Fottea, Olomouc, 17(1): 1–19, 2017 1 DOI: 10.5507/fot.2016.010

Selenastraceae (, ): rbcL, 18S rDNA and ITS–2 secondary structure enlightens traditional , with description of two new genera, Messastrum gen. nov. and Curvastrum gen. nov.

Thaís Garcia da Silva1*, Christina Bock2, Célia Leite Sant’Anna3, Inessa Lacati- va Bagatini1, Sabina Wodniok2 & Armando Augusto Henriques Vieira1

1Laboratory of Phycology, Department of Botany, Federal University of São Carlos, Graduate Program in Ecology and Natural Resources, CEP 13560–590, São Carlos, SP, Brazil; * Corresponding author e–mail: [email protected] 2Department of Biodiversity, University of Duisburg–Essen, Essen, D–45141, Germany 3Institute of Botany, Nucleus of Phycology, CEP 04301–902, São Paulo, SP, Brazil

Abstract: The phylogeny of the family was investigated by light microscopy, 18S rDNA, rbcL and ITS–2 analyses. Various morphological features traditionally used for and genera identification were investigated. All selenastracean strains studied have naked pyrenoids within the chloroplast, except the Chlorolobion, which presented starch envelope. The molecular analyses showed that no morphological criterion considered so far is significant for the systematics of the Selenastraceae, but a set of features may be suitable to identify the genera and Chlorolobion. Phylogenetic analyses showed the genera , and were not monophyletic and not distinguishable as separate genera. The polyphyly of the genus Selenastrum led to the description of two new genera, Curvastrum gen. nov and Messastrum gen. nov.

Key words: Ankistrodesmus, Chlorolobion, Kirchneriella, molecular systematics, morphology, phylogeny, Selenastrum

Introduction varies from the solitary to colonial forms (Komárek & Fott 1983; Korshikov 1987; Komárková–Legne- Selenastraceae Blackman & Tansley 1903 (Chloro- rová 1969; Comas 1996; Hindák 1977; Hindák 1980; phyceae, Sphaeropleales) is a green algae family com- Hindák 1984; Hindák 1988; Hindák 1990; Sant’Anna mon in freshwater bodies all over the world, presenting 1984). Their reproduction is exclusively by autospore a high morphological diversity (Krienitz et al. 2001). formation, in which the cytokinesis of the mother cell This family includes the most common members of protoplasm gives rise to 2–4–8 young cells (Komárek phytoplankton in nearly all types of inland waters. & Fott 1983). The combination of cell size, shape, so- They can produce mass developments in lakes, ponds, litary or colonial lifestyle, the releasing process of the pools and rivers (Messyasz 2003; Tas & Gonulol autospores and special habitat preferences, are consi- 2007). Although they commonly occur in freshwater dered to be species–specific (Hindák 1977; Komárek habitats, some species tolerate moderate saline habitats & Fott 1983). Based on these criteria, up to 100 spe- as well, i.e. some taxa are reported from brackish and cies were described in various genera and included in low saline areas of the Baltic Sea (Pankow 1990). The this family (Hindák 1977; Hindák 1984; Komárek & Selenastraceae are valuable indicator organisms for Fott 1983; Fawley et al. 2006; Krienitz et al. 2011). ecosystem health, and several species of this family are Since its description in 1903, the family Sele- regularly used as indicator species, e.g. in the frame of nastraceae has passed by many taxonomic changes, the European Water Framework Directive (Mischke & being recognized as: Bohlin 1904, Kusber 2009). The morphology of this group compri- Selenastraceae West & Fritsch 1927, Ankistrodesma- ses a variety of shapes: from coccoid to elongated, cy- caeae Korshikov 1953, Oocystaceae Bourrelly 1972, lindrical to fusiform, sickle–shaped to spirally curved, Chlorellaceae – Ankistrodesmoidea Komarek & Fott with sharp or rounded ends, where cell arrangements 1983. However, first studies in the 18S rDNA of the 2 da Silva et al.: Two new genera, Messastrum gen. nov. and Curvastrum gen. nov.

commonly observed genera in this family, e.g. Ankis- rRNA genes (Wolf et al. 2013). trodesmus, Selenastrum, Monoraphidium, , In response to the difficulty in accurately identi- Podohedriella and Kirchneriella, show that they form fying Selenastraceae species worldwide, this study ai- a monophyletic group within the Chlorophyceae (Fa- med to clarify the taxonomic status of some members wley et al. 2006; Krienitz et al. 2011; Krienitz et al. of this algae family and contribute to the knowledge 2001), apart from other members of Scenedesmaceae about their diversity. The present study is the first (Fawley et al. 2006; Krienitz et al. 2001), Oocystaceae attempt to evaluate Selenastraceae combining mor- [which is now placed within the Class Trebouxiophy- phology, gene sequences of 18S rRNA and rbcL, and ceae (Friedl 1995)] and Chlorellaceae (Friedl 1995; ITS–2 secondary structure. Krienitz et al. 2001). Since the onset of molecular phy- logeny, several genera were excluded from the family due to their molecular traits, e.g., Closteriopsis was transferred to the Chlorellaceae and Hyaloraphidium is Material and Methods in fact a fungus (Luo et al. 2010; Ustinova et al. 2001). Despite the monophyly of the family, the gene- Algal cultures and microscopy. Forty five Selenastraceae ra still need revision, since morphological features are strains were investigated (Table S2). The algal cultures were usually not in accordance with molecular data (Krie- obtained from Freshwater Microalgae Culture Collection from Universidade Federal de São Carlos (CCMA – UF- nitz et al. 2001; Krienitz et al. 2011). For example, SCar, WDCM 835) and from an author personal collection defined genera cluster polyphyletic on different clades (CB strains). All the strains were grown in WC medium within the Selenastraceae, e.g. Selenastrum bibraia- (Guillard & Lorenzen 1972) and maintained at of 23 ± 1 num (type species of Selenastrum), and Selenastrum ºC, under photoperiod 12/12 hours light/dark, and luminous gracile belong to different phylogenetic lineages based intensity of ~200 µmol.m–2.s–1. on 18S rDNA phylogeny (Fawley et al. 2006; Krienitz The whole life cycles of cultured strains were exam- et al. 2001) but no taxonomic changes were made in ined using an Axioplan 2 Imaging Zeiss or Nikon Eclipse the genus, since the authors suggested further studies E600 light microscope with differential interference contrast. with the family to ensure these findings. Micrographs were taken using an AxioCam with software Due to the difficulties in their identification and AxioVision 4.6 (Carl Zeiss Group, Oberkochen, Germany) and a Nikon digital camera DS–Fi1 with Nikon software taxonomy, the current knowledge of species diversity NIS–Elements D (Nikon Corporation, Tokyo, Japan). The and ecology of Selenastraceae is still poorly unders- algal strains were identified according to the published keys tood worldwide (Fawley et al. 2006; Krienitz et al. (Korshikov 1987; Komárek & Fott 1983; Komárková– 2001). In addition, previous studies were focused on Legnerová 1969; Comas 1996; Hindák 1977; Hindák 1980; temperate northern hemisphere isolates, whereas the Hindák 1984; Hindák 1988; Hindák 1990; Sant’Anna 1984). molecular diversity of tropical Selenastraceae remains Before Scanning Electron Microscopy (SEM) and unknown. Transmission Electron Microscopy (TEM) cells were fixed Phylogenetic inference in green algae is mainly in 2.5%, glutaraldehyde in culture medium (Hegewald et al. 1994) for 24 hours at –5ºC, and dehydrated as follows. The based on 18S rDNA sequences (Booton et al. 1998; cells were washed three times with culture medium and de- Buchheim et al. 2001; Fawley et al. 2006; Hegewald hydration series was taken in a graded acetone series: 20, 35, & Hanagata 2000; Krienitz et al. 2011; Krienitz et al. 50, 70, 90% (twice) for 15 minutes each step, and 100% kept 2003; Krienitz et al. 2001; Lewis 1997). Nevertheless, overnight. Samples were washed three times with culture several studies have shown that the 18S rDNA is in medium and postfixed with 1% osmium tetroxide for 2 hours. some cases too conserved to distinguish between clo- For SEM, a Critical Point Dryer (BAL–TEC 030, sely related genera and species (Luo et al. 2010). Diffe- Germany) was used at 80–90 bars and 30–34 °C. The sam- rent studies take a second marker into account as well, ples were placed on a gold–palladium–coater (High Resolu- tion Ion Beam Coater Model 681, Germany) and then 2 depo- to gain a higher resolution (Rindi et al. 2011). The gene sitions of palladium were made (±1 kÅ). SEM images were rbcL is being widely used mainly because of its higher taken with ESEM Quanta 400 FEG (FEI, The Netherlands). variations and better resolution than the 18S rDNA at TEM was performed according to Krienitz et al. lower taxonomic levels (Fučíková et al. 2011) and is (2011) with infiltration in epon. Thin sections were prepared also used as a DNA barcode in marine green macroal- on a Reichert UltraCut S (Reichert Inc., Depew, NY, USA), gae (Saunders & Kucera 2010). The ITS–2 has proven with no poststain and examined in a Hitachi S–4000 Scan- to be a suitable marker for small scale phylogenies and ning Electron Microscope (IMCES–Imaging Center Essen). it is commonly applied among species within the same genus (Bock et al. 2011b; Coleman 2003; Coleman DNA extraction, PCR and sequencing. For DNA extrac- tion, the algae cultures were grown in the conditions de- 2007; Coleman & Vacquier 2002; Schultz et al. 2005; scribed above for microscopy analyses. The cell suspen- Young & Coleman 2004) or for the resolution of clo- sion was centrifuged at 16.000 ×g for 10 minutes and the sely related genera (Luo et al. 2010; Luo et al. 2011b). pellet stored at –80 °C until the next step. The cells were The highly divergent properties and the rapid evolu- disrupted using glass beads (150–212 µm, Sigma–Aldrich), tion legitimate ITS–2 to discriminate closely related vortex briefly, and extracted using Invisorb® Spin Mini organisms, which exhibit nearly identical sequences in (STRATEC Biomedical AG; Germany) or My–Budget DNA Fottea, Olomouc, 17(1): 1–19, 2017 3 DOI: 10.5507/fot.2016.010

Mini Kit® (Bio–Budget Technologies GmbH; Krefeld; Ger- by calculating 1000 bootstrap values for NJ, MP, ML crite- many). ria. For all datasets, Bayesian analyses were performed using PCR amplification was carried out using - estab MrBayes version 3.1. (Huelsenbeck & Ronquist 2001). Two lished target–specific primers and some primers proposed runs with four chains of Markov chain Monte Carlo (MCMC) in this study. For 18S rDNA, the primers used were 18SF1 iterations were performed (3 million generations for rbcL (Katana et al. 2001), 1F, 300F, 528F, 690F, 920F, 1055F, and 8 million generations for 18S rDNA). The stationary dis- 1400F, 920R, 1200R, 1520R (Huss et al. 1999), 18SR1 (5’ tribution was assumed when the average standard deviations – TGATCCTTCTGCAGGTTCACCTA – 3’) modified from of split frequencies between two runs were lower than 0.01 Katana et al. (2001). Four new primers were designed for and Tracer V1.4 (Rambaut & Drummond 2007) was used the rbcL: rbcL 320 mod (5’ – TATTYGAASAAGGTTCW- to check the stationary phase and to identify an appropriate GTWAC– 3’, modified from Rindi (2008), Selenastraceae burn–in value. The first 25% of the calculated trees were dis- rbcL F (5’ – CGYTACAAAGGDCGTTGYT – 3’), rbcL carded as burn–in. 50% majority–rule consensus trees were Orb modified (5’ – CTGGNGCRTTACCCCAAGG – 3’), calculated for posterior probabilities (PP). The trees were modified fromP ažoutová (unpublished), and Selenastraceae edited using TreeGraph 2 (Stöver & Müller 2010). Previ- rbcL R (5’ – RTTACCCCAWGGGTGHCCTA – 3’). These ous publications indicated that Bracteacoccus (Krienitz et al. proposed primers were used in association with the follow- 2001) and Pediastrum (Fučíková et al. 2014) members were ing published primers, rbcL1, rbcL 1181, 1421 (Nozaki et suitable as an outgroup for the phylogeny of Selenastraceae. al. 1995), rbcL 320 (Rindi et al. 2008), rbcL RH1 (Manhart 1994), rbcL 1385 (McCourt et al. 2000), and rbcL ORB ITS–2 secondary structure prediction. The ITS–2 model (Pazoutova unpublished). For ITS–2 the primers used were for Scenedesmus, proposed by van Hannen et al. (2002), 1420F (Rogers et al. 2006), NS7m and LR1850 (An et al. was used as a template and adapted by hand. The secondary 1999) and ITS055R (Marin et al. 1998). For more details structure was obtained using the RNAfold Webserver (Gru- about primers, see Table S3 (supplementary material). The ber et al. 2008) where the minimum free energy (MFE) of the PCR amplifications for rbcL gene were performed using the secondary structure of single sequences and the equilibrium following reaction conditions: 95 °C for 5 min followed by base–pairing probabilities were predicted. The RNA secon- 25 cycles, each including 1 min at 95 °C, 1 min at 52 °C, and dary structures were visualized with Pseudoviewer 3 (Byun 2 min at 72 °C with Taq DNA Polymerase QIAGEN® or & Han 2009). DreamTaq DNA Polymerase Thermo Scientific®.18S rDNA PCR amplifications were conducted according to Katana et al. (2001) and Krienitz et al. (2011). ITS–2 PCR ampli- fications were performed according to Bock et al. (2011). Each PCR product was electrophoresed in a 1% agarose gel, stained with ethidium bromide. Results Purification of the PCR products was conducted using the polyethylene glycol protocol (PEG) according to Genera and species descriptions Rosenthal et al. (1993). The PCR products were sequenced by Macrogen Inc. (ABI 3130–Genetic–Analyzer, Applied Biosystems GmbH, Darmstadt, Germany) with the same Messastrum gen. nov. T. S. Garcia primers used for amplification. Part of the genomic DNA is Description: Green, planktonic microalgae. Narrow, stored at the Phycology Lab – UFSCar, and Department of fusiform to semilunate cells, ends gradually pointed, Biodiversity – University Duisburg–Essen. arcuate. Colonies with 2–4–8 or multi irregularly ar- Twenty three new 18S rDNA sequences, 34 new rbcL ranged cells, mostly with the convex side towards the sequences and 8 ITS–2 new sequences were amplified and center of the colony. One parietal chloroplast, contain- obtained on this study, totalizing 65 new entries in GenBank ing a pyrenoid, no starch cover observed. (National Center for Biotechnology Information [NCBI], Asexual reproduction by autosporulation (2– http://www.ncbi.nlm.nih.gov/). The whole dataset used for phylogenetic analyses with accession numbers are reported 4–8 autospores per sporangium), sexual reproduction in Table S2, including 29 reference sequences acquired from not known. An unseparated fragment of the mother cell GenBank. membrane remains and is covered by a thin mucous layer. Cells single or on 2–4–8 or multi–celled colony Phylogenetic analyses. Sequences were manually aligned formation. Cells often single celled in culture. A dif- using Align – Manual Sequence Alignment Editor (Hepperle fuse thin layer of mucilage is often concentrated as a 2004). For the phylogenetic analyses, two different datasets ring on the middle of the cells on both colonies and were prepared. The rbcL analyses contained a dataset of 40 free individuals. Genus differs from other genera in the sequences with 767 base positions. For the 18S rDNA ana- Selenastraceae based on differences in 18S rDNA and lyses, 46 sequences with 1511 base positions were acquired. The two genes (18S rDNA and rbcL) were analyzed separa- rbcL gene sequences. tely, both in maximum likelihood (ML) on Treefinder J( obb 2008), distance (neighbor joining; NJ) and maximum parsi- Typus generis: Messastrum gracile comb. nov. mony (MP) using PAUP* (portable version 4.0b10) (Swo- Etymology: From the Latin mess (= mess) and astrum fford 2002). For ML and Bayesian analyses, the evolutive (= star). model for both genes (18S rDNA and rbcL) (GTR[Optimum, Empirical]: G[optimum]:5) was applied as suggested by Mr- Modeltest (Nylander 2004), with tree sampling every 100 generations. The confidence of the tree topology was tested 4 da Silva et al.: Two new genera, Messastrum gen. nov. and Curvastrum gen. nov.

Messastrum gracile comb. nov. (Reinsch) T. S. Gar- Curvastrum pantanale sp. nov.T. S. Garcia cia Description: Cells narrow, fusiform to semilunate, Synonym: Ankistrodesmus gracilis (Reinsch) Korshikov 1953, Se- ends gradually pointed, arcuate. Planktonic, solitary lenastrum westii G.M.Smith 1920, Dactylococcopsis pannonicus or 4–celled colonies with 4 irregularly arranged cells, Hortobágyi 1943. Basyonym: Selenastrum gracile Reinsch 1866: 65, pl. IV: Fig. III. mostly with the convex sides towards the center of the Description: Cells narrow, fusiform to semilunate, colonies. Reproduction by autospore formation, where ends gradually pointed, arcuate. Planktonic, solitary the sporangium gives rise to 4 young cells, with zigzag or 2–4–8 or multi–celled colonies with irregularly ar- orientation.Pyrenoid without starch cover. One parietal ranged cells, mostly with the convex sides towards chloroplast. Cell wall covered by a diffuse thin layer of the center of the colonies. Reproduction by autospore mucilage on both colonies and free individuals. Cells formation, where the sporangium gives rise to 2–4–8 8–21 × 1.9–3.5 µm, distance between the opposite cell young cells, with parallel or zigzag orientation. Pyre- ends: 4–14 µm. noid without starch cover, observed just under TEM. One parietal chloroplast. Cell wall covered by a dif- Holotype: A formaldehyde fixed sample of this strain fuse thin layer of mucilage on both colonies and free is deposited at the Botanical Institute at São Paulo, individuals. A diffuse thin layer of mucilage is often Brazil, under the designation SP 469320. concentrated as a ring on the middle of the cells on Iconotype: our figure number 3–4. both colonies and free individuals. Cells19–55 × 1–6 Isotype: Material of the authentic strain CCMA–UFS- µm, distance between the opposite cell ends 6–34 µm. Car 350, maintained at the Culture Collection of Fresh- water Microalgae, Federal University of São Carlos, São Carlos, Brazil. Holotype: Selenastrum gracile Reinsch 1866: 65, pl. IV: Fig. III. Type locality: Isolated from a water fountain used as Epitype (designated here): A formaldehyde fixed a supply for animals on Pantanal (Table S2), a Bra- sample of strain CCMA–UFSCar 622 is deposited at zilian wetland, in October 2011. GPS 19°17'59.0"S the Botanical Institute at São Paulo, Brazil, under the 55°47'45.0"W. designation SP 469319. Etymology: The species epithet was derived from the Isotype: Material of the authentic strain CCMA–UFS- place of origin of the first isolate of this genus, “Panta- Car 622 (Fig. 1), maintained at the Culture Collection nal”, which means “great Swamp”. of Freshwater Microalgae, Federal University of São Carlos, São Carlos, Brazil. Notes: Also collected inside the state of São Paulo (CCMA–UFSCar 608). Etymology: The species epithet is based on a morpho- logical feature, “thin, slender”, kept on this nomencla- Selenastrum bibraianum (Reinsch) Korshikov 1953 tural change. Synonym: Ankistrodesmus bibraianus (Reinsch) Korshikov 1953 Basyonym: Selenastrum bibraianum Reinsch 1866: 65, pl. IV: Fig. Notes: Epitype isolated from a pond in Conchas, in the II. country side of the state of São Paulo, in August 2013. Description: Cells narrow, fusiform to semilunate, More strains were collected inside the state of São Pau- ends gradually pointed, arcuate. Planktonic, solitary or lo (CCMA–UFSCar 5 and CCMA–UFSCar 470 (For 2–4–8 or multi celled colonies with regularly arranged GPS see Table S2). cells, mostly with the convex sides towards the center of the colonies, by their convex side. Reproduction by Curvastrum gen. nov. T. S. Garcia autospore formation, where the sporangium gives rise Description: Green, planktonic microalgae. Narrow, to 2–4–8 young cells, with parallel or zigzag orienta- fusiform to semilunate cells, ends gradually pointed, tion. One parietal chloroplast. Cell wall covered by a arcuate. Colonies with 4 irregularly arranged cells. strong layer of mucilage on colonies. A diffuse thin One parietal chloroplast, containing a pyrenoid with- layer of mucilage is often concentrated as a ring on the out starch cover, observed just under TEM. middle of the cells on colonies and free individuals. Asexual reproduction by autosporulation (four au- Cells 16–40 × 2–8 µm, distance between the opposite tospores per sporangium), sexual reproduction not cell ends 2–20 µm. known. Cells single or on 4–celled colony formation. Cells often single celled in culture. Cell wall covered Holotype: Selenastrum bibraianumReinsch 1866: 65, by a diffuse thin layer of mucilage on both colonies and pl. IV: Fig. II. free individuals. Genus morphologic similar to Mes- Epitype (designated here): A formaldehyde fixed sastrum, distinguishing for the numbers of cells on col- sample of strain CCMA–UFSCar 47 is deposited at the ony and 18S rDNA, ITS–2 rDNA and rbcL sequences. Botanical Institute at São Paulo, Brazil, under the des- ignation SP 469321. Typus generis: Curvastrum pantanale sp. nov. Isotype: Material of the authentic strain CCMA–UFS- Etymology: From the Latin curvus (= curved) and as- Car 47, maintained at the Culture Collection of Fresh- trum , (= star). water Microalgae, Federal University of São Carlos, Fottea, Olomouc, 17(1): 1–19, 2017 5 DOI: 10.5507/fot.2016.010

Figs 1–5. (1) Messastrum gracile, original picture of strain CCMA–UFSCar 622 showing a frontal view of colony; (2) Selenastrum bibrai- anum, original picture of strain CCMA–UFSCar 125 showing a frontal view of colony; (3–4) Curvastrum pantanale, (3) original picture of strain CCMA–UFSCar 350, showing free cells and colony, (4) original picture of strain CCMA–UFSCar 350, showing cells in autospore liberation presenting a cell wall remnant (arrowhead) and protoplasm cleavage (star); (5) Ankistrodesmus arcuatus, original picture of strain CCMA–UFSCar 24, showing free cells and colony. Note autospore formation (star) and mucilaginous lump (arrowhead). Scale bar 10 μm.

São Carlos, Brazil. los (Represa do Monjolinho), in the country side of the Etymology: no reference. state of São Paulo, in November 2008. More strains were collected inside the state of São Paulo (CCMA– Notes: Epitype – isolated from a reservoir in São Car- UFSCar 125 and CCMA–UFSCar 630), two strains 6 da Silva et al.: Two new genera, Messastrum gen. nov. and Curvastrum gen. nov. were isolated from Germany (CB 2009/39 and CB to semilunate isopolar cells, 2–4–8 autospores per spo- 2009/43) and one from Sweden (CB 2012/47) (Table rangium, mother cells ruptures in the meridian part S2). with 4–8 autospores per sporangium. All strains exhib- ited a great mucus layer a parenthesis both in colony Ankistrodesmus arcuatus Korshikov 1953 and in solitary cells. Synonym: Monoraphidium arcuatum (Korshikov) Hindák 1970: 24, Strains CCMA–UFSCar 47, CCMA–UFSCar Figs 19, 10, Ankistrodesmus pseudomirabilis Korshikov 1953: 297, 125, CCMA–UFSCar 630, CB 2009/39, CB 2009/41 Fig. 258 a–f, Ankistrodesmus sabrinensis Belcher &Swale 1962: 131, Fig. 1:H. and CB 2012/47 differed only slightly in their mor- Basyonym: Ankistrodesmus arcuatus Korshikov 1953: 296, Fig. phology. They resembled the morphological criteria 257a, b. for Selenastrum bibraianum with fusiform to semilu- Emend diagnosis: Cell solitary or in colonies, fusi- nate cell shape, lengths of 18–40 long × 2–8 µm large. form, narrow or acute at the ends, arched or curved. They occured in big colonies with up to 19 cells and Four celled colony where cells are arranged by one end with an arcuated chain of cells, regularly arranged in attached in mucilage. Presence of mucilage. One pari- colony. Strains CCMA–UFSCar 5, CCMA–UFSCar etal chloroplast. Reproduction by autospore formation, 470, CCMA–UFSCar 622, CB 2009/3 and CB 2009/35 where the sporangium gives rise to 4–8 young cells, had similar morphological features as the strains of S. with parallel orientation. Mother cell wall ruptures bibraianum but differed in their colony formation. The along the whole length to autospore liberation. Pyre- morphological criteria to the newly combined Messas- noid without starch cover, observed just under TEM trum gracile was the fusiform to semilunate cell shape, analyses. Dimensions: 26–60 × 0.8–4.4 µm, distance lengths of 19–55 long × 1–6 µm large. They frequently between the cell ends: 30 µm. showed big colonies with an arcuated chain of cells, irregularly arranged in this case, where colonies up to Holotype: Ankistrodesmus arcuatus Korshikov 1953: 11 cells were observed. The strains that belong to the 296, Fig. 257a, b. new species Curvastrum pantanale (CCMA–UFSCar Epitype (designated here): A formaldehyde fixed 350 and CCMA–UFSCar 608) exhibited colonies with sample of strain CCMA–UFSCar 24 is deposited at the no more than 4 cells, irregularly arranged in colony, Botanical Institute at São Paulo, Brazil, under the de- which lengths observed were of 8–21 long × 2–3.5 µm signation SP 469322. large (Fig. 8a). Isotype: Material of the authentic strain CCMA–UF- Cells with fusiform and isopolar cells shape SCar 24 (Fig. 5), maintained at the Culture Collection (solitary cells and in colony), autospores arranged in of Freshwater Microalgae, Federal University of São parallel and mother cell wall remnants cone–shaped Carlos, São Carlos, Brazil. could be observed in the strains CB2009/9, CB Etymology: The species epithet is based on a morpho- 2012/3, CB2012/6, CB2012/29, CCMA–UFSCar 24, logical feature, “arched”, kept on this nomenclatural CCMA–UFSCar 277, CCMA–UFSCar 278, CCMA– change. UFSCar 423, CCMA–UFSCar 593, CCMA–UFSCar 611. Due to the characteristics in morphological keys, Note: According to the original diagnosis (Korshikov these strains were identified as members of the genus 1953), the author adds a suggestion that the cells may Ankistrodesmus. The identified and studied species belong to decomposed colonies of Messastrum graci- were: Ankistrodesmus spiralis (Turner) Lemmermann le, differing only by the absence of mucus. Komárko- (CB2012/29; colonies with central twisted bundles, vá–Legnerová (1969) considered a possible colony mucilage only observed around colonies); Ankistrodes- formation and mucilage presence but did not make a mus stipitatus (Chodat) Komárková–Legnerová nomenclatural recombination. Epitype: isolated from (CCMA–UFSCar 277, CCMA–UFSCar 278 cells fre- Broa Reservoir, Itirapina, in the country side of the sta- quently arranged in parallel in free floating fascicular te of São Paulo, in 1979 (Table S2). colonies, some cells were attached to the walls of the glass tubes, producing basal mucilage on their ends, Light microscopy. This section is focused on morpho- autospores seemed to be attached to each other by the logical traits that conducted to species differentiation middle of the cell even before the mother cell rup- based on light microscopy. For identification keys and ture); Ankistrodesmus fusiformis Corda (CB 2012/6, diacritical features of established species see Material CCMA–UFSCar 423, CCMA–UFSCar 611, CCMA– and methods. A summary is provided in Table S1. UFSCar 593; cruciform or stellate colonies joined by a Seven strains were analyzed based on light mi- central mucilaginous area); Ankistrodesmus fascicula- croscopy which match the morphological description tus (Lundberg) Komárková–Legnerová (CB 2012/3; for the genus Selenastrum according to Reinsch (1866) presented 2–4 celled colonies connected to each other and Komárek & Fott (1983). The strains CB 2009/39, by their convex side, revealing a fasciculate shape, sur- CB 2009/41, CB 2009/43, CB 2012/47, CCMA–UFS- rounded by a mucus layer. Ankistrodesmus sigmoides Car 47, CCMA–UFSCar 125 and CCMA–UFSCar had sigmoid colony formation. Ankistrodesmus arcu- 630 had similar cell shape, with narrow and fusiform atus (Korshikov) Hindák (CCMA–UFSCar 24) arcu- Fottea, Olomouc, 17(1): 1–19, 2017 7 DOI: 10.5507/fot.2016.010

Fig. 6. Maximum–likelihood (ML) phylogenetic tree inferred from rbcL gene sequences of some members of Selenastraceae. Support values correspond to Bayesian PP (Posterior Probability), ML BP (Bootstrap), MP (Maximum Parsimony)BP, NJ (Neighbor–Joining)BP. Hyphens correspond to values <50% for BP and <0.95 for PP. Scale represents the expected number of substitutions per site. Strain numbers used as mentioned in Table S2. ated cells were arranged frequently in parallel in free autospore formation was peculiar and very discernible floating fascicular colonies. in this species. Chlorolobion braunii (Nägeli) Komárek microscopica (Nygaard) Mar- (CCMA–UFSCar 137, CCMA–UFSCar 455, CCMA– van, Komárek et Comas (CB 2009/6, CB 2009/18 and UFSCar 462 and CCMA–UFSCar 476) had fusiform, CB 2012/39) formed colonies with mucilage and very heteropolar to isopolar cells, showing some cell asym- small cells (3–5 long × 1–2 µm large) irregularly dis- metry which lengths observed were of 13–52 long x tributed on the colony. 3–8 µm large. It was observed starched pyrenoid on All Kirchneriella Schmidle species were similar this species, with elliptic to rounded shape. Many cells in cell shape and in the colony morphology, varying were attached to the walls of the glass tubes, producing on cell size and on the next exposed features. Kirch- basal mucilage on their ends, as in A. stipitatus. The neriella pseudoaperta Komárek (CCMA–UFSCar 8 da Silva et al.: Two new genera, Messastrum gen. nov. and Curvastrum gen. nov.

Fig. 7. Maximum–likelihood (ML) phylogenetic tree inferred from 18S rDNA gene sequences of some members of Selenastraceae. Support values correspond to Bayesian PP (Posterior Probability), ML BP (Bootstrap), MP (Maximum Parsimony) BP, NJ (Neighbor–Joining)BP. Hyphens correspond to values <50% for BP and <0.95 for PP. Scale represents the expected number of substitutions per site. Strain numbers used as mentioned in Table S2.

346) presented visible pyrenoid under LM, and cell presented mucilage when in colony and solitary cells size were 1.6–9.6 long × 2.4–4.2 µm large. Mucilage were bigger in size than the colony living ones. Cell was exhibited when in colony, where mainly 4–celled size varied from 6–16 long × 2–9.5µm large. Kirchne- colonies were observed. Some cells kept connected to riella aperta Teiling (CCMA–UFSCar 482) exhibited each other before leaving the mother cell. Kirchneriel- mucilage when in colony but not when in solitary cells, la obesa (West) West etWest (CCMA–UFSCar 345) where cell size observed was 6–12 long × 12 µm large. Fottea, Olomouc, 17(1): 1–19, 2017 9 DOI: 10.5507/fot.2016.010

Fig. 8. Scanning electron micrographs of Curvastrum pantanale (CCMA–UFSCar 350) in culture: (a) typical colony formation, (b) young cells detaching from each other, (c) young cells, note the autospores position. Scale bar, 10 µm.

Kirchneriella lunaris (Kirchner) Möbius (CCMA– Car 24, CCMA–UFSCar 349 and CCMA–UFSCar UFSCar 443) had acute cell apex, with solitary cells 350. A large parietal and cup–shaped chloroplast was bigger than the colonies one, always presenting py- a typical feature between the observed strains (as seen renoids and with cell dimensions from 4–15 long to on Fig. 9, d; Fig.10 b and e). The parietal chloroplast 1.2–2.8 µm large. Kirchneriella contorta var. elegans was filled with a large number of thylakoids situated (Playfair) Komárek (CCMA–UFSCar 447) had circu- in parallel to the cell wall (Fig.9, c–e; Fig.10, b, d, e). lar, crescent–shaped to sigmoid cells with rounded cell In addition, some polyphosphate vacuoles (Fig.9, a, apex, with cell lengths from 6–8 long to 2 µm large. c; Fig.10, b, d, e) were found on cells on stationary Kirchneriella irregularis (Smith) Korshikov (CCMA– phase and often some lipid drops (Fig.9, a, b; Fig.10, UFSCar 348) cells were fusiform to strongly twisted, d).The nucleus could be observed at the opposite direc- very small in size (from 6–21 long to 3–6 µm large) tion of the pyrenoid (Fig.9, d, e; Fig.10, a, b, e) and, in and without mucilage. between them, there is a dictyosome (Fig. 10c). Mito- Genus Monoraphidium Komárková–Leg- chondria (Fig.9, c, e) was observed at the inner surface nerová always presented solitary and pyrenoid–less of the chloroplast. cells. Monoraphidium komarkovae Nygaard (CCMA– UFSCar 353 and CCMA–UFSCar 632) had very long Phylogenetic analyses and thin cells (from 25–182 long to 1.4–3.5 µm large), Representatives of Selenastraceae were included in without mucilage. Monoraphidium contortum (Th- our rbcL (Fig. 6) and 18S rRNA gene sequence ana- uret) Komárková–Legnerová (CCMA–UFSCar 306, lyses (Fig. 7). Due to the low support of some internal CCMA–UFSCar 329, CCMA–UFSCar 349 and CB branches in both phylogenies, the relationships among 2009/10) presented strongly twisted cells (from 7–40 some lineages were not clearly resolved. However, long to 1–5.2 µm large), with basal mucilage join- some well–supported clades can be identified in both ing young cells after autospores liberation, like in A. trees: (i) Selenastrum gracile, (ii) Selenastrum bibra- stipitatus. Monoraphidium pseudobraunii (Belcher et ianum, (iii) Raphidocelis, (iv) Curvastrum pantanale Swale) Heynig (CCMA–UFSCar 325) showed slight- nov. gen. et. sp., (v) Kirchneriella, (vi) Chlorolobion ly heteropolarity, with no mucilage and 2 autospores (including Podohedriella falcata on the 18S rDNA per sporangium, with cells dimensions from 8–25 tree), (vii) Ankistrodesmus, (viii) Monoraphidium. For long to 1–2.5 large. Monoraphidium indicum Hindák Tetranephris, Quadrigula, Rhombocystis, Nephrochla- (CCMA–UFSCar 549) cells were c–shaped to twisted mys, one clade for each genera was obtained on the (from 120–260 long to 3–5 µm large), with two auto- 18S rDNA tree, with no rbcL sequences for them. The spores per sporangium and the mother cell wall was major clades resolved in our trees correspond well to constantly found in culture. the main genera traditionally included in Selenastra- In our study, except for Chlorolobion braunii ceae, that are, Ankistrodesmus and the distinguishable (Nägeli) Komárek, all Selenastracean taxa studied had species of genus Selenastrum gracile and Selenastrum naked pyrenoids (without starch envelopes), both ob- bibraianum (Reinsch) Korshikov. Due to the different served by LM or TEM. number of strains inside 18S rDNA and rbcL trees, di- fferent results were obtained for some genera such as, TEM microscopy Monoraphidium, Raphidocelis and Kirchneriella. The micrographs of members of the genera Ankis- RbcL phylogenetic analyses (Fig. 6) showed trodesmus, Monoraphidium and Curvastrum showed Selenastrum gracile (syn. Ankistrodesmus gracilis) similarities among the studied strains, CCMA–UFS- strains closely related to Ankistrodesmus (Ankistrodes- 10 da Silva et al.: Two new genera, Messastrum gen. nov. and Curvastrum gen. nov. mus clade). 18S rDNA phylogeny (Fig. 7) reveal An- were placed on two different clades on both phyloge- kistrodesmus distributed in two clades: Ankistrodesmus netic analyses. nannoselene Skuja (KF673373) (Kirchneriella I) and Therefore some strains were only represented Ankistrodesmus fusiformis (type species, Ankistrodes- in one of the interfered phylogenies, which were, in mus I). Selenastrum gracile strains (Selenastrum gra- general, congruent (see Figs. 6–7). The rbcL proved to cilis clade) (syn. Ankistrodesmus gracilis) and Selenas- be a difficult gene to amplify in Selenastraceae. trum bibraianum (syn. Ankistrodesmus bibraianum) Strains with the Selenastrum–like morphology

Fig. 9. Transmission electron micrographs of Curvastrum pantanale (CCMA–UFSCar 350) in culture [key to labeling: (CW) cell wall, (C) chloroplast, (D) dictyosome, (ER) endoplasmic reticulum, (L) lipid drop, (M) mitochondrion, (N) nucleus, (P) pyrenoid, (PV) polyphosphate vacuole, (S) starch grain]: (a) longitudinal section, cell presenting lipid drops, polyphosphate vacuoles (arrowhead), chloroplast penetrated with starch grains, and a central pyrenoid; (b) detail of figure a, where an endoplasmic reticulum (arrowhead) can be observed; (c) longitudinal section; cell presenting polyphosphate vacuoles on both cell apexes, mitochondria, chloroplast filled with starch grains and a central pyrenoid; (d) cross section; chloroplast containing starch grains and a pyrenoid situated at the left, a central nucleus can be observed; (e) longitudinal section; mature cell containing a central nucleus, mitochondrion and pyrenoid (upper part). All the cell content is surrounded by a cell wall. Scale bar 1 µm. Fottea, Olomouc, 17(1): 1–19, 2017 11 DOI: 10.5507/fot.2016.010

(M. gracile, C. pantalale and S. bibraianum; details see ment (Fig. 8b) and presented parallel arrangement section morphology) form three separate clades in both of autospores (Fig. 8c). The likeness to Messastrum analyses with weak statistical support, except for the was distinguished by molecular data (18S rDNA and Curvastrum–clade within the rbcL phylogeny which rbcL sequences). ITS–2 Secondary structure analyses had high statistical support in all analyses (1.0 for BI; demonstrated that strains of the type species Curvas- 98 BS for ML; 100 BS for NJ and MP). trum pantanale (CCMA–UFSCar 350) when com- Morphologically similar to Selenastrum and pared to another strain belonging to the same species Messastrum, the fusiform to semilunate cells of Cur- (CCMA–UFSCar 608) differed from each other in 5 vastrum were organized in colonies with no more than base changes, demonstrating intraspecific variations, 4 irregularly arranged cells (see description). The ir- with 2 CBC on helix II and 1 on helix III (for ITS–2 regularly arranged cells in Curvastrum pantanale secondary structure see Supplementary Fig. A–D). The (CCMA–UFSCar 350) formed its colony (Fig. 3, 1.8a), phylogenetic inference of rbcL (Fig. 6) allocated Cur- where cells were grouped in a loose fascicle arrange- vastrum near Ankistrodesmus clade. ITS–2 Secondary

Fig. 10. Transmission electron micrographs of two Selenastraceae in culture: (a–c) Ankistrodesmus arcuatus (CCMA – UFSCar 24), (a) longi- tudinal section, cell with a nucleus, dictyosome and a chloroplast, (b) cross section, a cup–shaped chloroplast penetrated by a pyrenoid (star). On the opposite direction of the pyrenoid, the nucleus is situated. An arrowhead indicates a polyphosphate vacuole. (c) detail of figure a, where a dictyosome can be observed; (d–e) Monoraphidium contortum (CCMA–UFSCar 349), (d) longitudinal section; cells on different life cycle phase. The upper 3 cells are young indidivuals presenting starch grains (arrowhead), polyphosphate vacuoles (arrowhead), chloroplast and nucleus. The lower cell is a mature individual containing many starch grains on the chloroplast, some polyphosphate vacuoles (arrowhead), nucleus and big lipid drops. (e) cross section, dense chloroplast with starch grains. Two big polyphosphate vacuoles (arrowhead) and a nucleus can be observed. All the cell content is surrounded by a cell wall. Scale bar 1 µm. 12 da Silva et al.: Two new genera, Messastrum gen. nov. and Curvastrum gen. nov. structure analyses showed that strains of the type spe- cies within the Selenastraceae using molecular data cies S. bibraianum differ from M. gracile type species enabled pointing enhanced morphological features to in 34 base changes and 3 base insertions, where helix distinguish the members of this complex group for tra- I has 1 base insertion and 6 CBC, helix II has 4 CBC, ditional taxonomy, as further discussed. helix III has 8 CBC and helix IV has 2 base insertions and 4 CBC (for ITS–2 secondary structure see Supple- Morphological criteria with high taxonomic value mentary Fig. A–D). in traditional systematics of Selenastraceae Species with Ankistrodesmus designation formed two clades based on rbcL (CCMA–UFSCar Pyrenoids 423; CB 2012/3; CB2012/6; CCMA–UFSCar 593; The presence or absence of pyrenoids is an important CCMA–UFSCar 611; CB2012/29; CCMA–UFSCar problem for the taxonomy of the studied genera, sin- 278; EF113406) and one clade on 18S rDNA phylogeny ce using conventional light microscopy (LM), naked (CCMA–UFSCar 277; X97352; CB2009/9; CCMA– pyrenoids are almost impossible to detect, whereas UFSCar 278; X56100). For the strain CCMA–UFSCar starch covered pyrenoids are easily visualized. For 24, based on differences in rbcL gene sequences and on that reason, most Selenastraceae were considered to the colony formation observed, we decided to consider be pyrenoid–less (Komárek & Fott 1983; Korshikov this species as belonging to genus Ankistrodesmus. 1953) until the study of Eloranta (1979) that reported Species of the genus Monoraphidium were dis- the presence of pyrenoids in Monoraphidium griffi- tributed in different clades. Although using different thii (Berkeley) Komárková–Legnerová, using TEM strains, Raphidocelis was located near Monoraphidium images. Later studies detected pyrenoids in all taxa clade I for both rbcL and 18S rDNA analyses (Figs. 6 studied, presenting a matrix naked or covered by starch and 7). One clade constituted by M. contortum (strains grains (Hegewald et al. 1994; Krienitz et al. 2011; Kri- CCMA–UFSCar 349 and CB 2009/10) was closely enitz et al. 1985; Krienitz et al. 2001). Thus, TEM is related to Raphidocelis microscopica. A well sup- necessary for the observation of details on the pyrenoid ported clade for Monoraphidium contortum (2 strains), structure (Krienitz et al. 2001). Furthermore, presen- Monoraphidium indicum and Monoraphidium pseudo- ce or absence of starch–covered pyrenoids may be not braunii was presented on rbcL phylogeny. Also, M. ko- a valuable character to the Selenastraceae taxonomy markovae, a needle–shaped representative, constituted when not in controlled conditions (Krienitz & Klein an independent lineage on both phylogenies. 1988; Miyashi et al. 1986). Kirchneriella species, including the type spe- In our study, except for Chlorolobion braunii cies Kirchneriella lunaris (CCMA–UFSCar 443), were (Nägeli) Komárek, all Selenastracean taxa studied distributed on some branches on both phylogenetic have ellipsoidal or spherical naked pyrenoids (without analyses, always close to Monoraphidium species. starch envelopes), both investigated by LM or TEM, Chlorolobion braunii (Chlorolobion clade) but they can also be spherical to irregular in Selena- formed a well–supported clade based on rbcL and straceae (see also Krienitz et al. 2011). C. braunii pre- made up the clade that includes sequences designated sented starched and rounded pyrenoids, as reported by as Podohedriella falcata (X91263) and Monoraphid- Komárek & Fott (1983). ium neglectum (AJ300526) based on 18SrRNA gene Regarding to the pyrenoid differences between inference with strong bootstrap support. genera, the thylakoid membranes permeated the py- The species Quadrigula closterioides (Boh- renoid matrix in Curvastrum pantanale and Ankis- lin) Printz (Y17924), Tetranephris brasiliensis Leite trodesmus arcuatus (Fig. 9–10), but this character & Bicudo (HM565929; HM565927), Rhombocystis demonstrated to be highly variable in the genus An- complanata Komárek (HM483518) and Nephrochla- kistrodesmus and Monoraphidium. Similar variation in est orshikov mys subsolitaria (W ) K (HM560960) were pyrenoid structures were observed by Krienitz et al. distributed at one branch each, on the 18S rDNA tree (2001). (Fig.7). Colony formation Traditionally used to delimit genera, colony forma- Discussion tion is a questionable criterion. Despite the presence of colonies, many solitary cells and intermediates in This study has presented a large dataset of Selenastra- the formation of colonies can be found, both in fast ceae species, including many strains of tropical envi- growing cultures (Krienitz et al. 2001) and in environ- ronments, enlightening the phylogeny of this family. mental samples. Many Ankistrodesmus (as an example, The polyphyly of some genera as Monoraphidium, see Komarek & Fott, 1983, page 689, plate 193–1), Kirchneriella and Selenastrum have been confirmed, members of Kirchneriella and Chlorolobion detaches as already suggested by Krienitz et al.(2011, 2001) and from the colonies when the reproduction begins, due respectively two new genera could be described. to the lack of mucilage and the protoplasm differen- The taxonomical placement of genera and spe- tiation process. Similarly, colonies of Ankistrodesmus Fottea, Olomouc, 17(1): 1–19, 2017 13 DOI: 10.5507/fot.2016.010 frequently disintegrate into solitary cells and can har- and size were valuable for the taxonomy of the selena- dly be differentiated from Monoraphidium species. An- stracean strains. kistrodesmus arcuatus was described as solitary cells, and for this reason, later transferred to genus Monora- Remarks on genera phidium (Syn. Monoraphidium arcuatum). Observati- Selenastrum, Messastrum and Curvastrum ons on strain CCMA–UFSCar 24 on culture conditions The phylogenetic inference of rbcL and 18S rDNA (see demonstrated that this species is able to form colonies Figs 6, 7) showed that Selenastrum bibraianum (syn. and should be realocated to genus Ankistrodesmus, Ankistrodesmus bibraianus), Selenastrum gracile (syn. data corroborated by rbcL phylogeny. Ankistrodesmus gracilis) and Curvastrum pantanale represent unrelated lineages. Previous works had pre- Arrangement of autospores sented similar data for Selenastrum and Messastrum A parallel arrangement of autospores was observed (Fawley et al. 2006; Krienitz et al. 2001; Krienitz et for the genera Ankistrodesmus, Chlorolobion, Me- al. 2011), but no taxonomical change was made based ssastrum, Selenastrum and Curvastrum. Monoraphidi- on molecular data. The differences between the ITS–2 um and Kirchneriella presented both parallel and serial secondary structure, 18S rDNA and rbcL of the type arrangement of autospores. For Raphidocelis species species S. bibraianum, M. gracile and C. pantanale studied, Raphidocelis microscopica, the literature does (for ITS–2 secondary structure see supplemental ma- not describe this feature, but it was observed serial ar- terial) were enough to suggest the new genera. Sele- rangement of autospore. nastrum is distinct from Messastrum as indicated by While studying the ontogenesis of genera An- phylogenetic interference and by eight CBCs in the kistrodesmus and Monoraphidium, Komárková–Leg- conserved region of Helix III of the ITS2, which has nerová (1969) observed that the position of autospores been considered as reliable criterion for differentiation was found to be an invariable and stable qualitative at species (Coleman 2009) and higher taxonomic le- characteristic both in nature and in culture. vels (Coleman 2007; 2010). Curvastrum pantanale sp. nov. is a new genus, Cell shape and size proposed by the 18S rDNA and rbcL inference and Due to the high variability of these organisms on envi- only one diacritical morphological character (number ronmental samples and in culture (Fawley et al. 2006; of cells in colony). Komárková–Legnerová 1969), criteria as cell shape and size are problematic. For some species, as Selena- Ankistrodesmus strum bibraianum for example, cell shape vary during All Ankistrodesmus–like strains studied were fusiform, life cycle, detaching from the colony, and changing isopolar, arcuate or needle–shaped, with parallel ar- from semilunate to fusiform, with an increase in pro- rangement of autospores. Both phylogenies (Figs 6, 7) toplasm content. Cell size is also a doubtful characte- closely related all Ankistrodesmus strains. It was po- ristic. The cell dimension described for A. fusiformis, ssible to recognize the genus using both phylogenies for example, is big enough to accommodate A. fasci- (Figs 6, 7), but not species within it, since the strains culatus inside the same description (Komárek & Fott share very similar morphological traits and present a 1983). low number of variable positions in the sequences of The diacritic features of Selenastraceae have rbcL and 18S rDNA. been extensively discussed. According to the numeri- The strain Ankistrodesmus nannoselene (SAG cal investigation of Marvan et al. (1984) several gene- 202.6), a halfmoon–shaped or croissant–shaped soli- ra inside Selenastraceae differ from each other by only tary representative, seem to be related to Kirchneriella one of the above mentioned characters. As example, species and is not clearly positioned in Selenastraceae Selenastrum differs from Ankistrodesmus by the curva- (Bock et al. 2011a; Krienitz & Bock 2012). ture of the cells (Komárek & Comas González 1982), Molecular data revealed that Monoraphidium the presence of cell wall incrustations in Raphidocelis arcuatus (syn. Ankistrodesmus arcuatus), here repre- differs it from Kirchneriella (Hindák 1977), and Mo- sented by strain CCMA–UFSCar 24, should be reco- noraphidium differs from Chlorolobion by the absence gnized as belonging to genus Ankistrodesmus (Fig.6), of starched pyrenoid (Heynig & Krienitz 1982; Komá- since it shows a typical colony formation when atta- rek 1979). Indeed, these examples could be observed ched to a mucilage aggregate (Fig 5 – arrowhead). on rbcL inference (Fig.6). Morphological features associated with 18S Monoraphidium rDNA in Selenastraceae showed that a morphotype can On our rbcL inference, Monoraphidium contortum represent different phylotypes, suggesting that the di- showed the same morphotype for different phylotypes, versity of the family has been considerably underesti- feature also found by Fawley et al.(2006) for Monora- mated (Fawley et al. 2006). phidium. Overall a combination of colony formation, The morphology diversity inside Monoraphidi- presence/absence of starched pyrenoid and cell shape um has a huge range of variations, including fusiform 14 da Silva et al.: Two new genera, Messastrum gen. nov. and Curvastrum gen. nov. to straight cells, spiral to thin crescent–shaped orga- well–supported clade, with no sequences for P. falcata nisms. The absence of typus generis Monoraphidium and M. neglectum. Thus, since there is few molecular griffthii on this study did not allow the identification of data and the morphology is too similar, not enforcing real Monoraphidium clade. Further studies including a nomenclatural change, we maintained our strains as more representatives of Monoraphidium are necessary belonging to genus Chlorolobion. The numbers of va- to elucidate inter and intraspecific relations and desig- riable sequence positions on 18S rDNA among these nate new genera. The diacritic characters settled for the six taxa were 0–2. other genera in Selenastraceae could not distinguish all The main morphological differences are that Monoraphidium species. Chlorolobion braunii own starch sheathed pyrenoids Two strains of Monoraphidium convolutum, (Komárek & Fott 1983), is fusiform, with serial ar- one (Korshikov) Nygaard, rangement of autospores; Podohedriella falcata is ne- Komárek, Kristiansen & Skulberg and Monoraphidi- edle–shaped, representing one of the few periphytic um dybowskii (Woloszynska) Hindák & Komárkova– Selenastraceae (Düringer 1958; Hindák 1988), and Legnerová were closely related and these relationships M. neglectum have naked pyrenoids serially arranged were supported statistically. Recent study (Krienitz et (Krienitz et al. 2001). al. 2011) placed M. convolutum and Monoraphidium Thus, based on morphology and rbcL analysis, dybowskii as insertae sedis in Selenastraceae, emphasi- we maintained our strains as belonging to genus Chlo- zing that future studies should be done. rolobion. As well as for the genus Monoraphidium, other genera as Quadrigula and Nephroclamys need General view further studies based on larger taxon sampling. Selenastraceae, at this moment, is composed of eleven genera. Except for Ourococcus Grobéty representati- Raphidocelis and Kirchneriella ves of all the other ten genera were represented in our On our rbcL inference, Raphidocelis microscopica was phylogenetic study as well as in other previous studies closely related to M. contortum (strains CCMA–UF- (Fučíková et al. 2014; Krienitz & Bock 2012). SCar 349 and CB 2009/10). This species presents co- A joined analysis of rbcL and 18S rRNA gene nical, isopolar and capricornutum shape differing from data sets is used in some studies of algal phylogeny M. contortum by cell shape, autospore arrangement, (Rindi et al. 2007), since 18S rRNA gene is too con- and colony formation. served to elucidate the phylogenetic trends at genus Further investigations for the genus Kirchnerie- and species levels. Accordingly, due to the lower varia- lla is needed due to the lack of molecular data. Only tion of the 18S rRNA sequences observed in this study, two sequences for the same strain (K. aperta – SAG a better resolution of this group was achieved by using 2004) were available on NCBI. As observed on both a more variable marker, the rbcL. This gene had higher phylogenies, Kirchneriella species present diverse phy- sequence divergence in Selenastraceae, and, therefore, lotypes inside one morphotype, always closely related was more useful than the 18S rDNA gene for phylo- to Monoraphidium–like members, suggesting that the genetic inference at the genus and species levels. In different morphotypes applied for both genera hides most algal groups this is commonly the case (Gurgel similar genotypes. Similar findings were obtained for & Fredericq 2004; Hayden & Waaland 2002; Hepper- Dictyosphaerium, which morphotypes formed distinct le et al. 1998; Hohamet al. 2002; Müller et al. 2002). lineages inside Chlorella and Parachlorella, presenting Using both genes, the substantial number of variable independent evolution and confirming the polyphyletic sequence positions among the studied taxa (0–76 for origin of the Dictyosphaerium morphotype within the 18S rRNA and 0–86 for rbcL) revealed the relationship Chlorellaceae (Luo et al. 2010). patterns within Selenastraceae. An usual problem in the systematics of Selenas- Chorolobion traceae was the absence or a low bootstrap support for On the 18S rDNA tree, Chlorolobion braunii cluste- the basis of the trees and a good support only of smaller red in one well supported branch, with Podohedrie- groups of species or genera (Fawley et al. 2006; Krien- lla falcata and . We could itz et al. 2001). However, our clusters are in agreement observe that the strains SAG 202–2, SAG 48.87 with previous studies in Selenastraceae (Fawley et al. analyzed by Krienitz et al. (2001), and our strains 2006; Krienitz et al. 2011; Krienitz et al. 2001), sup- CCMA–UFSCar 137, 462, 477 and 476 refer to three porting the probability of the phylogenetic information different morphological species. The small difference on the branches, despite some low bootstrap values. between these three taxa (up to 20 variable sequence Since the species concept proposed by Mayr (1942) positions) on 18S rDNA was not enough to separate cannot be applied to coccoid green algae, limnologists the different morphotypes. Based on 18S rDNA, the adopted the morphological species concept using mor- distinction among Podohedriella, Monoraphidium, phology based diacritical characteristics as a way to and Chlorolobion remains unclear. However, for rbcL deal with this limitation (Krienitz & Bock 2012). inference, the four Chlorolobion braunii formed a In addition to the phenotype, discussions about Fottea, Olomouc, 17(1): 1–19, 2017 15 DOI: 10.5507/fot.2016.010 species concepts should consider intraspecific genetic ticular taxon could actually represent numerous taxa variation (Hilt 2006). The description of well–estab- (Komárková–Legnerová 1969; Nygaard & Komarek lished species, with known phenotypic population, 1986). should be completed by molecular, genetic, physi- Despite the possible phenotypical plasticity, the ological, and biochemical features of all those cul- molecular analyses associated with morphological re- tured strains that follow to the original type description view in different growth phases of the phytoplankton (Wood & Leatham 1992), leading to the resolution of strains, showed that some morphological criteria are taxonomic position of morphological variants from the important for the systematics of Selenastraceae on ge- type. nus and species level. Cell shape and size, colony for- Taxonomical value of diacritic features was dis- mation, and arrangement of autospores in the mother cussed based on morphology or ultrastructure (Krien- cell wall demonstrated to be useful for genera sepa- itz et al. 2001) suggesting that the concept of “small ration, but should be applied carefully inside species. genera” (Komárek & Fott 1983) including only a few The observation of a substantial number of colonies species for the Selenastraceae is not appropriated, since and solitary organisms (10–50 individuals) and obser- the existence of many transitional forms, and many vation of the whole life cycle, with special emphasis to diacritic features are considered not reliable (Fawley autospores liberation and the initial colony formation, et al. 2006). Since it was detected real phylogenetic should be done in order to identify a taxon. lineages in this family, conceiving ‘small’ genera was Unfortunately, some species description were considered uncertain for Selenastraceae (Krienitz et al. published with missing information on the original 2001). However, some genera presented a set of dia- publication, regarding morphological information (in- critic characteristics. For its wide variability, the estab- cluding plates) not covering all the necessary informa- lishment of mucilage as diacritic feature is of limited tion to identify many taxa (for morphological referenc- taxonomic value (Krienitz et al. 2012). Our findings es see material and methods). Although 18S rDNA and suggest that the small genera concept could be applied rbcL were important, morphological characteristics to Monoraphidium, Kirchneriella and also for Selenas- were observed and considered individually (morphol- trum and Messastrum. ogy or genetic data) for taxonomical decisions. Although establishing smaller genera, differen- However, Monoraphidium and Kirchneriella tiated from other genera by only a few diacritic charac- seem to be polyphyletic genera and therefore these teristics and containing only a few numbers of species, traditional traits could not be employed, otherwise the is a trend (Luo et al. 2010). On the last decades some identification would not be reliable. studies have divided previous morphological well es- Evidently, there might be a huge number of li- tablished genus in many smaller ones, as proposed for neages within the Selenastraceae that are not covered Scenedesmus (An et al. 1999; Krienitz et al. 2003) and by our study. Unfortunately, no cultures are available Pediastrum (Buchheim et al. 2005). Scenedesmus Mey- for hundreds of taxa designated in this cosmopolitan en has been differentiated in seven genera: Scenedes- family and some strains are difficult to obtain all the mus, Acutodesmus (Hegewald) Tsarenko, Desmodes- target genes. mus Chodat, Hylodesmus Eliáš, Němcová, Škaloud, Thus, more efforts and new studies with iso- Neustupa, Kaufnerová & Šejnohová, Neodesmus lated and cultivable strains are necessary. The many Hindák, Verrucodesmus Hegewald and Chodatodes- sequences from taxonomic and phylogenetic studies mus Hegewald, Bock & Krientz (An et al. 1999; Eliáš submitted to databases, are fundamental to be used as et al. 2010; Krienitz et al. 2003; Hegewald et al. 2013). references for future practical applications and scien- Studies on Chlorellaceae (Luo et al. 2010) differenti- tific studies J( i et al. 2013), including metabarcoding ated six genera within the Chlorella clade of Chlorel- approaches. The increasingly community studies using laceae, mostly by molecular criteria (18S rDNA and high–throughput sequencing still lacks references for ITS–1 and –2) and revealed that several morphological phytoplankton species (Eiler et al. 2012; Pawlowski criteria (mucilage and connecting strands, colonial ver- et al. 2012). sus solitary life form and bristle formation) are pheno- The coccoid green algae are important in eco- typic characteristics, representing adaptive responses system studies but little information is available about to environmental factors such as grazing pressure, en- its geographic distributions, even with the improve- dosymbiosis or edaphic life strategies. ment of the taxonomy (Norton et al. 1996; Padisák If in one hand some species may present phe- et al. 2015). It is estimated that exists around 14900 notypic plasticity, on the other hand, analogous mor- freshwater phytoplankton species (Bourrelly 1990). photypes could hide high genotypic diversity. Cres- Species richness in freshwater lakes has been clai- cent–shaped Selenastraceae isolates of belonging to med to be significantly higher in temperate lakes than the same morphospecies were found by Fawley et al. in tropical ones (Lewis Jr 1978), but this result could (2006), with differences regarding 18S rDNA (Krien- be also a consequence of the poorly exploration and itz et al. 2011) for the same morphotypes studied. High understanding about green microalgae diversity and levels of variability among isolates concerned to a par- phylogeny in the tropics. 16 da Silva et al.: Two new genera, Messastrum gen. nov. and Curvastrum gen. nov.

Acknowledgements merkungen über einige grüne Algen. – Plant Syste- The authors would like to thank Fundação de Amparo à Pesqui- matics and Evolution 105: 1–43. sa do Estado de São Paulo (FAPESP, Processes: 2011/50054–4, Eiler, A.; Drakare, S.; Bertilsson, S.; Pernthaler, J.; Peu- 2012/19520–1, 2013/17457–3 and 2013/18083–0) for financial and ra, S.; Rofner, C.; Simek, K.; Yang, Y. ;Znachor, scientific support and scholarships granted and the staff at the IM- P. & Lindström E. S. (2013): Unveiling distribution CES imaging facility for their help and assistance with imaging. patterns of freshwater phytoplankton by a next gen- eration sequencing based approach. – PLoS ONE 8: e53516. Eliáš, M.; Nĕmcová, Y.; Škaloud, P.; Neustupa, J.; Kauf- References nerová, V. & Šejnohová., L. (2009): Hylodesmus singaporensis gen. et sp. nov., a novel autosporic An, S.; Friedl, T. & Hegewald, E. (1999): Phylogenetic Re- subaerial green alga (Scenedesmaceae, ) lationships of Scenedesmus and Scenedesmus–like from Singapore. –International Journal of Systematic Coccoid Green Algae as Inferred from ITS–2 rDNA and Evolutionary Microbiology 60: 1224–1235. Sequence Comparisons. – Plant Biology 1: 418–428. Eloranta, V. (1979): The compound internal pyrenoid in Bock, C.; Krienitz, L. & Pröschold, T. (2011a): Taxonomic cultured cells of the green alga Monoraphidium reassessment of the genus Chlorella (Trebouxiophy- griffithii (Berkel.) Komar.–Legner. – Protoplasma ceae) using molecular signatures (barcodes), includ- 99: 229–235. ing description of seven new species. – Fottea 11: Fawley, M.W.; Dean, M.L.; Dimmer, S.K. & Fawley, K.P. 293–312. (2006): Evaluating the morphospecies concept in Bock, C.; Pröschold, T. & Krienitz, L. (2011b): Updating the Selenastraceae (Chlorophyceae, Chlorophyta).– the genus Dictyosphaerium and description of Mu- Journal of Phycology 42: 142–154. cidosphaerium gen. nov. (Trebouxiophyceae) based Friedl, T. (1995): Inferring taxonomic positions and test- on morphological and molecular data. – Journal of ing genus level assignments in coccoid green lichen Phycology 47: 638–652. algae: a phylogenetic analysis of 18s ribosomal rna Booton, G.C.; Floyd, G.L. & Fuerst, P.A. (1998): Origins sequences from Dictyochloropsis reticulata and and affinities of the filamentous green algal orders from members of the genus Myrmecia (Chlorophyta, Chaetophorales and Oedogoniales based on 18S Trebouxiophyceae Cl. Nov.). – Journal of Phycology rRNA gene sequences. – Journal of Phycology 34: 31: 632–639. 312–318. Fučíková, K; Lewis, P.O.; González–Halphen, D. & Lew- Bourrelly, P. (1990): Les Algues d’eau douce. Initation a la is, L. A. (2014): Gene arrangement convergence, Systématique, I: Les Algues Vertes. – 572pp. N Bou- diverse intron content, and genetic code modifica- bée & Cie, Paris. tions in mitochondrial genomes of Sphaeropleales Buchheim, M.; Buchheim, J.; Carlson, T.; Braband, A.; He- (Chlorophyta). – Genome biology and evolution 6: pperle, D.; Krienitz, L.; Wolf, M. & Hegewald, E. 2170–2180. (2005): Phylogeny of the Hydrodictyaceae (Chloro- Fučíková, K.; Rada, J.C.; Lukešová, A. & Lewis, L.A. phyceae): inferences from rDNA data. – Journal of (2011): Cryptic diversity within the genus Pseudo- Phycology 41: 1039–1054. muriella Hanagata (Chlorophyta, Chlorophyceae, Buchheim, M.A.; Michalopulos, E.A. & Buchheim, J.A. Sphaeropleales) assessed using four barcode mark- (2001): Phylogeny of the Chlorophyceae with special ers. – Nova Hedwigia 93: 29–46. reference to the Sphaeropleales: a study of 18S and Gruber, A. R.; Lorenz, R.; Bernhart, S.H.; Neuböck, R.; & 26S rDNA data. – Journal of Phycology 37: 819–835. Hofacker, I.L. (2008): The vienna RNA websuite. – Byun, Y. & Han, K. (2009): PseudoViewer3: generating Nucleic acids research 36: W70–W74. planar drawings of large–scale RNA structures with Guillard, R.R. & Lorenzen, C.J. (1972): Yellow–green al- pseudoknots. – Bioinformatics 25: 1435–1437. gae with chlorophyllide C1, 2. – Journal of Phycol- Coleman, A.W. (2003): ITS2 is a double–edged tool for eu- ogy 8: 10–14. karyote evolutionary comparisons. – Trends in Ge- Gurgel, C.F.D. & Fredericq, S. (2004): Systematics of the netics 19: 370–375. Gracilariaceae (Gracilariales, Rhodophyta): a critical Coleman, A.W. (2007): Pan– ITS2 homologies re- assessment based on rbcL sequence analyses. – Jour- vealed by RNA secondary structure. – Nucleic Acids nal of Phycology 40: 138–159. Research 35: 3322–3329. Hayden, H.S. & Waaland, J.R. (2002): Phylogenetic sys- Coleman, A.W. (2009): Is there a molecular key to the level tematics of the Ulvaceae (Ulvales, Ulvophyceae) us- of “biological species” in ? A DNA guide. ing chloroplast and nuclear dna sequences. – Journal – Molecular Phylogenetics and Evolution 50: 197– of Phycology 38: 1200–1212. 203. Hegewald, E.; Bock, C. & Krienitz, L. (2013): A phyloge- Coleman, A.W. & Vacquier ,V.D. (2002): Exploring the phy- netic study on Scenedesmaceae with the description logenetic utility of ITS sequences for animals: a test of a new species of Pectinodesmus and the new gen- case for abalone (Haliotis). – Journal of molecular era Verrucodesmus and Chodatodesmus (Chlorophy- evolution 54: 246–257. ta, Chlorophyceae). – Fottea 14: 149–164. Comas, A. (1996): Las dulciacuícolas de Hegewald, E. & Hanagata, N. (2000): Phylogenetic stud- Cuba. Bibliotheca Phycologica 99. – 192pp., J. Cra- ies on Scenedesmaceae (Chlorophyta). – Archiv für mer, Berlin. Hydrobiologie Supplementband, Algological Studies Düringer, I. (1958): Über die Verteilung epiphytischer Algen 136: 29–49. auf den Blättern wasserbewohnender Angiospermen Hegewald, E.; Krienitz, L. & Schnepf, E. (1994): Studies sowie systematisch–entwicklungsgeschichtliche Be- on Scenedesmus costato–granulatus Skuja. – Nova Fottea, Olomouc, 17(1): 1–19, 2017 17 DOI: 10.5507/fot.2016.010

Hedwigia 59: 97–128. G. (ed.): Das Phytoplankton des Süßwassers 7. Teil, Hepperle, D. (2004): Align Ver. 07/04©. Multisequence 1. Hälfte. – 1044 pp., Schweizerbart’sche Verlags- alignment–editor and preparation/manipulation of buchhandlung (Nägele u. Obermiller), Stuttgart. phylogenetic datasets. Win32–Version. Distributed Komárková–Legnerová, J. (1969): The systematics and on- by the author via: http://www. sequentix. de. togenesis of the genera Ankistrodesmus Corda and Hepperle, D.; Nozaki, H.; Hohenberger, S.; Huss, V.; Mori- Monoraphidium gen. nov. – In: Fott, B. (ed.): Stu- ta, E. & Krienitz, L. (1998): Phylogenetic position dies in Phycology. – pp. 75–122, Academia, Praha. of the Phacotaceae within the Chlamydophyceae as Korshikov, O.A. (1953): Viznachnik prisnovodnihk vodo- revealed by analysis of 18S rDNA and rbcL sequenc- rostey Ukrainsykoi RSR [Vyp] V. Pidklas Protokoko- es. – Journal of Molecular Evolution 47: 420–430. vi (Protococcineae). Bakuol‘ni (Vacuolales) ta Pro- Heynig, H. & Krienitz, L. (1982):Monoraphidium neglectum tokokovi (Protococcales). – 439 pp., Akad. NAUK n. sp. sowie einige Bemerkungen zu den Gattungen URSR, Kyjev. Monoraphidium, Chlorolobion und Keratococcus Korshikov, O. (1987): The freshwater algae of the Ukrainian (Chlorococcales). – Archiv für Protistenkunde 125: SSR– V. Sub–class Protococcineae: Vacuolales and 335–344. Protococcales (translated by J. WG. Lund & W. Ty- Hilt, S. (2006): Allelopathic inhibition of epiphytes by sub- lka). – 412 pp., Koeltz Scientific Books, Köenigstein. merged macrophytes. – Aquatic Botany 85: 252–256. Krienitz, L. & Bock, C. (2012): Present state of the systemat- doi: DOI 10.1016/j.aquabot.2006.05.004. ics of planktonic coccoid green algae of inland wa- Hindák, F. (1977): Studies on the chlorococcal algae (Chlo- ters. – Hydrobiologia 698: 295–326. rophyceae). Biol. Práce 23: 1–292. Krienitz, L.; Bock, C.; Kotut, K. & Pröschold, T. (2012): Hindák, F. (1980): Studies on the chlorococcal algae (Chlo- Genotypic diversity of Dictyosphaerium–morpho- rophyceae) – II.Biol. Práce 26: 1–296. species (Chlorellaceae, Trebouxiophyceae) in Afri- Hindák, F. (1984): Studies on the chlorococcal algae (Chlo- can inland waters, including the description of four rophyceae) – III.Biol. Práce 30: 1–263. new genera. – Fottea 12: 231–253. Hindák, F. (1988): Studies on the chlorococcal algae (Chlo- Krienitz, L.; Bock, C.; Nozaki, H. & Wolf, M. (2011): SSU rophyceae). – IV. Biol. Práce 34: 1–320. rRNA gene phylogeny of morphospecies affiliated to Hindák, F. (1990): Studies on the chlorococcal algae (Chlo- the bioassay alga “Selenastrum capricornutum” re- rophyceae). – V.Biol. Práce 36: 1–227. covered the polyphyletic origin of crescent–shaped Hoham, R.W.; Bonome, T.A.; Martin, C.W. & Leebens‐ Chlorophyta. – Journal of Phycology 47: 880–893. Mack, J.H. (2002): A combined 18s rDNA and rbcL Krienitz, L.; Hegewald, E.; Hepperle, D. & Wolf, M. (2003): phylogenetic analysis of Chloromonas and Chlam- The systematics of coccoid green algae: morphology ydomonas (Chlorophyceae, Volvocales) emphasizing versus 18S rRNA gene phylogeny. – Biológia 58: snow and other cold‐temperature habitats. – Journal 437–446. of phycology 38: 1051–1064. Krienitz, L. & Klein, G. (1988): Morphologie und Ultra- Huelsenbeck, J.P. & Ronquist, F. (2001): MRBAYES: struktur einiger Arten der Gattung Monoraphidium Bayesian inference of phylogenetic trees. – Bioinfor- (Chlorellales) III. Monoraphidium terrestre Bristol matics 17: 754–755. nov. comb. – Algological Studies/Archiv für Hydro- Huss, V.A.; Frank, C.; Hartmann, E.C.; Hirmer, M.; Klo- biologie, Supplement Volumes: 447–463. boucek, A.; Seidel, B. M.; Wenzeler, P. & Kessler, Krienitz, L.; Klein, G. & Böhm, H. (1985): Zur Morphologie E. (1999): Biochemical taxonomy and molecular und Ultrastruktur von Selenastrum gracileReinsch phylogeny of the genus Chlorella sensu lato (Chlo- 1867 (Chlorellales). – Archiv für Protistenkunde rophyta). – Journal of Phycology 35: 587–598. 130: 79–92. Ji, Y.; Ashton, L.; Pedley, S.M.; Edwards, D.P.; Tang, Y.; Krienitz, L.; Ustinova, I.; Friedl, T. & Huss, V.A. (2001): Nakamura, A.; Kitching, R.; Dolman, P.M.; Wood- Traditional generic concepts versus 18S rRNA gene cock, P. & Edwards, F.A. (2013): Reliable, verifiable phylogeny in the green algal family Selenastraceae and efficient monitoring of biodiversity via metabar- (Chlorophyceae, Chlorophyta). – Journal of Phycol- coding. – Ecology letters 16: 1245–1257. ogy 37: 852–865. Jobb, G. (2008): TREEFINDER version of October 2008. Lewis Jr, W.M. (1978): A compositional, phytogeographical Munich, Germany Distributed by the author at www. and elementary structural analysis of the phytoplank- Treefinder.de. ton in a tropical lake: Lake Lanao, Philippines. – The Katana, A.; Kwiatowski, J.; Spalik, K.; Zakryś, B.; Sza- Journal of Ecology: 213–226. lacha, E. & Szymańska, H. (2001): Phylogenetic Lewis, L.A. (1997): Diversity and phylogenetic placement of position of Koliella (Chlorophyta) as inferred from Bracteacoccus Tereg (Chlorophyceae, Chlorophyta) nuclear and chloroplast small subunit rDNA. – Jour- based on 18s ribosomal RNA gene sequence data. – nal of Phycology 37: 443–451. Journal of Phycology 33: 279–285. Komárek, J. (1979): Änderungen in der Taxonomie der Chlo- Luo, W.; Pröschold, T.; Bock, C. & Krienitz, L. (2010): rokokkalalgen. – Algological Studies/Archiv für Hy- Generic concept in Chlorella‐related coccoid green drobiologie, Supplement Volumes: 239–263. algae (Chlorophyta, Trebouxiophyceae). – Plant Bi- Komárek, J. & Comas González, A. (1982): Taxonomical ology 12: 545–553. definition of the genera and several species of Ankis- Manhart, J.R. (1994): Phylogenetic analysis of green plant trodesmus and Selenastrum (Chlorococcales). – Al- rbcL sequences. – Molecular phylogenetics and evo- gological Studies/Archiv für Hydrobiologie, Supple- lution 3: 114–127. ment Volumes: 259–277. Marin, B.; Klingberg, M. & Melkonian, M. (1998): Phy- Komárek, J. & Fott, B. (1983): Chlorophyceae (Grünalgen) logenetic relationships among the Cryptophyta: Ordnung: Chlorococcales. – In: Huber–Pestalozzi, analyses of nuclear–encoded SSU rRNA sequences 18 da Silva et al.: Two new genera, Messastrum gen. nov. and Curvastrum gen. nov.

support the monophyly of extant plastid–containing 1529–1540. lineages. – Protist 149: 265–276. Rindi, F.; McIvor, L.; Sherwood, A.R.; Friedl, T.; Guiry, Marvan, P.; Komárek, J. & Comas, A. (1984): Weighting and M.D. & Sheath, R.G. (2007): Molecular phylogeny scaling of features in numerical evaluation of coccal of the green algal order Prasiolales (Trebouxiophy- green algae (genera of the Selenastraceae). – Algo- ceae, Chlorophyta). – Journal of Phycology 43: logical Studies/Archiv für Hydrobiologie, Supple- 811–822. ment Volumes: 363–399. Rindi, F.; Mikhailyuk, T.I.; Sluiman, H.J.; Friedl, T. & Mayr, E. (1942): Systematics and the origin of species, from López–Bautista, J.M. (2011): Phylogenetic relation- the viewpoint of a zoologist. – 334 pp., Columbia ships in Interfilum and Klebsormidium (Klebsormid- University Press, Columbia. iophyceae, Streptophyta). – Molecular Phylogenetics McCourt, R.M.; Karol, K.G.; Bell, J.; Helm‐Bychows- and Evolution 58: 218–231. ki, K.M.; Grajewska, A.; Wojciechowski M.F. & Rogers, J.E.; Leblond, J.D.&Moncreiff, C.A. (2006): Phy- Hoshaw, R.W. (2000): Phylogeny of the conjugating logenetic relationship of Alexandrium monilatum green algae (Zygnemophyceae) based on rbcL se- (Dinophyceae) to other Alexandrium species based quences. – Journal of Phycology 36: 747–758. on 18S ribosomal RNA gene sequences. – Harmful Messyasz, B. (2003): Spatial distribution of chlorococcalean Algae 5: 275–280. genera in the phytoseston of the Wena and Nielba riv- Rosenthal, A.; Coutelle, O. & Craxton, M. (1993): Large– ers. – Oceanological and Hydrobiological Studies 2: scale production of DNA sequencing templates by 33–44. microtitre format PCR. – Nucleic Acids Research 21: Mischke, U. & Kusber,W. (2009): Die harmonisierte Taxa- 173–174. liste des Phytoplanktons für Seen und Flüsse in Sant’Anna, C.L. (1984): Chlorococcales (Chlorophyceae) Deutschland. Excel–Datei–Liste mit ausführlichen do Estado de São Paulo, Brasil. – In: Bibliotheca Anmerkungen.Stand 2009/05/20. Available in: http:// Phycologica 67. – 348 pp., J. Cramer, Berlin. www.igb–berlin.de/datenbanken.html. Saunders, G.W. & Kucera, H.. (2010): An evaluation of Miyashi, T.; Kamata M. & Mukai, T. (1986): Oxygenation rbcL, tufA, UPA, LSU and ITS as DNA barcode and [3+ 2]–cycloaddition of methylenecyclopro- markers for the marine green macroalgae. – Cryp- panes through electron donor–acceptor complexes togamie Algologie 31: 487–528. with tetracyanoethylene by photoexcitation and in Schultz, J.; Maisel, S.; Gerlach, D.; Müller, T. & Wolf, M. the dark. – Journal of the American Chemical Society (2005): A common core of secondary structure of the 108: 2755–2757. internal transcribed spacer 2 (ITS2) throughout the Müller, K.M.; Sherwood, A.R.; Pueschel, C. M.; Gutell, Eukaryota. – RNA 11: 361–364. R.R. & Sheath, R.G. (2002): A proposal for a new Stöver, B. C. & Müller, K.F. (2010): TreeGraph 2: combin- red algal order, the Thoreales. – Journal of Phycology ing and visualizing evidence from different phyloge- 38: 807–820. netic analyses. – BMC bioinformatics 11: 7. Norton, T.A.; Melkonian, M. & Andersen, R.A. (1996): Al- Swofford, D.L. (2002): Phylogenetic analysis using parsi- gal biodiversity. – Phycologia 35: 308–326. mony (* and other methods), version 4.0b 10. – Si- Nozaki, H.; Itoh, M.; Sano, R.; Uchida, H.; Watanabe, nauer Associates, Sunderland, Massachusetts. M.M. & Kuroiwa, T. (1995): Phylogenetic relation- Tas, B. & Gonulol, A. (2007). An ecologic and taxonomic ships within the colonial Volvocales (Chlorophyta) study on phytoplankton of a shallow lake, Turkey. – inferred from rbcL gene sequence data. – Journal of Journal of Environmental Biology 28: 439. Phycology 31: 970–979. Ustinova, I.; Krienitz, L. & Huss, V.A. (2001): Closteriopsis Nygaard, G.; Komárek, J. & Skulberg, O. (1986): Taxo- acicularis (G. M. Smith) Belcher & Swale is a fusi- nomic designations of the bioassay alga, NIVA–CHL form alga closely related to Chlorella kessleri Fott & I (Selenastrum capricornutum) and some related Nováková (Chlorophyta, Trebouxiophyceae). – Eu- strains. – Opera Botanica 90: 1–46. ropean Journal of Phycology 36: 341–351. Nylander, J. (2004): MrModeltest v2. Program distributed van Hannen, E.; FinkGodhe, P. & Lurling, M. (2002): A re- by the author. – Evolutionary Biology Centre, Up- vised secondary structure model for the internal tran- psala University. scribed spacer 2 of the green algae Scenedesmus and Padisák, J.; Vasas, G. & Borics, G. (2015): Phycogeography Desmodesmus and its implication for the phylogeny of freshwater phytoplankton: traditional knowledge of these algae. – European Journal of Phycology 37: and new molecular tools. – Hydrobiologia 764: 3–27. 203–208. Pankow, H. (1990): Ostsee–Algenflora. – 648 pp., Gustav Wolf, M.; Chen, S.; Song, J.; Ankenbrand, M. & Müller,T. Fischer, Jena. (2013): Compensatory base changes in ITS2 secon- Pawlowski, J.; Audic, S.; Adl, S.; Bass, D.; Belbahri, L.; dary structures correlate with the biological species Berney, C.; Bowser, S.S.; Cepicka, I.; Decelle J. & concept despite intragenomic variability in ITS2 se- Dunthorn, M. (2012): CBOL protist working group: quences–a proof of concept. – PloS one 8: e66726. barcoding eukaryotic richness beyond the animal, Wood, A.M. & Leatham, T. (1992): The species concept in plant, and fungal kingdoms. – PLoS Biology 10: phytoplankton ecology. – Journal of Phycology 28: e1001419. 723–729. Rambaut, A. & Drummond, A. (2007): Tracer (http://beast. Young, I. & Coleman, A.W. (2004): The advantages of the bio. ed. ac. uk/Tracer). Version 1.4. ITS2 region of the nuclear rDNA cistron for analysis Rindi, F.; Guiry, M.D. & López‐Bautista, J.M. (2008): Dis- of phylogenetic relationships of insects: a Drosophi- tribution, morphology, And phylogeny of Klebsor- la example. – Molecular phylogenetics and evolution midium (Klebsormidiales, Charophyceae) in urban 30: 236–242. environments in Europe. – Journal of Phycology 44: Fottea, Olomouc, 17(1): 1–19, 2017 19 DOI: 10.5507/fot.2016.010

Supplementary material

the following supplementary material is available for this article:

Figs A–D. (A) ITS–2 model for the type strain of Selenastrum bi- braianum (CCMA–UFSCar 125); (B) ITS–2 model for the type strain of Selenastrum bibraianum (CCMA–UFSCar 125); (C) ITS–2 model for the type strain of Messastrum gracile (CCMA– UFSCar 622); (D) ITS–2 model for the type strain of Curvastrum pantanale (CCMA–UFSCar 350).

Table S1. List of all strains, used to the construction of the tree in Fig. 3, with their accession numbers in GenBank and locali- ties of origin.

Table S2. List of studied strains with origin information and GenBank accession numbers for 18S rDNA and rbcL genes and ITS–2 secondary structure.

Table S3. 18S rDNA, rbcL and ITS primers used for amplificati- on and sequencing of Selenastraceae.

This material is available as part of the online article (http:// fottea.czechphycology.cz/contents)

© Czech Phycological Society (2017) Received November 30, 2015 Accepted March 23, 2016