807J. Phycol. 38, 807–820 (2002)A PROPOSAL FOR A NEW RED ALGAL ORDER, THE THOREALES1Kirsten M. MüllerDepartment of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1Alison R. SherwoodDepartment of Botany, 3190 Maile Way, University of Hawaii, Honolulu, HI 96822, USACurt M. PueschelDepartment of Biological Sciences, State University of New York at Binghamton, Binghamton, New York, 13901, USARobin R. GutellInstitute for Cellular and Molecular Biology, Section of Integrative Biology, University of Texas at Austin, Austin, Texas, 78712, USAandRobert G. Sheath2Provost’s Office, California State University San Marcos, San Marcos, California 92096, USARepresentatives of the freshwater red algal familyThoreaceae were studied to resolve their taxonomicand phylogenetic status. Three specimens of Nema-lionopsis and five collections of Thorea were exam-ined for pit plug ultrastructure and analyzed for thesequences of the genes coding for the large subunitof RUBISCO (rbcL) and the small subunit of rRNA(18S rRNA). The phylogenetic trees generated fromthe two genes, and a combined tree all showed theThoreaceae to be contained in a well-supportedmonophyletic clade that is separate from the othertwo families currently classified in the Batrachosper-males, the Batrachospermaceae and the Lemanea-ceae. In addition, secondary structure elements ofthe 18S rRNA gene were observed at positions 650and 1145 (Escherichia coli numbering system) that arenot present in other members of the Rhodophyta.The pit plugs of the gametophytic and chantransiastages of the Thoreaceae contain two cap layers, theouter one of which is typically plate-like, though oc-casionally inflated ones have been seen. No pit plugcap membrane has been observed. These findingsindicate the Thoreaceae has been misclassified inthe Batrachospermales and should be placed in itsown order, the Thoreales. This order is character-ized by having freshwater representatives with multi-axial gametophytes, a uniaxial chantransia stage, andpit plugs with two cap layers, the outer one of whichis usually plate-like.Key index words: 18S rRNA gene; Batrachospermales;Nemalionopsis; rbcL gene; Thorea Thoreales ord. nov.The freshwater red algal genera Thorea and Nemalion-opsis are currently classified in the family Thoreaceaeof the Order Batrachospermales (Sheath et al. 1993,Pueschel et al. 1995, Necchi and Zucchi 1997, Entwisleand Foard 1999). This family is delineated from otherfreshwater Rhodophyta by having gametophytes that aremultiaxial, with a colorless central medulla and determi-nate lateral assimilatory filaments (Starmach 1977). Theother families of the order, the Batrachospermaceaeand Lemanaceae, have uniaxial gametophytes (Sheath1984). The thoreacean genera are largely separatedfrom each other based on the positioning of the mono-sporangia. In Thorea they are on short branches andhence are axial in their position (Sheath et al. 1993); inNemalionopsis they are at the tips of assimilatory fila-ments, localized in the thallus periphery.Our main objective is to resolve the taxonomic andphylogenetic relationships of the Thoreaceae to otherred algal lineages. In particular, it is necessary to deter-mine whether this family is properly classified in theBatrachospermales because both ultrastructural andDNA sequence analyses have raised doubt about its tax-onomic status. For example, Schnepf (1992) notedthat Thorea hispida (Thore) Desvaux (as T. ramosissimaBory de Saint-Vincent) contained pit plugs with a dis-tinct plate-like outer cap layer. This observation differsfrom those of Lee (1971) and Pueschel (1989) for Tho-rea violacea Bory de Saint-Vincent (as T. riekei Bischoff)in which the outer pit plug cap layer is domed, a char-acteristic that is used to distinguish the Batrachosper-males. The Batrachospermales are also delineated byhaving pit plugs with no cap membrane (Pueschel1994); however, this feature has not been observed inThorea or Nemalionopsis. Hence, observations of pitplugs of the Thoreaceae using freeze substitutionmethods would be helpful in resolving the classifica-tion of this family. In terms of molecular data, Vis et al.(1998) demonstrated, using sequence analysis of the1Received 20 March 2001. Accepted 1 May 2002.2Author for correspondence: e-mail firstname.lastname@example.org.
808 KIRSTEN M. MÜLLER ET AL.large subunit of RUBISCO (rbcL) and small subunit ofrRNA (18S rRNA) genes, that Thorea violacea is posi-tioned on a branch separate from other members ofthe Batrachospermales, a result that is well supportedby bootstrap resampling in the combined gene tree.These authors concluded that Thorea should be re-moved from the Batrachospermales and should benoted as incertae sedis until further studies of the Tho-reaceae could be undertaken. Subsequent molecularanalyses using rbcL and 18S rRNA genes supported theconclusions of Vis et al. (1998) (e.g. Acrochaetiales:Harper and Saunders 1998, Balbianiales: Sheath andMüller 1999). Clearly more representatives of the Tho-reaceae need to be examined for pit plug ultrastruc-ture and sequence analyses of the rbcL and 18S rRNAgenes before the phylogenetic status of the family canbe conclusively determined. The present work has at-tempted to add as many samples as available for eachgenus. A secondary objective of this investigation is toascertain whether the separation of Nemalionopsis andThorea, based primarily on the positioning of themonosporangia, is supported by molecular analyses ofthe rbcL and 18S rRNA genes. A third goal is to deter-mine if the Nemalionopsis and Thorea species used in thisstudy are supported as being distinct taxa in moleculartrees of the rbcL and 18S rRNA genes.materials and methodsSamples of Thorea and Nemalionopsis were obtained for studyas follows:1. Culture Collection of Algae at the University of Göttingen(SAG):• 41.94 Nemalionopsis shawii Skuja f. caroliniana Howard etParker, Lower Barton Creek, Wake Co., NC, USA, iso-lated by F. D. Ott, 1973.• 42.94 Nemalionopsis tortuosa Yagi et Yoneda, Hanabusa atKikuchi-City, Kumamota Prefecture, Japan, isolated by F.D. Ott, 1973 (SAG).• 46.94 Thorea hispida (as T. ramosissima), River Thames atCookham, England, collected by D.M. John, isolated byF. D. Ott, 1992.• 45.94 Thorea violacea (as T. okadai Yamada), Kikuchi River,Yamaga City, Kumamota, Prefecture, Japan, isolated by F.D. Ott, 1973 (SAG 1).• 47.94 Thorea violacea (as T. riekei), New Braunfels, Texas,USA, isolated by F. D. Ott, 1973 (SAG 2).2. Field collections:• Nemalionopsis tortuosa, Tangipahoa River in Kentwood, Loui-siana, USA, 24.iv.2000, collected by R. G. Sheath (LA).• Thorea violacea, Hudson River near Thompson Island, Sa-ratoga County, New York State, USA, 29.ix.1994,29.x.1996 collected by C. M. Pueschel (NY).• Thorea violacea, San Marcos River, San Marcos, Texas,USA, 1.xii.1994, collected by R. G. Sheath (TX7b).Samples for LM and EM were fixed and processed as previ-ously described (e.g. Sheath and Müller 1999). To determinewhether a cap membrane is present in the pit plugs, the freezesubstitution method of Pueschel (1994) was followed.Some sequences of both the rbcL and 18S rRNA genes ofThorea and Nemalionopsis were obtained; GenBank accessionnumbers are noted in Table 1. In addition, samples wereground in liquid nitrogen, and the DNA was extracted either bythe protocol outlined by Saunders (1993) with modificationsgiven in Vis and Sheath (1997) or using the Qiagen DneasyPlant Mini Kit (Qiagen, Mississauga, Canada). The primer com-bination F160 (5Ј-CCT CAA CCA GGA GTA GAT CC-3Ј) andrbcLR (5Ј-ACA TTT GCT GTT GGA GTC TC-3Ј) was used toamplify a 1272-base pair fragment of the 1467-base pair rbcLgene (Vis et al. 1998). The reaction volume of 95 L consistedof 2 L of genomic DNA; 20 M each of dATP, dCTP, dGTPand dTTP; 0.4 M of each primer; 2 mM MgCl2; 10 L 10ϫ re-action buffer (Perkin Elmer, Norwalk, CT, USA); and 2 units ofAmplitaq. The 18S rRNA gene was amplified using the primerpair G01 and G07 (Saunders and Kraft 1994). Double-strandedPCR products were amplified in a Perkin Elmer (Norwalk, CT,USA) Gene Amp 2400 Thermal Cycler as follows: initial dena-turation at 95Њ C for 2 min, followed by 35 cycles of denaturationat 93Њ C, primer annealing at 47ЊC (rbcL) or 58Њ C (18S rRNA) for1 min, extension at 72Њ C for 4 min, and a final extension at 72Њ Cfor 6 min. All PCR products were subsequently purified using theQIAquick PCR Purification Kit (Qiagen, Mississauga, Canada) ac-cording to the protocols of the manufacturer. The double-stranded PCR products were sequenced using an ABI PRISM TaqFS Cycle Sequencing Ready Reaction Kit (Applied Biosystems,Foster City, CA, USA) and an ABI 377 Automated DNA Se-quencer. Sequencing reactions were performed using the origi-nal amplification primers as well as internal primers (Table 2).Sequences of the rbcL and 18S rRNA gene sequences wereassembled using DNA sequencing software (ABI). Alignment ofthe rbcL gene was easily accomplished because of the absenceof insertions or deletions. The 18S rRNA gene sequences weremanually aligned using the alignment editor AE2 (developedby T. Macke, see Larsen et al. 1993) on the basis of primarystructure similarity and previously established eukaryotic sec-ondary structure features (Gutell 1993). The aligned databasewas then subjected to a process of comparative sequence analy-sis (Gutell et al. 1985) as follows: 1) searches were conductedfor compensating base changes using computer programs, 2)the resulting information was used to infer additional second-ary structural features which refined the sequence alignment,and 3) the revised alignment was reanalyzed and the entire pro-cess was repeated until the proposed structures were entirelycompatible with the alignment. Secondary structure diagramswere generated interactively with the computer program XRNA(developed by B. Weiser and H. Noller, University of CaliforniaSanta Cruz and are available at http://www.rna.icmb.utexas.edu/).Table 1. GenBank accession numbers, culture, and collection information for members of the Thoreaceae used in this study.Taxon Collection informationGenBank accession numberrbcL 18S rRNANemalionopsis shawii Skuja SAG 41.94 AF506266 AF506272Nemalinonopsis tortuosa Yagi et Yoneda SAG 42.94 AF506267 —Thorea hispida (Thore) Desvaux SAG 46.94 (ϭ Thorea ramosissima) AF506270 AF506273Thorea violacea Bory de Saint Vincent (as T. violacea[SAG1] in figures) SAG 45.94 (ϭ Thorea okadae) AF506269 AF506274Thorea violacea (as T. violacea [SAG2] in figures) SAG 51.94 AF506271 —Thorea violacea (as T. violacea [NY] in figures) New York State, USA, collected by C. M. Pueschel AF506268 AF506275
809NEW RED ALGAL ORDERGenBank accession numbers for previously published red algalsequences used in the analyses are listed in Appendix 1. Mem-bers of the Bangiophycidae were used as outgroups for the phy-logenetic analyses, and the GenBank accession numbers forthese taxa are also given in Appendix 1.Parsimony trees for the rbcL and 18S rRNA genes were gen-erated with the heuristic search option under the conditions ofrandom sequence addition (100 replicates), steepest descent,and tree bisection-reconnection branch swapping using theprogram PAUP (version 4.0b 4a; Swofford 2000). The data werethen subjected to bootstrap resampling (1000 replicates). Anal-yses of the rbcL gene were performed with each base weightedequally as well as weighting first, second, and third positions ofthe codon, following the criteria of Albert et al. (1993), and a par-simony analysis of amino acid sequences was performed. Neigh-bor-joining (NJ) trees (Saitou and Nei 1987) were constructedusing PHYLIP (Phylogeny Inference Package; Felsenstein 1993)from a matrix of distance values estimated according to theKimura two-parameter model (Kimura 1980). A transition/trans-version ratio of 2.0 and a single category substitution rate wereused. The data were also bootstrap resampled (1000 replicates)according to PHYLIP. Number of base pair changes between se-quences was calculated using the program MEGA (MolecularEvolutionary Genetics Analysis; Kumar et al. 1993).Maximum likelihood analysis of the 18S rRNA gene align-ment was carried out using the puzzle function in PAUP (version4.0b 4a) as described by Strimmer and von Haeseler (1996) with1000 puzzling steps. This method applies maximum likelihoodtree reconstruction to all possible quartets that can be formedfrom all sequences that serve as starting points to reconstruct aset of optimal n-taxon trees (Strimmer and von Haeseler 1996).This method has been shown to be equally or better able to re-construct the true tree than NJ methods (Strimmer and von Hae-seler 1996). The values above the branches represent the per-centage of times that a particular cluster was found among the1000 intermediate trees (quartet puzzling step [QPS] values).resultsUltrastructure of pit plugs. We examined in detail thepit plugs of T. violacea (NY) gametophyte and chantran-sia phase, T. violacea (TX 7b) gametophyte, and Nema-lionopsis tortuosa (LA) gametophyte (Fig. 1, A–E). Inaddition, we also surveyed cultures of N. shawii, N. tor-tuosa, T. hispida, and T. violacea, the life history phaseof which is uncertain but appears to be the chantran-sia (not shown). All pit plugs observed (over 100) con-tained two cap layers, the inner of which is quite thinand typically electron translucent. The outer cap layer istypically plate-like but more electron dense and abouttwice as thick as the inner layer (Fig. 1, A and C–E).Although most of the outer cap layers are not domed,a small number were found to be inflated, such asthose of the N. tortuosa (LA) gametophyte (Fig. 1E).Freeze-substituted specimens of T. violacea (NY) wereanalyzed for the presence of a pit plug cap mem-brane. Neither the gametophyte (not shown) nor thechantransia stage (Fig. 1F) was observed to have a capmembrane.rbcL gene and 18S rRNA gene sequence analyses. Se-quence data for all taxa included in this study weresubmitted to GenBank, and accession numbers aregiven in Table 1 and in the Appendix. The rbcL genewas successfully amplified and sequenced for six sam-ples of the Thoreaceae. The gene contained no inser-tions or deletions, which facilitated the constructionof a final alignment of 1201 nucleotides (out of 1467nt). A NJ tree from analysis of the rbcL gene is de-picted in Figure 2, and parsimony analysis of 537 phy-logenetically informative characters resulted in threemost parsimonious trees with a length of 4275 and aconsistency index (CI) of 0.2271, one of which isshown in Figure 3. The quartet-puzzling tree is similarin topology, and hence the branch support is given onthe parsimony tree (Fig. 3, second value). One of themost notable features of these trees (Figs. 2 and 3) isthat members of the Thoreaceae form a well-sup-ported clade (maximum parsimony [MP], 99% boot-strap; NJ, 100% bootstrap; QPS, 85%). The Tho-reaceae forms a well-supported clade (97% bootstrap)with two other red algal orders, the Balliales and Bal-bianiales, in the NJ tree (Fig. 2). However, this rela-tionship is not supported in the parsimony or quartetpuzzling analyses (Fig. 3). In fact, relationships amongthe various florideophyceaen orders are not well re-solved in any of the analyses. The exception is the as-sociation between the Palmariales and the one se-quence of Audouinella hermannii (Roth) Duby of theAcrochaetiales (MP, 73% bootstrap; NJ, 90% bootstrap;QPS, 80%). The Batrachospermales forms a moderatelysupported entity (MP, 78% bootstrap; NJ, 97% boot-strap; QPS, 61%) and the family Thoreaceae is onlyweakly associated (MP, 61% bootstrap; QPS, Ͼ50%)with this order in the parsimony and quartet puzzlinganalyses (Fig. 3) and moderately associated (82% boot-strap) along with the Balbianiales and Balliales in theNJ analysis (Fig. 2).Table 2. Primers used to amplify and sequence the rbcL and 18S rRNA genes.Primer Gene Sequence (5Ј to 3Ј) ReferenceG01 18S rRNA CAC CTG GTT GAT CCT GCC AG Saunders and Kraft 1994G02.1 18S rRNA CGA TTC CGG AGA GGG AGC CTG Modified from Saunders and Kraft 1994G04.1 18S rRNA GTC AGA GGT GAA ATT CTT GG Modified from Saunders and Kraft 1994G06 18S rRNA GTT GGT GGT GCA TGG CCG TTC Saunders and Kraft 1994G07 18S rRNA TCC TTC TGC AGG TTC ACC TAC Saunders and Kraft 1994G08.1 18S rRNA GAA CGG CCA TGC ACC ACC AAC Modified from Saunders and Kraft 1994G09 18S rRNA ATC CAA GAA TTT CAC CTC TG Saunders and Kraft 1994F160 rbcL CCT CAA CCA GGA GTA GAT CC Rintoul et al. 1999F949 rbcL CTG TAA GTG GAT GCG TAT GGC Rintoul et al. 1999R897 rbcL CGA GAA TAA GTT GAG TTA CCT GC Rintoul et al. 1999rbcLR rbcL ACA TTT GCT GTT GGA GTC TC Rintoul et al. 1999
810 KIRSTEN M. MÜLLER ET AL.Within the Thoreaceae both Nemalionopsis shawiiand N. tortuosa are closely associated, but on a branchdistinct from the Thorea samples (MP, 100% boot-strap; NJ, 100% bootstrap, QPS, 97%). In addition, allfour collections of T. violacea (SAG1, SAG2, TX7, andNY) and the one sample of T. hispida form a solid clus-ter that is well supported by bootstrap and decay anal-ysis (MP, 99% bootstrap; QPS, 99%; NJ, 97% boot-strap) (Figs. 2 and 3). Interestingly, Thorea hispida isstrongly associated with T. violacea (SAG1) in the NJ(100% bootstrap) and QPS (99% support, not shown)and identical to T. violacea (SAG1) when only phylo-genetically informative sites are considered in the par-simony analysis. In addition, T. violacea (NY) is alsoclosely positioned with these two collections (MP, 99%;NJ, 100%; QPS, 97%). The two remaining collectionsof T. violacea (SAG2 and TX7), which are both fromTexas, are well supported as being distinct from T. vi-olacea (NY, SAG1) and T. hispida; thus, T. violacea isparaphyletic (Figs. 2 and 3).The corrected sequence divergence values of therbcL gene between members of the Thoreaceae andthe other orders of red algae ranged from 16.6% (be-tween Batrachospermum atrum and N. tortuosa) to 25.9%(between T. violacea [TX7] and Rhodogorgon carriebowen-sis Norris et Butcher). The Thoreaceae differed fromother members of the Batrachospermales by 16.6%–25.0% corrected sequence divergence. Within the Thor-eaceae the sequence divergence ranged from 5.42%to 16.3%. In the genus Thorea, the sequence diver-Fig. 1. Transmission electron micrographs of the pit plugs of the Thoreaceae. (A and B) Nemalionopsis tortuosa (LA) gameto-phyte. (A) Outer cap layer is plate-like (arrowheads). (B) Outer cap layer is slightly domed (arrowheads). (C and D) Thorea violacea(NY). (C) Gametophyte pit plug with flat plate-like outer cap layer (arrowheads). (D) Chantransia stage pit plug with a plate-likeouter cap layer (arrowheads). (E) Thorea violacea (TX7b) gametophyte with pit plug having a plate-like outer cap layer (arrowheads).(F) Thorea violacea (NY) chantransia state freeze substitution image of pit plug with no discernible pit plug cap membrane.
811NEW RED ALGAL ORDERFig. 2. Neighbor-joining tree from analysis of 1201 nucleotides of the rbcL gene. Numbers above the branches represent bootstrapresampling results (% of 1000 replicates). Branches lacking values had less than 50% support. Batrachospermum1 ϭ Batrachospermumvirgato-decaisneanum. Members of the subclass Bangiophycidae are used as outgroup taxa.
812 KIRSTEN M. MÜLLER ET AL.Fig. 3. One of three most parsimonious trees from analysis of the rbcL gene sequence data containing 537 phylogenetically infor-mative characters. The first of the numbers above the branches represents bootstrap values from maximum parsimony analysis (% of1000 replicates). The second value is the reliability value of each internal branch determined by the quartet puzzling method and in-dicates in percent the frequency that the corresponding cluster was found among the 1000 intermediate trees. An asterisk or branchlacking values had less than 50% bootstrap support. Members of the subclass Bangiophycidae are used as outgroup taxa.
813NEW RED ALGAL ORDERgence ranged from 5.42% (between T. violacea [SAG1]and T. hispida) to 13.3% (between T. violacea [TX7] andT. violacea [SAG1]). The two genera, Thorea and Nema-lionopsis, differed by 0 (SAG1 and NY) to 8 (N. tortuosaand T. hispida) amino acids and differed from the re-maining Rhodophyta in the analysis by 12 to 32 aminoacids (out of a total 400 codons). Parsimony analysisof amino acid sequences resulted in 1436 most parsi-monious trees (not shown) (l ϭ 179, CI ϭ 0.49).Members of the Thoreaceae again formed a well-sup-ported (100% bootstrap) clade in the amino acid treein which the relationship was unresolved with the re-maining taxa in the Batrachospermales and the otherorders (not shown).An NJ tree from analysis of the 18S rRNA gene isdepicted in Figure 4 and parsimony analysis of 732phylogenetically informative characters resulted in100 most parsimonious trees with a length of 3871and a CI of 0.4324, of which a strict consensus isshown in Figure 5. These 100 most parsimonious treesdiffered due to low resolution among the variousflorideophyte orders and within the Acrochaetialesand the Batrachospermales. The quartet puzzling treeis similar in topology, and hence the branch supportis given on the parsimony tree (Fig. 5, second value).As seen in the rbcL analyses, the Thoreaceae forms awell-supported entity (MP, 100% bootstrap; QPS,98%; NJ, 96% bootstrap) that is not strongly associ-ated with the remaining florideophyte orders or theBatrachospermales (Figs. 4 and 5). In fact, the Tho-reaceae appears to be more closely associated with theAcrochaetiales, Balbianiales, Nemaliales, and Palmari-ales than with the Batrachospermales (Figs. 4 and 5)(albeit this is not well supported by parsimony boot-strap [57%] or quartet puzzling [52% QPS] and onlymoderately supported by NJ bootstrap [77%]). Clus-tering within the Thoreaceae is similar to that ob-served in the rbcL gene sequence analyses; T. violaceais paraphyletic with the collection T. violacea (SAG1),being strongly associated with T. hispida. In addition,the genus Nemalionopsis is associated with the two col-lections of T. violacea (SAG2 and TX7). This relation-ship is well supported by MP bootstrap (100%) and NJbootstrap (99%) and weakly supported by QPS (68%)(Figs. 4 and 5).The sequence divergence between N. shawii and N.tortuosa was 2.88%. Within the genus Thorea, the se-quence divergence ranged from 2.50% (T. hispidaand T. violacea [SAG1]]) to 6.36% (T. violacea [SAG2]and T. hispida). These taxa differed from the genusNemalionopsis by 2.18% (T. violacea [SAG2]]) to 4.72%(T. hispida) corrected sequence divergence.Secondary structure signatures. Figure 6 depicts a sec-ondary structure model diagram of the 18S rRNAgene from T. violacea (TX7) highlighting two regionsA and B (Escherichia coli numbering: 650 and 1145 re-gions, respectively) in which structural elements existthat separate members of the Thoreaceae from the re-maining Rhodophyta (based on an alignment of over400 18S rRNA gene sequences, representing all rho-dophyte orders, Müller et al. 2000, unpublished data).Figure 7 depicts a gallery (collection of secondary struc-ture elements from different samples) of the 650 re-gion (E. coli numbering, also represented as A in Fig.6) in which there is an additional A-U rich helix rang-ing from 10 to 25 nucleotides in sequences of the ge-nus Thorea. This helix is not found in either the genusNemalionopsis (depicted also in Fig. 7) or any otherRhodophyta sequences examined (not shown). Themost striking example of a structural signature is thelarge extended helix in the 1145 region (Fig. 8), ofwhich there is no homologous structure in all otherRhodophyta 18S rRNA gene sequences examined.This extended helix is found in all members of theThoreaceae sequenced thus far and ranges from 21(N. shawii and N. tortuosa) to 124 (T. violacea [SAG2])additional nucleotides and is characterized by a con-served tetra-loop (GCAA) at the end of the helix anda conserved sequence in the beginning of this helix.The phylogenetic analyses depicted in Figures 4and 5 excluded these regions because they would beconsidered autapomorphies for the delineation ofthis group. However, phylogenetic analyses were car-ried out incorporating these regions, and there waslittle or no difference in the topology or supportwithin the Thoreaceae or with its relationship withother clades. Thorea violacea (SAG2) and T. violacea(TX7) were depicted in Figures 2, 3, 4, and 5 andform a strongly supported group; in fact, both thesetaxa were found to contain longer extended helices(121–124 nt). On the other hand, T. hispida and T. vi-olacea (SAG1) had smaller helices (73–75 nt, respec-tively) and the two sequences of the genus Nemalionop-sis had identical helices of only 21 nucleotides. Thesestriking features, in addition to the position of themembers of the Thoreaceae on a clearly separatebranch from other members of the Batrachosper-males, justifies the separation of the Thoreaceae fromthis order into a new order, the Thoreales.discussionUltrastructure of pit plugs. Ultrastructural observationsof pit plugs of members of the Thoreaceae have beenreported by several authors, nearly all of whom agreeon the existence of an outer cap layer. However, thecharacterization of this layer as a plate or as a dome hasbeen the subject of dispute. Bischoff (1965) did not de-scribe a plug cap in the gametophytic phase of T. viola-cea (as T. riekei), but a thin cap layer is discernible in hismicrograph of a pit plug. By contrast, Lee (1971) dem-onstrated domes in the cultured chantrasia phase ofthe same species. In two surveys of pit plug structure(Pueschel and Cole 1982, Pueschel 1989), the outercap layers of members of the Thoreaceae were catego-rized as domes, and in a cytochemical study of thechemical composition of outer cap layers (Trick andPueschel 1991), a dome-shaped cap of the Thoreachantrasia stage was demonstrated by LM. In the onlydetailed ultrastructural study of a member of the Tho-reaceae, Schnepf (1992) clearly demonstrated pit plugs
814 KIRSTEN M. MÜLLER ET AL.Fig. 4. Neighbor-joining tree from analysis of 2486 nucleotides (including alignment gaps) of the 18S rRNA gene. Numbersabove the branches represent bootstrap resampling results (% of 1000 replicates). Branches lacking values had less than 50% support.Members of the subclass Bangiophycidae are used as outgroup taxa.Fig. 5. Strict consensus of 100 most parsimonious trees derived from analysis of the 18S rRNA gene sequence data containing 752phylogenetically informative characters. The first of the numbers above the branches represents bootstrap values from maximum par-simony analysis (% of 1000 replicates). The second value is the reliability value of each internal branch determined by the quartetpuzzling method and indicates in percent the frequency that the corresponding cluster was found among the 1000 intermediatetrees. An asterisk or branch lacking values had less than 50% bootstrap support. Batrachospermum1 ϭ Batrachospermum virgato-decais-neanum. Members of the subclass Bangiophycidae are used as outgroup taxa.
816 KIRSTEN M. MÜLLER ET AL.Fig. 6. Secondary structure model determined by comparative sequence analysis (using Ͼ 5000 eukaryotic 18S rRNA gene se-quences, see http://www.rna.icmb.utexas.edu) of the 18S rRNA gene in Thorea violacea (TX7). (A) Region 650 (Escherichia coli num-bering) depicted in Figure 7. (B) Region 1145 (E. coli numbering) depicted in Figure 8. ᭺, A:G pairings; ᭹, noncanonical (non-Wat-son Crick) pairings; ؒ, G:U pairings.
817NEW RED ALGAL ORDERFig. 7. Gallery (collection of secondary structure elements from different samples) of secondary structure diagrams of the 650 re-gion (Escherichia coli numbering) in the 18S rRNA gene, highlighting differences among members of the Thoreaceae, (depicted as“A” in Fig. 6). ᭺, A:G pairings; ᭹, noncanonical (non-Watson Crick) pairings; ؒ, G:U pairings.
818 KIRSTEN M. MÜLLER ET AL.with thin plates in field-collected gametophytes of Tho-rea hispida. Our current study includes both field-col-lected gametophytes and cultured chantransia stages ofboth Nemalionopsis and Thorea, with the vast majorityhaving plate-like outer cap layers that are up to threetimes wider than the inner layer (Fig. 1, A and C–E). Inthe other members of the Batrachospermales in whichthe domed outer cap layers are typical, the dome isconsiderably wider than the inner cap layer by a factorof 10–15 (e.g. Brown and Weier 1970, Aghajanian andHommersand 1978, Pueschel and Cole 1982, Trick andPueschel 1991, Sheath and Whittick 1995, Sheath et al.1996a,b). Among the small number of cases in theThoreaceae with an inflated outer cap, the outer caplayer is up to four times wider than the inner layer (e.g.Fig. 1B). Hence, the outer cap layer appears to be agood characteristic to delineate the Thoreaceae fromother members of the Batrachospermales.Demonstrating the presence or absence of a capmembrane is made more difficult by the fact that theedges of the outer cap layer can produce an appear-ance that is easily mistaken for a membrane (Pueschel1987, 1994). Freeze substitution has clearly demon-strated cap membranes in Cumagloia andersonii (Far-low) Setchell et Gardner and Nemalion helminthoides(Velley) Batters (Nemaliales) (Pueschel 1994) andconfirmed the absence of cap membranes in somemembers of the Batrachospermaceae (Pueschel 1994).In the present study, freeze substitution showed nocap membranes in either the chantrasia or gameto-phytic phase of Thorea violacea, a finding that is consis-tent with an absence of cap membranes in a variety ofspecimens of the Thoreaceae prepared by conven-tional chemical fixation.In summary, the ultrastructural study of the pitplugs demonstrated that Nemalionopsis and Thorea arenot well classified in the Batrachospermales in thatthey differ in the outer cap layer largely being a flatplate.Molecular analyses. All the gene trees generatedshow the Thoreaceae to be a well-supported mono-phyletic clade that is a distinct lineage from that ofthe other batrachospermalean families, the Batracho-spermaceae and Lemaneaceae. These findings con-firm those of previous molecular studies, which in-cluded only one member of the Thoreaceae, such asHarper and Saunders (1998), Vis et al. (1998), andSheath and Müller (1999). These findings furthersubstantiate that Nemalionopsis and Thorea should beremoved from the Batrachospermales.The three molecular trees generated all exhibit adistinct separation of the two genera of the Tho-reaceae. Hence, the current basis for distinguishingNemalionopsis from Thorea primarily by peripheral ver-sus axial positioning of monosporangia (Starmach1977, Sheath et al. 1993) is supported by this study. Bycontrast, the distinction of T. hispida from T. violaceabased on degree of branching (Sheath et al. 1993) isnot supported in the gene trees. Thorea hispida(Thore) Desvaux 1818 has precedence over T. violaceaBory de Saint-Vincent 1808 because the basionym forT. hispida is Conferva hispida Thore 1799. However, atthis time there are not enough samples or moleculardata to justify the synonymy of T. hispida and T. viola-cea. This is despite the little support in the moleculartrees for the use of primary or secondary branching asa defining taxonomic character.The separation of the Thoreaceae from the Batra-chospermales is further substantiated by the findingof unique secondary structure signatures in the 18SrRNA gene of Nemalionopsis and Thorea. Over 400 se-quences of this gene were examined within the Rho-dophyta, including representatives of all orders to makethis finding (Müller et al. 2000, unpublished data).Fig. 8. Gallery (collection of secondary structure elements from different samples) of secondary structure diagrams of the 1145 re-gion (Escherichia coli numbering) in the 18S rRNA gene, highlighting differences among members of the Thoreaceae (depicted as “B”in Fig. 6). ᭺, A:G pairings; ᭹, noncanonical (non-Watson Crick) pairings; ؒ, G:U pairings.
819NEW RED ALGAL ORDERWoese (1987) justified using the higher order structurefrom a phylogenetic perspective, and Winker and Woese(1991) delineated three domains of organisms basedon a combination of homologous and non-homolo-gous signature sequences of the 16S rRNA gene. Theunique helices in members of the Thoreaceae in thepositions 650 and 1145 of the 18S rRNA gene are suchsignature sequences. The A:U rich helix at position 650in Thorea has not been found in any other eukaryotic18S rRNA gene sequences examined to date (Ͼ5000,Gutell unpublished data). The large extended helixin the 1145 region of both genera is not present inother red algal 18S rRNA gene sequences (Müller et al.2000, unpublished data). These nonhomologous sig-natures appear to be diagnostic of members of the ge-nus Thorea (position 650) and the family Thoreaceae(position 1145), respectively.Taxonomic conclusions. This study has shown that theThoreaceae is well separated from other members ofthe Batrachospermales based on the following charac-teristics: 1) multiaxial gametophyte thallus; 2) pit plugswith typically plate-like outer cap layers; 3) position inrbcL, 18S rRNA, and combined gene trees; and 4)presence of signature elements in the 18S rRNA gene,particularly at position 1145. Based on the fact thatthe Thoreaceae is well supported as a separate mono-phyletic grouping in the gene trees, we propose that anew order be described for members of this clade asfollows:Thoreales ord. nov. Müller, Sheath, Sherwoodet PueschelFilum multiaxialis cum gametangia, filum chanstra-nium uniaxialialis, obturamentum lacunum cum stratumexternum planum, in aquis dulcibus.Multiaxial gametophytic filaments, alternating withuniaxial chantransia stage, pit plugs with plate-likeouter cap layers, freshwater habitats.Type family: Thoreaceae (Reichenbach) Hassallwith same characteristics as order.Type genus: Thorea Bory de St.-VincentSupported by NSERC grant 0183503 to R. G. S., OGS scholar-ship to K. M. M., NSERC PGSB scholarship to A. R. S. and NIHGM48207 and the University of Texas start-up funds to R.R.G.We thank the Culture Collection of Algae at the University ofGöttingen (SAG) for supplying five cultures used in this studyand Dana Couture, Gary Sullivan, and John Titus for help infield collections and Jaime Cannone for assitance with rRNA se-quence and structural analyses. 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Biogeography of Batrachospermum ge-latinosum (Batrachospermales, Rhodophyta) in North Americabased on molecular and morphological data. J. Phycol. 33: 520–6.Winker, S. & Woese, C. R. 1991. A definition of the domainsArchea, Bacteria and Eucarya in terms of small subunit riboso-mal RNA characteristics. Syst. Appl. Microbiol. 14:205–10.Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221–71.Appendix 1rbcL: Ahnfeltia plicata U04168, Amphiroa fragilissima U04039, Antithamnion sp. X54532, Audouinella hermannii U04033, Balbianiainvestiens AF132293, Ballia callitricha AF149029, Bangia atropurpurea AF043370, B. fuscopurpurea AF043366, Batrachospermum atrumAF029139, B. gelatinosum AF026045, B. helminthosum AF029142, B. louisianae AF029144, B. macrosporum AF029145, B. turfosumAF029147, B. virgato-decaisneanum AF029148, Bonnemaisonia hamifera U04044, Chondrus crispus U02984, Compsopogon coeruleusAF087116, Dumontia contorta U26823, Erythrotrichia carnea AF087118, Gelidium elegans AB030623, Gracilaria tikvahiae U04172,Gracilariopsis sp. U04170, Grateloupia filicina AB038612, Halosaccion glandiforme U04173, Halymenia dilatata AB038604, Lemaneafluviatilis AF029150, Nothocladus nodosus AF029152, Palmaria palmata U04186, Paralemanea catenata AF029154, Porphyra miniataAF168663, Porphyridium aerugineum X17597, Psilosiphon scoparum AF029155, Ptilophora subcostata U16835, Rhododraparnaldiaoregonica AF029156, Rhodogorgon carriebowensis U04183, Rhodymenia pseudopalmata U04182, Sirodotia suecica AF029158, Thoreaviolacea(TX7) AF029160, Tuomeya americana AF029352.18S rRNA gene: Ahnfeltia plicata Z14139, Amphiroa fragilissima U60744, Antithamnionella floccosa AF236788, Audouinella amphiroaeAF079785, A. arcuata AF079784, A. asparagopsis AF079795, A. caespitosa AF079787, A. dasyae L26181, A. daviesii AF079788, A.endophytica AF079789, A. hermannii AF026040, A. macrospora (1) AF199505, A. macrospora (2) AF199506, A. pectinata AF079790, A.proskaueri AF079791, A. rhizoidea AF079792, A. secundata AF079784, A. tenue AF079796, A. tetraspora AF079793, Balbiania investiensAF132294, Ballia callitricha AF236790, Bangia atropurpurea AF043365, B. fuscopurpurea AF043365 AF043355, Bangiopsis subsimplexAF168627, Batrachospermum boryanum AF026044, B. gelatinosum AF026045, B. helminthosum AF026046, B. louisianae AF026047, B.macrosporum AF026048, B. turfosum AF026049, B. virgato-decaisneanum AF026050, Bonnemaisonia hamifera L26182, Bostrychiamoritziana AF203893, Camontagnea oxyclada AF079794, Champia affinis U23951, Chantransia sp. AF199507, Chondrus crispus Z14140,Chroodactylon ornatum AF168628, Compsopogon coerulus AF087124, Corallina elongata U60946, Dumontia contorta AF317099,Erythrotrichia carnea L26189, Galaxaura marginata AF006090, Gelidium elegans AB017670, Gracilaria gracilis L26205, Gracilariopsis sp.L26208, Grateloupia filicina U33132, Halosaccion glandiforme L26193, Halymenia plana U33133, Hildenbrandia rivularis AF208830, H.rubra AF108413, Lemanea fluviatilis AF026051, Meiodiscus spetsbergensis U23814, Nemalion helminthoides L26196, Nemalionopsis tortuosaAF342743, Nothocladus nodosus U23815, Palmaria palmata Z14142, Paralemanea catenata AF026052, Plocamiocolax pulvinata U09618,Plocamium cartilagineum U09619, Porphyra miniata AF175540, Porphyridium aerugineum L27635, Psilosiphon scoparum AF026041,Ptilophora subcostata U60348, Rhodochorton purpureum U23816, Rhododraparnaldia oregonica AF026043, Rhodogorgon carriebowensisAF006089, Rhodymenia leptophylla U09621,Sirodotia suecica AF026053, Sirodotia huillensis AF026054, Thorea violacea (SAG2)AF342744, Thorea violacea (TX7) AF026042, Tuomeya americana AF026055.