J. Phycol. 46, 346–362 (2010) 2010 Phycological Society of America DOI: 10.1111/j.1529-8817.2010.00814.x

MOLECULAR PHYLOGENY AND REVISION OF THE GENUS NETRIUM (ZYGNEMATOPHYCEAE, STREPTOPHYTA): NUCLEOTAENIUM GEN. NOV.1

Andrey A. Gontcharov2,3 and Michael Melkonian Botanisches Institut, Lehrstuhl I, Universita¨tzuKo¨ln, Gyrhofstr. 15, D-50931 Ko¨ln, Germany

Nuclear-encoded SSU rDNA, chloroplast LSU Molecular phylogenetic studies have significantly rDNA, and rbcL genes were sequenced from 53 changed our perception of the relationships among strains of conjugating green algae (Zygnematophy- algal taxa and challenged the importance of many ceae, Streptophyta) and used to analyze phyloge- phenotypic characters traditionally used in algal sys- netic relationships in the traditional order tematics. As a result, the composition and taxo- Zygnematales. Analyses of a concatenated data set nomic structure of many algal taxa are uncertain, (5,220 nt) established 12 well-supported clades in requiring revision based on new monographic work. the order; seven of these constituted a superclade, Species richness and morphological diversity often termed ‘‘Zygnemataceae.’’ Together with genera complicate taxonomic studies, but morphologically (Zygnema, Mougeotia) traditionally placed in the fam- simple taxa revealing only few distinguishing charac- ily Zygnemataceae, the ‘‘Zygnemataceae’’ also ters often provide equal challenges for taxonomic included representatives of the genera Cylindrocystis revision. Both situations apply to the systematics of and Mesotaenium, traditionally placed in the family the conjugating green algae (Zygnematophyceae, Mesotaeniaceae. A synapomorphic amino acid Streptophyta), which comprise >4,000 species of uni- replacement (codon 192, cysteine replaced by cellular, filamentous, or colonial algae currently valine) in the LSU of RUBISCO characterized this classified into 55 genera, six families, and two superclade. The traditional genera Netrium, Cylindro- orders (Mix 1972, 1975, Ru˚ˇzicˇka 1977, Brook and cystis, and Mesotaenium were shown to be para- or Johnson 2002, Gerrath 2003). The large number of polyphyletic, highlighting the inadequacy of pheno- species described sets this class apart from the other typic traits used to define these genera. Species of relatively species-poor lineages of streptophyte green the traditional genus Netrium were resolved as three algae. Members of the Zygnematophyceae are fur- well-supported clades each distinct in the number of ther distinctive in their peculiar mode of sexual chloroplasts per cell, their surface morphology reproduction (i.e., conjugation) and in the absence (structure and arrangement of lamellae) and the of flagellate reproductive stages throughout their position of the nucleus or nuclear behavior during life history. cell division. Based on molecular phylogenetic analy- The orders and families of the Zygnematophy- ses and synapomorphic phenotypic traits, the genus ceae were established on the basis of cell wall ultra- Netrium has been revised, and a new genus, Nucleo- structure and cellular organization (i.e., filamentous taenium gen. nov., was established. The genus Plano- or unicellular), whereas most of the genera have taenium, also formerly a part of Netrium, was been defined by characters of cell morphology, such identified as the sister group of the derived as cell and semicell shape, ornamentation of the cell Roya ⁄ Desmidiales clade and thus occupies a key wall, degree of cell constriction, and, more rarely, position in the evolutionary radiation leading to the chloroplast shape. Most of these morphological most species-rich group of streptophyte green algae. characters are apparently late innovations that occur only in the derived order Desmidiales, which is Key index words: cp LSU rDNA; Netrium; Nucleotae- characterized by ornamented and porous cell walls nium;nuSSUrDNA;phylogeny;Planotaenium; composed of two or more parts (McCourt et al. rbcL; Zygnematales; Zygnematophyceae 2000, Gontcharov et al. 2003, 2004, Hall et al. Abbreviations: BI, Bayesian inference; BP, boot- 2008). The morphological diversity of the Desmidi- strap percentages; ML, maximum likelihood; MP, ales has led to the description of >3,000 species dis- maximum parsimony; NJ, neighbor joining; PP, tributed over 35 genera. Most of these genera, posterior probabilities; rbcL, LSU of RUBISCO however, have previously been regarded as artificial. gene Recent molecular phylogenetic studies that specifi- cally addressed the phylogeny of the Desmidiales and its most species-rich family, Desmidiaceae, fully supported this notion (Denboh et al. 2001, Nam 1Received 3 March 2009. Accepted 17 September 2009. 2Permanent address: Institute of Biology and Soil Science, RUS- and Lee 2001, Gontcharov et al. 2003, Moon and 690022, Vladivostok-22, Russia. Lee 2003, Gontcharov and Melkonian 2008, Hall 3Author for correspondence: e-mail [email protected]. et al. 2008).

346 MOLECULAR PHYLOGENY AND REVISION OF NETRIUM 347

In contrast, the order Zygnematales, comprising pected because Netrium, a genus with only 10 850 species classified in 17 genera that are split described species (Ohtani 1990, Gerrath 1993), is between two families, has received comparatively lit- morphologically relatively uniform, and its identity tle attention. Zygnematalean taxa are indistinct in has never been seriously questioned (West and West their cell wall ultrastructure (the simple cell wall is 1904, Kossinskaja 1952, Prescott et al. 1972, Brook nonfragmented without ornamentation or pores). and Johnson 2002, Gerrath 2003). The taxonomic The two families (Mesotaeniaceae, Zygnemataceae) history of the genus Netrium is briefly summarized were distinguished based on cellular organization: here: originally, Netrium taxa were included in the unicellular forms comprising the family Mesotaenia- genus Penium (Ralfs 1848). Subsequently, they were ceae (five genera and 65 species); and filamentous transferred to the genus Closterium as subgenus Netri- forms, the Zygnemataceae (12 genera with 800 um (Na¨geli 1849) and finally raised to genus level species). Chloroplast shape (stellate, laminate, or by Itzigsohn and Rothe (1856). However, Ralfs’s ribbon-like) was used as the major criterion to dis- classification was often favored (Bre´bisson 1856, de tinguish genera in both families (West and West Bary 1858, Archer 1861, Turner 1892) until 1904, Czurda 1932, Kolkwitz and Krieger 1941, Kos- Lu¨tkemu¨ller (1902) showed that Netrium differs sinskaja 1952, Kadlubowska 1984, Brook and John- from Penium by its smooth, one-piece cell wall lack- son 2002, Gerrath 2003). An apparent similarity in ing pores and the different mode of cell division, chloroplast shapes between filamentous and unicel- observations later confirmed by EM and life-history lular forms was recognized early, but relationships studies (Biebel 1964, Mix 1972). between the corresponding taxa remained specula- Each of the Netrium lineages established through tive. It was assumed that each chloroplast shape had sequence comparisons was distinct in the number of evolved only once, and unicellular (or filamentous, chloroplasts per cell (one, two, and four, respec- depending on the presumed ancestral state) forms tively), a feature affecting the position and behavior arose independently in each lineage characterized of the nucleus during cell division (Pickett-Heaps by a specific chloroplast shape, thus rendering 1975, Jarman and Pickett-Heaps 1990). These find- either the Mesotaeniaceae or the Zygnemataceae ings suggested that the traditional genus Netrium polyphyletic (Randhawa 1959, Yamagishi 1963, Ho- comprises several only distantly related and pheno- shaw and McCourt 1988). Alternatively, others typically distinct lineages (Gontcharov et al. 2004). favored a monophyletic origin of the unicellular or Here, we reinvestigated the molecular phylogeny filamentous taxa in the Zygnematales (West and and taxonomic status of Netrium using an extended West 1904, Prescott et al. 1972), implying extensive taxon sampling of unicellular Zygnematales (Netrium homoplasy in chloroplast shapes. s. l., Cylindrocystis, and Mesotaenium), mostly by isolat- Molecular phylogenetic analyses demonstrated ing new strains from natural populations. To the paraphyletic nature of the order Zygnematales improve phylogenetic resolution, we also extended and rejected the traditional concept of its two fami- the data set by including the chloroplast-encoded lies, Zygnemataceae and Mesotaeniaceae, neither of (cp) LSU rRNA genes in addition to the previously which is monophyletic as their taxa intermixed in a used nuclear-encoded SSU rDNA and rbcL in a con- basal clade of the Zygnematophyceae (McCourt catenated data matrix. Using this new data set et al. 2000, Gontcharov et al. 2003). It was also (5,220 positions), we confirmed that members of revealed that the genera Roya and Netrium, tradi- the traditional genus Netrium formed three robust tional members of Mesotaeniaceae, were only dis- clades in the phylogenetic tree of the Zygnemato- tantly related to other Zygnematales and instead phyceae and fully resolved the relationships among showed affinity to the derived order Desmidiales these clades. A detailed analysis of morphological (McCourt et al. 2000, Gontcharov et al. 2003). Due traits by conventional and confocal LM identified to their plastid morphology (axial, laminate chlo- morphological synapomorphies that characterize roplasts as in many Desmidiales), Roya and Netrium each clade. In consequence, we recognize one novel have sometimes been regarded as transitional forms lineage as a new genus and in addition describe between saccoderm (Mesotaeniaceae) and placo- three new species. derm desmids (Desmidiales; West and West 1904, Yamagishi 1963, Brook 1981), and the molecular MATERIALS AND METHODS data supported this hypothesis (McCourt et al. 2000, Cultures. Fifty-three strains of Zygnematophyceae used for Gontcharov et al. 2004, Hall et al. 2008). this study were obtained from different sources (see Table S1 Previous molecular phylogenetic analyses also in the supplementary material) and grown in modified WARIS- indicated that the genus Netrium may not be mono- H culture medium (McFadden and Melkonian 1986) at 15C )2 )1 phyletic, because in combined analyses of nuclear- with a photon fluence rate of 40 lmol Æ m Æ s in a 14:10 encoded SSU rDNA and rbcL, four Netrium light:dark (L:D) cycle. The taxonomic designation of all strains was verified by LM prior to DNA extraction (Prescott et al. sequences were distributed among three branches 1972, Brook and Johnson 2002, Coesel and Meesters 2007). of the tree (N. interruptum, N. oblongum SVCK 255, DNA extraction, amplification, and sequencing. After mild and the ‘‘N-clade’’) that formed an unresolved poly- ultrasonication to remove mucilage, total genomic DNA was tomy (Gontcharov et al. 2004). This result was unex- extracted using the Qiagen (Hilden, Germany) DNeasy Plant 348 ANDREY A. GONTCHAROV AND MICHAEL MELKONIAN

Mini Kit. Nuclear-encoded (nu) SSU rDNA and chloroplast- Markov chain Monte Carlo (MCMC) runs, each with four encoded (cp) rbcL and LSU rDNA were amplified by PCR using Markov chains lasting for two million generations, were published protocols and 5’-biotinylated PCR primers (Marin performed. Trees and parameters were sampled every 100th et al. 1998, 2005, Gontcharov et al. 2004). PCR products were generation for a total of 20,000 samples. Convergence of the purified with the Dynabeads M-280 system (Dynal Biotech, two cold chains was checked; burn-in was determined using the Oslo, Norway) and used for bidirectional sequencing reactions. ‘‘sump’’ command, and the remaining samples were analyzed Gels were run on a Li-Cor IR2 DNA sequencer (Li-Cor Inc., using the ‘‘sumt’’ command. The robustness of the trees was Lincoln, NE, USA). estimated by bootstrap percentages (BP; Felsenstein 1985) Sequence alignments and tree reconstructions. Sequences were using 1,000 (NJ and MP) or 100 (ML) replications and by manually aligned using the SeaView program (Galtier et al. posterior probabilities (PP) in BI. BP < 50% and PP < 0.95 1996). For coding regions of the nu SSU rDNA and cp LSU were not taken into account. In MP, the stepwise addition rDNA, the alignment was guided by primary and secondary option (10 heuristic searches with random taxon input order) structure conservation (Wuyts et al. 2000, 2001, Gillespie et al. was used for each bootstrap replicate. The ML bootstrap used a 2006). All three codon positions of the rbcL gene were used for single heuristic search (starting tree via stepwise addition) per analyses (Gontcharov et al. 2004). The alignment is available replicate. from TreeBASE (http://www.treebase.org/treebase/) under Combined analyses. For concatenated ML and NJ analyses, the accession number M4539. partitions were fused and analyzed using a single ‘‘concate- The amount of phylogenetic signal versus noise in the nu nated’’ model with averaged parameters. Prior to that, models SSU rDNA, rbcL, and cp LSU rDNA data was assessed by for individual partitions (Table 1), ML topologies, and plotting the uncorrected distances against the corrected ML ⁄ NJ(ML) ⁄ MP bootstrap support (Table 2) were obtained distances determined with the respective model of sequence and compared to reveal significant discrepancies. We also evolution estimated by Modeltest 3.06 (Posada and Crandall assessed incongruence between the data sets by the incongru- 1998). The models selected and the model parameters are ence length difference (ILD) test (Farris et al. 1994) in PAUP summarized in Table 1. In addition, the measure of skewness (partition homogeneity test with 1,000 replicates). The concat- (g1-value calculated for 10,000 randomly selected trees in enated data set was analyzed in BI using specific model PAUP 4.0b10; Swofford 2002) was compared with the empirical parameters for each partition. threshold values (Hillis and Huelsenbeck 1992) to verify the Topology tests. User-defined trees were generated by man- nonrandom structuring of the data. To quantify the extent of ually modifying the treefile of the ‘‘best tree’’ using TreeView substitutional saturation in the data sets, the Iss statistic was 1.6.2 (Page 1996). To compare user-defined topologies with calculated with DAMBE (Xia and Xie 2001) for the individual the ‘‘best tree,’’ site-wise log-likelihoods were calculated for and combined data sets. each topology in PAUP and used as input for CONSEL Phylogenetic trees were inferred with maximum-likelihood (Shimodaira and Hasegawa 2001), which calculates the (ML), distance (neighbor joining, NJ), and maximum-parsi- probability values according to the Kishino–Hasegawa test mony (MP) optimality criteria using PAUP 4.0b10 and Bayesian (KH; Kishino and Hasegawa 1989), the Shimodaira–Hasega- inference (BI) using MrBayes 3.1.2 (Huelsenbeck and Ronquist wa test (SH; Shimodaira and Hasegawa 1999; both weighted 2001). Evolutionary models (for ML and NJ analyses) were [w] and unweighted), and the approximately unbiased test selected by the Akaike information criterion in Modeltest. ML (AU) using the multiscale bootstrap technique (Shimodaira and MP analyses used heuristic searches with a branch- 2002). swapping algorithm (tree bisection-reconnection); distances Apomorphy analysis. To find all molecular synapomorphies for NJ analyses were calculated by ML. In BI, two parallel of clades, an exhaustive apomorphy analysis was performed as

Table 1. Evolutionary models, log likelihood values ()lnL), and model parameters identified by Modeltest for individual and combined data sets.

Model nu SSU cp LSU nu SSU + rbcL+cp parameter ⁄ data set rDNA rbcL rDNA LSU rDNA (Fig. 2) GTR+I+G GTR+I+G GTR+I+G GTR+I+G )lnL 11,937.776 22,384.956 18,037.221 54,414.706 I 0.4812 0.5387 0.5377 0.5097 G 0.4555 1.2456 0.5922 0.5660 Base frequencies: A 0.2466 0.2861 0.3201 0.2780 C 0.2240 0.1444 0.1771 0.1724 G 0.2772 0.1527 0.2598 0.2420 T 0.2522 0.4169 0.2429 0.3077 GC 0.5012 0.3071 0.4369 0.4144 Rate matrix ([GMT = 1.00]): [AMC] 1.3301 1.3111 1.1841 1.6911 [AMG] 2.4113 6.5273 4.2995 5.0804 [AMT] 1.4097 2.2167 1.5083 2.4784 [CMG] 0.6965 1.6221 0.2854 0.8640 [CMT] 7.2488 11.2904 7.8236 10.6497 Aligned nt 1,749 1,338 2,133 5,220 Constant nt 1,179 746 1,394 3,319 MP-informative 427 538 613 1,578 MP-uninformative 143 54 126 323 Measure of skewness (g1-value) )0.9573 )0.8623 )0.9261 )1.1014 Iss statistic (Iss ⁄ Iss +c,P-value of 0.193 ⁄ 0.781; 0.418 ⁄ 0.766; 0.274 ⁄ 0.792; 0.258 ⁄ 0.792; 32 taxa data subsets) P < 0.001 P < 0.001 P < 0.001 P < 0.001 MOLECULAR PHYLOGENY AND REVISION OF NETRIUM 349

Table 2. Support values [bootstrap values, posterior probabilities; ML ⁄ NJ(ML) ⁄ MP ⁄ BI] for the clades and branches (encircled numbers in Fig. 2) with different data sets (partitions).

Clade (branch) ⁄ data set nu SSU rDNA rbcL cp LSU rDNA Combined Desmidiaceae without A. Cruciferum – ⁄ – ⁄ – ⁄ –56⁄ 88 ⁄ 69 ⁄ 1.00 59 ⁄ 80 ⁄ 71 ⁄ 1.00 95 ⁄ 98 ⁄ 91 ⁄ 1.00 Closteriaceae 100 91 ⁄ 95 ⁄ 86 ⁄ 1.00 92 ⁄ 97 ⁄ 81 ⁄ 1.00 100 Gonatozygaceae 100 57 ⁄ 60 ⁄ 55 ⁄ – 100 100 Desmidiales 86 ⁄ 95 ⁄ – ⁄ 1.00 – ⁄ – ⁄ – ⁄ ––⁄ – ⁄ – ⁄ –93⁄ – ⁄ – ⁄ 1.00 Roya ⁄ Desmidiales (1) – ⁄ – ⁄ – ⁄ – – ⁄ – ⁄ – ⁄ – – ⁄ – ⁄ – ⁄ – 95 ⁄ 94 ⁄ – ⁄ 1.00 Planotaenium 100 98 ⁄ 100 ⁄ 100 ⁄ 1.00 100 100 Planotaenium + Roya ⁄ Desmidiales (2) 67 ⁄ – ⁄ – ⁄ 1.00 55 ⁄ – ⁄ 51 ⁄ – – ⁄ – ⁄ – ⁄ – 94 ⁄ 90 ⁄ 70 ⁄ 1.00 Nucleotaenium 98 ⁄ 97 ⁄ 100 ⁄ 1.00 100 100 100 Netrium 100 100 100 100 Netrium + Nucleotaenium – ⁄ – ⁄ – ⁄ ––⁄ – ⁄ – ⁄ –82⁄ – ⁄ – ⁄ 1.00 97 ⁄ – ⁄ 72 ⁄ 1.00 Nucleotaenium ⁄ Netrium + Planotaenium ⁄ – ⁄ – ⁄ – ⁄ 1.00 – ⁄ 64 ⁄ 64 ⁄ –66⁄ – ⁄ – ⁄ 1.00 97 ⁄ – ⁄ 55 ⁄ 1.00 Roya ⁄ Desmidiales (3) Spirogyra 100 80 ⁄ 83 ⁄ 80 ⁄ 1.00 100 100 Mougeotia 95 ⁄ 100 ⁄ 100 ⁄ 1.00 100 100 100 Mesotaenium caldariorum 96 ⁄ 98 ⁄ 98 ⁄ 1.00 – ⁄ – ⁄ – ⁄ –79⁄ 92 ⁄ 64 ⁄ 1.00 88 ⁄ 94 ⁄ 99 ⁄ 1.00 Mougeotia + Mesot. caldariorum – ⁄ – ⁄ – ⁄ –90⁄ – ⁄ – ⁄ 0.96 88 ⁄ 97 ⁄ 64 ⁄ 1.00 100 ⁄ 83 ⁄ 100 ⁄ 1.00 ‘‘Cylindrocystis’’ 99 ⁄ – ⁄ – ⁄ 1.00 – ⁄ – ⁄ – ⁄ –70⁄ 53 ⁄ 59 ⁄ 1.00 90 ⁄ 58 ⁄ – ⁄ 1.00 MZC 71 ⁄ 84 ⁄ 100 ⁄ 0.97 97 ⁄ 70 ⁄ – ⁄ 1.00 96 ⁄ 80 ⁄ 98 ⁄ 1.00 100 ⁄ 99 ⁄ 91 ⁄ 1.00 671⁄ – ⁄ – ⁄ –90⁄ – ⁄ – ⁄ 1.00 90 ⁄ 77 ⁄ 64 ⁄ 1.00 100 ⁄ 99 ⁄ 100 ⁄ 1.00 Zygnema + Zygogonium – ⁄ 69 ⁄ – ⁄ –97⁄ 99 ⁄ 99 ⁄ 1.00 100 100 5–⁄ – ⁄ – ⁄ –73⁄ – ⁄ – ⁄ 0.99 – ⁄ – ⁄ – ⁄ – 63 ⁄ – ⁄ – ⁄ – Cylindrocystis brebissonii 99 ⁄ 100 ⁄ 100 ⁄ 1.00 100 100 100 4–⁄ – ⁄ – ⁄ ––⁄ – ⁄ – ⁄ –94⁄ 90 ⁄ 52 ⁄ 1.00 99 ⁄ 85 ⁄ – ⁄ 1.00 Mesotaenium 1–⁄ – ⁄ – ⁄ –81⁄ 87 ⁄ – ⁄ 1.00 93 ⁄ 50 ⁄ – ⁄ 1.00 100 ⁄ 82 ⁄ – ⁄ 1.00 ‘‘Zygnemataceae’’ – ⁄ – ⁄ – ⁄ –74⁄ – ⁄ – ⁄ ––⁄ – ⁄ – ⁄ –92⁄ 85 ⁄ – ⁄ 1.00 Mesotaenium 292⁄ 51 ⁄ 71 ⁄ 1.00 99 ⁄ 98 ⁄ 71 ⁄ 1.00 88 ⁄ 92 ⁄ 100 ⁄ 1.00 100 BI, Bayesian inference; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbor joining. 100 = 100 ⁄ 100 ⁄ 100 ⁄ 1.00. described previously (Marin et al. 2003). A synapomorphy positioned pyrenoids, and radiating longitudinal characterized by (1) absence of convergent evolution outside lamellae that are conspicuously notched at the mar- the clade and (2) strict conservation within the clade analyzed gins. Large spherical nucleus occupies an area is designated a nonhomoplasious synapomorphy (NHS; Marin et al. 2003). NHSs for lineages of the Zygnematophyceae are between the chloroplasts in the center of the cell. defined as ‘‘NHS within the streptophyte green algae,’’ Type species: Netrium digitus (Ralfs) Itzigs. et Rothe allowing for homoplasies in embryophytes, Chlorophyta or (Penium digitus Ralfs, Brit. Desm.: 150, pl. 25: 3. 1848). eight other eukaryotes. Sequence data of streptophyte green Nucleotaenium gen. nov. algae and other eukaryotes (400 reliable sequences) defined Diagnosis: Cellulae cylindricae, apicibus rotundis the plesiomorphic character states. et chloroplasto singulo cum incisura media; nucleus Microscopic observation. Fresh (3–4 weeks after inoculation) applanatus, locatus lateraliter in medio cellulae. cultures were observed with a Zeiss IM inverted microscope (Carl Zeiss AG, Oberkochen, Germany) and photographed Chloroplastus cum 2–8 pyrenoidibus parvis et 8 ± 2 with a Canon EOS 40D camera (Canon Deutschland GmbH, laminis longitudinalibus; laminae rectae vel obli- Krefeld, Germany). For confocal laser scanning fluorescence quae marginesque incisae vel leves. microscopy, the cells were immobilized in low-temperature Diagnosis: Cells cylindrical, with rounded apices; gelling agarose (Reize and Melkonian 1989) and observed with chloroplast one per cell, with a notch (in the mid- a Leica TCS SP2 (Leica Mikrosysteme GmbH, Wetzlar, Ger- dle) that harbors an asymmetrical, flattened many) using a 488 nm excitation line. Series of optical sections of chloroplasts, mostly 1 lm in thickness, were collected and nucleus. Chloroplast with 2–8 relatively small pyre- used to reconstruct their three-dimensional morphology with noids and provided with 8 ± 2 radiating, longitudi- Leica Confocal Software (Leica Microsystems, Heidelberg, nal lamellae, the lamellae are straight or oblique, Germany). To visualize the position of the nucleus, cells were notched at the margins or smooth. stained with DAPI (Sigma Inc., St. Louis, MO, USA) after Type species: Nucleotaenium eifelense sp. nov. fixation with 3.7% formaldehyde solution and permeabilization Etymology: The generic name Nucleotaenium is com- with Triton X-100 and observed with a Nikon Eclipse E800 fluorescent microscope (Nikon GmbH, Du¨sseldorf, Germany). posed from the Latin nucleus and taenia, in refer- ence to the unusual position of the nucleus in the cell and the lamellated chloroplasts. Nucleotaenium eifelense sp. nov. RESULTS Diagnosis: Cellulae ellipticae vel cylindricae, apici- Taxonomic revisions. Netrium (Na¨geli) Itzigs. et Rothe. bus rotundis 11.5–14.4 lm latae, 23.6–46.1 lm lon- Emended diagnosis: Vegetative cells oblong-elliptical gae; chloroplastus singulus, divisus in partes duas to cylindrical, chloroplasts axial, two in the cell, the lateraliter connectas. Singula pars cum 1–2 pyre- chloroplasts with rod-shaped or spherical, centrally noidibus et 8 ± 2 laminis longitudinalibus, obliquis 350 ANDREY A. GONTCHAROV AND MICHAEL MELKONIAN vel rectis marginibusque undulatis et incisis. Type locality: Czech Republic, Doksy, peat bog area Nucleus applanatus, locatus lateraliter in medio cell- at the SE corner of lake Machovo jezero (Velky Ryb- ulae. Zygosporae incognitae. nik); collected and isolated in June 2007 by Andrey Diagnosis: Cells elliptical to cylindrical, with Gontcharov. rounded apices, 11.5–14.4 lm in width, 23.6– Etymology: The name denotes the cylindrical shape 46.1 lm in length, chloroplast one in each cell, of the cell, characteristic for the new species. divided into two large laterally connected halves. Planotaenium ohtanii sp. nov. Each half of the chloroplast has 1–2 pyrenoids and Diagnosis: Cellulae vegetativae cylindricae, apici- is provided with 8 ± 2 longitudinal lamellae, straight bus rotundis, 15.6–18.4 lm latae, 45.3–69.5 lm or oblique, margins wavy, notched. Asymmetrical, longae, cum vacuolis apicalibus. Chloroplasti duo in flattened nucleus occupies small pocket between cellula, cum 9–12 laminis longitudinalibus, rectibus the chloroplast halves. Zygospores unknown. vel paulum obliquis, pyrenoidibus singulis. Zygospo- Type: Permanent slide of strain M3006 deposited rae incognitae. at the Culture Collection of Algae at the University Diagnosis: Vegetative cells cylindrical, with of Cologne (CCAC), Germany, under the designa- rounded apices, 15.6–18.4 lm in width, 45.3– tion ZYG.H.002 (hic designatus). 69.5 lm in length, with apical vacuoles. Chloroplasts Figure 4e has been chosen to represent the type in two in each cell with 9–12 straight or somewhat obli- accordance to fulfill article 39.1 of the International que longitudinal lamellae and one pyrenoid. Zy- Code of Botanical Nomenclature (ICBN). The strain gospores unknown. M3006 and a DNA sample of this strain are kept at Type: Permanent slide of strain M2697 deposited CCAC. DNA sequences of strain M3006 have been at the CCAC, under the designation ZYG.H.001(hic submitted to the European Molecular Biology designatus). Laboratory (EMBL) ⁄ GenBank ⁄ DNA Data Bank of Figure 4c has been chosen to represent the type Japan (DDBJ) databases under the accession num- in accordance to fulfill article 39.1 of the ICBN. bers FM992324 (nuclear SSU rDNA), FM992553 (par- The strain M2697 and a DNA sample of this strain tial chloroplast LSU rDNA), and FM992348 (rbcL). are kept at CCAC. DNA sequences of strain M2697 Type locality: Germany, Eifel near Monschau, aero- have been submitted to the EMBL ⁄ GenBank ⁄ DDBJ phytic, collected and isolated in June 2006 by databases under the accession numbers FM992322 Andrey Gontcharov. (nuclear SSU rDNA), FM992549 (partial chloroplast Etymology: The species name refers to the type LSU rDNA), and FM992339 (rbcL). locality, the low mountain range Eifel, where it was Type locality: Germany, Cologne (Wahner Heide), collected. temporary puddle, collected and isolated in January Nucleotaenium cylindricum sp. nov. 2005 by Michael Melkonian. Diagnosis: Cellulae cylindricae apicibus rotundis, Etymology: The epithet honors the Japanese phy- 8.5–8.9 lm latae, 26.2–34.8 lm longae; chloropla- cologist Shuji Ohtani, who first described section stus singulus, divisus in partes duas lateraliter con- Planotaenium of the genus Netrium. nectas; nucleus applanatus, locatus lateraliter in Taxon sampling. For this study, 15 new SSU rDNA, medio cellulae. Chloroplastus cum 2–4 pyrenoidibus 27 rbcL, and 45 cp LSU rDNA sequences were parvis et 8 ± 2 laminis longitudinalibus; laminae rec- obtained from 53 strains of the Zygnematophyceae, tae vel obliquae marginesque leves, rarum incisae. representing mostly the traditional order Zygnema- Zygosporae incognitae. tales (see Table S1). For three nonmonophyletic Diagnosis: Cells cylindrical, 8.5–8.9 lm in width genera (Netrium, Mesotaenium, and Cylindrocystis; and 26.2–34.8 lm in length with rounded apices, Gontcharov et al. 2004, Hall et al. 2008) all available chloroplast one in each cell, provided with a notch strains ⁄ species were included in the analyses, in the middle that harbors an asymmetrical, flat- whereas for the monophyletic genera Spirogyra, tened nucleus. Chloroplast has 2–4 relatively small Mougeotia, and Zygnema, only two or three represen- pyrenoids and provided with 8 ± 2 longitudinal tative strains (the most divergent in rbcL phyloge- lamellae, straight or oblique, margins mostly nies) were selected. To cover the derived family smooth, rarely notched. Zygospores unknown. Desmidiaceae (containing 35 genera and >2,500 Type: Permanent slide of strain M3003 deposited species), 17 species representing recently established at the CCAC, under the designation ZYG.H.003 (hic clades were selected (Gontcharov and Melkonian designatus). 2008). Since the position of the Zygnematophyceae Figure 4g has been chosen to represent the type among other streptophytes as well as the affiliation in accordance to fulfill article 39.1 of the ICBN. of the genus Spirotaenia to the class is not yet settled The strain M3003 and a DNA sample of this strain (Karol et al. 2001, Gontcharov and Melkonian 2004, are kept at CCAC. DNA sequences of strain M3003 McCourt et al. 2004, Turmel et al. 2005, 2007, Adam have been submitted to the EMBL ⁄ GenBank ⁄ DDBJ et al. 2007) we performed only unrooted analyses databases under the accession numbers FM992323 and excluded Spirotaenia from the data sets. (nuclear SSU rDNA), FM992552 (partial chloroplast Concatenated analyses of nu SSU rDNA, rbcL, and cp LSU rDNA), and FM992347 (rbcL). LSU rDNA. Despite differences among the partitions MOLECULAR PHYLOGENY AND REVISION OF NETRIUM 351

(e.g., the alignment length, base and substitution Table 1) yielded the phylogenetic tree shown in Fig- frequencies, number of parsimony-informative sites, ure 2. Fifteen terminal clades, mostly corresponding the distribution pattern of substitutions [G-parame- to genera (Planotaenium, Nucleotaenium, Netrium, Spi- ter], and the proportion of invariable sites), the rogyra, Mougeotia, ‘‘Cylindrocystis,’’ and Zygnema)or most complex GTR + I + G model of sequence evo- families (Closteriaceae and Gonatozygaceae) of the lution was always selected by Modeltest as the best Zygnematophyceae were resolved. Two clades com- model fitting the data (Table 1). A test of individual prised multiple representatives of the same species data sets for substitutional saturation (distribution (i.e., Mesotaenium caldariorum and Cylindrocystis brebis- of the uncorrected vs. corrected distances; Fig. 1) sonii), and the MZC clade included members of revealed a nearly linear correlation in the SSU three traditional zygnematalean genera, Cylindrocystis, rDNA and cp LSU rDNA data, indicating low satura- Mesotaenium, and Zygnemopsis. In addition, five tion levels. The saturation plot of rbcL leveled off species of the genus Mesotaenium were split between somewhat, suggesting some saturation that would be two clades, here termed Mesotaenium 1 and 2 expected from the third codon position of this pro- (Fig. 2). The largest clade of the tree consisted of tein-coding gene (Gontcharov et al. 2004). However, representatives of two families, Desmidiaceae and according to the Iss statistics, none of the data sets Peniaceae. Penium (Peniaceae) species branched pa- were saturated (P < 0,001; Table 2). raphyletically at the base of this clade, and one of Comparison of the skewness of the tree length the Penium subclades that also included Actinotaeni- distribution (g1-value) of random trees of all data um cruciferum was monophyletic with the Desmidia- sets with the empirical threshold values (Hillis and ceae (93%–98% BP and 1.00 PP; Fig. 2). Most of Huelsenbeck 1992) showed that the length distribu- the terminal clades and internal branches of the tions were considerably left skewed, indicating that tree attained high support in our analyses (>90 BP the alignments were significantly more structured and 1.00 PP; Table 2). than random data and likely contained a strong Roya was resolved as a sister to the Desmidiales su- phylogenetic signal (Table 1). perclade (93% BP in ML and 1.00 PP, but no sup- Since a comparison of evolutionary models port in NJ and MP) that comprised the basally selected for individual genes (66 taxa data set) diverging Gonatozygaceae (79% BP in ML and 1.00 (Table 1), tree topologies (results not shown), and PP), the Closteriaceae, and the Peniaceae ⁄ Desmidia- bootstrap support values of the trees (Table 2) ceae clade (Fig. 2). The Roya ⁄ Desmidiales clade was showed general agreement among markers, we con- well supported (‡94% BP in ML and NJ, 1.00 PP). catenated nu SSU rDNA, rbcL, and cp LSU rDNA The former Netrium species formed three mono- sequences obtained from the same strains (i.e., phyletic lineages whose relationships to each other using a strictly congruent taxon sampling) to and the Roya ⁄ Desmidiales clade were largely enhance phylogenetic resolution (Hillis 1996). ML resolved (Fig. 2). The genus Planotaenium (with two analyses of the concatenated data set (5,220 nt, species) was resolved as a clade with a sister group relationship to the Roya ⁄ Desmidiales clade (‡90% BP in ML and NJ, and 1.00 PP). The genera Nucleo- taenium and Netrium were sisters (their common branch received 97% BP in ML, 72% BP in MP, and 1.00 PP) and together diverged prior to the Plano- taenium ⁄ Roya ⁄ Desmidiales lineage (Fig. 2). The three genera formerly constituting Netrium plus the Roya ⁄ Desmidiales clade also formed a clade well sep- arated from the rest of the Zygnematophyceae [BP support in ML (97%) and PP (1.00); BP support in NJ and MP was low (MP) or nonexistent (NJ); Fig. 2]. The remaining taxa of the traditional order Zygnematales formed three monophyletic lineages whose interrelationships were not resolved (Fig. 2), namely, the long-branched Spirogyra clade; a novel superclade here provisionally termed ‘‘Zygnemata- ceae,’’ containing the bulk of the Zygnematales (receiving 92% BP in ML, 85% BP in NJ, and 1.00 PP); and a clade containing two species of Mesotaeni- um (Mesotaenium 2: M. endlicherianum and M. brau- nii). The phylogenetic relationships among taxa within the ‘‘Zygnemataceae’’ superclade were largely Fig. 1. Analyses of saturation in the nu SSU rDNA, rbcL, and cp LSU rDNA data (uncorrected vs. corrected distances). Cor- resolved, although support for some internal rected distances were calculated with the GTR + I + G model esti- branches was only moderate. The clade Mesotaenium mated by Modeltest for each partition (Table 1). 1 constituted the basal divergence of the 352 ANDREY A. GONTCHAROV AND MICHAEL MELKONIAN

Fig. 2. Phylogeny of Zygne- matophyceae based on the com- bined analyses of nu SSU rDNA, rbcL, and cp LSU rDNA sequences (66 taxa, 5,220 nt, ML topology; for model and model parameters, see Table 1). Nodes are characterized by bootstrap percentages (BP ‡ 50%) and Bayesian posterior probabilities (PP ‡ 0.95): ML ⁄ NJ(ML) ⁄ MP ⁄ BI. Branches with 100% BP in all methods and 1.00 PP are shown bold; • = 100% BP or 1.00 PP. Strain data are provided for those strains for which new sequences were obtained during this study. New taxa described in this study are in bold. Numbers in paren- theses after some taxon names indicate positions of group I in- trons in their cp LSU rDNA rela- tive to Escherichia coli 23S rDNA. Asterisks denote branches sup- ported by nonhomoplasious syna- pomorphies in SSU rDNA or rbcL (Fig. 3). Encircled numbers (1– 6) below some branches refer to clades that were also subjected to phylogenetic analyses using single gene data sets (see Table 2). BI, Bayesian inference; ML, maxi- mum likelihood; MP, maximum parsimony; NJ, neighbor joining.

‘‘Zygnemataceae’’ superclade, whereas a well-sup- strains per genus). In contrast, genera comprising ported clade comprising Mesotaenium caldariorum ⁄ unicellular members of the order (i.e., Cylindrocystis, Mougeotia, Cylindrocystis spp. (excluding C. brebisso- Mesotaenium, and the traditional Netrium) were not nii), and MZC represented a late divergence monophyletic. The genera represented by the larg- (Fig. 2). The clades Cylindrocystis brebissonii and Zyg- est number of strains (Cylindrocystis [13 strains] and nema ⁄ Zygogonium occupied intermediate positions in Mesotaenium [eight strains]) were resolved as poly- the superclade ‘‘Zygnemataceae.’’ The branching phyletic, their members being distributed among order of the latter two clades was not resolved three and four clades, respectively. Seven Cylindrocys- (Fig. 2). tis strains formed a moderately supported clade Within the traditional order Zygnematales, only (‘‘Cylindrocystis’’; 90% BP in ML and 1.00 PP); one genera consisting of filamentous forms (members of strain (Cylindrocystis sp. M3009) was excluded from the traditional family Zygnemataceae) were mono- this lineage, and one more strain (Cylindrocystis sp. phyletic, that is, Mougeotia, Zygnema, and Spirogyra UTEX 1926) was a member of the well-supported (taxon sampling, however, was low; two to three MZC clade (Fig. 2). The remaining four highly MOLECULAR PHYLOGENY AND REVISION OF NETRIUM 353 similar sequences constituted the Cylindrocystis brebis- Desmidiales from the traditional Zygnematales, only soniii clade. In contrast, three strains assigned to the one character in the SSU rDNA represented an same species, Mesotaenium caldariorum, formed two NHS for the order Desmidiales: the fifth nucleotide genetically quite dissimilar subgroups but were still in the spacer between helices 25 and 26 (U==>A; monophyletic (Fig. 2). Fig. 3a). The clade consisting of Planotaenium User-defined trees (UD trees). Several phylogenetic plus the Roya ⁄ Desmidiales clade was also supported hypotheses about relationships among taxa were by a synapomorphic compensatory base change tested. The first UD tree addressed a possible sister (CBC) in Helix 43 of the SSU rDNA (base pair 18, group relationship between Planotaenium and the G–C ==>C–G; Fig. 3b). Nucleotaenium ⁄ Netrium clade (i.e., monophyly of the An exhaustive apomorphy search for rbcL traditional genus Netrium), which was rejected by revealed only a single synapomorphy at the amino the AU and KH tests (Table 3). Testing another acid level without homoplasious character changes conflict case, the position of the Mesotaenium 2 clade reflecting two NHSs at the nucleotide level. In outside the ‘‘Zygnemataceae’’ superclade, by placing codon 192 (a1 helix; Kellogg and Juliano 1997), the it as a sister to Mesotaenium 1 (UD -tree 2), was not plesiomorphic cysteine typical for the Zygnemato- significantly different from the best tree (Fig. 2). In phyceae and green plants in general (codon TGY) UD trees 3 and 4, an affinity of C. brebissonii and Cyl- changed into valine (codon GTN) in all members indrocystis sp. M3009 to the ‘‘Cylindrocystis’’ clade was of the ‘‘Zygnemataceae’’ clade analyzed (Fig. 3c). probed. Monophyly with the ‘‘Cylindrocystis’’ clade Morphological observations. Stellate chloroplasts was rejected for C. brebissonii by all tests but not for displaying an axial core with pyrenoid(s) and radiat- Cylindrocystis sp. M3009, which could be a member ing longitudinal lamellae or ribs are a common fea- of the ‘‘Cylindrocystis’’ clade. The position of the ture of Planotaenium, Nucleotaenium, and Netrium s. Zygnema ⁄ Zygogonium clade is unstable in the phylog- str. that differentiates these genera from other uni- eny; placing it as a sister to the C. brebissonii clade cellular members of the traditional order Zygnema- was not significantly different from the best tree tales. Each genus was distinct in the number of (UD tree 5; Table 3). chloroplasts per cell and features of chloroplast Nonhomoplasious synapomorphies (NHSs). The morphology. Planotaenium had either four (P. inter- exhaustive apomorphy analysis was limited to the ruptum) or two (P. ohtanii) chloroplasts; Netrium s. nuclear-encoded SSU rDNA and rbcL and was str., only two; and Nucleotaenium spp., only one applied to selected branches (marked in Fig. 2 with (Figs. 4 and 5). The single chloroplast in all mem- an asterisk). Among 24 characters separating the bers of the Nucleotaenium clade consisted of two halves appearing as two chloroplasts connected by a fine central strand (strain SVCK 255; Fig. 4, i and Table 3. Comparison of the maximum-likelihood trees j), or the halves were connected laterally leaving lit- with user-defined trees by Kishino–Hasegawa and Shimo- tle space between them (N. eifelense and N. cylindri- daira–Hasegawa tests. cum; Figs. 4, e and g; and 5, e, h, and i). In the latter case, the longitudinal lamellae were discontin- Pb uous in the region in which the two chloroplast Tree no Diff-lnLa AU KH SH wSH halves were connected (Figs. 4, f and h; and 5, e and h). Best Continuous longitudinal chloroplast lamellae 1 18.5 0.028 0.048 0.351 0.125 2 16.9 0.147 0.117 0.384 0.259 with a smooth margin characterized the clade 3 102.9 6e-061 0 0 0 Planotaenium (Figs. 4, b and d; and 5, a–d). In 4 10.0 0.234 0.183 0.585 0.433 Netrium s. str. as well as in strain SVCK 255 (Nucleo- 5 3.4 0.426 0.280 0.853 0.727 taenium clade), the longitudinal lamellae were inter- AU, the P-value of the approximately unbiased test calcu- rupted with notched margins (Figs. 4, i–l; and 5o). lated from the multiscale bootstrap; KH, the Kishino–Hasega- The remaining small-celled species of Nucleotaenium wa test; SH, the Shimodaira–Hasegawa test; wSH, the had small riblike lamellae with a less-elaborated weighted Shimodaira–Hasegawa test. structure. In N. eifelense, the lamellae had a wavy User-defined tree significantly worse than the best trees at appearance with their margins notched (Fig. 5i), P £ 0.05 are indicated by grey shadow. The tree topologies tested: 1. Planotaenium is a sister to the whereas N. cylindricum had longitudinal lamellae clade Nucleotaenium + Netrium. 2. Clades Mesotaenium 1 and that were straight or oblique with their margins Mesotaenium 2 are sisters. 3. Cylindrocystis brebissonii is a sister mostly smooth, and only rarely interrupted or to the clade ‘‘Cylindrocystis’’. 4. Cylindrocystis sp. M3009 and notched (Fig. 5, e and h). the clade ‘‘Cylindrocystis’’ are sisters. 5. Clades Zygnema ⁄ Zygogo- In the genera Planotaenium and Netrium, a large nium and Cylindrocystis brebissonii are sisters. spherical nucleus with an easily detectable nucleolus aDifference in –log-likelihood between the best tree and the user-defined tree. was positioned symmetrically between the chlorop- bProbability of obtaining a more extreme T-value under the lasts in the center of the cell (Fig. 4, a, c, and k). In null hypothesis of no difference between the two trees (one- contrast, in the Nucleotaenium spp., the nucleus was tailed test). located asymmetrically in the cell, occupying a posi- 354 ANDREY A. GONTCHAROV AND MICHAEL MELKONIAN

Fig. 3. Nonhomoplasious synapomorphies (Marin et al. 2003) in the SSU rRNA molecule and in rbcL characterizing major clades of the Zygnematophyceae. Alignments shown contain representative species only. Taxa and nucleotides characterized by the synapomorphy are in bold. SSU rRNA secondary structure after Wuyts et al. (2000); diagrams shown are based upon the last taxon in the alignment. The nomenclature of nucleotides (nt, single stranded spacers and loops) and base pairs (bp, stem regions of helices) depends on the polarity of the RNA: increasing numbers generally indicate the 5¢fi3¢ direction. tion lateral from a central strand connecting the shape typical for Planotaenium, Netrium, and most chloroplast halves (strain SVCK 255; Fig. 4, i and j) other Zygnematophyceae: the nucleus was flattened or in a lateral notch of the chloroplast (N. eifelense with a thin extension into the area where the two and N. cylindricum; Fig. 4, e and g). In the latter chloroplast halves were interconnected mimicking case, the confocal images of the fluorescing chlo- the outline of the chloroplast ‘‘pocket’’ (Fig. 5, f roplasts clearly revealed the small asymmetrical and j). Viewed perpendicular to this cell orienta- ‘‘pocket’’ (notch) between the chloroplast halves tion, the nucleus displayed the shape of a narrow (Fig. 5, e and i; asterisks). Staining with DAPI finally disk (Fig 5, g and k). proved that in N. eifelense and N. cylindricum, the Two apical vacuoles were observed in the two Pla- nucleus was displaced from the rotational axis of notaenium species but not in strains of Nucleotaenium the cell and moreover deviated from the spherical or Netrium. MOLECULAR PHYLOGENY AND REVISION OF NETRIUM 355

Fig. 4. Differential interference contrast micrographs showing chloroplast morphology and nucleus position (arrow) in the genera Planotaenium, Nucleotaenium, Netrium s. str., and Cylindrocystis cushleckae: (a, b) Planotaenium interruptum SVCK335, late interphase cell with four chloroplasts and apical vacuoles (V); (c, d) P. ohtanii M2697, late interphase cell with two cleaved (arrowheads) chloroplasts and large apical vacuoles (V); (e, f) Nucleotaenium eifelense, M3006, a single chloroplast, composed of two connected at the side halves (black arrowhead) and small lateral pocket harboring the nucleus; (g, h) Nucleotaenium cylindricum M3003, chloroplasts composed of two halves connected at the side (black arrowhead) with a small lateral pocket harboring the nucleus. The chloroplast surface is provided with smooth longitudinal ribs inter- rupted in the middle; (i, j) [Netrium oblongum] SVCK255, note a subtle bridge between two massive chloroplast halves (black arrowhead) and laterally positioned nucleus; (k, l) Netrium oblongum ASW07201, two chloroplasts with notched lamellae and large centrally positioned nucleus; (m, n) Cylindrocystis cushleckae M2158, two chloroplasts provided with two to three stout overlapping ridges. Scale bars, 10 lm. 356 ANDREY A. GONTCHAROV AND MICHAEL MELKONIAN

Fig. 5. Features of the chloroplast structure and nucleus morphology in the genera Planotaenium, Nucleotaenium, Netrium s. str., and Cylindrocystis cushleckae. (a, b) Planotaenium interruptum SVCK335: (a) late interphase cell with four chloroplasts having smooth lamellae; (b) young cell with two cleaved chloroplasts (arrowhead). (c, d) P. ohtanii M2697: (c) late interphase cell with two cleaved (arrowheads) chloroplasts having smooth lamellae; (d) young cell with two chloroplasts. (e–h) Nucleotaenium cylindricum M3003: (e) A single chloroplast composed of two laterally connected large parts having smooth longitudinal ribs. The chloroplast has a small transverse pocket (asterisk) harboring the nucleus. (f) A side view of the laterally positioned asymmetrical and flattened nucleus. (g) A front view of the flattened nucleus. (h) Chloroplast from the side of its halves joining. (i–l) Nucleotaenium eifelense M3006: (i) a single chloroplast with a small trans- verse pocket (asterisk) harboring the nucleus and longitudinal wavy or interrupted ribs; (j) a side view of the laterally positioned asymmet- rical and flattened nucleus; (k) a front view of the flattened nucleus; (l) transverse section of the chloroplast showing central core with ribs. (m, n) Cylindrocystis cushleckae: (m) cell with two chloroplasts, each provided with two stout ridges; (n) spherical centrally positioned nucleus. (o, p) Netrium oblongum ASW07201: (o) two large chloroplasts provided with stout notched lamellae; (p) large centrally positioned spherical nucleus. MOLECULAR PHYLOGENY AND REVISION OF NETRIUM 357

DISCUSSION gence in nu SSU rDNA slightly exceeded that of cp Two ribosomal RNA genes (nu SSU rDNA and cp LSU rDNA, to the zygnematalean lineages in which LSU rDNA) and the protein-coding rbcL were used the impact of the nu SSU rDNA was the least in this study to assess phylogenetic relationships among the three markers (Fig. 6). among the basal lineages of the conjugating green Although the chloroplast genes (rbcL and LSU algae (the traditional order Zygnematales). rDNA) were most divergent within and between Comparison of tree topologies and their support most lineages and provided high support for most values showed that the three molecular markers of the clades, they were unable to resolve a major used in this study contained a similar phylogenetic split in the tree between the order Desmidiales and signal and yielded largely congruent phylogenetic the traditional Zygnematales (Table 2). The mono- relationships between zygnematophycean taxa phyly of the Desmidiales was supported only in the despite markedly differing evolutionary rates among SSU rDNA phylogeny (and in the combined analy- genes and lineages (Table 1). An assessment of the ses; Table 2) and by a synapomorphic substitution input of each gene to the sequence diversity within in the spacer between helixes 25-26 of the SSU the data set [uncorrected mean pair-wise genetic rRNA (Fig. 3a). The chloroplast markers, however, divergence (%) between any two sequences within contributed specifically to resolve phylogenetic rela- the clades] revealed that the rbcL nucleotide tionships among the basal branches of the tree in sequences contributed most significantly to the over- which the nu SSU rDNA provided little phyloge- all divergence between the species in all clades netic information (Table 2). (Fig. 6). Only in the Spirogyra clade elevated evolu- The ‘‘Zygnemataceae’’ superclade. Our analyses con- tionary rates in the SSU rDNA (Gontcharov et al. firmed the complex phylogenetic structure of the 2003, 2004) resulted in comparable divergence of traditional order Zygnematales as reported previ- SSU rDNA and rbcL sequences. In most other lin- ously (McCourt et al. 2000, Gontcharov et al. 2003, eages, the SSU rDNA sequence divergence was rela- 2004, Gontcharov 2008, Hall et al. 2008) and tively low and did not exceed 2.5%. The impact of resolved seven major lineages within the traditional the cp LSU rDNA on sequence diversity varied in Zygnematales—Roya, Planotaenium, Nucleotaenium, different parts of the tree. There was a clear trend Netrium, Mesotaenium 2, Spirogyra, and a superclade toward increasing diversity in the cp LSU rDNA that we termed ‘‘Zygnemataceae’’ (Fig. 2; Table 2). sequences from the Desmidiales, in which diver- It had previously been hypothesized that either

Fig. 6. Mean pair-wise genetic divergence (%) between any two sequences within the clades estab- lished in phylogenetic analyses (Fig. 2) computed with nu SSU rDNA, rbcL, and cp LSU rDNA data. Divergence was calculated as uncorrected p-distance using Mega4 (Tamura et al. 2007) with gaps and missing data treated with pair-wise deletion. 358 ANDREY A. GONTCHAROV AND MICHAEL MELKONIAN

Spirogyra or Mesotaenium 2 (then represented only by distinguish this clade from the C. brebissoniii clade the taxon M. endlicherianum), which formed an and Cylindrocystis UTEX 1926, a member of the unresolved polytomy in the present analyses, were MZC clade. Revision of the genus Cylindrocystis must likely the most basal divergence in the class, therefore await refined analyses of phenotypic traits although support for either scenario had been weak of the different clades containing taxa with a ‘‘Cylin- (Gontcharov et al. 2004, Hall et al. 2008). Since we drocystis’’-like phenotype. The latter is characterized performed only unrooted analyses, we cannot by a comparatively small, cylindrical cell with mostly address this question here. two stellate (in cross section) chloroplasts (de Bary The remaining taxa in the traditional Zygnema- 1858, West and West 1904, Prescott et al. 1972). tales fell into two groups: the traditional genus Netri- Based on this simple circumscription, some mem- um (Planotaenium, Nucleotaenium gen. nov., and bers of the genus Cylindrocystis share similarities with Netrium s. str.) and Roya formed a clade together small-celled Netrium species (Brook and Johnson with the Desmidiales, whereas the other taxa consti- 2002), but the less regular structure of the chloro- tuted the well-supported ‘‘Zygnemataceae’’ superc- plast (without prominent longitudinal lamellae) dis- lade. The latter (termed ‘‘crown Zygnematales’’ in tinguishes them from the latter (and molecular Gontcharov et al. 2004) was further characterized by phylogenetic analyses disprove any specific relation- an NHS in the rbcL protein (cystein ==> valine in ship between the two genera, see Results). Cylindro- codon 192) that presumably originated in the com- cystis has often been regarded as closely related and mon ancestor of the clade (Fig. 3c). Within the likely ancestral to the filamentous Zygnema because Viridiplantae, valine in codon 192 in the LSU of of obvious similarities in chloroplast morphology RUBISCO is almost unique [>400 rbcL sequences (Palla 1894, Randhawa 1959, Yamagishi 1963). A were analyzed, and only one exception was found: sister group relationship between the Zygnema and Halimeda AB038489 (Caulerpales); in the seed Cylindrocystis brebissonii clades cannot be ruled out plants, Kellogg and Juliano (1997) recorded only a based on the present study but should not be single taxon with a valine in the same position]. deduced based on the similarities in chloroplast This superclade apparently relates to the tradi- morphology (see above). Revision of the genus tional family Zygnemataceae Ku¨tz. because it Cylindrocystis is further complicated by the fact that includes Zygnema as well as some other members of many of the formally described species can be iden- the family, such as the filamentous Zygogonium, Zyg- tified only based on zygospore structure, a character nemopsis, and Mougeotia. However, inclusion of some that usually cannot be evaluated in cultures. unicellular forms classified traditionally in the family Mesotaenium. Species of this zygnematalean Mesotaeniaceae (i.e., Cylindrocystis spp. and most genus were also distributed among four well-sup- Mesotaenium spp.) in this superclade requires revi- ported clades of the tree—Mesotaenium 1, Mesotaeni- sion of the taxon (at either family or order level). um 2, MZC, and Mesotaenium caldariorum. These Cylindrocystis. Although the genus Cylindrocystis clades were even less related to each other than the was confined to the ‘‘Zygnemataceae’’ superclade, it representatives of the genus Cylindrocystis, strongly was not monophyletic, corroborating previous stud- suggesting that the traditional genus Mesotaenium ies (Gontcharov et al. 2003, 2004, Hall et al. 2008). also cannot be maintained. Strain SAG 12.97, desig- The 13 Cylindrocystis strains investigated here, split nated as Mesotaenium endlicherianum, the type species into four lineages (the C. brebissonii, MZC, and of the genus, was a member of clade Mesotaenium 2 ‘‘Cylindrocystis’’ clades, and the single strain Cylindro- that is likely positioned outside the ‘‘Zygnemata- cystis sp. M3009). The well-supported MZC clade ceae’’ superclade (see Results). The clade Mesotaeni- contained representatives of two other genera um 1 was identified as the first divergence in the (Zygnemopsis sp. and Mesotaenium kramstai), whereas ‘‘Zygnemataceae’’ superclade; the remaining Meso- the other lineages comprised only nominal taenium sequences were grouped with representa- ‘‘Cylindrocystis’’ taxa. The type species of the genus, tives of other genera. M. kramstai was a member of C. brebissonii, represented by four strains, was only the heterogenous but robust MZC clade (Gontcha- distantly related to the other Cylindrocystis spp. (e.g., rov et al. 2004, Hall et al. 2008), and three M. calda- UD tree topology tests failed to position the C. breb- riorum strains formed a strongly supported sister issonii clade as sister to the ‘‘Cylindrocystis’’ clade; see lineage to the Mougeotia clade. Our analyses also Results). This suggests that the genus Cylindrocystis revealed an affinity of Fottea pyrenoidosa (Ulvophy- should be restricted to members of the C. brebissonii ceae, Chlorophyta) to the class Zygnematophyceae clade. The ‘‘Cylindrocystis’’ clade, established in this and resolved it in the clade Mesotaenium 1 with study, thus likely represents a new genus that will strong support (see Results; Fig. 2). The assignment accommodate most species of the traditional genus of this strain to the ulvophycean genus Fottea was Cylindrocystis. Strains comprising this clade are quite based on morphological features of the colony, cell, diverse in cell shape, dimensions, and details and chloroplast (Broady 1976), and the strain has of chloroplast morphology (A. Gontcharov and apparently not been further investigated by ultra- M. Melkonian, unpublished data), and morphologi- structure or DNA sequence comparisons. Taking cal synapomorphies have yet to be discovered that the polyphyletic nature of the genus Mesotaenium MOLECULAR PHYLOGENY AND REVISION OF NETRIUM 359 and its uncertain taxonomic status into account, other unicellular Zygnematales, and this feature is transfer of F. pyrenoidosa into Mesotaenium is cur- also shared by the genera Planotaenium and Nucleo- rently not warranted. taenium, now segregated from Netrium s. str. How- The poor status of knowledge about the taxon- ever, chloroplasts of similar morphology occur in omy of Mesotaenium derives in part from the fact most clades of the Desmidiales (e.g., in Closterium, that its species occur mostly in subareal habitats Penium, and some Desmidiaceae), suggesting that that are rarely sampled by desmid researchers. this character state is plesiomorphic or even homo- The genus is defined by its unicellular life form plasious, thus questioning its diagnostic value. and the presence of one or two ribbon-like, cen- However, other features of chloroplast morphol- trally positioned chloroplasts (Na¨geli 1849, West ogy were found to be suitable to circumscribe the and West 1904, Prescott et al. 1972, Brook and genera: Planotaenium, Nucleotaenium, and Netrium s. Johnson 2002). However, Mesotaenium includes a str. differ from each other in the number of chlo- number of species with more or less parietal chlo- roplasts per cell (two or four in Planotaenium, one roplast(s) (Buech 1967, Prescott et al. 1972). In in Nucleotaenium, and two in Netrium) and, related to addition, at least in some small-celled species, cen- this, in the position of the nucleus in the cell trally positioned chloroplasts may display a helicoi- and ⁄ or in the behavior of the nucleus during cell dal structure (Wartenberg and Dorscheid 1964). division, and finally in the surface pattern (i.e., the Mesotaenium species form several groups according structure and arrangement of the lamellae) of the to cell size and shape, pattern of chloroplast divi- chloroplast. Two large chloroplasts with profusely sion (in relation to cell division), and features of notched longitudinal lamellae and a large nucleus zygospore morphology, but application of these located centrally between the chloroplasts, diagnos- characters has been restricted to species discrimi- tic features of the traditional genus Netrium, are typi- nation (Buech 1967). Our results suggest that the cal only for the genus Netrium s. str. Its sister clade differences between Mesotaenium taxa could reflect Nucleotaenium is distinct in displaying only one chlo- a much deeper phylogenetic divergence that may roplast per cell with either notched or smooth even transcend genus boundaries (e.g., Mesotaeni- lamellae. A fine central strand connecting the chlo- um 2 clade). roplast halves (in strain SVCK 255) or a lateral Phylogenetic relationships between representatives of the broad connection between the chloroplast halves traditional genus Netrium. Netrium was previously (N. eifelense and N. cylindricum; Fig. 4) results in a lat- shown to occupy a key phylogenetic position eral position of the nucleus in these taxa and likely between the species-poor basal zygnematalean lin- is the reason for the unusual flattened, asymmetrical eages and the derived, morphologically diverse, and shape of the nucleus in the latter species. These syn- species-rich order Desmidiales (McCourt et al. 2000, apomorphic characters unambiguously characterize Gontcharov et al. 2003, 2004). The large data set the new genus Nucleotaenium and distinguish it from and dense taxon sampling used here provided all other Zygnematophyceae. Smooth, straight ⁄ strong evidence that the traditional genus Netrium is oblique chloroplast lamellae differentiate the genus paraphyletic. N. interruptum and N. oblongum SVCK Planotaenium. 255, which had been excluded from the generic The distinct chloroplast features of P.(Netrium) clade in a previous analysis (Gontcharov et al. interruptum and some related species had been 2004), represent members of the previously unrec- previously recognized as taxonomically important ognized genus Planotaenium and the new genus Nu- and were used to establish the section Planotaeni- cleotaenium described here, respectively (Fig. 2; um within the genus Netrium (Ohtani 1990). Early Results). Using three molecular markers from two molecular phylogenetic analyses corroborated this genomes (nuclear-encoded SSU rDNA, and chloro- conclusion but could not resolve the relationship plast-encoded rbcL and LSU rDNA) the relation- between P.(Netrium) interruptum and other species ships between these lineages were fully resolved: the of the traditional genus Netrium because the genera Nucleotaenium and Netrium s. str. are sisters, branch separating these lineages received only whereas the genus Planotaenium has only distant weak support (Gontcharov et al. 2004). With an relationships to the Nucleotaenium ⁄ Netrium clade. extended data set and improved taxon sampling, Instead, Planotaenium was resolved with strong sup- we resolved Planotaenium as a clade independent port as a sister group to the Roya ⁄ Desmidiales clade. from Nucleotaenium ⁄ Netrium (94% BP in ML, 1.00 A synapomorphic CBC revealed in the SSU rRNA of PP), confidently identified it as a sister to the Planotaenium (see Results; Fig. 3b) and shared with Roya ⁄ Desmidiales clade (95% BP in ML, 1.00 PP), the Roya ⁄ Desmidiales clade corroborated this sister and therefore substantiated the recent taxonomic group relationship. Results of UD-tree topology tests rearrangement of Palamar-Mordvintseva and Pet- further rejected affiliation of Planotaenium with the lovany (2009), who raised the section Planotaenium Nucleotaenium ⁄ Netrium clade (Table 3). of Netrium to genus level. Stellate chloroplasts consisting of an axial core The new species in this genus, P. ohtanii, is dis- with pyrenoid(s) and radiating longitudinal lamellae tinct in the presence of only two chloroplasts in differentiate the traditional genus Netrium from the cell, apical vacuoles, and the smaller cell size. 360 ANDREY A. GONTCHAROV AND MICHAEL MELKONIAN

Chloroplast number and cell dimensions link P. above) and much larger cell size. Although strain ohtanii with P. minus (Prescott) Petlov. et Pal.-Mordv. SVCK is clearly a member of the genus Nucleotaeni- and Netrium pseudactinotaenium Coes., but absence of um, we abstain from describing it formally here, the apical vacuoles and the ellipsoid cell shape dif- because the strain was lost in the SVCK collection ferentiate these species from P. ohtanii.InPlanotae- and is thus not available for investigation. nium, apical vacuoles were previously known only in The genus Netrium s. str. was represented in the P. interruptum and its varieties, all with four chlorop- global analyses by only two strains (see Results; lasts per cell. Fig. 2). A preliminary assessment of the genetic The presence of only one chloroplast per cell diversity within the genus using rbcL sequence com- associated with the lateral position of the nucleus parisons of 10 strains showed that the additional sets Nucleotaenium apart from other Netrium s. l. taxa. eight Netrium strains sequenced formed a well-sup- Particularly noteworthy is the position of the ported sister group to N. digitus var. latum (Fig. 7). nucleus in strain SVCK 255. Unlike N. eifelense and Although these eight strains (except for strain N. cylindricum where the halves of the chloroplast CCAC0141, designated ‘‘N. oblongum’’) would all fit are interconnected laterally, in this strain, the two the morphospecies N. digitus, it is obvious that dif- massive halves of the chloroplast are connected by a ferent genotypes exist, which require further analy- thin axial strand so that the nucleus is located on sis using additional molecular markers, such as the one side of the strand. A ‘‘vacuole’’ was always seen ITS1 and ITS2 of the rDNA operon. opposite the nucleus at the other side of the strand (Fig. 4, i and j). In every other respect, this alga is very similar to typical Netrium spp., and it had thus CONCLUSIONS been identified as N. oblongum because of its cell The molecular phylogenetic analyses of the tradi- shape. Since there were no published reports on Ne- tional Zygnematales presented here demonstrated trium species with a single chloroplast and lateral that unicellular representatives traditionally classi- position of the nucleus, a literature search was per- fied in the genera Netrium, Cylindrocystis, and Meso- formed to investigate whether other ‘‘Netrium oblon- taenium were either para- or polyphyletic. This result gum’’ images ⁄ illustrations exist that would fit the highlights the deficiency of the morphological char- characteristics of strain SVCK 255. Examination of acters used to date to define these genera and calls images of Netrium oblongum at the ‘‘Protist Informa- for a fundamental taxonomic revision of the Zyg- tion Server’’ (http://protist.i.hosei.ac.jp) yielded a nematales. This revision has been initiated here number of images showing the same type of cell with respect to the genus Netrium. To extend the and chloroplast morphology with a laterally posi- results to other genera within the Zygnematales in tioned nucleus as in strain SVCK 255. This alga thus our estimation requires (i) a much denser taxon appears to be widespread in Japan, in addition to its sampling that should include new isolates to better occurrence in Europe (Germany) where strain represent the genetic diversity of the taxa in ques- SVCK 255 had been isolated. Apparently, Ohtani tion; (ii) a large enough data set that increases the (1990), who thoroughly studied Netrium populations power of phylogenetic resolution to a level appro- in Japan and Nepal, did not encounter this species. priate to the problem to be solved; and (iii) finally, The Wests (1904) also depicted Netrium cells with and most importantly, a new and unbiased one chloroplast per cell (their plate VII: 2) and with approach to the analyses of phenotypic traits (i.e., a laterally positioned nucleus (plate V: 7), but they morphological characters) that takes advantage of did not provide further details. A single chloroplast of somewhat different morphology is typical for the genus Roya and has also been reported in Cylindro- cystis cushleckae (Brook 1994), but neither of these taxa showed an affinity to Nucleotaenium in the phy- logenetic analyses reported here (see Results; Fig. 2). Moreover, confocal laser scanning micros- copy of fluorescing chloroplasts revealed that C. cushleckae in fact has two chloroplasts per cell, each provided with two ridges seen in front view (Figs. 4n and 5m). Overlapping ridges mask a space between the chloroplasts (Fig. 4m). DAPI staining revealed that in these taxa, the laterally (Roya; results not shown) or centrally (C. cushleckae; Figs. 4m and 5n) positioned nucleus displays a spherical shape, which is typical for the Zygnematophyceae. Fig.7. RbcL phylogeny of Netrium s. str. and Nucleotaenium rooted with two Planotaenium sequences (15 taxa, 1,332 nt, ML Strain SVCK 255 is phylogenetically closely topology). Nodes are characterized by ML bootstrap percentages related to N. eifelense and N. cylindricum but differs (BP ‡ 50%). Branches with 100% BP are shown in bold. ML, from both species in its chloroplast features (see maximum likelihood. MOLECULAR PHYLOGENY AND REVISION OF NETRIUM 361 comparison among cultures and sophisticated meth- Gontcharov, A. A., Marin, B. & Melkonian, M. 2003. Molecular odology. phylogeny of conjugating green algae (Zygnemophyceae, Streptophyta) inferred from SSU rDNA sequence compari- This study was supported by grants from the DFG (ME- sons. J. Mol. Evol. 56:89–104. 658 ⁄ 26-1), RFBR (09-04-00270a and -00621a), and FEB RAS Gontcharov, A. A., Marin, B. & Melkonian, M. 2004. Are combined (09-III-A-06-167). We thank Thomas Pro¨schold and Karl- analyses better than single gene phylogenies? A case study using SSU rDNA and rbcL sequence comparisons in the Zyg- Heinz Linne von Berg for help with the Latin diagnoses and nematophyceae (Streptophyta). Mol. Biol. Evol. 21:612–24. two anonymous reviewers for providing helpful comments. Gontcharov, A. A. & Melkonian, M. 2004. Unusual position of the genus Spirotaenia (Zygnematophyceae) among streptophytes Adam, Z., Turmel, M., Lemieux, C. & Sankoff, D. 2007. Common revealed by SSU rDNA and rbcL sequence comparisons. intervals and symmetric difference in a model-free phyloge- Phycologia 43:105–13. nomics, with an application to streptophyte evolution. Gontcharov, A. A. & Melkonian, M. 2008. In search of mono- J. Comput. Biol. 14:436–45. phyletic taxa in the family Desmidiaceae (Zygnematophy- Archer, W. 1861. Sub-group Desmidieae or Desmidiaceae. In Prit- ceae, Viridiplantae): the genus Cosmarium. Am. J. Bot. chard, A. [Ed.] A History of Infusoria, Including the Dismidiace 95:1079–95. and Diatomace, British and Foreign, 4th ed. Whittaker & Co, Hall, J. D., Karol, K. G., McCourt, R. M. & Delwiche, C. F. 2008. London, pp. 715–51. Phylogeny of the conjugating green algae based on chloroplast de Bary, A. 1858. Untersuchungen u¨ber die Familie der Conjugaten and mitochondrial nucleotide sequence data. J. Phycol. 44:467– (Zygnemeen und Desmidieen).A.Fo¨rstnersche Buchhandlung, 77. Leipzig, Germany, 91 pp. Hillis, D. M. 1996. Inferring complex phylogenies. Nature 383:130–1. Biebel, P. 1964. The sexual cycle of Netrium digitus. Am. J. Bot. Hillis, D. M. & Huelsenbeck, J. P. 1992. Signal, noise and reliability 51:697–704. in molecular phylogenetic analyses. J. Hered. 83:189–95. Bre´bisson, A. 1856. Liste des Desmidie´es, observe´es en Basse-Nor- Hoshaw, R. W. & McCourt, R. M. 1988. The Zygnemataceae mandie. Mem. Soc. Sci. Nat. Cherbourg 4:113–62, 301–4. (Chlorophyta) – a 20-year update of research. Phycologia Broady, P. A. 1976. Six new species of terrestrial algae from Signy 27:511–48. Island, South Orkney Islands, Antarctica. Br. Phycol. J. Huelsenbeck, J. P. & Ronquist, F. 2001. MrBayes: Bayesian infer- 11:387–405. ence of phylogenetic trees. Bioinformatics 17:754–5. Brook, A. J. 1981. The Biology of Desmids. Botanical monographs. Vol. Itzigsohn, H. & Rothe. 1856. Netrium digitus. In Rabenhorst, L. 16. Blackwell Sci. Publ., Oxford, UK, 276 pp. [Ed.] Die Algen Sachsens. No. 508. C. Heinrich, Dresden, Ger- Brook, A. J. 1994. Cylindrocystis cushleckae, a new species of sacco- many. derm desmids (Family Mesotaeniaceae). Queckett J. Microsc. Jarman, M. & Pickett-Heaps, J. 1990. Cell division and nuclear 37:225–31. movement in the saccoderm desmid Netrium interruptum. Pro- Brook, A. J. & Johnson, L. R. 2002. Order Zygnemales. In John, D. M., toplasma 157:136–43. Whitton, B. A. & Brook, A. J. [Eds.] The Freshwater Algal Flora of Kadlubowska, J. Z. 1984. Su¨sswasserflora von Mitteleuropa. Bd. 16, the British Isles. An Identification Guide to Freshwater and Terrestrial Chlorophyta VIII, Conjugatophyceae I, Zygnematales. Gustav Algae. Cambridge Univ. Press, Cambridge, UK, pp. 479–593. Fischer Verlag, Stuttgart, Germany, 531 pp. Buech, G. 1967. Zur Taxonomie und Cytologie der Gattung Karol, K. G., McCourt, R. M., Cimino, M. T. & Delwiche, C. F. Mesotaenium Na¨geli. Inaug. diss., University of Saarbru¨cken, 2001. The closest living relatives of land plants. Science Saarbru¨cken, Germany, 221 pp. 294:2351–3. Coesel, P. F. M. & Meesters, K. J. 2007. Desmids of the Lowlands. Kellogg, E. A. & Juliano, N. D. 1997. The structure and function of Mesotaeniaceae and Desmidiaceae of the European Lowlands. KNNV RuBisCO and their implications for systematic studies. Am. J. Publishing, Utrecht, the Netherlands, 352 pp. Bot. 84:413–28. Czurda, V. 1932. Zygnematales. In Pascher, A. [Ed.] Die Su¨swasser- Kishino, H. & Hasegawa, M. 1989. Evaluation of the maximum Flora von Deutschland, O¨sterreich und der Schweiz. Heft 9. Gustav likelihood estimate of the evolutionary tree topologies from FischerVerlag, Jena, Germany, pp. 1–232. DNA sequence data, and the branching order of the Homi- Denboh, T., Hendrayanti, D. & Ichimura, T. 2001. Monophyly of noidea. J. Mol. Evol. 29:170–9. the genus Closterium and the order Desmidiales (Charophy- Kolkwitz, R. & Krieger, H. 1941. Zygnemales. In Kolkwitz, R. ceae, Chlorophyta) inferred from nuclear small subunit rDNA [Ed.] Rabenhorst’s Kryptogamenflora von Deutschland, O¨sterreich data. J. Phycol. 37:1063–72. und der Schweiz. 13(2). Becker & Erler, Leipzig, Germany, Farris, J. S., Kallersjo, M., Kluge, A. G. & Bult, C. 1994. Testing 499 pp. significance of incongruence. Cladistics 10:315–19. Kossinskaja, C. C. 1952. Flora Plantarum Cryptogamarum URSS. Vol. II Felsenstein, J. 1985. Confidence limits on phylogenies: an approach Conjugatae 1. Mesotaeniales et Gonatozygales. Nauka, Moscow, using the bootstrap. Evolution 39:783–91. Leningrad, 162 pp. Galtier, N., Gouy, M. & Gautier, C. 1996. SeaView and Phylowin, Lu¨tkemu¨ller, J. 1902. Die Zellmembran der Desmidiaceen. Beitr. two graphic tools for sequence alignment and molecular Biol. Pflanz. 8:347–414. phylogeny. Comput. Appl. Biosci. 12:543–8. Marin, B., Klingberg, M. & Melkonian, M. 1998. Phylogenetic Gerrath, J. F. 1993. The biology of desmids: a decade of pro- relationships among the Cryptophyta: analysis of nuclear-en- gress. In Round, F. E. & Chapman, D. J. [Eds.] Progress in coded SSU rRNA sequences support the monophyly of extant Phycological Research, Vol. 9. Biopress Ltd., Bristol, UK, pp. plastid-containing lineages. Protist 149:265–76. 79–192. Marin, B., Nowack, E. C. M. & Melkonian, M. 2005. A plastid in the Gerrath, J. F. 2003. Conjugating green algae and desmids. In Wehr, making: evidence for a second primary endosymbiosis. Protist J. D. & Sheath, R. G. [Eds.] Freshwater Algae of North America, 156:425–32. Ecology and Classification. Academic Press, San Diego, Califor- Marin, B., Palm, A., Klingberg, M. & Melkonian, M. 2003. Phylog- nia, pp. 353–81. eny and taxonomic revision of plastid-containing eugleno- Gillespie, J. J., Johnston, J. S., Cannone, J. J. & Gutell, R. R. 2006. phytes based on SSU rDNA sequence comparisons and Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and synapomorphic signatures in the SSU rRNA secondary struc- mitochondrial (12S and 16S) rRNA genes of Apis mellifera ture. Protist 154:99–145. (Insecta: Hymenoptera): structure, organization, and retro- McCourt, R. M., Delwiche, C. F. & Karol, K. G. 2004. Charophyte transposable elements. Insect Mol. Biol. 15:657–86. algae and land plant origins. Trends Ecol. Evol. 19:661–6. Gontcharov, A. A. 2008. Phylogeny and classification of Zygne- McCourt, R. M., Karol, K. G., Bell, J., Helm-Bychowski, K. M., matophyceae (Streptophyta): current state of affairs. Fottea Grajewska, A., Wojciechowski, M. F. & Hoshaw, R. W. 2000. 8:87–104. 362 ANDREY A. GONTCHAROV AND MICHAEL MELKONIAN

Phylogeny of the conjugating green algae (Zygnemophyceae) Swofford, D. L. 2002. PAUP* Phylogenetic Analysis Using Parsimony based on rbcL sequences. J. Phycol. 36:747–58. (and Other Methods). Beta version 10. Sinauer Associates, Sun- McFadden, G. I. & Melkonian, M. 1986. Use of Hepes buffer for derland, Massachusetts. microalgal culture media and fixation for electron micros- Tamura, K., Dudley, J., Nei, M. & Kumar, S. 2007. MEGA4: copy. Phycologia 25:551–7. molecular evolutionary genetics analysis (MEGA) software Mix, M. 1972. Die Feinstruktur der Zellwa¨nde bei Mesotaeniaceae version 4.0. Mol. Biol. Evol. 24:1596–9. und Gonatozygaceae mit einer vergleichenden Betrachtung Turmel, M., Otis, C. & Lemieux, C. 2005. The complete chloroplast der verschiedenen Wandtypen der Conjugatophyceae und DNA sequences of the charophycean green algae Staurastrum u¨ber deren systematischen Wert. Arch. Mikrobiol. 81:197– and Zygnema reveal that the chloroplast genome underwent 220. extensive changes during the evolution of the Zygnematales. Mix, M. 1975. Die Feinstruktur der Zellwa¨nde der Conjugaten und BMC Biol. 3:22. ihre systematische Bedeutung. Nova Hedwigia Beih. 42:179– Turmel, M., Pombert, J. F., Charlebois, P., Otis, C. & Lemieux, C. 94. 2007. The green algal ancestry of land plants as revealed by the Moon, B. & Lee, O.-M. 2003. A phylogenetic significance of chloroplast genome. Int. J. Plant Sci. 168:679–89. several species from genus Cosmarium (Chlorophyta) of Turner, W. B. 1892. Algae aquae dulcis Indiae orientalis. The Korea based on mitochondrial coxIII gene sequences. Algae freshwater algae (principally Desmidiaceae) of East India. 18:199–206. Kungliga Svenska Vetenskapsakademiens Handlingar 25:1–187. Na¨geli, C. W. 1849. Gattungen einzelliger Algen physiologisch und Wartenberg, A. & Dorscheid, T. 1964. Die helicoidale Struktur des systematisch bearbeitet. Neue Denkschr. Allg. Schweiz. Ges. Ges- Chloroplasten von Mesotaenium violascens De Bary. Arch. ammt. Nat. 10:1–147. Mikrobiol. 49:291–304. Nam, M. & Lee, O.-M. 2001. A comparative study of the morpho- West, W. & West, G. S. 1904. A Monograph of the British Desmidiaceae, logical characters and sequence data of rbcL gene in Cosmarium Vol. 1. Ray Soc., London, 224 pp. species. Algae 16:349–61. Wuyts, J., De Rijk, P., Van de Peer, Y., Pison, G., Rousseeuw, P. & De Ohtani, S. 1990. A taxonomic revision of the genus Netrium (Zyg- Wachter, R. 2000. Comparative analysis of more than 3000 nematales, Chlorophyceae). J. Sci. Hiroshima Univ. Ser. B Div. 2 sequences reveals the existence of two pseudoknots in area V4 23:1–51. of eukaryotic small subunit ribosomal RNA. Nucleic Acids Res. Page, R. D. M. 1996. TREEVIEW: an application to display phylo- 28:4698–708. genetic trees on personal computers. Comput. Appl. Biosci. Wuyts, J., Van de Peer, Y. & De Wachter, R. 2001. Distribution of 12:357–8. substitution rates and location of insertion sites in the ter- Palamar-Mordvintseva, G. M. & Petlovany, O. A. 2009. Flora algarum tiary structure of ribosomal RNA. Nucleic Acids Res. 29:5017– Ucrainicae. Vol. 12. Streptophyta. Fas. 1. Fam. Mesotaeniaceae. Ve- 28. les, Kiev, Ukraine, 157 pp. Xia, X. & Xie, Z. 2001. DAMBE: data analysis in molecular biology Palla, E. 1894. U¨ ber eine neue, pyrenoidlose Art und Gattung der and evolution. J. Hered. 92:371–3. Conjugaten. Ber. Deutsch. Bot. Ges. 12:228–36. Yamagishi, T. 1963. Classification of the Zygnemataceae. Sci. Rep. Pickett-Heaps, J. D. 1975. Green Algae: Structure, Reproduction and Tokyo Kyoiku Daigaku B 11:191–210. Evolution in Selected Genera. Sinauer Associates, Sunderland, Massachusetts, 606 pp. Posada, D. & Crandall, K. A. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–8. Supplementary Material Prescott, G. W., Croasdale, H. T. & Vinyard, W. C. 1972. A Synopsis of The following supplementary material is avail- North American Desmids. Part I: Desmidiales: Saccodermae, Mesota- eniaceae. North American Flora Series II (6). New York able for this article: Botanical Garden, Bronx, New York, 84 pp. Ralfs, J. 1848. The British Desmidieae. Reeve, Benham and Reeve, Table S1. Strain information and EMBL ⁄ Gen- London, 226 pp. Bank accession numbers for taxa used in this Randhawa, M. S. 1959. Zygnemaceae. Indian Council of Agriculture study. New sequences are in bold. Research, New Delhi, India, 478 pp. Reize, I. B. & Melkonian, M. 1989. A new way to investigate living This material is available as part of the online flagellated ⁄ ciliated cells in the light microscope: immobiliza- tion of cells in agarose. Bot. Acta 102:145–51. article. Ru˚ˇzicˇka, J. 1977. Die Desmidiaceen Mitteleuropas. Band 1(1). Schwe- izerbart’sche Verlagsbuchhandlung, Stuttgart, Germany, 292 Please note: Wiley-Blackwell are not responsi- pp. ble for the content or functionality of any supple- Shimodaira, H. 2002. An approximately unbiased test of phyloge- mentary materials supplied by the authors. Any netic tree selection. Syst. Biol. 51:492–508. queries (other than missing material) should be Shimodaira, H. & Hasegawa, M. 1999. Multiple comparisons of log- likelihoods with applications to phylogenetic inference. Mol. directed to the corresponding author for the Biol. Evol. 16:1114–16. article. Shimodaira, H. & Hasegawa, M. 2001. CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 17:1246–7.