J. Phycol. 47, 164–177 (2011)  2011 Phycological Society of America DOI: 10.1111/j.1529-8817.2010.00949.x POLYPHYLY OF CHAETOPHORA AND STIGEOCLONIUM WITHIN THE CHAETOPHORALES (CHLOROPHYCEAE), REVEALED BY SEQUENCE COMPARISONS OF NUCLEAR-ENCODED SSU rRNA GENES 1 Lenka Caisová 2,3 Institute of Botany v.v.i., Academy of Sciences of the Czech Republic, Dukelská 135, CZ – 379 82 Třeboň, Czech Republic Faculty of Science, University of South Bohemia, Branišovská 31, CZ – 370 05 České Budějovice, Czech Republic Birger Marin3, Nicole Sausen Biozentrum Köln, Botanisches Institut, Universität zu Köln, Zülpicher Str. 47b, 50674 Köln, Germany Thomas Pro¨schold Department Limnology, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria and Michael Melkonian Biozentrum Köln, Botanisches Institut, Universität zu Köln, Zülpicher Str. 47b, 50674 Köln, Germany Previously published molecular phylogenetic analyses of the Chaetophorales (Chlorophyceae) suffered from limited taxon sampling (six genera with only a single species per genus). To test the monophyly of species-rich genera, and to analyze the phylogenetic relationships among families and genera in the Chaetophorales, we determined nuclear-encoded SSU rDNA sequences from 30 strains of Chaetophorales, performed phylogenetic analyses using various methods, and screened clades for support by unique molecular synapomorphies in the SSU rRNA secondary structure. The Schizomeridaceae and the weakly supported Aphanochaetaceae were recovered as basal lineages. The derived family Chaetophoraceae diverged into two clades: the ‘‘Uronema clade’’ containing unbranched filaments, and a sister clade designated as ‘‘branched Chaetophoraceae’’ comprising Chaetophora, Stigeoclonium, Draparnaldia, Caespitella, and Fritschiella. Although some terminal clades corresponded to genera described (e.g., Caespitella and Draparnaldia), other clades were in conflict with traditional taxonomic designations. Especially, the genera Stigeoclonium and Chaetophora were shown to be polyphyletic. The globose species Chaetophora elegans was unrelated to lobate Chaetophora spp. (e.g., Chaetophora lobata). Since the original description of Chaetophora referred to a lobate thallus organization, the latter clade represented Chaetophora sensu stricto. In consequence, C. lobata was designated as lectotype of Chaetophora. Two Stigeoclonium species, Stigeoclonium farctum Berthold and Stigeoclonium ‘Longipilus’, diverged independently from the type species of Stigeoclonium, Stigeoclonium tenue (C. Agardh) Kütz. These results indicated that some commonly used taxonomic characters are either homoplasious or plesiomorphic and call for a reevaluation of the systematics of the Chaetophorales using novel morphological and molecular approaches. Key index words: 18S rDNA; Aphanochaete; Caespitella; Chaetophora; Chaetophorales; Draparnaldia; Fritschiella; phylogeny; polyphyly; Stigeoclonium Abbreviations: CBC, compensatory base change; CRW, comparative RNA Web site and project; ERRD, the European Ribosomal RNA Database; ICBN, International Code of Botanical Nomenclature; INA, Index Nominum Algarum; MCMC, Markov chain Monte Carlo; ML, maximum likelihood; MP, maximum parsimony; NHS, nonhomoplasious synapomorphy; NJ, neighbor joining; OCC, Oedogoniales, Chaetopeltidales, and Chaetophorales; RAxML, randomized accelerated maximum likelihood; rbcL, gene encoding RUBISCO LSU; UTC, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae The order Chaetophorales (Chlorophyceae) comprises predominantly freshwater taxa, and only a few genera are known from terrestrial habitats (Berthold 1878, Aziz and Islam 1962, Ettl and Gärtner 1995). Most members of the Chaetophorales have been described from temperate and tropical regions (Saxena 1962, Islam 1963, Printz 1964, Bourrelly 1972, Tupa 1974, Prescott 1979, Sarma 1986). Traditionally, unbranched and branched filamentous as well as parenchymatous green algae containing a single parietal chloroplast per cell were 1 Received 28 January 2010. Accepted 20 July 2010. Author for correspondence: e-mail lcaisova@gmail.com. 3 These authors contributed equally to this work. 2 164 P O L Y P H Y L Y O F C H A E T O P H O R A A N D S T I G EO C L O N I U M classified within the order Chaetophorales sensu Wille (1901). Some authors (e.g., Hazen 1902, West and Fritsch 1927) considered unbranched genera as members of the Ulotrichales Borzi, regarding only branched taxa as genuine Chaetophorales. On the basis of ultrastructural analyses of mitosis-cytokinesis (summarized by Stewart and Mattox 1975) and motile cell ultrastructure (Manton 1964, Melkonian 1975, Floyd et al. 1980) in selected taxa, Silva (1982) redefined the order Chaetophorales to include unbranched or branched filaments (occasionally parenchymatous) with the ultrastructural characteristics of the Chlorophyceae (i.e., a collapsing telophase spindle, phycoplast, and cruciate microtubular flagellar root system with basal bodies exhibiting slight clockwise displacement without overlap) recognizing three families (Chaetophoraceae, Aphanochaetaceae, Schizomeridaceae). With this restricted circumscription, many of the traditional genera of the Chaetophorales (e.g., Coleochaete, Chaetosphaeridium, Microthamnion, and Trentepohlia) were transferred to other green algal orders or classes (summarized by Mattox and Stewart 1984, O’Kelly and Floyd 1984, Melkonian 1990). Molecular phylogenetic analyses performed since the 1990s corroborated these conclusions (Friedl and Zeltner 1994, O’Kelly et al. 1994; reviews by Melkonian and Surek 1995, Lewis and McCourt 2004, and Pröschold and Leliaert 2007). Currently, the order Chaetophorales sensu John (1984) includes unbranched and branched filamentous taxa. Cytokinesis involves a phycoplast-associated cell plate; daughter cells remain in contact through plasmodesmata, thus resulting in true multicellularity of the filaments. During cytokinesis, centrioles remain in the position of the former spindle poles (i.e., they are not associated with the division plane). Asexual reproduction is by quadriflagellate zoospores. Of the three families, only the Chaetophoraceae contains several genera with highly differentiated morphologies and life histories (Godward 1942, Forest 1956, Abbas and Godward 1963, Printz 1964, Cox and Bold 1966, Sarma and Jayaraman 1980, John 1984, Michetti et al. 2010). Within the Chaetophoraceae, definition of genera and species has been traditionally based on characters (e.g., extent of prostrate and upright systems, hair formation, degree of branching) that are known to be sensitive to environmental factors (Vischer 1933, Tupa 1974, Harding and Whitton 1978, Johnstone 1978, Francke and Ten Cate 1980, Gibson and Whitton 1987, Van Beem and Simons 1988, Pawlik-Skowrońska 2003). Some authors even hypothesized that the genera Schizomeris, Caespitella, and Draparnaldia might represent simplified growth forms of different Stigeoclonium spp., albeit without conclusive experimental evidence (Uspenskaja 1930, Cox and Bold 1966, Bourrelly 1972, Campbell and Sarafis 1972, Johnstone 1978). Nevertheless, the species-rich genera of the Chaetophoraceae are gener- 165 ally regarded as monophyletic—that is, Chaetophora (defined by mucilaginous thalli with long, multicellular hairs), Stigeoclonium (presence of prostrate and erect filament systems, the latter terminating in multicellular hairs, with only a thin layer of mucilage), and Draparnaldia (differentiation into an erect main axis and subordinate lateral branch systems, again terminating with multicellular hairs). Among these genera, Stigeoclonium and Draparnaldia contain the largest number of described species (each 80), although some critical revisions of Stigeoclonium (Islam 1963, Cox and Bold 1966, Francke and Simons 1984, Simons et al. 1986) using the morphology of prostrate filaments and type of zoospore germination as major taxonomic characters have reduced the number of species to 23 (Islam 1963) or even only three (Simons et al. 1986). The first molecular phylogenetic analysis of four genera ⁄ species of the Chaetophoraceae, using nuclear-encoded SSU rDNA sequence comparisons, confirmed the monophyly of this family as well as its placement within the class Chlorophyceae (Booton et al. 1998). Buchheim et al. (2001) added Schizomeris and Aphanochaete, thus ‘‘completing’’ the order Chaetophorales and, by analyzing nuclear-encoded SSU + partial LSU rDNA data, provided the first evidence for a sister-group relationship between Chaetophorales and Chaetopeltidales. Recent multigene analyses of partial and complete chloroplast genomes from six Chlorophyceae (including Stigeoclonium, Floydiella, and Oedogonium) gave further evidence for phylogenetic relationships among Oedogoniales, Chaetopeltidales, and Chaetophorales (Brouard et al. 2008, Turmel et al. 2008, 2009). Although previous molecular phylogenetic analyses have been performed with six chaetophoralean genera, these analyses included only one or two (Uronema) representatives of each genus (Booton et al. 1998, Buchheim et al. 2001). Thus, the relationship between the families Schizomeridaceae and Aphanochaetaceae remains unknown, as well as the phylogenetic status of the species-rich genera of the Chaetophoraceae (Chaetophora, Draparnaldia, and Stigeoclonium). In the present contribution, we extended the taxon sampling and determined 30 new nuclear-encoded SSU rDNA sequences from the Chaetophorales (i.e., two strains of Schizomeris, five strains of the family Aphanochaetaceae, and 23 strains of the Chaetophoraceae). Molecular phylogenetic analyses unexpectedly revealed polyphyly of well-established genera and challenge the taxonomic value of their definitions based on morphological characters. MATERIALS AND METHODS Cultures, DNA isolation, gene amplification, and sequencing. Thirty strains of Chaetophorales were used for determination of new sequence data (Table S1 in the supplementary material; taxa in bold in Fig. 1). Sequence data of five of these strains were already available. However, these sequences 166 L E N K A C A I S O V Á E T A L . contained ambiguities as well as apparent errors when compared with the conserved 18S rRNA secondary structure and thus had to be resequenced. The following abbreviations were used for strain designations: CCALA, Culture Collection of Autotrophic Organisms, CZ (http://www.butbn.cas.cz/ccala/ index.php); CCAP, Culture Collection of Algae and Protozoa (http://www.ccap.ac.uk/); SAG, Sammlung von Algenkulturen, University of Göttingen, Germany (http://www.epsag. uni-goettingen.de/html/sag.html); ACOI, Coimbra Collection of Algae (http://acoi.ci.uc.pt/). Four strains isolated for this study by L. C. (abbr. LC_L; Table S1) were identified using species descriptions of Pascher (1914), Islam (1963), and Starmach (1972); these strains are available through Culture Collection of Algae at the University of Cologne, Germany (CCAC; http://www.ccac.uni-koeln.de/). In addition, all other strains listed in Table S1 were investigated by LM (Olympus BX 51; Olympus, Tokyo, Japan) to ensure their correct identification. Total genomic DNA was extracted with the DNeasy Plant Mini Kit from Qiagen (Hilden, Germany; http://www.qiagen. com), used for gene amplification by PCR, and sequenced as described earlier Marin et al. (2003). Newly determined sequences of 30 strains are available under the accession numbers FN824371–FN824400 (Table S1). Alignments and phylogenetic analyses. Together with 53 published sequences representing the chlorophyte classes Ulvophyceae, Trebouxiophyceae, and Chlorophyceae (UTC clade), 30 new chaetophoralean SSU rDNA sequences were subjected to manual alignment procedures guided by the conserved secondary structural architecture of the SSU rRNA (see below), using SeaView 4.2 (http://pbil.univ-lyon1.fr/software/seaview. html). BLAST searches (http://blast.ncbi.nlm.nih.gov/Blast. cgi) with new SSU rDNAs as Query revealed eight previously published chaetophoralean homologs. Only four of them were integrated into our alignment, whereas the remaining sequences were omitted since we resequenced the same strains—that is, Schizomeris SAG 44.84; C. lobata CCAP 413 ⁄ 1; and Stigeoclonium helveticum CCAP 477 ⁄ 1, SAG 477-2, and CCALA 499 (Table S1 including taxonomic authors). The taxon sampling of nonchaetophoralean green algae for phylogenetic analyses was guided by preanalyses involving >300 Viridiplantae (trees not shown). To represent the diversity within the UTC clade, 49 basal, short-branched divergences of major subclades were selected. We prepared two data sets: an 83 taxa alignment covering the UTC clade with 1,704 unambiguously aligned nucleotide positions and a smaller alignment containing 40 taxa of Oedogoniales (3), Chaetopeltidales (3), and Chaetophorales (34 taxa) (= OCC clade) with 1,743 unambiguously aligned characters. Phylogenetic analyses were performed by several methods: randomized accelerated maximum likelihood (RAxML), maximum likelihood (ML), PhyML, distance (neighbor joining, NJ), maximum parsimony (MP), and Bayesian analyses. For ML, PhyML, and NJ analyses, the appropriate model of sequence evolution including model parameters was determined with ModelTest 3.7 (Posada 2008), resulting in GTR + I + G as selected by the Akaike criterion. The same model was used for RAxML and MrBayes with parameters estimated by these programs. Analyses used RAxML 7.0.3. (Stamatakis 2006), PAUP*4.0b10 (for ML, NJ, MP; Swofford 2000), PhyML (Guindon and Gascuel 2003), and MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). The tree topology in Figure 1 was obtained by heuristic search under the ML criterion, starting with an NJ tree. ML analyses reached the ‘‘optimal’’ tree after 1,000 rearrangements, and therefore, ML bootstrap analyses (100 replicates, starting trees obtained by randomized sequence addition) could here be constrained toward 4,000 rearrangements per replicate. Distance (NJ, 1,000 replicates) and MP bootstrap analyses (1,000 replicates, each with 10 randomized sequence addition replicates) have not been constrained. For 1,000 RAxML bootstrap replicates, 10 multiple searches per replicate were defined. Bayesian analyses used two Markov chain Monte Carlo (MCMC) chains with 2,000,000 generations, and 640,000 generations were discarded as ‘‘burn-in.’’ Bootstrap values <50% and Bayesian posterior probabilities <0.95 were considered as ‘‘no support.’’ In Figure 2, the Bayesian analysis used 1,000,000 generations (20,000 = ‘‘burn-in’’); for other methods, see above. Molecular synapomorphies of the order Chaetophorales and nested subclades. The order Chaetophorales was analyzed for molecular ‘‘nonhomoplasious (= unique) synapomorphies’’ (NHS) to define clades unambiguously. For this study, an NHS is defined as ‘‘unique within the Viridiplantae.’’ Methods to identify NHS using an exhaustive search has been described previously (Marin et al. 2003, 2005). To find NHS of Chaetophorales, a taxon-rich SSU rDNA alignment was used to obtain an MP tree that contained five glaucophytes, 28 rhodophytes, and 1,226 Viridiplantae (tree not shown). Synapomorphies were described by their positions (Fig. 2; Table 1) according to two different rRNA secondary structure models and nomenclature (helix numbering) systems: (i) the European Ribosomal RNA Database (ERRD; http://bioinformatics.psb.ugent. be/webtools/rRNA/) and (ii) the Gutell Lab comparative RNA Web site and project (CRW) site (http://www.rna.ccbb. utexas.edu/). The following ‘‘reference’’ secondary structure diagrams may be used to find NHS-type synapomorphies: ERRD, Chlamydomonas reinhardtii (http://bioinformatics.psb. ugent.be/webtools/rRNA/secmodel/Crei_SSU.html); CRW, Escherichia coli helix numbering system (http://www.rna.ccbb. utexas.edu/CAR/1A/Structures/h.16.b.E.coli.hlxnum.pdf); CRW, Staurastrum (http://www.rna.ccbb.utexas.edu/RNA/ Structures/d.16.e.Staurastrum.sp.M752.pdf); CRW, Apis mellifera, including an E. coli–based helix numbering system (Gillespie et al. 2006). Formalized analysis and ancestral state reconstruction of morphological characters in the Chaetophorales. For 40 taxa (as in Fig. 2), 18 morphological characters with two to five character states were combined within a data matrix in nexus format (Appendix S1 in the supplementary material). The data matrix was first used for a cladistic analysis via heuristic searches under the MP criterion implemented in PAUP, resulting in several equally parsimonious trees. Second, morphological data were mapped upon the 18S rDNA tree topology by loading the ML treefile from Figure 2, with branch lengths reflecting morphological character changes. In both cases, synapomorphy searches were performed as described above for molecular characters. Unique morphological synapomorphies for selected clades were then reanalyzed via ancestral state reconstruction implemented in MrBayes by mapping this morphological character onto Bayesian trees based on molecular data. In practice, morphological as well as molecular data of 40 taxa were combined within a partitioned nexus file (mixed ‘‘standard ⁄ DNA’’ model; files not shown), with the clade of interest defined by constraint, and used for ancestral state reconstruction as described in the manual of MrBayes. In all cases, both MCMC chains congruently supported only one character state by high probability values. Both probabilities were integrated as mean values (without burn-in generations) in Figure 4. Topology tests of user-defined trees. User-defined trees were generated by modifying the ML topology of the OCC clade analysis (40 taxa), by (i) running ML analyses with constraints, using PAUP, or (ii) by collapsing or moving single branches through manual edition of the original treefile, using TreeView (Page 1996; http://darwin.zoology.gla.ac.uk/~rpage/ treeviewx/). The original (best) treefile and six user-defined trees were compared by the approximately unbiased test, the 167 P O L Y P H Y L Y O F C H A E T O P H O R A A N D S T I G EO C L O N I U M UTEX 2012, AB110439 Ulvophyceae SAG 29.93, Z28970 Acrosiphonia arcta, AY303600 SAG 2022, AJ416104 Oltmannsiellopsis geminata MBIC10525, AB183610 72/64/89/88/1.00 Oogamochlamys gigantea SAG 21.72, AJ410468 Volvocales Lobochlamys segnis SAG 9.83, AJ410459 -/-/-/-/0.97 98/98/98/96/1.00 CC-1952, AY665727 (CW-clade) 56/-/-/56 Heterochlamydomonas inaequalis UTEX 1705, AF367857 /0.98 NIES 1715, AB248247 -/-/-/-/1.00 SAG 29.83, AJ410448 93/93/83/81/1.00 Asteromonas gracilis UTEX 635, M95614 77/79/73/66/1.00 Dunaliella salina CCAP 19/30, EF473749 84/88/ Haematococcus pluvialis SAG 34-1b, AF159369 66/79/1.00 Stephanosphaera sp. UTEX 2409, AB360751 71/59/75/61/1.00 SAG 16.99, AY009897 -/-/ Chlamydomonas monadina SAG 8.87, AY220559 55/-/53/-/-/-/0.99 Carteria radiosa NIES 432, D86500 70/54/-/50/1.00 Carteria crucifera NIES 421, D86501 Spermatozopsis similis SAG 1.85, X65557 Golenkinia sp. UTEX 931, AF499924 Desmodesmus communis UTEX 76, X73994 87/82/75 Sphaeropleales Scenedesmus obtusus Hegewald 1980-9, AB037091 -/-/-/-/1.00 -/-/59/-//71/1.00 SAG 236-1b, AY780660 (DO-clade) UTEX 138, M62861 Polyedriopsis spinulosa SAG 31.81, AF288362 65/50/-/-/0.95 Kirchneriella aperta SAG 2004, AJ271859 72/54/ 51/-/ Selenastrum capricornutum UTEX 1648, AF169628 -/-/1.00 -/-/0.95 75/70/65 Pseudodictyosphaerium sp. Itas 6/3 M-2w, AY543062 /55/1.00 Bracteacoccus aerius UTEX 1250, U63101 90/82/79/89/Oedogonium brevicingulatum, DQ078299 Oedogoniales Oedogonium cardiacum UTEX 40, U831333 Bulbochaete hiloensis UTEX 952, U83132 96/94/99/100/1.00 Pseudulvella americana UTEX 2852, DQ242477 Chaetopeltidales UTEX 422, U83125 52/-/-/-/0.98 UTEX 104, U83126 Schizomeris leibleinii SAG 44.84 Schizomeridaceae Schizomeris leibleinii SAG 24.88 70/60/54/53/Aphanochaete repens SAG 21.91 Aphanochaetaceae Aphanochaete confervicola SAG 27.91 91/89/98/91/1.00 Aphanochaete magna UTEX 1909, AF182816 58/-/68/57/Aphanochaete ('Dilabifilum') sp. SAG 450-1b Aphanochaete ('Dilabifilum') sp. SAG 450-1a 95/89/96/86/1.00 Aphanochaete elegans SAG 4.91 100/97/99/98/1.00 94/94/67/95/1.00 Uronema confervicolum CCAP 386/2 100/100/99/100/1.00 Uronema-clade 77/-/-/65/'Ulothrix' fimbriata CCAP 384/2 (unbranched Chaetophoraceae) 88/83/94/94/1.00 Uronema ('Hormidiella') sp. CCAP 334/1 Uronema acuminatum UTEX 1178, U83128 Uronema trentonense CCAP 386/5 Uronema belkae UTEX 1179, AF182821 99/84/90/90/1.00 Caespitella pascheri SAG 410-1 Fritschiella-clade Caespitella pascheri CCAP 410/2 84/75/62/91/1.00 Chaetophora elegans M3254 (LC L5) Chaetophora elegans CCAP 413/2 100/100/99/100/1.00 Fritschiella tuberosa SAG 112.80 75/53/52/75/CCAP 477/10A 89/78/-/82/1.00 Fritschiella tuberosa UTEX 1821, U83129 98/73/62/80/1.00 Draparnaldia glomerata CCAP 418/2 Chaetophora-clade 92/91/99/87/1.00 Draparnaldia plumosa CCAP 418/1A Chaetophora draparnaldioides (S. 'Longipilus') M3257 (LC L3) 67/56/-/73/0.96 Chaetophora lobata ACOI 447 97/92/98/98/0.97 Chaetophora lobata CCAP 413/1 56/-/-/-/CCAP 477/13 M3255 (LC L1) M3256 (LC L4) CCAP 477/11B 98/97/99/98/1.00 CCALA 499 SAG 477-2 CCAP 477/1 CCAP 477/11A 99/98/98/99/1.00 Pseudochlorella pyrenoidosa SAG 18.95, AM422985 Trebouxiophyceae SAG 397-1b, AJ416107 Leptosira terrestris SAG 463-3, Z28973 60/66/67 Coccomyxa dispar NIES 2252, AB488787 /50/1.00 Botryococcus braunii CCAP 807/1, AJ581913 ASIB S234, AB006051 Myrmecia israeliensis UTEX 1181, M62995 93/86/79/83/1.00 Trebouxia arboricola SAG 219-1a, Z68705 Microthamnion kuetzingianum UTEX 1914, Z28974 Chlorella vulgaris SAG 211-11b, X13688 62/57/ 54/52/Eremosphaera viridis UTEX 34, AF387154 -/-/0.99 /-/0.99 88/81/75/-/1.00 0.02 Planctonema sp. J45-9, AF387149 Geminella minor SAG 22.88, AF387150 64/63/61/-/0.99 Chlorophyceae Chaetophorales branched Chaetophoraceae Chaetophoraceae Fig. 1. Maximum-likelihood (ML) phylogeny of 83 Chlorophyta using nuclear-encoded SSU rDNA sequence comparisons; 1,704 aligned characters were used for analyses. Newly determined sequences are in bold (for accession numbers, see Table S1 in the supplementary material); taxon names are combined with strain designations and (for published sequences) accession numbers. Support values at branches are bootstrap partitions from randomized accelerated maximum likelihood (RAxML), ML, neighbor joining (NJ), maximum parsimony (MP), and Bayesian posterior probabilities. Bold branches were maximally supported by all methods (= 100 ⁄ 100 ⁄ 100 ⁄ 100 ⁄ 1.00). Interrupted branches ( ⁄ ⁄ ) have been shortened to 50% of their original length. The branch separating the Trebouxiophyceae from the remaining classes was defined as root of the tree. 168 L E N K A C A I S O V Á E T A L . 100/100/97/100/1.00 66/67/-/60/0.95 98/99/98/100/0.98 0.01 Oedogoniales Chaetopeltidales Schizomeridaceae Aphanochaete confervicola SAG 27.91 Aphanochaetaceae Aphanochaete magna UTEX 1909, AF182816 Aphanochaete ('Dilabifilum') sp. SAG 450-1b -/55/-/59/Aphanochaete ('Dilabifilum') sp. SAG 450-1a Aphanochaete elegans SAG 4.91 Aphanochaete repens SAG 21.91 95/96/73/79/1.00 62/67/-/66/-(0.85) 76/77/69/91/0.95 Uronema-clade 80/73/82/89/0.99 Fritschiella-clade 65/74/-/75/0.98 98/97/100/99/1.00 99/99/99/99/1.00 Draparnaldia glomerata, D. plumosa Stigeoclonium 'Longipilus', Chaetophora lobata Stigeoclonium variabile CCAP 477/13 Fig. 2. Maximum-likelihood phylogeny of the Oedogoniales, Chaetopeltidales, and Chaetophorales (OCC clade) using 1,743 aligned characters of the 18S rRNA gene. Significances as in Figure 1. Note that the family Aphanochaetaceae forms a monophyletic divergence, in contrast to Figure 1, albeit with low support. The branching pattern in graphically reduced clades (triangles) is identical to Figure 1. Stigeoclonium amoenum, S. protensum, S. helveticum, S. tenue Table 1. Synapomorphy support for derived Chaetophorales and nested subclades. Taxon ⁄ character Evolutionary change Chaetophoraceae Helix 8: bp 4 [H122: bp 5] Helix 24 [H655: bp 672 ⁄ 743] Branched Chaetophoraceae Spacer Helices 9-10 [spacer H144 -H184a] Helix E10_1 [H184b-1]: bp 5 Internal loop of Helix 13 [H289] (reverse strand): nt 2 Chaetophora clade Helix 18 [H441]: bp 1 Draparnaldia, Chaetophora lobata, Chaetophora draparnaldioides (= Stigeoclonium ‘Longipilus’) Spacer Helices 18-19 [H441 -H500]: nt 3 C. lobata, C. draparnaldioides (= S. ‘Longipilus’) Helix 43 [H1118]: sixth from last bp Chaetophora elegans Helix 24: last bp [H655: bp 680 ⁄ 736] Fritschiella tuberosa, Stigeoclonium farctum Helix 25 [H673]: bp 2 A-U ==> G-C [U-G ==> UsU] Characterization Exceptions: G-C in Friedlia irregularis nom. nud. (Trebouxiophyceae) U = NHS (A in Dictyochloris and Halicoryne, C in Heterochlamydomonas) 8 nt’s ==> 9 nt’s [6 nt’s ==> 7 nt’s] U-G ==> A-U A ==> G NHS within Chlorophyta A = NHS within Chlorophyceae G = NHS G-C ==> U-A U-A = NHS G ==> U U = NHS within Chlorophyceae U-A ==> C-G C-G = NHS within Chaetophorales A-U ==> U-A U-A = NHS G-C ==> A-U Almost unique within Chlorophyceae (A-U in Chlamydomonas sordida) Nomenclature of rRNA secondary structures after (i) the European Ribosomal RNA Database (http://bioinformatics. psb.ugent.be/webtools/rRNA/), and (ii) [nomenclature in brackets] the Gutell Lab comparative RNA Web site and project site (http://www.rna.ccbb.utexas.edu/); for reference structure diagrams, see Materials and Methods. Unique signatures without known parallel changes in other Viridiplantae are flagged as nonhomoplasious synapomorphy (NHS). Shimodaira–Hasegawa test, and the Kishino–Hasegawa test using CONSEL (Shimodaira and Hasegawa 2001, Shimodaira 2002; http://www.is.titech.ac.jp/~shimo/prog/consel/). RESULTS The Chaetophorales in the context of the UTC clade. To analyze the Chaetophorales together with representatives of all derived classes of the Chlorophyta (i.e., the UTC clade), a nuclear-encoded SSU rDNA alignment consisting of 83 sequences (13 Trebouxiophyceae, five Ulvophyceae, and 65 Chlorophyceae, including 34 Chaetophorales) was used for tree reconstructions (Fig. 1). Within the Chlorophyceae, the Chaetophorales showed a sister-group relationship with the Chaetopeltidales, supported by low-tomoderate-bootstrap percentages (70% by RAxML), but without Bayesian support (Fig. 1). These two orders formed a nonsupported assemblage with the Oedogoniales, representing the OCC clade sensu Turmel et al. (2008). The monophyly of the Chaetophorales was strongly supported, as was the basal divergence of Schizomeris leibleinii (= family Schizomeridaceae) as sister of the remaining Chaetophorales (families Aphanochaetaceae and Chaetophoraceae; Fig. 1). The six strains of the genus Aphanochaete investigated were not recovered as monophyletic (Fig. 1); instead they diverged paraphyletically as three independent branches whose interrelationships could not be resolved in the global UTC analysis: (i) Aphanochaete repens, (ii) Aphanochaete confervicola and Aphanochaete magna, and (iii) Aphanochaete elegans together with two strains (SAG 450-1a, SAG 450-1b) previously labeled ‘‘Dilabifilum sp.’’ (Fig. 1, P O L Y P H Y L Y O F C H A E T O P H O R A A N D S T I G EO C L O N I U M Table S1). In contrast, the family Chaetophoraceae gained maximal support by all methods applied and showed a basal split into the well-supported ‘‘Uronema clade’’ (consisting of unbranched filamentous taxa) and the remaining genera (5) of the Chaetophoraceae (termed ‘‘branched Chaetophoraceae’’ in Fig. 1), which gained maximal support. Relationships within the ‘‘Uronema clade’’ (six strains investigated) remained largely unresolved, and only the monophyly of ‘‘Ulothrix’’ fimbriata and Uronema confervicolum (the type species of Uronema) was strongly supported (Fig. 1). The ‘‘branched Chaetophoraceae’’ diverged into two well-supported sister clades. One was named the ‘‘Chaetophora clade’’ since it contained C. lobata, the lectotype species of Chaetophora (designated here). The other clade was labeled the ‘‘Fritschiella clade’’ because it included the type species of Fritschiella, Fritschiella tuberosa (Fig. 1). Interestingly, the two most common and well-known genera of the Chaetophorales, Stigeoclonium and Chaetophora, were dispersed over these two clades, intermixed with the remaining genera, and therefore, both represented polyphyletic taxa (Fig. 1). More specifically, two Stigeoclonium spp., S. farctum and S. ‘Longipilus’ (an illegitimate name, synonymous to Chaetophora draparnaldioides; see below), were clearly separated from the type species of the genus (S. tenue), and two strains of C. elegans belonged to the Fritschiella clade and were thus unrelated to C. lobata (Fig. 1). Seven strains of Stigeoclonium, representing four species (S. tenue, Stigeoclonium amoenum, S. helveticum, Stigeoclonium protensum), diverged at the base of the ‘‘Chaetophora clade,’’ albeit without synapomorphic signals in the SSU rDNA and thus without a common branch. Therefore, we cannot presently recognize a ‘‘Stigeoclonium s. str. clade’’ (S. tenue is the type species). Although sequence diversity among these strains was low, S. helveticum CCAP 477 ⁄ 1 differed from two putatively identical strains (SAG 477-2, CCALA 499, the same isolate from Vischer; Table S1) by three substitutions in the SSU rRNA, two of which represented a single autapomorphic compensatory base change (CBC) in a conserved base pair of Helix 29 (the next to last pair, usually U-A) toward C-G (not shown). The well-supported ‘‘crown’’ lineage of the ‘‘Chaetophora clade’’ comprised Draparnaldia, S. ‘Longipilus’ (= C. draparnaldioides), and C. lobata (Fig. 1). S. ‘Longipilus’ and both strains of C. lobata studied had identical SSU rDNA sequences; however, it should be noted that the morphology of S. ‘Longipilus’ strain M3257 corresponded to its description in the literature and therefore differed from C. lobata (results not shown). Although S. ‘Longipilus’ belonged to the earliest species of Stigeoclonium described (Kützing 1843, 1845), its original name was C. draparnaldioides, also described by Kützing (1834) 9 years before he established the genus Stigeoclonium (http://ucjeps. berkeley.edu/INA.html). In light of its phylogenetic 169 position and due to nomenclatural reasons (unnecessary change of the epithet by Kützing), we consider S. ‘Longipilus’ to be a synonym of the earlier described C. draparnaldioides (Fig. 1). Another species of Stigeoclonium (Stigeoclonium variabile) branched in an intermediate position between the basal Stigeoclonium polytomy, and the ‘‘crown’’ lineage, though this position received only moderate support (Fig. 1). The ‘‘Fritschiella clade’’ comprised Caespitella pascheri, C. elegans, both represented by two strains with identical SSU rDNA sequences, and a subclade consisting of F. tuberosa and S. farctum. The moderate sequence diversity in the latter subclade (five positions differed between CCAP 477 ⁄ 10A and SAG 112.80) is reflected in the considerably different morphology of F. tuberosa and S. farctum (results not shown). Phylogenetic analysis of the OCC clade. Thirty-four Chaetophorales, together with their closest relatives (Chaetopeltidales, Oedogoniales), were analyzed without other green algae to increase the number of aligned characters (1,743 vs. 1,704 in the global analysis) and to test the robustness of chaetophoralean clades in a modified taxonomic environment (i.e., OCC clade only) (Fig. 2). Tree topology and support values were largely congruent (compare Figs. 1 and 2); however, we encountered one prominent difference to the global analysis, concerning Aphanochaete (= family Aphanochaetaceae). Whereas the six strains of Aphanochaete were not resolved as monophyletic in the global analysis (Fig. 1), they formed a weakly supported clade in the OCC analysis (‡62% support by RAxML, ML, and MP; Fig. 2), thus rendering the family Aphanochaetaceae monophyletic. When the OCC analysis was repeated with only 1,704 characters (as in Fig. 1), Aphanochaete again collapsed and formed paraphyletic subclades (tree not shown). Synapomorphy support for clades. To further substantiate chaetophoralean clades and to present molecular characters for future taxonomic revisions, all clades ⁄ branches of the Chaetophorales were analyzed for presence of unique (= nonhomoplasious) synapomorphies in the nuclear-encoded SSU rRNA molecule. The order Chaetophorales as well as the basally branching families Schizomeridaceae and Aphanochaetaceae gained no support by molecular synapomorphies. However, the derived family Chaetophoraceae and six of its subclades could be unambiguously characterized by NHS, as summarized in Table 1 and Figure 3. Only two subclades lacked NHS support, the ‘‘Uronema clade,’’ and the ‘‘Fritschiella clade.’’ Most synapomorphies listed in Table 1 represented CBCs in rRNA helices, characterizing six clades unambiguously. Three unique synapomorphies occurred in single-stranded parts of the rRNA molecule, that is, substitutions in internal loop and spacer regions, or an insertion (Table 1). The polyphyly of the genera Chaetophora and Stigeoclonium was clearly mirrored by several unique synapomorphies. Among several branches that 170 L E N K A C A I S O V Á E T A L . separate C. lobata from C. elegans, four were not only supported by high bootstrap values ⁄ posterior probabilities (Fig. 1), but also by unique synapomorphies (Table 1). C. lobata together with closely (C. draparnaldioides) and more distantly related taxa (Draparnaldia) as well as the whole ‘‘Chaetophora clade’’ all gained NHS support to the exclusion of C. elegans (Table 1, Fig. 3). Moreover, both strains of C. elegans displayed a unique CBC in Helix 24, representing an NHS to the exclusion of all Viridiplantae including C. lobata (Fig. 3). Similarly, the type species of Stigeoclonium (S. tenue) was clearly differentiated from S. ‘Longipilus’ (both members of the ‘‘Chaetophora clade’’) and also from S. farctum (within the ‘‘Fritschiella clade’’) by NHS (Table 1, Fig. 3). Synapomorphy analyses of user-defined trees that enforced either Chaetophora or Stigeoclonium as monophyletic (see below) revealed absence of any nuclear-encoded SSU rRNA synapomorphies that would support these artificially generated clades. Evolution of morphological characters within the Chaetophorales. Comparing results from cladistic analyses of morphological characters alone (Fig. 4A) with the tree topology that was favored by molecular data (Figs. 2 and 4B) revealed several clades, which were congruently recovered by both data sets (1–8 in Fig. 4). However, several branches of the cladistic tree were in conflict with 18S rDNA analyses (dashed lines in Fig. 4A). Only three clades, which were highly supported by 18S rDNA data (dashed lines in Fig. 4B), showed incongruence with the morphological tree. First, the Chaetophoraceae (clade 9 in Fig. 4) gained no support by cladistic analyses (Fig. 4A), although pyrenoid structure was revealed as a unique morphological synapomorphy of this clade (Fig. 4C). Second, the clade comprising Draparnaldia and two species of Chaetophora (excluding C. elegans; clade 10 in Fig. 4B), suggesting two independent origins of thick mucilage within the Chaetophoraceae, was in conflict with the cladistic analysis (Fig. 4A), which favored monophyly of all Chaetophora species and a unique gain of thick mucilage. Third, the Fritschiella ⁄ S. farctum clade (no. 11 in Fig. 4B), displaying secondarily reduced branching of filaments, formed two independent lineages at the base of the ‘‘branched Chaetophoraceae’’ clade in Figure 4A. It should be noted that both analyses (Fig. 4, A and B) recovered Stigeoclonium as nonmonophyletic, in contrast to the conflicting case of Chaetophora. Bayesian ancestral state reconstruction of morphological synapomorphies of selected clades (4, 9, 10, and 11 in Fig. 4) largely confirmed interpretations that resulted from cladistic synapomorphy searches (summarized in Fig. 4C) by high probability values (P > 0.94), except for lower probabilities for the only synapomorphy of the Chaetophoraceae (clade 9). In addition, the ‘‘mucilage’’ character was investigated by Bayesian ancestral state reconstruction for the common ancestor of the ‘‘branched Chaetophoraceae’’ lineage, resulting in high support (P = 0.99 ⁄ 0.99) for the character state ‘‘thin mucilage,’’ which often was used for defining a single genus (i.e., Stigeoclonium; see Discussion). Topology tests of user-defined trees. To reevaluate the major results of this study by ML-based topology tests, we generated artificial (user-defined) trees, which addressed alternative hypotheses to our results. Various topology tests (au, kh, sh) were applied to estimate whether user-defined trees are ‘‘significantly worse’’ than the ‘‘best’’ tree at the significance level 0.05 (Table 2). Two user-defined trees were not significantly worse than the original tree and thus could not be rejected by statistical comparisons using the SSU rDNA data: (i) the tree with the common branch of Aphanochaete collapsed, and (ii) the tree ‘‘Stigeoclonium monophyletic A’’ with an enforced clade comprising the four ‘‘main’’ Stigeoclonium species together with S. variabile (Table 2). In contrast, trees enforcing a monophyletic genus Stigeoclonium including the more-distant species (S. farctum and ⁄ or S. ‘Longipilus’), as well as trees enforcing a monophyletic genus Chaetophora (C. lobata, C. draparnaldioides and C. elegans), were rejected by nearly all topology tests with P-values well below 0.05 (Table 2). Therefore, topology tests clearly confirmed the nonmonophyly of the genera Stigeoclonium and Chaetophora. Taxonomic revision. Chaetophora Schrank 1783, Naturforscher (Halle) 19: p. 125. Lectotype (designated here): Chaetophora lobata Schrank 1783, Naturforscher (Halle) 19: p. 125, tab. VII, figs. 2, 3. Synonyms: Chaetophora incrassata (Hudson) Hazen 1902, Mem. Torrey Bot. Club 11: p. 214 (Basionym: Ulva incrassata Hudson 1778, Flora Anglica, Tomus II: p. 572; non U. incrassata O. F. Müller 1775, Flora Danica 4, fasc. II: p. 7, tab. DCLIII); Chaetophora endiviifolia (Roth) Agardh 1812, Dispositio Algarum Sueciae p. 42 [Basionym: Rivularia endiviaefolia Roth 1798, in Roemer (ed) Archiv für die Botanik 1 ⁄ 3: p. 51]; Chaetophora cornudamae (Roth) Bory de SaintVincent 1823, Dict. Class. Hist. Nat. 3: p. 431 (Basionym: Rivularia cornudamae Roth 1797, Catalecta Botanica 1: p. 212, tab. VI, fig. 2). DISCUSSION In the present study, we demonstrated that the most common chaetophoralean genera, Chaetophora and Stigeoclonium, as currently circumscribed, are polyphyletic taxa. Evidence for this conclusion included not only branching patterns and support values in phylogenetic trees but also unique synapomorphies in the SSU rRNA molecule. In addition, user-defined trees generated to restore monophyly of Chaetophora and Stigeoclonium were significantly worse than the best tree confirming polyphyly of these taxa based on topology tests. Although the 171 P O L Y P H Y L Y O F C H A E T O P H O R A A N D S T I G EO C L O N I U M G G U A AC H722 U U H673 A U CU A A UUC U GC G UUA UGA A A GA U G A A G GUUA CUUUA U A C A CG AA U GGA G U U A C U Fritschiella tuberosa U U 5' 3' G G U A AC H722 U U H673 A A CU A A UUC UUA UGA A A GA CG U GC G A A G GUUA CUUUA UGC A CG AA U GGA G C U A C U Chaetophora elegans U U H655 Fritschiella-clade (F. tuberosa and Stigeoclonium farctum) Helix 25: [H673]: bp 2 G-C ==> A-U (parallel change in Chlamydomonas sordida) Chaetophora elegans Helix 24: last bp [H655: bp 680/736] A-U ==> U-A U A C G H655 G G G G C U G A C A G G 5' A A G A G G C C A A G U A U Chaetophoraceae G U Helix 24 [H655: bp C U U-G ==> U U U C A U 3' 672/743] -AAGA-GGGACAGUCGGGGGC-U-UC--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACA-UCU-GC-GAAA-GC--A--UU-A-GCCAAG-U-AU-GUCUUC-AU-U- Chaetophora_elegans Fritschiella_tuberosa Stigeoclonium_farctum -AAGA-GGGACAGUCGGGGGC-A-UU--C--A-UAUUUC-/-GUGAAA-/-UGAAAGA-U--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--A-UAUUUC-/-GUGAAA-/-UGAAAGA-U--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U- Stigeoclonium_tenue Stigeoclonium_helveticum Stigeoclonium_variabile Chaetophora_lobata Chaetophora_draparnaldioides Draparnaldia_plumosa Caespitella_pascheri Uronema_confervicola Aphanochaete_magna Dilabifilum_sp. Aphanochaete_repens Schizomeris_leibleinii Position_Ecoli Escherichia_coli Helices_CRW Helices_ERRD Pseudulvella_americana Oedogonium_cardiacum Hydrodictyon_reticulatum Kirchneriella_aperta Scenedesmus_obtusus Chlamydomonas_sordida Chlamydomonas_reinhardtii Carteria_crucifera -AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-U-AU-GUCUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACA-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U660 670 680 710 720 730 740 750 | | | | | | | | -CUUG-AGUCUCGUAGAGGGG-G-GU--A--G-AAUUCC-/-GUGAAA-/-UGGAGGA-A--U-ACCG-GUG-GC-GAAG-GC--G--GC-C-CCCUGG-A-CG-AAGACU-GA-C-----[--H655--------------][------H673--/]------[/--H673'------]----[-H722-]----[H722'][-------H655'-----------]----[-------24-------------]---[------25----/]------[/--25'--------]--------[26]----[26']-----[----24'------------------] -AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAUA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-ACU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-ACU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-ACU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--A-UAUUUC-/-GUGAAA-/-UGAAAGA-U--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGGUAGUCGGGGGC-A-UU--C--G-UAUUCC-/-GUGAAA-/-CGGAAGA-C--G-AACA-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-ACUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U- Gloeotilopsis_planctonica Oltmannsiellopsis_geminata Ignatius_tetrasporus -AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUCC-/-GUGAAA-/-UGGAAGA-C--G-AACA-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGU-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-ACCAAG-G-AU-GUUUUC-AU-U- Ulvophyceae Trebouxia_arboricola Stichococcus_bacillaris Microthamnion_kuetzingianum Chlorella_vulgaris Geminella_minor -AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-ACU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-ACU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U- Trebouxiophyceae Tetraselmis_striata -AAGA-GGGACAGUCGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GUCAAG-G-AU-GUUUUC-AU-U- Chlorodendrophyceae Picocystis_salinarum Nephroselmis_olivacea Pseudoscourfieldia_marina Mamiella_gilva Pyramimonas_tetrarhynchus Prasinococcus_capsulatus -AAUA-GGGACAGUUGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAUA-GGGACAGUUGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-CG--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUUGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGAACAGUUGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAGA-GGGACAGUUGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAUA-GGGACAGUCGGGGGC-A-UC--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U- basal Chlorophyta Mesostigma_viride Coleochaete_scutata Chara_globularis Staurastrum_M752 -AAUA-GGGACAGUUGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAUA-GGGACAGUUGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U-AAUA-GAGACGGUUGGGGGC-A-UU--C--G-UAUUCC-/-GUGAAA-/-UGGAUGA-C--G-AACU-UCU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AC-GUUCUC-AU-U-AAUA-GGGACAGUUGGGGGC-A-UU--C--G-UAUUUC-/-GUGAAA-/-UGAAAGA-C--G-AACU-ACU-GC-GAAA-GC--A--UU-U-GCCAAG-G-AU-GUUUUC-AU-U- streptophyte green algae A U A 5' A CUCGG C U U C G U G C AU A A ACUGUAG U A A CGACAUC U U U H144 Chaetophoraceae without Uronema-clade AA C U U Spacer Helices [H144]-[H184a]: UCCC C 6 nucleotides ==> 7 nucleotides GGU C A GGG G 3' G A A H184a AA Draparnaldia_plumosa Chaetophora_lobata Chaetophora_draparnaldioides Stigeoclonium_tenue Stigeoclonium_helveticum Stigeoclonium_variabile Stigeoclonium_farctum Fritschiella_tuberosa Caespitella_pascheri Chaetophora_elegans Uronema_confervicola Aphanochaete_magna Dilabifilum_sp. Aphanochaete_repens Schizomeris_leibleinii Helices_CRW Helices_ERRD Pseudulvella_americana Oedogonium_cardiacum Hydrodictyon_reticulatum Kirchneriella_aperta Scenedesmus_obtusus Chlamydomonas_sordida Chlamydomonas_reinhardtii Carteria_crucifera Gloeotilopsis_planctonica Oltmannsiellopsis_geminata Ignatius_tetrasporus Trebouxia_arboricola Stichococcus_bacillaris Microthamnion_kuetzingianum Chlorella_vulgaris Geminella_minor Tetraselmis_striata Picocystis_salinarum Nephroselmis_olivacea Pseudoscourfieldia_marina Mamiella_gilva Pyramimonas_tetrarhynchus Prasinococcus_capsulatus Mesostigma_viride Coleochaete_scutata Chara_globularis Staurastrum_M752 Chaetophorales other Chlorophyceae A U A C Draparnaldia, Chaetophora lobata, A C and Chaetophora draparnaldioides U A C U U A A Spacer Helices [H441]-[H500]: nt U A G ==> U H441 C A U A A GA Major Draparnaldia-clade U UU A A CC Helix 18 [H441]: bp 1 G AA U A G - C ==> U- A 3' A 5' G 3 A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-U-A-ACAAU-CUAAAUC-CAUU-A-U-A-GA-U-UACCAA A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-U-A-ACGAU-CUAAAUC-CACU-A-U-A-GA-U-UACCAA A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-U-A-ACGAU-CUAAAUC-CACU-A-U-A-GA-U-UACCAA A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-U-A-ACGAU-CUAAACC-CACU-A-U-A-GA-G-UAUCCA A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-U-A-ACGAU-CUAAACC-CACU-A-U-A-GA-G-UAUCCA A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-U-A-ACGAU-UUAAAUC-CACU-A-U-A-GA-G-UAUCCA A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAACC-CAUU-A-A-C-GA-G-UACCAA A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAACC-CAUU-A-A-C-GA-G-UACCAA A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAACC-CAUU-A-A-C-GA-G-UACCAA A-CU-CGGAUAACU-GUAG-UAAUU-CUACAGCUAAUACG-UG-CUUCAAG-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAACC-CAUU-A-A-C-GA-G-UACCAA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GCAAC-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-UAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-UUU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-UAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-CAC---GAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-UAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GCAAA-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-UAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-UAUCCA -[---H144----------]-----[----H144'--------]-------[--H184a-]----[-H184a'-]---/-------[H441-----]-------[-----H441']--------------[--9-----------]-----[----9'--------]----------[--10----]----[-10'----]---/-------[18-]--------------------[18']----------A-CU-CGGAUAACC-GUAG-GAAAA-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-UAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-A-ACAAU-CUANAUC-CCUU-A-U-C-GA-G-GAUCCA A-CU-CGGAUACCC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-ACCCGACU-UCU--GGAAGGGU-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-UAUCCA A-CA-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA A-UU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-CCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GCAAA-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GCACA-ACCCGACU-UCU--GGAAGGGU-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-GAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCAACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-CAC---GAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GCAAA-UCCCGACU-CAC---GAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCUA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-AUCCA-UCCCGACU-CAC---GGAGGGA-CG-/-GAAUGA-G-U-ACAAU-UUAAAUC-CCUU-A-A-C-GA-G-UAUCAA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GCACA-UCCCGACU-CAC---GAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCAA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GCAAA-UCCCGACU-UCC--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCAA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCAA A-CU-CGGAUACCC-GUAG-UAAAU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAACC-CCUU-A-A-C-GA-G-GAUCAA A-CU-CGGAUACCC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUACA-GCCCGACU-UCU--GGAAGGGC-CG-/-GAAUGA-G-U-ACAAU-CUAAACC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAACAACCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUACCC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAC-UCCCGACU-UCU--GGAAGGGA-UG-/-GAAUGA-G-U-ACAAC-GUAAAAC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GCAAC-UCCCGACU-UCG---GAAGGGA-CG-/-GAAUGA-G-A-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCUCGACU-UCG---GAAGAGA-CG-/-GAAUGA-G-A-ACAAU-CUAAAUC-CCUU-A-U-C-GA-G-GAUCAA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GUAAA-UCCCGACU-UCG---GAAGGGA-CG-/-GAAUGA-G-A-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-GCAAC-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-A-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-AUAAA-UCCC-----AGCA-----GGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAACC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUACCC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-CACCAAG-UCCCGACU-CUC--GGAAGGGA-UG-/-GAAUGA-G-U-ACAAU-CUAAACC-CCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-CACCGAC-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-UCUU-A-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUGG-UAAUU-CUAGAGCUAAUACG-UG-CAGCAUA-UCCCGACU-UCU--GGAAGGGA-UG-/-GAAUGA-G-A-ACAGUCCUAAACU-CCUUCA-A-C-GA-G-GAUCCA A-CU-CGGAUAACC-GUAG-UAAUU-CUAGAGCUAAUACG-UG-C-ACAAC-UCCCGACU-UCU--GGAAGGGA-CG-/-GAAUGA-G-U-ACAAU-CUAAAUC-CCUU-A-A-C-GA-G-GAUCCA Fig. 3. Evidence for the polyphyly of Chaetophora and Stigeoclonium by unique molecular synapomorphies characterizing selected clades within the Chaetophoraceae. Secondary-structure diagrams based upon the first taxon intercepted by black lines. For details, see Table 1. 172 L E N K A C A I S O V Á E T A L . Table 2. Topology tests upon six user-defined trees derived from Figure 2 (= best tree), with a focus on the genera Aphanochaete, Chaetophora, and Stigeoclonium. Best tree (Fig. 2) Aphanochaete not monophyletica Stigeoclonium monophyletic Ab Stigeoclonium monophyletic Bc Stigeoclonium monophyletic Cd Chaetophora monophyletic Ae Chaetophora monophyletic Bf Method Observation au TreeView PAUP PAUP PAUP PAUP TreeView )2.7 2.7 3.2 29.7 129.6 95.0 108.0 0.857 0.170 0.316 0.016* <0.000* <0.000* <0.000* kh 0.844 0.156 0.278 0.018* 0* 0* 0* sh 0.986 0.801 0.791 0.103 0* 0* 0* User-defined trees were either generated manually (TreeView), or by running maximum-likelihood (ML) analyses with constraints, using PAUP (see Materials and Methods). Trees are characterized by the observed difference in )ln Lik (obs), and P-values of the approximately unbiased test (au), Kishino–Hasegawa test (kh), and the Shimodaira–Hasegawa test (sh). P-values <0.05, indicating significant rejection at the 5% level, are flagged with an asterisk (*). a Common branch of Aphanochaete collapsed. b ‘‘Main’’ Stigeoclonium (= Stigeoclonium tenue, amoenum, helveticum, protensum) plus Stigeoclonium variabile. c ‘‘Main’’ Stigeoclonium plus S. variabile and Stigeoclonium ‘Longipilus’. d ‘‘Main’’ Stigeoclonium plus S. variabile, S. ‘Longipilus’, and Stigeoclonium farctum. e Chaetophora elegans sister of Chaetophora lobata ⁄ draparnaldioides in the Chaetophora clade. f Chaetophora elegans sister of C. lobata ⁄ draparnaldioides in the Fritschiella clade. Chaetophorales have been studied for almost two centuries, the concepts and definitions of ‘‘old’’ genera (Chaetophora, Draparnaldia, and Stigeoclonium), established by Schrank (1783), Bory de SaintVincent (1808), and Kützing (1843), respectively, have remained virtually unchanged since about 1850. In particular, Chaetophora and Stigeoclonium have generally been regarded as two separate taxonomic units, and we found almost no reference for the notion that either of these genera could be para- or polyphyletic and may need revision at the genus level (see, however, Forest 1956). Notably, species of Chaetophora have not been reassigned to other genera, except for the transfer of marine species to phaeophyte and rhodophyte genera— for example, Chaetophora maritima to Ectocarpus (Rosenvinge 1910) and Kolderupia (Lund 1959), C. marina to Leathesia (Decaisne 1842), Chaetophora lumbricalis to Nemalion (Bornet 1892), and Chaetophora pellita to Cruoria (Fries 1835). Newly discovered taxa with a Stigeoclonium-like morphology were generally attributed to Stigeoclonium. Two genus descriptions by Pascher (1905) and Vischer (1933) represent the only exceptions. Pascher (1905) transferred Stigeoclonium terrestre to his new genus Iwanoffia, due to its terrestrial lifestyle and biflagellate zoospores; unfortunately, the phylogenetic position of Iwanoffia cannot be investigated since no cultures are available. Vischer (1933) discovered and isolated a new Stigeoclonium-like taxon with unusual grasslike growth and, rather than describing it as a new species of Stigeoclonium, created the new genus Caespitella (type: C. pascheri). Interestingly, this genus was not accepted by later authors (Fritsch 1935, Cox and Bold 1966, Shyam and Sarma 1980) and merged with Stigeoclonium, the species designated as S. pascheri by Cox and Bold (1966). Fortunately, we could investigate the authentic strain of C. pascheri (i.e., the culture established by Vischer). Our results based on molecular synapomorphies as well as phylogenetic reconstructions clearly favor Vischer’s concept of Caespitella as a separate genus, independent of Stigeoclonium. Polyphyly of Chaetophora and its taxonomic history. The polyphyly of ‘‘old’’ genera in their broad meaning (sensu lato; s. l.) is often related to the choice of plesiomorphic or homoplasious diagnostic characters (e.g., in Pleurastrum, Chamydomonas, Chloromonas; Friedl 1996, Pröschold et al. 2001). When polyphyly of a genus is demonstrated, it is necessary to select one clade as ‘‘genus sensu stricto’’ (s. str.) by (i) identifying the type species of the genus in question and (ii) analyzing morphological characters that were used in the original diagnosis. In the present study, we established Chaetophora as polyphyletic, diverging into one clade with C. elegans forming globose thalli, and another clade containing C. lobata in which the thallus has an irregularly lobed shape (Schrank 1783, Hazen 1902). The taxonomic history of Chaetophora is very confused. Currently, it is even unclear which Chaetophora species must be regarded as type, and three databases (Index Nominum Algarum [INA], http:// ucjeps.berkeley.edu/INA.html; Index Nominum Genericorum, http://botany.si.edu/ing/; Algaebase, http://www.algaebase.org/) yield three different results. When Chaetophora was established (Schrank 1783), the genus contained only two species, Chaetophora globosa and C. lobata, without designation of a type species. However, the first of these species resulted from transferring Conferva stellaris Müller to Chaetophora as C. globosa (as an illegitimate change of the epithet—the valid name would have been ‘‘Chaetophora stellaris’’; Art. 11.4. in International Code of Botanical Nomenclature [ICBN]; http:// ibot.sav.sk/icbn/main.htm). Hazen (1902) identified C. globosa as lectotype species of Chaetophora, thereby following the ‘‘American Code,’’ that is, 173 P O L Y P H Y L Y O F C H A E T O P H O R A A N D S T I G EO C L O N I U M A.) One of 50 most parsimonious trees resulting from MP analyses of 18 morphological characters (total tree length = 40 character changes) Stigeoclonium helveticum CCAP 477/1 Stigeoclonium helveticum SAG 477-2 Stigeoclonium helveticum CCALA 499 Stigeoclonium tenue CCAP 477/11A Stigeoclonium tenue CCAP 477/11B Stigeoclonium protensum LC L4 Stigeoclonium amoenum LC L1 Stigeoclonium variabile CCAP 477/13 Chaetophora lobata CCAP 413/1 Chaetophora lobata ACOI 447 Chaetophora draparnaldioides (S. 'Longipilus') LC L3 8 Chaetophora 'elegans' CCAP 413/2 Chaetophora elegans LC L5 6 Draparnaldia plumosa CCAP 418/1A Draparnaldia glomerata CCAP 418/2 Caespitella pascheri CCAP 410/2 Caespitella pascheri SAG 410-1 Stigeoclonium farctum CCAP 477/10A 4 5 Fritschiella tuberosa UTEX 1821 Fritschiella tuberosa SAG 112.80 Schizomeris leibleinii SAG 24.88 Schizomeris leibleinii SAG 44.84 Aphanochaete magna UTEX 1909 Aphanochaete confervicola SAG 27.91 2 Aphanochaete repens SAG 21.91 Aphanochaete ('Dilabifilum') sp. SAG 450-1a Aphanochaete ('Dilabifilum') sp. SAG 450-1b Aphanochaete elegans SAG 4.91 Uronema belkae UTEX 1179 Uronema trentonense CCAP 386/5 Uronema acuminatum UTEX 1178 Uronema ('Hormidiella') sp. CCAP 334/1 'Ulothrix' fimbriata CCAP 384/2 Uronema confervicolum CCAP 386/2 Hormotilopsis gelatinosa UTEX 104 Chaetopeltis orbicularis UTEX 422 Pseudulvella americana UTEX 2852 Oedogonium cardiacum UTEX 40 Oedogonium brevicingulatum 1 morphological Bulbochaete hiloensis UTEX952 character change 1 B.) ML tree topology resulting from 18S rDNA analyses (see Fig. 2) with branch lengths estimated upon 18 morphological characters (total tree length = 44 character changes) 10 4 Stigeoclonium helveticum CCAP 477/1 Stigeoclonium tenue CCAP 477/11A Stigeoclonium protensum LC L4 Stigeoclonium amoenum LC L1 Stigeoclonium variabile CCAP 477/13 7 Chaetophora lobata CCAP 413/1 Chaetophora lobata ACOI 447 Chaetophora draparnaldioides (S. 'Longipilus') LC L3 6 Draparnaldia plumosa CCAP 418/1A Draparnaldia glomerata CCAP 418/2 Stigeoclonium helveticum SAG 477-2 Stigeoclonium helveticum CCALA 499 Stigeoclonium tenue CCAP 477/11B 9 5 Fritschiella tuberosa UTEX 1821 Fritschiella tuberosa SAG 112.80 Stigeoclonium farctum CCAP 477/10A Caespitella pascheri CCAP 410/2 Caespitella pascheri SAG 410-1 11 50% 8 Chaetophora elegans CCAP 413/2 Chaetophora elegans LC L5 Uronema belkae UTEX 1179 Uronema trentonense CCAP 386/5 3 Uronema acuminatum UTEX 1178 Uronema ('Hormidiella') sp. CCAP 334/1 'Ulothrix' fimbriata CCAP 384/2 Uronema confervicolum CCAP 386/2 Aphanochaete magna UTEX 1909 Aphanochaete confervicola SAG 27.91 2 Aphanochaete ('Dilabifilum') sp. SAG 450-1a Aphanochaete ('Dilabifilum') sp. SAG 450-1b Aphanochaete elegans SAG 4.91 Aphanochaete repens SAG 21.91 1 Schizomeris leibleinii SAG 24.88 Schizomeris leibleinii SAG 44.84 Hormotilopsis gelatinosa UTEX 104 Chaetopeltis orbicularis UTEX 422 Pseudulvella americana UTEX 2852 Oedogonium cardiacum UTEX 40 Bulbochaete hiloensis UTEX952 1 morphological Oedogonium brevicingulatum character change C.) clade 1 (Schizomeris): - filament organization: uniseriate ==> multiseriate erect system - pyrenoid structure: matrix with cytoplasmic channels ==> traversed by several undulating thylakoids - flagellar root system: cruciate with 5-2-5-2 microtubules ==> 5-5-5-5 microtubules clade 2 (Aphanochaete): - vegetative growth form: only with erect filaments ==> predominantly prostrate - pyrenoid structure: matrix with cytoplasmic channels ==> penetrated shallowly by a few thylakoids - hairs: absent ==> unicellular hairs, basally swollen (parallel: Bulbochaete) clade 6 (Draparnaldia): - chloroplasts: parietal with straight edge ==> parietal with serrate edge - cell shape in the main axis of filaments: cylindrical ==> barrel-shaped clade 7 (Chaetophora lobata / draparnaldioides): - colony shape: no solid macroscopic colonies ==> solid lobate colonies clade 8 (Chaetophora elegans): - colony shape: no solid macroscopic colonies ==> solid globose colonies clade 3 (Uronema-clade): - branching: branched (with primary branches) ==> unbranched (parallel: Hormotilopsis, Oedogonium) clade 4 (branched Chaetophoraceae): - vegetative growth form: only with erect filaments ==> well developed erect and prostrate systems (p = 0.95 / 0.96) - branching: branched (with primary branches) ==> with more than two levels of branching (changed: Fritschiella-clade) (p = 0.94 / 0.94) clade 5 (Fritschiella): - filament organization: uniseriate ==> usually uniseriate, but occasonally with multiseriate prostrate system - mucilage: thin mucilage ==> without mucilage - orange/red secondary carotenoids: absent ==> present - habitat: attached in freshwater ==> also subaerial on soil clade 9 (Chaetophoraceae): - pyrenoid structure: matrix with a few to several thylakoids ==> matrix bounded by one peripheral thylakoid (p = 0.71 / 0.75) clade 10 (C. lobata, C. draparnaldioides, Draparnaldia): - mucilage: thin mucilage ==> always with thick mucilage (p = 0.97 / 0.96) (thick mucilage covering only older thalli in C. draparnaldioides) (parallel change: C. elegans) clade 11 (Fritschiella, Stigeoclonium farctum): - branching: with more than two levels of branching ==> with primary and secondary branches (p = 0.97 / 0.98) (parallel change: Aphanochaete strains SAG 450-1a, SAG 450-1b) Fig. 4. Cladistic analysis of morphological character evolution in the Chaetophorales. (A) Maximum-parsimony (MP) analysis of morphological characters using PAUP. (B) Morphological data matrix (Appendix S1 in the supplementary material) mapped upon the branching pattern that resulted from maximum-likelihood (ML) analyses of 18S rDNA data (treefile of Fig. 2). Note that all divergences collapsed that displayed no morphological character changes (e.g., the Fritschiella clade and the Chaetophora clade resolved in Fig. 2). Dashed lines in the morphological tree indicate noncongruence with the molecular tree and vice versa. Encircled numbers 1–9 indicate clades supported by both data sets; clades 9–11 (double circled) were recovered only by 18S rDNA data. (C) Morphological character changes (ancestral ==> derived character states) of 11 clades labeled in trees. Selected character states were analyzed by Bayesian ancestral state reconstruction and probabilities for both Markov chain Monte Carlo chains (P = chain1 ⁄ chain2). 174 L E N K A C A I S O V Á E T A L . selecting the first-mentioned species in Schrank’s original description (see Canon 15 in the American Code of Botanical Nomenclature [1907]; Torrey Botanical Club Bulletin 34:167–78; http://www.jstor. org/stable/2479237). This method of lectotype selection is now considered as ‘‘largely mechanical’’ and must not be followed (Art. 10.5 in ICBN). As Schrank’s (1783) conception of the new genus Chaetophora was largely based on his own observations on C. lobata, we herein designated C. lobata as lectotype of this genus. C. lobata was briefly described as ‘‘This species is lobed...the color is grass-green’’ (translated from German), and illustrated (Schrank 1783, figs. 2 and 3, tab. VII) as a deeply bilobed thallus with long, radiating hairs, growing on submerged Ceratophyllum. Schrank’s (1783, p. 125) generic diagnosis (‘‘Muscus frondibus setas longissimas ferentibus’’) can be translated as ‘‘a moss with fronds, carrying extremely long hairs.’’ Clearly, Schrank’s (1783) circumscription of Chaetophora was based upon species with distinctly lobed thalli (‘‘frondibus’’), characterizing not only C. lobata but also later described taxa (e.g., C. incrassata, Chaetophora atra, C. cornudamae, and C. endiviifolia). Later authors (Hazen 1902, Starmach 1972) regarded C. endiviifolia, C. cornudamae, and C. incrassata as synonymous. Hazen (1902) also listed Schrank’s (1783) C. lobata as synonym of C. incrassata, albeit contrary to the current rule of priority (Art. 11.4. in ICBN; http://ibot.sav.sk/icbn/main.htm). Moreover, for the new combination C. incrassata, she selected U. incrassata Hudson (1778) as basionym, which is an invalid homonym of U. incrassata Müller (1775). We therefore listed C. incrassata, C. cornudamae, and C. endiviifolia as synonyms of C. lobata. In our phylogenetic analyses, one of the two unrelated Chaetophora lineages combined the lobate species, C. lobata, with C. draparnaldioides, which is characterized by hemispherical lobes (‘‘Fronde hemisphaerica’’; Kützing 1834). Therefore, we regard the subclade containing these lobate species as Chaetophora sensu stricto. Chaetophora elegans, a species with globose thalli, branched independently from Chaetophora s. str. in our phylogenetic trees. C. elegans was the first globose species described in this genus (Agardh 1812) and was followed by others (e.g., C. attenuata, C. pisiformis, C. punctiformis, C. tuberculosa). To circumscribe the ‘‘extended’’ genus Chaetophora, inclusive of lobate forms, Kützing (1843, p. 325) introduced an emended diagnosis in which the shape of the thallus was no longer specified (‘‘heteromorphis’’). Instead, he placed emphasis on the presence of a mucilaginous envelope (‘‘Phycoma gelatinosum... mucosa involutis’’) surrounding the filaments to distinguish Chaetophora from its relatives (Stigeoclonium, Draparnaldia). This relaxed diagnosis of Chaetophora defined a polyphyletic genus, using a homoplasious character, that is, the copious production of mucilage, which apparently evolved independently in two lineages within the Chaetophorales (see Fig. 4). Polyphyly of Stigeoclonium. The genus Stigeoclonium s. l. represents the most species-rich genus of the Chaetophorales, together with Draparnaldia (both 80 species; Chaetophora: 60 species; http:// ucjeps.berkeley.edu/INA.html). One of the five original species introduced by Kützing (1843), S. tenue, is the currently accepted type species (ICBN) and should thus define Stigeoclonium s. str. (this is deferred to a later publication). Although our analyses included two strains of S. tenue, their SSU rDNA sequences were almost identical to those of three other species, which together formed a polytomy at the base of the Chaetophora clade. Other molecular markers (e.g., rbcL, internal transcribed spacer regions) will have to be analyzed in addition to rDNA genes to resolve the basal branches of the ‘‘Chaetophora clade’’ and to define Stigeoclonium s. str. unambiguously. However, our SSU rDNA phylogenies as well as formalized cladistic analyses of morphological characters resolved two additional species of Stigeoclonium as members of two lineages, which are clearly unrelated to each other and also to S. tenue (i.e., S. ‘Longipilus’ and S. farctum). Therefore, Stigeoclonium s. l. is polyphyletic, and it is reasonable to assume that the original description of the genus by Kützing (1843) was also based on homoplasious characters. In the diagnosis of Stigeoclonium, Kützing (1843, p. 253) listed several features, which also apply to Chaetophora but regarded the presence of a very thin layer of mucilage surrounding filaments (‘‘cellulae gelineae tenuissimae’’) as characteristic for Stigeoclonium. For S. tenue, Kützing (1843) described and illustrated zoosporangia in all filaments (main axis and lateral branches) and pointed out a difference to Chaetophora and Draparnaldia in which zoosporangia are confined to lateral branches. The presence of a thin layer of mucilage and the type of zoospore formation are still used to define this genus and apply to all Stigeoclonium species studied here as well as to C. pascheri. In molecular phylogenetic trees, these Stigeoclonium-like taxa represent mainly the basal divergences of both the ‘‘Fritschiella clade’’ and the ‘‘Chaetophora clade’’ but also occupy some derived positions (e.g., S. farctum). We conclude that the traditional diagnostic characters used to define Stigeoclonium represent plesiomorphies of the entire ‘‘branched Chaetophoraceae’’ clade and have been inherited from the last common ancestor of this major lineage of Chaetophorales (this view was confirmed by Bayesian ancestral state reconstruction; see Results). The same likely applies to other characters added later to descriptions of Stigeoclonium (e.g., intercalary vegetative cell division) (Islam 1963). The divergent position of S. ‘Longipilus’ as closest relative of C. lobata, though initially unexpected, gains support by selected morphological features P O L Y P H Y L Y O F C H A E T O P H O R A A N D S T I G EO C L O N I U M and is accordingly supported by the cladistic analysis performed here (Fig. 4). In contrast to ‘‘adult’’ filaments, ‘‘young’’ stages of S. ‘Longipilus’ have been described as ‘‘surrounded by mucilage matrix as with Chaetophora’’ (Islam 1963) and forming small tufts, again resembling Chaetophora (our own unpublished observations; see also fig. 104 in Pascher 1914), corroborating its placement in the phylogenetic analyses. Moreover, the short, cylindrical to globose cells, deeply constricted at the partition wall, which are characteristic of S. ‘Longipilus’, differ from most other Stigeoclonium species (Islam 1963). S. ‘Longipilus’ has initially been described under the name C. draparnaldioides (Kützing 1834), and our phylogenetic analyses provide clear support for its original designation. Phylogeny of Draparnaldia. The present paper contributed the first released nuclear-encoded SSU rDNA sequence data of Draparnaldia (i.e., of D. glomerata and D. plumosa). Phylogenetic analyses recovered Draparnaldia as a monophyletic sister clade of Chaetophora s. str. However, the monophyly of Draparnaldia still needs to be confirmed by addition of more species. Monophyly of Draparnaldia may reflect the fact that this genus appears well delimited from Chaetophora s. str. as well as from Stigeoclonium by differentiation into a large-celled main axis of unlimited growth, which never forms zoospores, and lateral filaments of limited growth, composed of smaller cells, capable of zoospore differentiation (Pascher 1914, Forest 1956). Character evolution in the Chaetophorales. Based on the present phylogenetic analyses, a first attempt can be made to trace the evolution of morphological characters during the diversification of the Chaetophorales (see Fig. 4). First, the following characters likely represent the ancestral state (plesiomorphies) within the order: multicellular, branched filaments consisting of uninucleate cells with a single, parietal chloroplast; filaments attached to a substrate; and reproduction by quadriflagellate zoospores. Only few morphological characters can presently be regarded as synapomorphies of the Chaetophorales to the exclusion of other Chlorophyceae—for example, features of the flagellar apparatus (upper and lower pairs of basal bodies in a clockwise [1 ⁄ 7 o’clock] arrangement, peripheral and terminal fibers between adjacent basal bodies; Melkonian 1975, Watanabe and Floyd 1989, Lewis and McCourt 2004) and the mode of cell division (closed mitosis, cytokinesis by formation of a phycoplast-associated cell plate with several plasmodesmata; Stewart et al. 1973, Mattox and Stewart 1984), which, however, has not yet been studied in detail in the Chaetopeltidales. Both basal chaetophoralean lineages, the Schizomeridaceae and Aphanochaetaceae, show divergent traits: Schizomeris developed a unique multiseriate filament system lacking hairs and a prostrate system of ‘‘creeping’’ filaments. In contrast, the thallus of Aphanochaete is dominated by 175 prostrate filaments with colorless hairs often present, usually unicellular, unsheathed, with a bulbous base. The derived family Chaetophoraceae shows a basal divergence into unbranched (Uronema clade) and branched morphotypes and thus cannot yet be circumscribed by morphological synapomorphies, except pyrenoid ultrastructure (pyrenoids bounded, but not traversed by thylakoids; Stewart and Mattox 1975, John 1984). Unbranched Chaetophoraceae mainly display ‘‘loss of’’ characters, such as branching, a prostrate filament system, and hairs. Branched Chaetophoraceae are well distinguished by differentiation into prostrate and upright filament systems, usually terminating with gradually tapering, multicellular hairs. Altogether, these synapomorphies may characterize a Stigeoclonium-like morphology (see above). The ‘‘branched Chaetophoraceae’’ lineage likely evolved toward mucilaginous (Chaetophora) and highly differentiated thalli (Fritschiella and Draparnaldia), which are traditionally also regarded as the most derived Chaetophorales (Forest 1956, Islam 1963). Rather than a single evolutionary series, molecular phylogenies suggest two independent, parallel progressions from Stigeocloniumlike ancestors toward more structurally complex genera such as Fritschiella and Draparnaldia. Our study clearly revealed that several morphological characters previously used for genus and species descriptions are insufficient to determine taxonomic and evolutionary relationships in the Chaetophorales. As a next step, it seems necessary to combine more detailed molecular phylogenetic analyses, using a more taxon-rich multigene approach, in combination with careful reevaluation of morphological traits under both natural and controlled conditions. We thank the culture collections CCAP, CCALA, and SAG for providing strains. L. C. was supported by the grant CR no. 206 ⁄ 09 ⁄ 0697, JU 038 ⁄ 2008P, and by the University of Cologne. Further support came from NERC Oceans 2025 (NF3 *CCAP) and NERC Molecular sequencing grant NERC MGF 154. We thank Paul Silva for his valuable comments on nomenclatural issues. Abbas, A. & Godward, B. E. 1963. Cytology in relation to taxonomy in Chaetophorales. Bot. J. Linn. Soc. 58:499–507. Agardh, C. A. 1812. Dispositio Algarum Sueciae, Quam Publico Examini Subjiciunt Carol Adolph Agardh et Gustav Sannberg. Litteris Berlingianis, Lund, Sweden, pp. 27–45. Aziz, M. K. S. & Islam, N. 1962. A new species of Fritschiella Iyengar from East Pakistan Trans. Am. Microsc. Soc. 81:185–9. Berthold, G. 1878. Untersuchungen über die Verzweigung einiger Süsswasseralgen. Nova Acta Acad. Caesareae Leopoldino-Carolinae Germanicae Naturae Curiosorum 40:167–230. Booton, G. C., Floyd, G. L. & Fuerst, P. A. 1998. Origins and affinities of the filamentous green algal orders Chaetophorales and Oedogoniales based on 18S rRNA gene sequences. J. Phycol. 34:312–8. Bornet, E. 1892. Les algues de P. K. A. Schousboe. Me´m. Soc. Natl. Sci. Nat. Math. Cherbourg 28:165–376. Bory de Saint-Vincent, M. 1808. Mémoire sur le genre Draparnaldia, de la famille des Conferves (Voyez la planche 35). Ann. Mus. Hist. Nat. 12:399–409. 176 L E N K A C A I S O V Á E T A L . Bourrelly, P. 1972. Les Algues d¢Eau Douce. I. Les Algues Vertes. N. Boubée and Cie, Paris, 570 pp. Brouard, J.-S., Otis, C., Lemieux, C. & Turmel, M. 2008. Chloroplast DNA sequence of the green alga Oedogonium cardiacum (Chlorophyceae): unique genome architecture, derived characters shared with the Chaetophorales and novel genes acquired through horizontal transfer. BMC Genomics 9:290. Buchheim, M. A., Michalopulos, E. A. & Buchheim, J. A. 2001. Phylogeny of the Chlorophyceae with special reference to the Sphaeropleales: a study of 18S and 26S rRNA data. J. Phycol. 37:819–35. Campbell, E. O. & Sarafis, V. 1972. Schizomeris – a growth form of Stigeoclonium tenue (Chlorophyta: Chaetophoraceae). J. Phycol. 8:276–82. Cox, E. R. & Bold, H. C. 1966. Phycological studies. VII. Taxonomic investigations of Stigeoclonium. Univ. Texas Publ. 6612: 1–167. Decaisne, J. 1842. Essais sur une classification des Algues et des Polypiers calciféres de Lamouroux. Ann. Sci. Nat. Bot. Paris Se´r. 2 XVII:297–380. Ettl, H. & Gärtner, G. 1995. Syllabus der Boden-, Luft- und Flechtenalgen. Gustav Fischer Verlag, Stuttgart, Germany, 720 pp. Floyd, G. L., Hoops, H. J. & Swanson, J. A. 1980. Fine structure of the zoospore of Ulothrix belkae with emphasis on the flagellar apparatus. Protoplasma 104:17–31. Forest, H. S. 1956. A study of the genera Draparnaldia Bory and Draparnaldiopsis Smith and Klyver. Castanea J. 21:1–27. Francke, J. A. & Simons, J. 1984. Morphology and systematics of Stigeoclonium Kützing (Chaetophorales). In Irvine, D. E. G. & John, D. M. [Eds.] Systematics of the Green Algae. Academic Press, London, pp. 363–77. Francke, J. A. & Ten Cate, H. J. 1980. Ecotypic differentiation in response to nutrition factors in the algal genus Stigeoclonium Kütz. (Chlorophyceae). Br. Phycol. J. 15:343–55. Friedl, T. 1996. Evolution of the polyphyletic genus Pleurastrum (Chlorophyta): inferences from nuclear-encoded ribosomal DNA sequences and motile cell ultrastructure. Phycologia 35:456–69. Friedl, T. & Zeltner, C. 1994. Assessing the relationships of some coccoid green lichen algae and the Microthamniales (Chlorophyta) with 18S ribosomal RNA gene sequence comparisons. J. Phycol. 30:500–6. Fries, E. M. 1835. Corpus Florarum Provincialium Sueciae. I. Floram Scanicam Scripsit Elias Fries. Uppsala, Sweden, 192 pp. Fritsch, F. E. 1935. The Structure and Reproduction of the Algae. I. Cambridge, Univ. Press, Cambridge, UK, 791 pp. Gibson, M. T. & Whitton, B. A. 1987. Hairs, phosphatase activity and environmental chemistry in Stigeoclonium, Chaetophora and Draparnaldia (Chaetophorales). Br. Phycol. J. 22:11–22. Gillespie, J. J., Johnston, J. S., Cannone, J. J. & Gutell, R. R. 2006. Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements. Insect Mol. Biol. 15:657–86. Godward, M. B. 1942. The life-cycle of Stigeoclonium amoenum. New Phytol. 41:293–301. Guindon, S. & Gascuel, O. 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52:696–704. Harding, J. P. C. & Whitton, B. A. 1978. Resistance to zinc of Stigeoclonium tenue in the field and the laboratory. Br. Phycol. J. 11:417–26. Hazen, T. E. 1902. The Ulotrichaceae and Chaetophoraceae of the United States. Mem. Torrey Bot. Club 11:135–250. Hudson, W. 1778. Flora Anglica; Exhibiens Plantas per Regnum Britanniae Sponte Crescentes, Distributas Secundum Systema Sexuale: Cum Differentiis Specierum, Synonymis Auctorum, Nominibus Incolarum, Solo Locorum, Tempore Florendi, Officinalibus Pharmacopaeorum. Tomus II. Editio Altera, Emendata et Aucta. J. Nourse, London, pp. 335–690. Islam, A. K. M. N. 1963. A Revision of the Genus Stigeoclonium. Nova Hedwigia Beih. 10, Verlag J. Cramer, Weinheim, Germany, 164 pp. John, D. M. 1984. On the systematic of the Chaetophorales. In Irvine, D. E. G. & John, D. M. [Eds.] Systematics of the Green Algae. Academic Press, London, pp. 207–32. Johnstone, I. M. 1978. Phenotypic plasticity in Draparnaldia (Chlorophyta: Chaetophoraceae). I. Effects of the chemical environment. J. Phycol. 14:302–8. Kützing, F. T. 1834. Algarum Aquae Dulcis Germanicarum. Decas XI. Societ. Bot. Ratisbon, Solidalis, Halis Saxonum, in commissis C. A. Schwetschkii et fil., Halle, 23 pp. Kützing, F. T. 1843. Phycologia Generalis Oder Anatomie, Physiologie und Systemkunde der Tange. F. A. Brockhaus, Leipzig, Germany, 458 pp. Kützing, F. T. 1845. Phycologia Germanica, d.i. Deutschlands Algen in Bündigen Beschreibungen. Nebst Einer Anleitung zum Untersuchen und Bestimmen Dieser Gewächse für Anfänger. Wilh. Köhne, Nordhausen, 340 pp. Lewis, L. A. & McCourt, R. M. 2004. Green algae and the origin of land plants. Am. J. Bot. 91:1535–56. Lund, S. 1959. The marine algae of East Greenland. I. Taxonomical part. Meddel. Grønland 156:1–247. Manton, I. 1964. Observations on the fine structure of the zoospore and young germling of Stigeoclonium. J. Exp. Bot. 15:399–411. Marin, B., Nowack, E. C. M. & Melkonian, M. 2005. A plastid in the making: evidence for a second primary endosymbiosis. Protist 156:425–32. Marin, B., Palm, A., Klingberg, M. & Melkonian, M. 2003. Phylogeny and taxonomic revision of plastid-containing euglenophytes based on SSU rDNA sequence comparisons and synapomorphic signatures in the SSU rRNA secondary structure. Protist 154:99–145. Mattox, K. R. & Stewart, K. D. 1984. Classification of the green algae: a concept based on comparative cytology. In Irvine, D. E. G. & John, D. M. [Eds.] Systematics of the Green Algae. Academic Press, London, pp. 29–72. Melkonian, M. 1975. The fine structure of the zoospores of Fritschiella tuberosa Iyeng. (Chaetophorineae, Chlorophyceae) with special reference to the flagellar apparatus. Protoplasma 86:391–404. Melkonian, M. 1990. Phylum Chlorophyta: Class Chlorophyceae. In Margulis, L., Corliss, J. O., Melkonian, M. & Chapman, D. [Eds.] Handbook of Protoctista. Jones & Bartlett Publishers, Boston, pp. 608–16. Melkonian, M. & Surek, B. 1995. Phylogeny of the Chlorophyta: congruence between ultrastructural and molecular evidence. Bull. Soc. Zool. Fr. 120:191–208. Michetti, K. M., Leonardi, P. I. & Caceres, E. J. 2010. Morphology, cytology and taxonomic remarks of four species of Stigeoclonium (Chaetophorales, Chlorophyceae) from Argentina. Phycol. Res. 58:35–43. Müller, O. F. 1775. Icones Plantarum Sponte Nascentium in Regnis Daniae et Norvegiae, et in Ducatibus Slesvici, Holsatiae et Oldenburg: Ad Illustrandum Opus de Iisdem Plantis, Regio Jussu Exarandum, Florae Danicae Nomine Inscriptum. Volumen Quartum, Fasc. XI. Mart. Hallageri, Copenhagen, 8 pp., and tab. DCI–DCLX. O’Kelly, C. J. & Floyd, G. L. 1984. Correlation among patterns of sporangial structure and development. Life histories, and ultrastructural features in the Ulvophyceae. In Irvine, D. E. G. & John, D. M. [Eds.] Systematics of the Green Algae. Academic Press, London, pp. 121–56. O’Kelly, C. J., Watanabe, S. & Floyd, G. L. 1994. Ultrastructure and phylogenetic relationships of Chaetopeltidales ord. nov. (Chlorophyta, Chlorophyceae). J. Phycol. 30:118–28. Page, R. D. M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comp. Appl. Biosci. 12:357–8. Pascher, A. 1905. Zur Kenntnis der geschlechtlichen Fortpflanzung bei Stigeoclonium. Flora (Jena) 1:95–107. Pascher, A. 1914. Die Süsswasserflora Deutschlands, Österreichs und der Schweiz. Heft 6: Chlorophyceae III. Ulothrichales, Microsporales, Oedogoniales. Gustav Fischer, Jena, Germany, 250 pp. Pawlik-Skowrońska, B. 2003. Resistance, accumulation and allocation of zinc in two ecotypes of the green alga Stigeoclonium tenue Kütz. coming from habitats of different heavy metal concentrations. Aquat. Bot. 75:189–98. P O L Y P H Y L Y O F C H A E T O P H O R A A N D S T I G EO C L O N I U M Posada, D. 2008. ModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25:1253–6. Prescott, G. W. 1979. A Contribution to a Bibliography of Antarctic and Subantarctic Algae Together With a Checklist of Freshwater Taxa Reported to 1977. Bibliotheca Phycologica 45. J. Cramer in der A.R. Gantner Verlag Kommanditgesellschaft, Vaduz, Liechtenstein, 312 pp. Printz, H. 1964. Die Chaetophoralen der Binnengewässer (eine systematische Übersicht). Hydrobiologia 24:1–376. Pröschold, T. & Leliaert, F. 2007. Systematics oft the green algae: conflict of classic and modern approaches. In Brodie, J. & Lewis, J. [Eds.] Unravelling the Algae. The Past, Present and Future of Algal Systematics. Taylor & Francis, Boca Raton, Florida, pp. 124–53. Pröschold, T., Marin, B., Schlösser, U. G. & Melkonian, M. 2001. Molecular phylogeny and taxonomic revision of Chlamydomonas (Chlorophyta). I. Emendation of Chlamydomonas Ehrenberg and Chloromonas Gobi, and description of Oogamochlamys gen. nov. and Lobochlamys gen. nov. Protist 152:265–300. Ronquist, F. & Huelsenbeck, J. P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–4. Rosenvinge, L. K. 1910. On the marine algae from north-east Greenland (N of 76 N Lat) collected by the ‘‘Danmarkexpedition’’. Meddr. Grønland. 43:93–133. Sarma, P. 1986. The Freshwater Chaetophorales of New Zealand. Nova Hedwigia Beih. 58. J. Cramer, Berlin, 163 pp. Sarma, Y. S. R. K. & Jayaraman, S. 1980. Karyological studies on certain taxa of Stigeoclonium and Chaetophora (Chaetophorales, Chlorophyceae). Phycologia 19:253–9. Saxena, P. N. 1962. Algae of India-1, Chaetophorales. Bull. Nat. Bot. Gard. 57, National Botanic Gardens, Lucknow, India, 59 pp. Schrank, F. v. P. 1783. Botanische Rhapsodien. Der Naturforscher (Halle) 19:116–26. Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51:492–508. Shimodaira, H. & Hasegawa, M. 2001. CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 17:1246–7. Shyam, R. & Sarma, Y. S. R. K. 1980. Observations on the morphology, reproduction and cytology of Stigeoclonium pascheri (Vischer) Cox et Bold (Chaetophorales-Chlorophyceae) and their bearing on the validity of the genus Caespitella Vischer. Hydrobiologia 70:83–93. Silva, P. C. 1982. Chlorophycota. In Parker, S. [Ed.] Synopsis and Classification of Living Organisms, Vol. 1. McGraw-Hill, New York, pp. 133–61. Simons, J., Van Beem, A. P. & De Vries, P. J. R. 1986. Morphology of the prostrate thallus of Stigeoclonium (Chlorophyceae, Chaetophorales) and its taxonomic implications. Phycologia 25:210– 20. Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–90. Starmach, K. 1972. Chlorophyta III. Zielenice nitkowate: Ulothrichales, Ulvales, Prasiolales, Sphaeropleales, Cladophorales, Chaetophorales, Trentepohliales, Siphonales, Dichotomosiphonales. Flora Słodkowodna Pol. 10:1–750. Stewart, K. D. & Mattox, K. R. 1975. Comparative cytology, evolution and classification of the green algae with some consideration of the origin of other organisms with chlorophyll a and b. Bot. Rev. 41:104–35. Stewart, K. D., Mattox, K. R. & Floyd, G. L. 1973. Mitosis, cytokinesis, the distribution of plasmodesmata, and other cytological 177 characteristics in the Ulotrichales, Ulvales, and Chaetophorales: phylogenetic and taxonomic considerations. J. Phycol. 9:128–41. Swofford, D. L. 2000. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Tupa, D. D. 1974. An investigation of certain chaetophoralean algae. Nova Hedwigia Beih. 46:1–155. Turmel, M., Brouard, J.-S., Gagnon, C., Otis, C. & Lemieux, C. 2008. Deep division in the Chlorophyceae (Chlorophyta) revealed by chloroplast phylogenomic analyses. J. Phycol. 44:739–50. Turmel, M., Gagnon, M. C., O’Kelly, C. J., Otis, C. & Lemieux, C. 2009. The chloroplast genomes of the green algae Pyramimonas, Monomastix, and Pycnococcus shed new light on the evolutionary history of prasinophytes and the origin of the secondary chloroplasts of euglenids. Mol. Biol. Evol. 26:631–48. Uspenskaja, W. J. 1930. Über die Physiologie der Ernährung und die Formen von Draparnaldia glomerata Agardh. Z. Bot. 22:337– 93. Van Beem, A. P. & Simons, J. 1988. Growth and morphology of Draparnaldia mutabilis (Chlorophyceae, Chaetophorales) in synthetic medium. Br. Phycol. J. 23:143–51. Vischer, W. 1933. Über einige kritische Gattungen und die Systematik der Chaetophorales. Bot. Centralbl. Beih. 51:1–100. Watanabe, S. & Floyd, G. L. 1989. Ultrastructure of the quadriflagellate zoospores of the filamentous green algae Chaetophora incrassata and Pseudoschizomeris caudata (Chaetophorales, Chlorophyceae) with emphasis on the flagellar apparatus. Bot. Mag. Tokyo 102:533–46. West, G. S. & Fritsch, F. E. 1927. A Treatise on the British Freshwater Algae. Cambridge Univ. Press, Cambridge, UK, 534 pp. Wille, N. 1901. Algologische Notizen. VII, VIII. Nyt Mag. Naturvidenskab. 39:1–24. Supplementary Material The following supplementary material is available for this article: Table S1. Strain designations and origins for 30 strains of Chaetophorales examined in this study and accession numbers of newly determined nuclear-encoded SSU rRNA genes. Appendix S1. Data matrix of 18 morphological characters in the Chaetophorales used for cladistic analyses and ancestral state reconstructions in Figure 4. This material is available as part of the online article. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.