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Home , Chlorellaceae, Chlorellales, Chlorella sorokiniana, Chlorella vulgaris, Closteriopsis, Dicloster

Fottea 11(2): 293–312, 2011 293

Taxonomic reassessment of the genus () using molecular signatures (barcodes), including description of seven new species

Christina Bo c k 1,2, Lothar Kr i e n i t z 1* and Thomas Pr ö s c h o l d 3,4

1Leibniz–Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhütte 2, D–16775 Stechlin–Neuglobsow, Germany; *e–mal: krie@igb–berlin.de, tel.: 033082 69926, fax: 033082 69917 2University of Essen, Faculty of Biology, D–45141 Essen, Germany 3Culture Collection of Algae and , Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Dunbeg by Oban, Argyll, PA37 1QA, United Kingdom 4University of Vienna, Dept. Limnology, Althanstr. 14, A–1090 Vienna, Austria

Abstract: After the description of by Beijerinck, 120 years ago, members of the genus Chlorella belong to the best studied green algae worldwide. However, numerous open questions remained regarding their systematics. Recent molecular studies showed the polyphyly of the genus within the Chlorophyceae and Trebouxiophyceae. Chlorella–species were traditionally characterized by spherical to oval cell shape, solitary life– form and the absence of mucilaginous envelopes. The challenge in the past was how to distinguish species due to their high phylogenetic diversity combined with a limited amount of morphological characters. Using a polyphasic approach of SSU– and ITS rDNA phylogeny, secondary structure of the ITS and light microscopic observations, we were able to detect six lineages with –like strains in close relationship to C. vulgaris, here described or combined newly as C. coloniales sp. nov., C. pituita sp. nov., C. pulchelloides sp. nov., C. singularis sp. nov., C. elongata comb. nov. and C. chlorelloides comb. nov. Furthermore, three new species without mucilage were described as C. lewinii sp. nov., C. rotunda sp. nov. and C. volutis sp. nov. Using the 5.8S rRNA and part of the ITS–2 as molecular signature (barcode), we were able to distinguish not only the five already known species of Chlorella, C. vulgaris, C. sorokiniana, C. heliozoae, C. lobophora and C. variabilis but the seven new species and two new combinations as well. CBCs and hemi–CBCs within the secondary structure of the ITS–2 confirmed the separation of the species. Our study led to a new understanding of the evolution of morphology within the genus Chlorella and to an emendation of the generic description.

Key words: barcode, Chlorella, Dictyosphaerium, ITS, mucilage, phylogeny

Introduction mainly focused on their nutritional requirements (Sh r i f t & Sp r o u l 1963; Sh i h i r a & Kr a u s s 1965), Chlorella Be i j e r i n c k is one of the most famous morphological and structural features (Fo t t & microalgal genera worldwide. Although members No v á k o v á 1969; An d r e y e v a 1975; No z a k i et al. of the genus suffer from a scarcity of morphological 1995), serological cross–reactions (Sa n d e r s et al. characters, more than 100 Chlorella species have 1971), ultrastructural and chemical composition been named since the description of the type species of the cell wall (At k i n s o n et al. 1972; Ka p a u n Chlorella vulgaris Be i j e r i n c k in 1890. These taxa & Re i s s e r 1995; Nĕ m c o v á & Ka l i n a 2000), have been described from freshwater, marine, and pyrenoid ultrastructures (Ik e d a & Ta k e d a 1995; edaphic habitats or as (Ko m á r e k Nĕ m c o v á & Ka l i n a 2000), biochemical and & Fo t t 1983; Hu s s et al. 1989; Ni s h i h a r a et physiological characters (Ke s s l e r 1976, 1982; al. 1998; Ho s h i n a et al. 2005; Su m m e r e r et al. 1984, Ke s s l e r & Hu s s 1992) and molecular 2008; Šk a l o u d 2009; Kh a y b u l l i n a et al. 2010; phylogenetic characteristics (Hu s s et al. 1989, Pr ö s c h o l d et al. 2011). Over time, numerous 1999; Hu s s & So g i n 1990; Kr i e n i t z et al. 2004; studies aimed at revising the systematics of this El i a š & Ne u s t u p a 2009; Da r i e n k o et al. 2010). genus have been carried out. These studies have All these studies have shown that the genus 294 Bo c k et al.: Barcoding in Chlorella

is a heterogeneous assemblage of species and individual taxa (Et t l & Gä r t n e r 1995; Ne u s t u p a that there is an urgent need for a revision of the et al. 2009). genus. On the basis of biochemical and molecular The barcode initiative attempts to resolve data, the genus presently consists of only of five the problem of species delimitation by defining a “true” Chlorella species: Chlorella vulgaris, C. short and highly variable DNA region as barcode lobophora An d r e y e v a , C. sorokiniana Sh i h i r a et for the species (Ha j i b a b a e i et al. 2007). For Kr a u s s , C. heliozoae Pr ö s c h o l d et Da r i e n k o and the green algae, discussions are still going on C. variabilis Sh i h i r a et Kr a u s s (Hu s s et al. 1999; which part of the DNA is suitable for barcoding. Kr i e n i t z et al. 2004; Pr ö s c h o l d et al. 2011). Studies on diatoms have shown that the cox1 The separation of the genera and species region is short, variable and a useful marker for of has traditionally been based phylogenetic analyses in combination with other on morphological and cytological characters genes (Ev a n s et al. 2007; Ev a n s & Ma n n 2009). (Pr ö s c h o l d & Le l i a e r t 2007). However, the However, Mo n i z & Ka c z m a r s k a (2009) suggested hypothesis that similar morphology leads to a a different barcode for diatoms based on a segment close phylogenetic relationship has often been starting with the 5.8S start codon and ending in the proven to be inaccurate and misleading. Recent conserved motif of the Helix III of the ITS–2. The phylogenetic studies have demonstrated that the ITS–2 is a comparably fast evolving sequence, typical Chlorella morphology is shared with which has been widely used for phylogenetic other lineages of the Trebouxiophyceae and analyses at the generic and species levels (Ál v a r e z Chlorophyceae (Hu s s et al. 1999; Ne u s t u p a et & We n d e l 2003; Mi a o et al. 2008). In addition to al. 2009; Da r i e n k o et al. 2010). For example, the primary sequence, the secondary structure of Da r i e n k o et al. (2010) have shown in their the ITS–2 based on complementary base changes study three ellipsoid Chlorella–like species (CBC) has often been taken into account when that form a monophyletic lineage within the distinguishing between closely related species Trebouxiophyceae. The species previously known (Mü l l e r et al. 2007). Studies have shown that as Chlorella saccharophila (Kr ü g e r ) Mi g u l a , C. one CBC in a conserved region of the Helix II or ellipsoidea Ge r n e c k , and C. angusto–ellipsoidea III of the ITS–2 is in most cases associated to an N. Ha n a g a t a et M. Ch i h a r a have on the basis of inability for sexual reproduction (Co l e m a n 2007, their phylogenetic characteristics been placed in 2009; Mü l l e r et al. 2007). the genus Chloroidium Nadson. In this paper we investigated the phylogeny Members of the genus Chlorella belong and morphology of 20 different strains from the traditionally to the , which have genus Chlorella and described seven new species been recently divided in two different clades, and three new combinations. In addition, we the –clade and the Chlorella– applied the Barcoding concept after Moniz & clade (Kr i e n i t z et al. 2004; Lu o et al. 2010). Ka c z m a r s k a (2009) to 49 sequences of the genus Investigations focusing on the Dictyosphaerium– Chlorella and compared the results with CBCs morphotype (colonial, with connecting strands in the secondary structure of the ITS–2 and the between the cells and gelatinous envelope) traditional species delineation. showed an affiliation of this morphotype to the Chlorellaceae (Kr i e n i t z et al. 2010). Recent studies showed that Dictyosphaerium–like strains Materials and methods cluster independently in the Parachlorella–clade and in the Chlorella–clade of the Chlorellaceae, Algal cultures and morphology. Strains were some taxa among members of the genus Chlorella obtained either from the Culture Collection of Algae (Bo c k et al. 2010; Kr i e n i t z et al. 2010; Lu o et al. and Protozoa (CCAP, UK), Culture Collection of Algae 2010). at the University of Göttingen (SAG, Germany), the The current challenge in the study of this Culture Collection of Algae at the University of Texas (UTEX, USA), Coimbra Collection of Algae (ACOI, genus is how to distinguish individual species in the Portugal) or isolates from field material and deposited light of the extremely high phylogenetic diversity at CCAP (Table 1). All strains were grown at 15 °C of the Chorella like species combined with the under a 14/10h light/dark regime in modified Bourrelly limited number of morphological characters and medium (Kr i e n i t z & Wi r t h 2006). The morphology small dimensions of vegetative cells that hampers was analysed according to Ko m á r e k & Fo t t (1983) suitable identification and discrimination of and Ko m á r e k & Pe r m a n (1978). The Fottea 11(2):Fottea 2011293–312, Table 1. List of strains used in this study.

Strain Accession Species Origin a) Reference number Number

Plankton in pond Oxidationteich Neuglobsow, Bo c k et al. SAG 65.94 Catena viridis GU592792 Brandenburg, KR 1991/4, Germany (2010)

Lu o et al. CCAP 200/1 hantzschii Nakuru Sewage Pond, KR 2002/18, Kenya FM205882 (2010) 295 River Elbe near Aken, Sachsen-Anhalt, KR 1996/4, Lu o et al. SAG 2015 Actinastrum hantzschii FM205841 Germany (2010) Plankton in lake Pragsdorfer See, Mecklenburg CCAP 211/116 Chlorella chlorelloides HQ111432 this study Vorpommern, CB 2008/110, Germany

Lu o et al. UTEX 938 Chlorella coloniales J. Stein (146), 1958, origin unknown FM205862 (2010)

Lu o et al. CCAP 222/18 Chlorella elongata Fountain, Berlin, Steglitz, Wolf 2000/1, Germany FM205858 (2010) of the Heliozoa Acanthocystis turfacea, Lu o et al. SAG 3.83 Chlorella heliozoae Newfoundland, bog pool in Terra Nova Natl. Park, FM205850 (2010) Canada

Soil from the edge of a permanent freshwater pond in a Lu o et al. CCAP 211/90 Chlorella lewinii FM205861 crater, Easter Island, Chile (2010)

Soil from forest, Briamsk District, Krasmyj Rog, Lu o et al. SAG 37.88 Chlorella lobophora FM205833 Russia (2010)

Lu o et al. ACOI 856 Chlorella pituita Serra da Estrela (Manteigas), Portugal FM205856 (2010)

Kr i e n i t z et ACOI 311 Chlorella pituita Mira, trout nursery, M.F. Santos 865, 1984, Portugal GQ176853 al. (2010)

Lu o et al. SAG 222-2a Chlorella pulchelloides Pond near Amies, E.A. George, 1949, France FM205857 (2010) Xochimilico, EN 2003/25, Mexico CCAP 211/117 Chlorella pulchelloides HQ111430 this study 296 Table 1 Cont.

Plankton in lake Feldberger Haussee, CB 2008/50, CCAP 211/118 Chlorella pulchelloides HQ111431 this study Germany River Okawango, KR 2007/5, Angola CCAP 260/11 Chlorella rotunda HQ111433 this study

CCAP 211/119 Chlorella singularis Nakuru Sewage Pond, CB 2008/73, Kenya HQ111435 this study

Lu o et al. CCALA 260 Thermal spring near Piestany, Slovakia FM205860 (2010)

Austin, Texas, Waller Creek at University Campus, Lu o et al. SAG 211/8k Chlorella sorokiniana FM205859 USA (2010)

USA, endosymbiont of , Lu o et al. SAG 211-6 Chlorella variabilis FM205849 authentic strain of Chlorella variabilis (2010)

Endosymbiont of the Paramecium bursaria, Ho s h i n a et CCAP 211/84 Chlorella variabilis AB206549 NC64A, USA al. (2005) CCAP 211/120 Chlorella volutis Nakuru National Park, Rhinopool, CB 2008/69, Kenya HQ111434 this study

Eutrophic pond near Delft, authentic strain, Lu o et al. SAG 211-11b Chlorella vulgaris FM205854 Netherlands (2010)

Pond Salzteich near Trebbichau, Sachsen-Anhalt, KR Lu o et al. CCAP 211/81 Chlorella vulgaris FM205854 1979/36, Germany (2010) B

Lu o et al. k c o SAG 11.86 acicularis Plankton of Grömitzer See, Germany FM205847 (2010) et al.:

Kr i e n i t z et CCAP 222/1A Dictyosphaerium ehrenbergianum Pond near Cambridge, E.G. Pringsheim, 1940, UK GQ176854

al. (2010) Barcoding in

Kr i e n i t z et UTEX 731 Dictyosphaerium pulchellum Nova Scotia, R.A. Lewin (T1/3), 1952, Canada GQ176861 al. (2010)

Lu o et al. SAG 41.98 acuatus Pond at Krasne, Ukraine FM205848 (2010) Chlorella

Lu o et al. SAG 18.91 anomala River Rhein, near Linz, Hegewald 1990/11, Germany FM205839 (2010)

Fottea 11(2):Fottea 2011293–312, Table 1 Cont.

Lu o et al. SAG 30.92 Didymogenes palatina Water tank, Jülich, Hegewald 1982/83, Germany FM205840 (2010) Windermere, Cumbria, England/G. Jaworski (FBA Bo c k et al. CCAP 222/2D Heynigia dictyosphaerioides GQ487221 L246); 1972 (2010)

Bo c k et al. CCAP 222/47 Heynigia riparia River Kunene, KR 2007/12, Angola GQ487225 (2010) 297

Bo c k et al. CCAP 222/29 Hindakia fallax Lake Victoria, Dunga Beach, KR 2006/317, Kenya GQ487223 (2010) Lake Kleiner Tietzensee, Brandenburg, Bo c k et al. CCAP 222/80 Hindakia tetrachotoma GQ487233 CB 2008/48, Germany (2010) Lake Itasca, Itasca State Park, Minnesota, Itas224S1w, AY543040 Fa w l e y et al. CCMP 2446 Meyerella planctonica USA AY543045 (2005)

AY195973 Fa w l e y et al. CCMP 2259 Meyerella planctonica Lake Itasca, Itasca State Park, Minnesota USA AY543044 (2005)

Lu o et al. SAG 42.98 belenophorum Plankton in Lietzensee, Berlin, Germany FM205879 (2010)

Lu o et al. CCAP 248/5 Micractinium pusillum Nakuru Sewage Pond, Kenya FM205836 (2010)

A tributary of lake Tollensesee, Nonnenbach brook, Lu o et al. SAG 2046 Parachlorella beijerinckii FM205845 West Pomerania, Germany (2010)

Lu o et al. SAG 211-11g Parachlorella kessleri Pond at state New York, USA FM205846 (2010) 298 Bo c k et al.: Barcoding in Chlorella

Table 2. Sequences used for p calculation

Species Accession Reference Number

Chlorella chlorelloides HQ111432 this study Chlorella coloniales FM205862 Lu o et al. (2010) Chlorella elongata FM205858 Lu o et al. (2010) Chlorella heliozoae FM205850 Lu o et al. (2010) Chlorella lewinii FM205861 this study Chlorella lobophora FM205833 Lu o et al. (2010) Chlorella pituita FM205856 Lu o et al. (2010) Chlorella pituita GQ176853 Kr i e n i t z et al. (2010) Chlorella pulchelloides FM205857 Lu o et al. (2010) Chlorella pulchelloides HQ111430 this study Chlorella pulchelloides HQ111431 this study Chlorella rotunda HQ111433 this study Chlorella singularis HQ111435 this study Chlorella sorokiniana FM205860 Lu o et al. (2010) Chlorella sorokiniana FM205859 Lu o et al. (2010) Chlorella variabilis AB162913 Ho s h i n a et al. (2005) Chlorella variabilis AB162912 Ho s h i n a et al. (2005) Chlorella variabilis AB206546 Ho s h i n a et al. (2005) Chlorella variabilis AB206550 Ho s h i n a et al. (2005) Chlorella variabilis AB162914 Ho s h i n a et al. (2005) Chlorella variabilis AB162915 Ho s h i n a et al. (2005) Chlorella variabilis AB162916 Ho s h i n a et al. (2005) Chlorella variabilis AB162917 Ho s h i n a et al. (2005) Chlorella variabilis AB219527 Ho s h i n a et al. (2005) Chlorella variabilis FM205849 Lu o et al. (2010) Chlorella variabilis AB206549 Ho s h i n a et al. (2005) Chlorella volutis HQ111434 this study Chlorella vulgaris AY591508 Mü l l e r et al. (2005) Chlorella vulgaris AB162910 Ho s h i n a et al. (2005) Chlorella vulgaris AY591509 Mü l l e r et al. (2005) Chlorella vulgaris AY591510 Mü l l e r et al. (2005) Chlorella vulgaris AY591511 Mü l l e r et al. (2005) Chlorella vulgaris AY591512 Mü l l e r et al. (2005) Chlorella vulgaris AY591513 Mü l l e r et al. (2005) Chlorella vulgaris AY591500 Mü l l e r et al. (2005) Chlorella vulgaris AY591501 Mü l l e r et al. (2005) Chlorella vulgaris AY591502 Mü l l e r et al. (2005) Chlorella vulgaris AY591503 Mü l l e r et al. (2005) Chlorella vulgaris AY591504 Mü l l e r et al. (2005) Chlorella vulgaris AY591505 Mü l l e r et al. (2005) Chlorella vulgaris AY591506 Mü l l e r et al. (2005) Chlorella vulgaris AY591493 Mü l l e r et al. (2005) Chlorella vulgaris AY591494 Mü l l e r et al. (2005) Chlorella vulgaris AY591495 Mü l l e r et al. (2005) Chlorella vulgaris AY591496 Mü l l e r et al. (2005) Chlorella vulgaris AY591497 Mü l l e r et al. (2005) Chlorella vulgaris AY591498 Mü l l e r et al. (2005) Chlorella vulgaris AY591499 Mü l l e r et al. (2005) Chlorella vulgaris FM205854 Lu o et al. (2010) Fottea 11(2): 293–312, 2011 299

descriptions followed Fo t t & No v á k o v á (1969). A 50% majority–rule consensus tree was calculated for posterior probabilities using PAUP *. DNA isolation, PCR and sequencing. Algal cells were mechanically disrupted in the presence of glass beads Barcode and secondary structure analyses. A dataset (~ 0.5 mm in diameter, Carl Roth GmbH + Co. KG, of 49 sequences of Chlorella–strains was used for Karlsruhe, Germany) using the Tissuelyser II (Qiagen). analyzing the Barcode region (Table 2). The Barcoding Total Genomic DNA was isolated using the DNeasy region stretches from the start codon of the 5.8S rDNA Plant Mini Kit (Qiagen GmbH, Hilden, Germany) up to the conserved motif on Helix III (consisting of following the instructions given by the manufacturer. UGGU) near the tip of the helix and close to the end Genomic DNA will be stored at the BGBM DNA of the ITS–2 (Kr i e n i t z et al. 2004; Co l e m a n 2007). Network (Gemeinholzer et al. 2008). The SSU and ITS Sequences were aligned manually according to their rRNA gene were amplified and sequenced as previously secondary structure using SequentiX Alignment Editor reported (Bo c k et al. in press). The overlapping partial (He pp e r l e 2004). Completed alignments were imported sequences of each strain were assembled to a complete into PAUP*(4.0b10; Sw o ff o r d 2002) for estimating consensus sequencing consisting of SSU, ITS–1, 5.8S, divergence rates by using simple uncorrected pair– ITS–2 using the software SeqAssem (He pp e r l e 2004). wise (p) distance matrices. Genetic distances between sequences were given as substitutions (differences) per Phylogenetic analyses. Phylogenetic analyses were site (Li t a k e r et al. 2007). performed on a concatenated data set of SSU, 5.8S, To locate hemi–compensatory base changes ITS–1 and ITS–2 rRNA sequences. The alignement was (hemi–CBCs) and CBCs, the ITS–2 secondary structure constructed by adding sequences of the known Chlorella was constructed with the help of mfold (Zu k e r 2003) species (Lu o et al. 2010; Pr ö s c h o l d et al. 2011) to and 4SALE (Se i b e l et al. 2006, 2008). the newly obtained sequences. Representatives of the different genera of the Chlorella and Parachlorella clades were chosen according to Lu o et al. (2010) and Results Bo c k et al. (2010). Catena viridis Chodat was chosen as outgroup based on previous analyses by Kr i e n i t z et al. (2003) and Bo c k et al. (2010). The sequences Taxonomic revision were aligned using the SequentiX Alignment Editor The taxonomic revisions (see below) were (He pp e r l e 2004) according to their secondary structure based on the results such as morphology, the (see Figs S1 and S2 in Lu o et al. 2006) and manually phylogenetic tree, secondary structure of the adjusted by eye. The GenBank accession numbers for ITS–2 and the barcoding criteria. The new species all included strains are given in Table 1. A data set of and new combinations showed base changes at 39 strains with 2488 aligned bases positions was used the barcoding–region mentioned below. for the phylogenetic analyses, introns were excluded. Phylogenetic inference was based on Maximum Chlorella Be i j e r i n c k 1890 Likelihood (ML), Maximum Parsimony (MP), distan- Be i j e r i n c k , M.W. 1890, Botanische Zeitung 48: p. 758, ce (Neighbor Joining; NJ) and Bayesian Inference tafel VII, fig. 2 (MB). MP and NJ were calculated using PAUP* Emended Diagnosis: Class Trebouxiophyceae; (version 4.0b10; Sw o ff o r d 2002). The ML analyses were perfomed with Treefinder (Jo bb 2008) with cells spherical, subspherical or ellipsoid, single four partitions. Models and parameters proposed by or forming colonies with up to 64 cells, mucilage Treefinder under AICc criteria were as follows: SSU present or absent. Chloroplast single, parietal, (1701 bases; model J1), ITS–1 (343 bases; model J1), pyrenoid present, surrounded by grains. 5.8S (137 bases; model HKY) and ITS–2 (304 bases; Reproduction by autospores, zoospores lacking. model J2). To confine the tree topology, bootstrap Autospores released through disruption of mother analyses were calculated by distance (NJ; 1000 cell wall. Daughter cell can remain attached to replicates), parsimony (MP; 1000 replicates) and ML remnants of mother cell wall and form colonies (1000 replicates) criteria. For the MB analyses, the with mucilage envelopes. Planktonic, edaphic or dataset was partitioned as described above with the GTR+I+G settings, gamma shape parameters and endosymbiontic. proportion of invariable sits for all partitions using Type species: Chlorella vulgaris Be i j e r i n c k MrBayes version 3.1 (Hu e l s e n b e c k & Ro n q u i s t 2001). The parameters were unlinked and allowed to Chlorella pituita C. Bo c k , Kr i e n i t z et vary across the partitions. The stationary distribution Pr ö s c h o l d , sp. nov. (Figs 1, 8, 9) was verified (average standart deviations of split Latin diagnosis: Cellulae in coloniis vel solitariae, frequencies lower than 0.01) before stop of the analyses. planctonicae. Coloniae 4–32 cellularis, 40–50 µm The first 25% of the trees were discarded as burn–in. in diametro, cum tegumento mucilagineis vestitae. 300 Bo c k et al.: Barcoding in Chlorella

Figs 1–7. Drawings of light microscopical characters of Chlorella species. Iconotypes: (1) Chlorella pituita, authentic strain ACOI 311; (2) Chlorella pulchelloides, authentic strain CCAP 211/118; (3) Chlorella coloniales, authentic strain UTEX 938; (4) Chlorella singularis, authentic strain CCAP 211/119; (5) Chlorella rotunda, authentic strain CCAP 260/11; (6) Chlorella lewinii, authentic strain CCAP 211/90; (7) Chlorella volutis, authentic strain CCAP 211/120. Scale bars 10 μm.

Cellulae adultae rotundae vel leviter ovalis, or horizontally. Differs from other species of this 6.5–8.6 × 5.5–8 µm, cellulis funibus subtilibus genus by the order of nucleotides in ITS–1, ITS–2 hyalinis iunctis . Cellulae juvenilis ovalis vel prope and the barcoding signatures. sphericae, 6–8 × 4.3–5.3 µm, funibus junctus ad Holotype: An air–dried as well as a formaldehyde– apices latiusculis. Chloroplastus unicus, parietalis, fixed sample of strain ACOI 311 was deposited at poculiformis patelliformisve, pyrenoide granis amylis the Botanical Museum at Berlin–Dahlem under tecto. Reproductio asexualis 2–4 autosporum ope, e ruptura cellulis matricalis oblique vel horizontaliter. the designation B40 0040661 A speciebus ceteris generis ordine nucleotidorum in Type locality: Mira, trout nursery, Portugal. ITS–1, ITS–2 et signis molecularis differt. Ethymology: from Latin: pituita = mucilage Authentic strain: Material of the authentic strain ACOI 311 is maintained at the Coimbra Collection Cells colonial or single, planktonic. Colonies 4–32 of Algae, Portugal. celled, with mucilaginous envelope. Diameter of Iconotype: Figure 1 colonies up to 40–50 µm. Adult cells spherical to slightly oval, 6.5–8.6 × 5.5–8 µm, connected via Chlorella pulchelloides C. Bo c k , Kr i e n i t z et mucilaginous stalks. Young cells oval to almost Pr ö s c h o l d , sp. nov. (Figs 2, 10, 11) spherical, 6–8 × 4.3–5.3 µm, connected to the Latin diagnosis: Cellulae in coloniis, planctonicae, stalks more or less at the apices of the broader interdum tegumento gelatinoso vestitae. Coloniae side. Chloroplast single, parietal, cup– or saucer– 4–32 cellularis, 25–35 µm in diametro. Cellulae shaped with ellipsoid to spherical pyrenoid, adultae sphaericae, 4.5–6.5 µm diametro, cellulis covered by two starch grains. Reproduction by 2–4 funibus subtilibus hyalinis iunctis. Cellulae juvenilis autospores. Release of the autospores obliquely ovalis vel ovoides, 3.5–4.5 × 4–6 µm, funibus Fottea 11(2): 293–312, 2011 301

Figs 8–19. Micrographs of different Chlorella strains: (8–9) Chlorella pituita (ACOI 311); (10–11) Chlorella pulchelloides (CCAP 211/117); (12) Chlorella chlorelloides (CCAP 211/116); (13–14) Chlorella coloniales (UTEX 938) ; (15) Chlorella elongata (CCAP 222/18); (16) Chlorella singularis (CCAP 211/119); (17) Chlorella rotunda (CCAP 260/11); (18) Chlorella lewinii (CCAP 211/90); (19) Chlorella volutis (CCAP 211/120). Scale bars 10 µm. Arrowheads indicating connection of cells to mucilagenous stalks. 302 Bo c k et al.: Barcoding in Chlorella junctus ad apices latiusculis. Chloroplastus unicus, oval to ovoid, 3–4.5 × 2.5–3.5 µm. Chloroplast parietalis, poculiformis, pyrenoide granis amylis single, parietal, cup–or saucer–shaped with tecto. Reproductio asexualis 2–4 autosporum ope, e ellipsoid to spherical pyrenoid, covered by two ruptura cellulis matricalis oblique vel horizontaliter.. starch grains. Reproduction by autospores. Differs A speciebus ceteris generis ordine nucleotidorum in from other species of this genus by the order of ITS–1,ITS–2 et signis molecularis differt. nucleotides in ITS–1 and ITS–2 and the barcoding

signatures. Cells colonial, planktonic, with mucilaginous Holotype: An air–dried as well as a formaldehyde– envelope. Colonies 4–32 celled, diameter of fixed sample of the authentic strain UTEX 938 colonies 25–35 µm. Adult cells spherical 4.5–6.5 was deposited at the Botanical Museum at Berlin– µm, connected via mucilaginous stalks. Young Dahlem under the designation B40 0040665 cell oval to ovoid, 3.5–4.5 × 4–6 µm, attached Type locality: type locality unknown, studied to the stalks at their broader side. Chloroplast culture UTEX 938 isolated in 1958 from J. Stein single, parietal, cup– or saucer–shaped with Ethymology: from Latin: colonialis = colonial ellipsoid to spherical pyrenoid, covered by two Authentic strain: Material of the authentic strain starch grains. Reproduction by 2–4 autospores. UTEX 938 maintained at the Culture Collection Release of autospores after rupture of mother cell of Algae at the University of Texas, USA. wall horizontally or slightly obliquely. Differs Iconotype: Figure 3 from other species of this genus by the order of nucleotides in ITS–1 and ITS–2 and the barcoding signatures. Chlorella singularis C. Bo c k , Kr i e n i t z et Holotype: Material of the authentic strain CCAP Pr ö s c h o l d , sp. nov. (Figs 4, 16) 211/118 is cryopreserved at the Culture Collection Latin diagnosis: Cellulae solitariae, planctonicae. of Algae and Protozoa, Oban, Scotland. Cellulae adulate globose vel leviter ovalis, 6.7–9 Isotype: An air–dried as well as a formaldehyde– µm, interdum tegumento gelatinoso vestitae. Cellulae fixed sample of strain CCAP 211/118 was deposited juvenilis ovalis vel globosae, 5–7 µm. Chloroplastus unicus, parietalis, poculiformis, pyrenoide granis at the Botanical Museum at Berlin–Dahlem under amylis tecto. Reproductio asexualis autosporum ope, the designation B40 0040664 e ruptura cellulis matricalis in 4 partibus. A speciebus Type locality: Lake Feldberger Haussee, ceteris generis ordine nucleotidorum in ITS–1, ITS–2 Brandenburg, Germany (53°20’27,35’’N; et signis molecularis differt. 13°26’10,89’’E). Ethymology: from Latin: pulchella = nice Cells solitary, planktonic. Adult cells globose Authentic strain: CCAP 211/118 or slightly oval, 6.7–9 µm, with mucilaginous Iconotype: Figure 2 envelope. Young cells oval to spherical, 5–7 µm. Reproduction by autospores. Release of autospores Chlorella colonialis C. Bo c k , Kr i e n i t z et after ruptures of mother cell wall into four flaps. Pr ö s c h o l d , sp. nov. (Figs 3, 13, 14) Chloroplast parietal, cup– or saucer–shaped with Latin diagnosis: Cellulae in coloniis, planctonicae, ellipsoid to spherical pyrenoid, covered by two interdum tegumento gelatinoso vestitae. Coloniae starch grains. Differs from other species of this 4–32 cellularis, 25–35 µm in diametro. Cellulae genus by the order of nucleotides in ITS–1 and adultae late ellipsoidae, ovalis ad elongates 5.5–7.5 ITS–2 and the barcoding signatures. × 4–5 µm, cellulis funibus subtilibus hyalinis iunctis. Holotype: Material of the authentic strain CCAP Cellulae juvenilis ovalis vel ovoides, 3–4.5 × 2.5–3.5 µm Chloroplastus unicus, parietalis, poculiformis, 211/119 is cryopreserved at the Culture Collection pyrenoide granis amylis tecto. Reproductio asexualis of Algae and Protozoa, Oban, Scotland. autosporum ope. A speciebus ceteris generis ordine Isotype: An air–dried as well as a formaldehyde– nucleotidorum in ITS–1, ITS–2 et signis molecularis fixed sample of strain CCAP 211/119 was deposited differt. at the Botanical Museum at Berlin–Dahlem under the designation B40 0040666. Cells colonial, planktonic, with mucilagenous Type locality: Sewage pond, Nakuru–National envelope. Colonies 4–32 celled, diameter of Park, Nakuru District, Rift Valley Province, Kenya colonies 25–35 µm. Adult cells broadly ellipsoid, (0°19’18,42’’S; 36°04’38’’E). oval to elongate, 5.5–7.5 × 4–5 µm, connected via Ethymology: from Latin: singularis = single mucilaginous stalks at narrow ends. Young cells Authentic strain: CCAP 211/119 Fottea 11(2): 293–312, 2011 303

Table 3. Light microscopical characters of the newly described Chlorella species [(M) mucilage].

Connection Adult cell Adult cell No. of Colony of young Chlorella (young cell) M Release of (young cell) cells in size cells to species size autospores shape colony (µm) gelatinous (µm) stalks

spherical to 6.5–8.6 × at the apices slightly oval, 5.5–8 of the oblique or C. pituita 4–32 40–50 + (oval to almost ( 6–8 × broader side horizontal spherical) 4.3–5.3) of the cells

4.5–6–5 horizontal spherical (oval at the C. pulchelloides (3.5–4.5 × 4–32 25–35 + or slightly to ovoid) broader side 4–6) oblique

broadly 5.5–7.5 × at narrow oblique or C. colonialis ellipsoid, oval 4–5 (3–4.5 4–32 25–35 + ends horizontal to elongate × 2.5–3.5) globose or slightly oval 6.7–9 rupture of C. singularis 1 – – + (oval to (5–7) cell wall spherical) globose, egg rupture of C. rotunda 3.3–4.5 1 – – – shaped cell wall

oval, egg rupture of C. lewinii 4–6 1 – – – shaped cell wall

rupture of C. volutis globose 5–6.5 1 – – – cell wall

Iconotype: Figure 4 260/11 is cryopreserved at the Culture Collection of Algae and Protozoa, Oban, Scotland under the designation CCAP 260/11. Chlorella rotunda C. Bo c k , Kr i e n i t z et Isotype: An air–dried as well as a formaldehyde– Pr ö s c h o l d , sp. nov. (Figs 5, 17) fixed sample of strain CCAP 260/11 was deposited Latain Diagnosis: Cellulae solitariae planctonicae, at the Botanical Museum at Berlin–Dahlem under globose vel ovoideae, 3.3–4.5 µm diametro. Sine tegumento gelatinoso. Chloroplastus unicus, the designation B40 0040662. parietalis, olliformis patelliformisve, pyrenoide granis Type locality: Okawango, Angola. amylis tecto. Reproductio asexualis autosporum ope. Ethymology: from Latin: rotunda = spherical A speciebus ceteris generis ordine nucleotidorum in Authentic strain: CCAP 260/11 ITS–1, ITS–2 et signis molecularis differt. Iconotype: Figure 5

Cells solitary, planktonic, globose or egg shaped, Chlorella lewinii C. Bo c k , Kr i e n i t z et 3.3–4.5 µm. Mucilage absent. Chloroplast Pr ö s c h o l d , sp. nov. (Figs 6, 18) Latin diagnosis: Cellulae solitariae edaphicae, single, parietal, cup–, girdle– or saucer–shaped, ellipsoidae vel ovoideae, 4–6 µm. Sine tegumento with broadly ellipsoidal to spherical pyrenoid. gelatinoso. Chloroplastus unicus, parietalis, olliformis Reproduction by autospores. Differs from other patelliformisve, pyrenoide granis amylis tecto. species of this genus by the order of nucleotides Reproductio asexualis autosporum ope. A speciebus in ITS–1, ITS–2 and the barcoding signatures. ceteris generis ordine nucleotidorum in ITS–1, ITS–2 Holotype: Material of the authentic strain CCAP et signis molecularis differt. 304 Bo c k et al.: Barcoding in Chlorella

Cells solitary, edaphic, oval or egg shaped, 4–6 Kenya (00°23.421’ S, 36°53.831 E). µm. Mucilage absent. Chloroplast single, parietal, Ethymology: from Latin: volutis = rolling cup–, girdle– or saucer–shaped, with broadly Authentic strain: CCAP 211/120 ellipsoidal to spherical pyrenoid. Reproduction Iconotype: Figure 7 by autospores, zoospores not observed. Differs from other species of this genus by the order of Chlorella chlorelloides (Na u m a n n ) C. Bo c k , nucleotides in ITS–1, ITS–2 and the barcoding Kr i e n i t z et Pr ö s c h o l d , comb. nov. signatures. Basionym: Brachionococcus chlorelloides Na u m ., Ark. Holotype: Material of the authentic strain CCAP Bot. 16,2:15, 1919 211/90 is cryopreserved at the Culture Collection Synonym: Dictyosphaerium chlorelloides (Na u m .) of Algae and Protozoa, Oban, Scotland. Ko m á r e k et Pe r m a n Algol. Stud. 20, p. no. 252, 1978. Isotype: An air–dried as well as a formaldehyde– fixed sample of strain CCAP 211/90 was deposi- Holotype: Fig. 8–9, Naumann 1921. ted at the Botanical Museum at Berlin–Dahlem Epitype (designated here): Material of the strain under the designation B40 0040663 CB2008/110 was cryopreserved in metabolic Type locality: Permanent freshwater pond in a inactive state at the Culture Collection of Algae and crater, Easter Island, Chile Protozoa, Oban, Scotland under the designation Ethymology: The species is named in memory of CCAP 211/116. the late Ralph Lewin, who was a leading authority Emended diagnosis: Cells solitary or in four in green algae genetics and who collected the celled colonies, sourrounded by mucilagenous original soil sample from the Easter Islands, from envelope. Adult cells spherical 3.8–8 µm, which the strain was isolated. connected via mucilagenous stalks. Young cells Authentic strain: CCAP 211/90 oval to semilunate, 3–7 × 2–6 µm, connected Iconotype: Figure 6 to the stalks with their narrow end. Chloroplast parietal, cup– or saucer–shaped with ellipsoid to spherical pyrenoid, covered by two starch grains. Chlorella volutis C. Bo c k , Kr i e n i t z et Reproduction by 2–4 autospores. Release of the Pr ö s c h o l d , sp. nov. (Figs 7, 19) Latin diagnosis: Cellulae solitariae, planctonicae vel autospores after rupture of mother cell wall by edaphicae, globose, 5–6.5 µm. Chloroplastus unicus, slanting in 180°. parietalis. Sine tegumento gelatinoso. Chloroplastus unicus, parietalis, poculiformis, pyrenoide granis Chlorella elongata (Hindák) C. Bo c k , Kr i e n i t z amylis tecto. Reproductio asexualis autosporum ope. et Pr ö s c h o l d , comb. nov. A speciebus ceteris generis ordine nucleotidorum in Basionym: Dictyosphaerium elongatum Hi n d á k Biol. ITS–1, ITS–2 et signis molecularis differt. Práce 23:38, 1977 Synonym: Selenodictyon elongatum (Hi n d á k ) Co m a s Cells solitary, planktonic or edaphic, globose, et Ko m á r e k in Co m a s 1992, Algol. Stud. 65: p. 22. 5–6.5 µm, without mucilaginous envelope. Chloroplast parietal, cup– or saucer–shaped Epitype (designated here): Material of the with ellipsoid to spherical pyrenoid, covered by strain CCAP 222/18 was cryopreserved at the two starch grains. Reproduction by autospores, Culture Collection of Algae and Protozoa, Oban, zoospores not observed. Differs from other species Scotland. of this genus by the order of nucleotides in ITS–1 and ITS–2 and the barcoding signatures. Holotype: Material of the authentic strain CCAP Morphological observations by light micro- 211/120 is cryopreserved in metabolic inactive scope state at the Culture Collection of Algae and Within the newly analysed strains, seven species Protozoa, Oban, Scotland. were observed with a surrounding mucilaginous Isotype: An air–dried as well as a formaldehyde– envelope. The colonial life–form was often lost in fixed sample of strain CCAP 211/120 was deposited culture, disintegrating into single cells. However, at the Botanical Museum at Berlin–Dahlem under even in culture the strains kept their gelatinous the designation B40 0040733. indusium. C. pituita occurred in colonies with 16 Type locality: Rhinopool Nakuru–National Park, cells and more. The adult cells were spherical to small seasoning pond near the western border of slightly oval, 6.5–8.6 µm. The young cells showed the park, Nakuru District, Rift Valley Province, a oval cell shape, 6–8 × 4–5 µm. The release of Fottea 11(2): 293–312, 2011 305

Fig. 20. Maximum likelihood (ML) phylogenetic tree of the Chlorellaceae as inferred from concatenated rRNA gene sequence data set of SSU, ITS–1, 5.8S and ITS–2 . Support values correspond to Bayesian PP, Maximum Likelihood BP, Maximum Parsimony BP and Neighbor Joining BP. Hyphen correspond to values lower than 50% for BP and lower 0.95 for PP. Branch lengths represent substitutions per site. 306 Bo c k et al.: Barcoding in Chlorella the autospores happened after the rupture of the could be distinguished from C. lewinii by its mother cell wall horizontally or slightly tilted. much smaller cell size (3–4.5 µm) and the more The young cells were attached to the gelatinous spherical cells (Fig. 17). Another single celled stalks at their narrow end, but shifted to the upper taxon without a mucilage envelope was Chlorella end of their broader side (Figs 8–9). A similar volutis. This species showed spherical cells of morphology was observed by C. pulchelloides. 5–6.3 µm with spherical to slightly oval young However, the cells were smaller, 4.5–6.5 µm, in cells (Fig. 19). The morphological characteristics comparison to C. pituita and the oval young cells of the new described species are summarized in were attached at the broader side to the gelantinous Table 3. stalks (Figs 10–11). Both species showed a high resemblance to Dictyosphaerium pulchellum. A Phylogenetic analyses clear morphological criterion to distinguish them The genus Chlorella is highly supported in is the attachment of the young cells to the stalks, Bayesian inference (MB), but only moderate which occurred in case of D. pulchellum at the to weak supported in Maximum Likelihood tips of the cells. Strain CCAP 211/116 showed (ML), Neighbour Joining (NJ) and Maximum the typical features of D. chlorelloides with Parsimony (MP) analyses (Fig. 1). The tree re- four–celled colonies (Fig. 12). The cells easily vealed 14 distinct lineages within the genus, separated from each other. The adult cells were nevertheless the branching order of the lineages spherical, young cells oval to semilunate. The cell in most cases remained unresolved. Five of size of the adult cells were larger than previously the lineages belonged to the already known reported; 6.5–8 µm in our culture vs. 3.8–6 (7.5) Chlorella species: C. vulgaris, C. lobophora, C. µm according to Ko m á r e k & Pe r m a n (1978). The sorokiniana, C. heliozoae and C. variabilis. The autospores slanted after the rupture of the mother species with colonial life–form and surrounding cell wall in 180°. mucilage envelope evolved at five different The cells of C. coloniales were oval to positions within the genus Chlorella. Chlorella sometimes elongated, 5.5–7.3 × 4–6 µm. They pituita sp. nov. clustered as sister to C. vulgaris occurred in colonies with 16 to 32 cells. The with high statistical support in all analyses. The young cells were oval to ovoid, 3–4.5 × 2.5–3.5 three strains of C. pulchelloides sp. nov. clustered um. The cells were attached to the stalks at their next to C. chlorelloides comb. nov. with high to narrow end (Figs 13–14). This species showed a moderate support. Next to this lineage evolved C. resemblance with strain CCAP 222/18 (Fig. 15). coloniales sp. nov. and C. singularis (single celled This strain showed oval or elongated adult cells of with surrounding mucilage) with no support in 6–9 × 2–4 µm length. The cells were attached to any analyses. The relationship of C. elongata the stalks at their broader side. This strain could be comb. nov. to the others is also not supported identified as Dicytyosphaerium elongatum, and is in the analyses. Next to this species evolved the here new combined to Chlorella elongata comb. single celled C. volutis (without mucilage). C. nov. It can be distinuished from C. coloniales by lewinii evolved as sister to C. sorokiniana, and C. the different cell form and the cell size. Chlorella rotunda as sister to C. heliozoae with high support singularis showed spherical to sligtly oval cells in the analyses. with 6.7–9 µm (Fig. 16). The mother cell wall ruptured in four flaps. This species was mainly Barcoding and secondary structure analyses observed single celled, only young cells occurred We investigated the Barcoding region of 49 rarely as 4–celled colonies. The species showed Chlorella–sequences (published and new similarities to Parachlorella beijerinckii, but di- sequences; see Table 2) according to Mo n i z & ffered by more spherical cells, the four–celled Ka c m a r s k a (2009). Interspecific uncorrected colonies and the phylogenetic position within the genetic distances (p) between strains belonging to tree of our analyses (see below). Three new species the same species ranged from p = 0 to p = 0.0098 without mucilage and without colony life–form diff./site (Table 4). Distances among the five spe- could be observed in this study. Chlorella lewinii cies previously thought to be the only species in is an edaphic species that lacked a mucilage coat, the genus Chlorella: C. vulgaris, C. sorokiniana, did not form colonies and possessed spherical to C. lobophora, C. variabilis, C. heliozoae, ranged slightly oval cells of 4–6 µm (Fig. 18). Young from p = 0.0777 to p = 0.1542 diff./site. All the cells were more oval, 3–5 × 2–5 µm. C. rotunda 14 species described in this study together showed Fottea 11(2):Fottea 2011293–312, Table 4. Comparison of the different Chlorella species by barcoding and CBC-approach.

CBC/ hCBC P [diff./site] C. pituita C. volutis C. lewinii C. rotunda C. vulgaris C. elongata C. variabilis C. heliozoae C. singularis C. coloniales C. lobophora

uncorrected C. sorokiniana C. chlorelloides C. pulchelloides

0/0 C. vulgaris (n=22) 2/2 5/5 4/1 5/1 9/3 4/1 3/2 6/2 6/4 7/3 4/1 5/5 8/1 0-0.0098 0 307 C. pituita 0.0863 – 5/4 5/6 2/4 3/6 2/2 3/3 5/5 3/3 4/4 4/3 6/6 3/6 (n=2) 0.0896 0 0 C. lobophora 0.119 – 0.1282 5/2 3/3 3/3 2/1 5/3 3/4 2/3 3/5 3/2 5/3 3/5 (n=1) 0.1256 0 0 C. variabilis 0.1287- 0.1188 – 0.1094 – 3/0 4/2 3/1 2/3 6/2 2/2 2/4 2/3 7/3 2/2 (n=11) 0.1321 0.1196 0.1125 0 0 C. sorokiniana 0.1345 – 0.0777 – 0.1293 0.101 1/2 1/0 2/1 2/3 2/2 3/3 2/4 1/1 3/6 (n=2) 0.1379 0.0778 0 0.1024 0 C. lewinii 0.1473 – 0.1447 0.1245 - 0.0673 3/0 3/3 1/3 4/3 4/4 2/3 3/1 3/6 (n=1) 0.157 0.1025 0 0 C. rotunda 0.1417 - 0.0963 – 0.1348 0.1315 0.073 0.1043 2/2 2/1 2/1 5/2 6/2 2/0 3/4 (n=1) 0.1505 0.0992 0 0 C. heliozoae 0.1443 – 0.0955 – 0.1411 0.1114 0.0743 0.098 0.0421 2/3 3/3 7/4 4/4 6/3 3/6 (n=1) 0.1542 0.0956 0 C. pulchelloides 0.1187 0.1272- 0.0862 – 0.0896 0.0738 – 0.0827 – 0.1032 – 0.1061 – 0 2/3 4/4 4/1 3/1 2/4 (n=3) -0.1252 0.1305 0.0897 – 0.0898 0.0742 0.086 0.1066 0.1094 0.0033 0 C. chlorelloides 0.1285 – 0.0694 – 0.0396 – 0.1136 0.0867 0.0507 0.0827 0.0927 0.0926 1/3 2/2 4/1 7/3 (n=1) 0.1351 0.0696 0.0429 0 0.0973 0 C. singularis 0.1492 – 0.1364 0.1078 0.0888 0.0728 0.1045 0.1040 0.1071 – 0.0793 3/2 4/4 6/4 (n=1) 0.1527 0.1007 0 0 C. coloniales 0.1441 – 0.1741 0.1352 0.1248 0.0834 0.1383 0.1462 0.1485 0.1122 0.1023 0.1108 5/2 1/3 (n=1) 0.1507 0 0 C. volutis 0.1287 – 0.0759 – 0.086 – 0.1516 0.1219 0.0745 0.0983 0.1111 0.1017 0.0768 0.0879 0.1051 4/5 (n=1) 0.1355 0.076 0.0872 0 0 C. elongata 0.1342 – 0.0965 – 0.1688 0.1322 0.0925 0.0742 0.108 0.1081 0.108 0.0931 0.101 0.118 0.1052 (n=1) 0.1441 0.0998 0 308 Bo c k et al.: Barcoding in Chlorella a distance of between p = 0.073 and p = 0.1741 C. sorokiniana without problems. In addition, diff./site. To confirm the effectiveness of the two formerly Dictyosphaerium species, D. barcoding region, we calculated the secondary chlorelloides and D. elongatum showed a close structure of the ITS–2 from these strains and phylogenetic relationship to Chlorella and were compared all 14 species with each other. We found therefore transferred to the genus Chlorella (see that the species of the genus Chlorella differ in 1–9 above). Although the original species description CBCs in the Helices I–III and 0–6 hemi–CBCs of D. chlorelloides mentions a cell size of 3.8– respectively. An assumed threshold of p ≥ 0.04 6 (–7.5) µm, which is slightly smaller than strain for species delineation in Chlorella corresponded CCAP 211/116 cells (6.2–8.4 µm), all other well with the CBCs and hemi–CBCs of the helixes characters matched to the latter. Given that there I–III of the ITS–2 (see Table 4). A low p value was was no authentic strain available for this species, correlated with a low CBC/hemi–CBC content. and that cell size is often dependent on culture conditions and life cycle, we decided to transfer the species Dictyosphaerium chlorelloides to Discussion Chlorella. In addition to this, we described three new species with colonial life–form and gelatinous The focus of this study was to revise the envelope. Chlorella pituita, C. pulchelloides and species concept of Chlorella. We provided an C. colonialis showed the typical morphology of emendation of the genus including taxa which the genus Dictyosphaerium, but had no similarities form mucilaginous envelopes and colonies. This to the already known species. The type strain of systematic revision was made possible by use of C. pituita was labelled as D. tetrachotomum in molecular signatures. We tested the barcoding the ACOI collection. Although the description concept as described by Mo n i z & Ka c z m a r s k a of this species mentions oval cells, the strains (2009) in regard to members of the genus showed spherical cells. The strain ACOI 856, Chlorella. The concept was developed for diatoms which revealed the same sequence as the type and successfully tested for Mediophyceae and strain exhibited as single celled morphology Bacillariophyceae and had a success rate of 99.9% with a gelatinous indusium. A similar problem in separating biologically defined species (Mo n i z arose with the strain UTEX 938. The strain was & Ka c z m a r s k a 2009). The ITS is widely used for initially labelled as D. planctonicum Ti ff . et phylogenetic studies of different organisms and is Ah l s t r ., this was later transferred to Lobocystis under consideration as barcode region in several R.H. Th o m p s o n and was characterized by two cases. Li t a k e r et al. (2007) suggested using this autospores and broad gelatinous stalks (Ko m á r e k region as barcode for dinoflagellates, and Se i f e r t & Fo t t 1983). These characters did not match (2009) applied it as barcode for fungi. Co l e m a n the morphology of UTEX 938, and we erected (2009) pointed out that variation in this DNA the new species C. coloniales. The strain SAG region correlates with taxonomic classification. 222–2a was often referred as D. pulchellum. Our So far, there is no official code for green algae. observations showed considerable differences in However, CBCs in the ITS have been used in the morphology of the young cells. Strains with many taxonomic studies and have proven to be the D. pulchellum morphology cluster next to D. powerful tools for species separation (Kr i e n i t z et ehrenbergianum within the Parachlorella–clade al. 2004, 2010; Mü l l e r et al. 2007; Wo l f et al. (Bo c k et al. in press). Therefore, the new lineage 2007; Ru h l et al. 2009). As our study showed, the with the strains SAG 222–2a, CCAP 211/117 5.8S and ITS–2 region works well for Chlorella and CCAP 211/118 formed a new species, C. as a tool for species delineation. The separation pulchelloides even if they bear a high similarity according the barcode region correlates with the with D. pulchellum. CBCs and hemi–CBCs in the region. The independent evolution of Dictyo- The species delineation in Chlorella has sphaerium morphotype in different clades of always been a problem because the morphological the Chlorellaceae has been discussed by Bo c k characters separating individual species are scarce et al. (2010) and Kr i e n i t z et al. (2010). Our and not easy to observe. In applying the barcoding analyses confirmed the independent evolution regions, we were able to separate the already of Dictyosphaerium in different clades of the known “true” Chlorella species: C. vulgaris, Chlorellaceae and revealed a major change in the C. lobophora, C. variabilis, C. heliozoae and understanding of typical Chlorella species. Instead Fottea 11(2): 293–312, 2011 309 of the more or less uniform “green balls”, several References members with different morphologies now occur in Chlorella s. str. The type species Chlorella Al v a r e z , I. & We n d e l , J. F. (2003): Ribosomal ITS vulgaris was considered as the typical Chlorella: sequences and plant phylogenetic inference. – small, green and more or less spherical. Our Mol.Phylogenet.Evol. 29: 417–434. An d r e y e v a , V.M. (1975): Rod Chlorella: Morfologya, analyses showed that the generic circumscription Sistematika, Prinzipy Klassifikaziyi. –88 pp., was way too narrow and is now emended (see Nauka, Leningrad. above). The question that is raised by these At k i n s o n , A.W., Jo h n , P.C.L. & Gu n n i n g , B.E.S. findings is the extent to which morphology is (1972): Sporopollenin in the cell–wall of influenced by the environment. Chlorella and other Algae: ultrastructure, The investment of phytoplankton in chemistry, and incorporation of 14C–acetate, mucilage is a striking feature in nature. The studied in synchronous cultures. – Planta 107: presence or absence of mucilage is in most 1–32. cases taxon specific, but the thickness is variable Bo c k , C., Pr ö s c h o l d , T. & Kr i e n i t z L. (2010): Two and often influenced by environmental factors new phylogenetic lineages of Dictyosphaerium– morphotype within Chlorellaceae (Re y n o l d s 2007). Biological investigations (Trebouxiophyceae): Heynigia gen. nov. and on the role of the mucilage coat suggest that Hindakia gen. nov. – Eur.J.Phycol. 45: 267– there is no single, unambiguous function of 277. mucilage. Often discussed is the role played Bo c k , C., Pr ö s c h o l d , T. & Kr i e n i t z L. (in press): by the mucilage indusium and the colony size Updating the genus Dictyosphaerium and of algae in grazing protection and the factors description of Mucidosphaerium gen. nov. that influence its size and thickness. A common (Trebouxiophyceae) based on morphological assumption is that large phytoplankton cannot and molecular data. – J.Phycol. be grazed due to size mismatch. Several studies Co l e m a n , A.W. (2007): Pan– ITS2 on the colonial and gelatinous algae Phaeocystis homologies revealed by RNA secondary (Prymnesiophyceae) revealed an increase in structure. – Nucl.Acids Res. 35: 3322–3329. colony–size as a result of chemicals released from Co l e m a n , A.W. (2009): Is there a molecular key to the level of “biological species” in ? A grazer or associated microbes (Ja k o b s o n & Ta n g DNA guide. – Mol.Phylogenet.Evol.50: 197– 2002; Ta n g et al. 2008). Another example is the 203. well–studied chlorophyte genus Scenedesmus Co m a s , A. (1992): Taxonomical contributions to the Meyen. Biotests with Scenedesmus in culture green algal flora () of Cuba. – Arch. revealed an increase in the colony–size due to Hydrobiol. 93/Algological Studies 65: 11–21. “Daphnia–factors” (He s s e n & v a n Do n k 1993; Da r i e n k o , T., Gu s t a v s , L., Mu d i m u , O., Ra d Me n e n d e z , La m p e r t et al. 1994; Lü r l i n g 1998; von El e r t & C., Sc h u m a n n , R., Ka r s t e n , U., Fr i e d l , T. & Fr a n k 1999; Wi l t s h i r e & La m p e r t 1999) as well Pr ö s c h o l d , T. (2010): Chloroidium, a common as “Brachionus–factors” (Ve r s c h o o r et al. 2004). terrestrial coccoid green alga previously Another example of an environmentally induced assigned to Chlorella (Trebouxiophyceae, morphological change is the bristle formation by Chlorophyta). – Eur.J.Phycol. 45: 79–95. Micractinium, a close relative of Chlorella, which El i a š , M. & Ne u s t u p a , J. (2009): Pseudomarvania, gen. nov. (Chlorophyta, Trebouxiophyceae), a has been linked to the “Brachionus–factors” (Lu o new genus for “budding” subaerial green algae et al. 2006). Nevertheless, factors responsible aerophytica Neustupa et Šejnohová for the appearance of the Dictyosphaerium– and Stichococcus ampulliformis Handa. – Fottea morphotype are yet to be resolved. As to whether 9: 169–177. or not the mucilage production is a response to Et t l , H. & Gä r t n e r , G. (1995): Syllabus der Boden–, environmental factors remains to be confirmed. Luft– und Flechtenalgen. – 729 pp. Gustav Fischer, Jena, New York. Acknowledgements Ev a n s , K.M., Wo r t l e y , A.H. & Ma n n , D.G. (2007): This research was financially supported by the An assessment of potential diatom “barcode” Deutsche Forschungsgemeinschaft (KR 1262/11–1, genes (cox1, rbcL, 18S and ITS rDNA) and 2) and NERC Oceans 2025 and NERC MGF 154 their effectiveness in determining relationships sequencing grant. Eberto Novelo provided a strain of Chlorella pulchelloides from Mexico. We thank M. in Sellaphora (Bacillariophyta). – 158: Degebrodt and M. Papke for technical assistance and 349–364. Kiplagat Kotut for helpful discussions on early draft Ev a n s , K.M. & Ma n n , D.G. (2009): A proposed of this article. protocol for nomenclaturally effective DNA 310 Bo c k et al.: Barcoding in Chlorella

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© Czech Phycological Society (2011) Received November 24, 2010 Accepted January 19, 2011