasaki et al. (2001) to removethe Halosiphonaceae and Phyllariaceae from theLaminariales to a broader Tilopteridales. Theremaining Laminariales form a monophyletic line-age composed of two clades: the Pseudochordaceaeand Akkesiphycaceae, and the Chordaceae and theALL (Alariaceae, Costariaceae, Laminariaceae, andLessoniaceae). Interestingly, P. cryophila clusterswith the kelps rather than with taxa of the Tilop-teridales where it is currently classified. This alga isa late winter ⁄ early summer annual endemic toNewfoundland. It was first described by Hooperet al. (1988) and placed as a third genus of theTilopteridaceae (Tilopteridales sensu stricto)because of its morphological similarity to Haplos-pora. Previous rbcL results (Sasaki et al. 2001)placed P. cryophila in the Tilopteridales, but thissequence was probably an isolate of Haplosporarather than P. cryophila (H. Kawai, pers. comm.).Our results clearly place P. cryophila within, or atleast closely related to, the Laminariales, and cer-tainly not associated with the Tilopteridales. More-over, in terms of modes of growth, there are somepotential affinities between Phaeosiphoniella and theLaminariales. Phaeosiphoniella has intercalarygrowth, and the Laminariales have an intercalarymeristem possibly derived from intercalary growth.de Reviers and Rousseau (1999) and Rousseauet al. (2001) also pointed out the polyphyly of theTilopteridaceae, but the position of Phaeosiphoniellain the crown group was far from clear. The mor-phology and reproduction of P. cryophila is differ-ent from other taxa in the Ectocarpales andLaminariales, and, regardless of its ultimate ordinalaffinities, it deserves assignment to its own family.A diagnosis for Phaeosiphoniellaceae fam. nov. isprovided below.

While the included Sporochnales and Scytotham-nales resolve as monophyletic lineages, and theAscoseirales and Bachelotia antillarum are indepen-dent and distinct lines, their relationships to other

crown group lineages were not resolved; thus, littlemore can be stated about their phylogenetic associa-tions until further data are generated. In terms ofthe Tilopteridaceae, Phyllariaceae, and Cutleriaceae,our analyses clearly establish that the Cutleriales(Cutleriaceae) diverge from within the Tilopteri-dales (Tilopteridaceae and Phyllariaceae). This find-ing is not a new observation (Rousseau et al. 2001),but this association has not been well supportedstatistically until now. Both the Tilopteridales andCutleriales were established by Bessey (1907); thus,neither ordinal name has clear taxonomic prece-dence. However, the name Tilopteridales has beenwidely applied to this group. Thus, to minimizetaxonomic confusion and provide consistent taxo-nomic treatment, we choose Tilopteridales overCutleriales for this assemblage.

The final clade resolved here includes theFucales and its alliance with N. tingitanum (Ralf-siales). This clade is generally well supported in allour analyses. Our work represents one of the mostcomprehensive analyses for this assemblage, includ-ing two divergent gene systems and representativesof all of the putative orders and families, allowingfor a more reliable assessment of taxonomic issues.In traditional concepts, the Cystoseiraceae are con-sidered the ancestral family, whereas the Fucaceaeare regarded as the most derived (Nizamuddin1962, Clayton 1984). In sharp contrast to thistraditional concept, our data suggest that theSeirococcaceae diverged from an ancestor in com-mon with all of the remaining families, whereasboth the Cystoseiraceae (along with the Sargassa-ceae) and Fucaceae are relatively recently derivedfamilies.

The present results support the merging of theDurvillaeales and Notheiales within a broad con-cept of the Fucales, as well as the merger of theCystoseiraceae with the Sargassaceae, as suggestedby Rousseau and de Reviers (1999b). Xiphophorachondrophylla does not group with the Fucaceaewith which it is generally classified, but ratherresolves as a sister taxon of Hormosira banksii (Hor-mosiraceae) and the Fucaceae. A similar result wasobserved in Rousseau and de Reviers (1999b), withthe suggestion that X. chondrophylla be placed intoa new monogeneric family. Xiphopora is the onlyaustral member of the Fucaceae, and its spermato-zoids have many ultrastructural and morphologicaldifferences from boreal members of this family(Moestrup 1982, Clayton 1990). These differencesinclude differential lengths of the anterior andposterior flagella, a spine on the anterior of theposterior flagellum, and the lack of a proboscis. Inthese characteristics, Xiphophora has more similari-ties with Hormosira and Himanthalia. Additionally,altritol is present in Xiphophora, Hormosira,Himanthalia, and Bifurcariopsis, but not in theFucaceae (Chudek et al. 1984). On the basis of mor-phological differences and the molecular evidence,


we support the placement of Xiphophora into a newmonogeneric family, Xiphophoraceae, as proposedby Cho et al. (2006).

N. tingitanum is a small crustose brown alga(Kuckuck 1912) common in (sub) tropical regionsof the Atlantic Ocean and Mediterranean Sea(Ballesteros 1981, John et al. 2004). It is currentlyclassified in the Nemodermataceae, which, alongwith two other families of crustose brown algae,Ralfsiaceae and Lithodermataceae, is included inthe Ralfsiales (Nakamura 1972). Nemoderma differsfrom the Ralfsiaceae in having multiple plastidswithout pyrenoids, intercalary unilocular reproduc-tive organs, and an isomorphic life cycle withmarked anisogamy (Kuckuck 1912), rather than asingle plate-like plastid (lacking pyrenoids), term-inal unilocular reproductive organs, and a directlife cycle. With the Fucales it shares multiple plas-tids lacking pyrenoids. On the basis of our molecu-lar analyses and the anatomical features mentionedabove, we remove the monotypic family Nemoder-mataceae Feldmann (Feldmann 1937) from theRalfsiales to the Nemodermatales ord. nov. Unfor-tunately, insufficient data exist presently to evaluatethe placement of other taxa within the Ralfsiales,but further research into this issue should providefuture insights.

TAXONOMIC RECOMMENDATIONS

Onslowiales ord. nov. Draisma et Prud’hommeMarinae fuscae algae (Phaeophyceae) cum oligos-

tichis, irregulariter ramosis thallis et isomorphovitae cyclo. Cum prominentibus apicalibus celluliscrescentes; subapicales cellulae sine transversalespartes sunt. Cellulae cum numerosis rotundatis plas-tibus sine pyrenoides. Ramuli et reproductivaepartes latere thalli cellulis exorientes. Tres variaereproductivae partes sunt: uniloculata sporangia,pluriloculata sporangia, et vegetativae propapulaesine mediam apicalem cellulam.

Typa familia: Onslowiaceae Draisma et Prud’hommevan Reine 2001, J. Phycol. 37:648.

Marine phaeophytes with oligostichous, irregu-larly branched thalli and an isomorphic life cycle.Growth by a prominent apical cell without trans-verse division of subapical cells. Each cell withnumerous discoid chloroplasts without pyrenoids.Branches and reproductive structures arise laterallyfrom thallus cells. Three kinds of reproductive struc-tures, that is, unilocular sporangia, plurilocular spor-angia, and vegetative propagules without a centralapical cell.

Type family: Onslowiaceae Draisma et Prud’hommevan Reine 2001, J. Phycol. 37:648.

Phaeosiphoniellaceae fam. nov.Thalli filamentosi, irregulariter ramosi, in

partibus inferioribus multiseriati et intercalaribuscrescentes. Cellulae cum numerosis discoideischloroplastis sine pyrenoides. Reproductivae partes

intercalaria, pluriloculata sporangia, antheridia etoogonia sunt. Gamia incognita.

Genus typus: Phaeosiphoniella R. G. Hooper, E. C.Henry et Kuhlenkamp 1988: 395, 397, figs. 1–15,Phycologia 27(3):395–404.

Thalli made of irregularly branched filamentswith subapical intercalary growth, becoming multi-seriate in lower parts. Cells with numerous discoidplastids lacking pyrenoids. Reproductive structuresintercalary, made of plurilocular sporangia, as wellas oogonia and antheridia.

Type genus: Phaeosiphoniella R. G. Hooper, E. C.Henry et Kuhlenkamp 1988: 395, 397, figs. 1–15.This monotypic genus presently contains onlyP. cryophila R. G. Hooper, E. C. Henry et Kuhlen-kamp.

Nemodermatales ord. nov. M. Parente, R. A.Fletcher, F. Rousseau et N. Phillips

Marinae crustaceae fuscae algae (Phaeophyceae)cum uniseriatis heterotrichis filamentis, et ismorphovitae cyclo cum anisogamia. Cellulae cum numerosiselongatis discoideis chloroplastis sine pyrenoides.Lateralia pluriloculata et intercalairia uniloculatareproductivae partes.

Typa familia: Nemodermataceae Kuckuck 1912:146 ex J. Feldmann 1937:261, Les algues marines dela cote des Alberes. I-III, Cyanophycees, Chloro-phycees, Pheophycees. Revue Algologique 9:149–335.

Marine phaeophytes with encrusting heterotri-chous thalli and an isomorphic life cycle with aniso-gamy. Each cell with numerous discoid chloroplastswithout pyrenoids. Lateral plurilocular and interca-lary unilocular reproductive structures.

Type family: Nemodermataceae Kuckuck 1912:146ex J. Feldmann 1937:261 [121 in a book-form withseparate pagination], Les algues marines de la cotedes Alberes. I-III, Cyanophycees, Chlorophycees,Pheophycees. Revue Algologique 9:149–335. Thisfamily presently contains only the monotypic genusNemoderma with N. tingitanum Schousboe.

This study was supported by a National Science FoundationInternational Postdoctoral fellowship to N. P., NaturalSciences and Engineering Research Council of Canada andCanada Research Chair Program grants to G. W. S., as wellas infrastructure support from the Canada Foundation forInnovation and the New Brunswick Innovation Foundation.R. Burrowes’ research was supported by a grant by theMinistere de l’education nationale, de la Recherche et de latechnologie. The authors would like to thank colleagues whosupplied taxa for this work: S. Fredericq, G. Kraft, C. Hunter,S. Obrebski, and J. Smith. We are honored to have the ordi-nal designation of S. Draisma and W. Prud’homme van Reinepublished here. Technical support for R. B. was provided bythe Service de systematique moleculaire of the MNHN. Weare grateful for the technical assistance and kelp sequencesprovided by Chris Lane. We thank Denis Lamy for editingthe Latin diagnoses.

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Supplementary Material

The following supplementary material is avail-able for this article:

Table S1. Taxonomic details, location informa-tion, and GenBank accession numbers.

Figure S1. Bayesian phylogeny based on theglobal alignment of LSU rDNA sequences.

Figure S2. Bayesian phylogeny based on thelocal LSU rDNA alignment.

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RESOLVING EVOLUTIONARY RELATIONSHIPS AMONG THE BROWN ALGAE USING CHLOROPLAST AND NUCLEAR GENES