UPDATING THE GENUS DICTYOSPHAERIUM AND DESCRIPTION OF MUCIDOSPHAERIUM GEN. NOV. (TREBOUXIOPHYCEAE)...

UPDATING THE GENUS DICTYOSPHAERIUM AND DESCRIPTION OFMUCIDOSPHAERIUM GEN. NOV. (TREBOUXIOPHYCEAE) BASED ON

MORPHOLOGICAL AND MOLECULAR DATA1

Christina Bock2

Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhutte 2, D-16775 Stechlin-Neuglobsow, Germany

Thomas Proschold3

Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Dunbeg by

Oban, Argyll PA37 1QA, UK

and Lothar Krienitz

Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhutte 2, D-16775 Stechlin-Neuglobsow, Germany

Recent molecular analyses of Dictyosphaeriumstrains revealed a polyphyletic origin of this mor-photype within the Chlorellaceae. The type speciesDictyosphaerium ehrenbergianum Nageli formed anindependent lineage within the Parachlorella clade,assigning the genus to this clade. Our study focusedon three different Dictyosphaerium species to resolvethe phylogenetic position of remaining species. Weused combined analyses of morphology; moleculardata based on SSU and internally transcribed spacerregion (ITS) rRNA sequences; and the comparisonof the secondary structure of the SSU, ITS-1, andITS-2 for species and generic delineation. The phy-logenetic analyses revealed two lineages without gen-eric assignment and two distinct clades ofDictyosphaerium-like strains within the Parachlorellaclade. One clade comprises the lineages with theepitype strain of D. ehrenbergianum Nageli and twoadditional lineages that are described as new species(Dictyosphaerium libertatis sp. nov. and Dictyosphaeriumlacustre sp. nov.). An emendation of the genusDictyosphaerium is proposed. The second clade com-prises the species Dictyosphaerium sphagnale Hindakand Dictyosphaerium pulchellum H. C. Wood. On thebasis of phylogenetic analyses, complementary basechanges, and morphology, we describe Mucidosphae-rium gen. nov with the four species Mucidosphaeriumsphagnale comb. nov., Mucidosphaerium pulchellumcomb. nov., Mucidosphaerium palustre sp. nov., andMucidosphaerium planctonicum sp. nov.

Key index words: Dictyosphaerium; ITS; Mucidosp-haerium; Parachlorella; phylogeny; SSU; taxonomy

Abbreviations: BP, bootstrap percentages; CBC,compensatory base change; ITS, internally tran-scribed spacer region; ML, maximum likelihood;MP, maximum parsimony; NHS, nonhomoplas-ious synapomorphies; NJ, neighbor joining; PP,posterior probability; TBR, tree-bisection-recon-nection

Traditionally, genera and species of Chlorophytahave been distinguished based on morphologicaland ecological characters (Komarek and Fott 1983).In recent years, taxa defined morphologically havebeen reexamined using molecular data, and somegenera have been shown to be polyphyletic, forexample, the monadoid green algae Chlamydomonas(Proschold et al. 2001) and Chlorogonium (Nozakiet al. 2003, Nakada et al. 2008), and several coccoidgreen algae, such as Coelastrum (Hegewald et al.2010), Micractinium (Proschold et al. 2010), Mono-raphidium (Krienitz et al. 2001, Fawley et al. 2006),Pediastrum (Buchheim et al. 2005, McManus andLewis 2005), and Scenedesmus Meyen (An et al.1999). Based on these results, new species and gen-era have been erected.

The Chlorellaceae (Trebouxiophyceae) occupy abroad range of different habitats and are prominentmembers of the phytoplankton communities of lakesand rivers. Several studies based on molecular andmorphological data were carried out to revise thephylogeny and taxonomy of this family (Ustinovaet al. 2001, Krienitz et al. 2004, Aslam et al. 2007,Neustupa et al. 2009, Darienko et al. 2010). Krienitzet al. (2004) revealed a phylogenetic association ofsome members of the genus Dictyosphaerium Nageliwith the family Chlorellaceae (order Chlorellales,class Trebouxiophyceae). Following the traditionalmorphological classification, the genus Dictyosphaerium

1Received 21 April 2010. Accepted 2 November 2010.2Author for correspondence: e-mail [email protected] address: Department of Limnology, University of Vienna,

Althanstr. 14, A-1090 Vienna, Austria.

J. Phycol. 47, 638–652 (2011)� 2011 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2011.00989.x

638

was formerly placed within the Chlorophyceae(Komarek and Fott 1983). Since Nageli introducedthis genus in 1849 (Nageli 1849), 28 species havebeen described worldwide from different climaticzones, for example, temperate regions, the tropics,and even Antarctica (Korshikov 1953, Komarek andPerman 1978, Komarek and Fott 1983, Hindak 1984,1988, Comas 1996, Ling and Seppelt 1998). Komarekand Perman (1978) published a first revision of thegenus based on morphological and ecological charac-ters that reduced the number of species to 11. Newspecies descriptions and new combinations were sub-sequently published, changing the number further to16 (Hindak 1977, 1980, 1984, Komarek and Fott1983, Comas 1996, Ling and Seppelt 1998).

Molecular studies of the genus Dictyosphaerium arescarce, and therefore species circumscriptions so farhave mostly followed the traditional morphologicalconcept (Komarek and Perman 1978). The first evi-dence of a polyphyletic nature of the Dictyosphaeriummorphotype based on molecular data was presentedby Krienitz et al. (2010). These authors demonstratedthat this morphotype evolved independently in theChlorella and Parachlorella clades of the Chlorellaceae.A new epitype for the type species D. ehrenbergianumwas designated; this established the genus Dictyosphae-rium as a sister lineage to Parachlorella Krienitz, E. He-gewald, Hepperle, V. Huss, T. Rohr et M. Wolf withinthe Parachlorella clade. A subsequent study focusingon the Dictyosphaerium tetrachotomum Printz morpho-type revealed that this species belongs to the Chlorellaclade, thus establishing the new genus HindakiaC. Bock, Proschold et Krienitz (Bock et al. 2010).The phylogenetic positions of the remaining species[Dictyosphaerium chlorelloides (Naum.) Komarek et Per-man, Dictyosphaerium coacervatum A. Comas Gonzalez,Dictyosphaerium dichotomum Ling et Seppelt, Dictyosp-haerium elongatum Hind., Dictyosphaerium granulatumHind., Dictyosphaerium indicum Iyeng. et Ramanath.,D. pulchellum, Dictyosphaerium primarium Skuja, D.sphagnale Hind., and Dictyosphaerium subsolitarium vanGoor] are still unresolved. Hindak (1988) transferredthe species Dictyosphaerium anomalum Korshikov,Dictyosphaerium botrytella Komarek et Perman, Dictyosp-haerium densum Hindak, and Dictyosphaerium elegans H.Bachm. to the genus Pseudodictyosphaerium Hindak.He used the absence of a pyrenoid as the maincriterion to distinguish Pseudodictyosphaerium fromDictyosphaerium. Unfortunately, there are no authen-tic strains available for any previously described spe-cies of Dictyosphaerium. This lack makes it impossibleto clarify the phylogenetic position of the differentspecies based on molecular data from original material.

In the present study, we focused on the phylogenyand taxonomy of three different Dictyosphaerium spe-cies. We evaluated if the morphology of the speciesconcerned reflects their phylogenetic position withinthe Chlorellaceae. Using the polyphasic approach ofcombined analyses of SSU-ITS phylogeny, secondarystructure of the ITS, and light microscopic observa-

tions, we emend the genus Dictyosphaerium and estab-lish the new genus Mucidosphaerium gen. nov.,describing new species for both genera.

MATERIALS AND METHODS

Algal strains and culture conditions. We obtained the strainsused in this study either from the Culture Collection of Algaeand Protozoa (CCAP, UK), the Culture Collection of Algae atthe University of Gottingen (SAG, Germany), the CultureCollection of Algae at the University of Texas (UTEX, USA),the Coimbra Collection of Algae (ACOI, Portugal), the CultureCollection of Autotrophic Organisms (CCALA, Czech Repub-lic), or from isolation using microcapillaries from field material(for details of origin and isolator, see Table S1 in thesupplementary material). All strains were grown at 15�C undera 14:10 light:dark regime in liquid modified Bourrelly medium(Krienitz and Wirth 2006). For reestablishment of the coloniallife form, the cultures were transferred to agar in petri dishes.

Light microscopic observations. We analyzed cell shape, sizeand arrangement of cells, and connecting strands within thecolonies according to Komarek and Fott (1983), Komarek andPerman (1978), and Hindak (1977) using a light microscopeZEISS Axio Imager Z1 (Carl-Zeiss, Jena, Germany) with thesoftware Axio vision for recording.

DNA isolation, amplification, and sequencing. Algal cells weremechanically disrupted with glass beads using the Tissuelyser II(Qiagen, Hilden, Germany). Total Genomic DNA was isolatedusing the Lysozym ⁄ sodium phosphate method (Allgaier andGrossart 2006). PCR for the SSU rRNA gene and the ITS rRNAwas carried out with the Taq PCR Mastermix Kit (QiagenGmbH) using 0.2 lL of the enzyme with �10 ng of extractedDNA in 40 lL reaction volume. The SSU gene was amplified in agradient thermal cycler (Bio-Rad GmbH, Munich, Germany)with the primer set 18SF and 18SR (Katana et al. 2001) usingthe following profile: initial denaturation at 95�C for 5 min;followed by 33 cycles of denaturation at 94�C for 1 min, anneal-ing at 55�C for 2 min, elongation at 72�C for 3 min; and finalelongation at 72�C for 10 min. The ITS was amplified asdescribed above with the primer set NS7m and LR1850 (Anet al. 1999), using an annealing temperature of 45�C. PCRproducts were purified with the polyethylene glycol precipita-tion after Rosenthal et al. (1993). PCR products were sequencedusing Applied-Biosystems 3130-Genetic-Analyzers (Applied Bio-systems GmbH, Darmstadt, Germany) (for details on usedprimers, see Table 1). The overlapping SSU and ITS partialsequences of each strain were assembled to a complete consen-sus sequence consisting of SSU, ITS-1, 5.8S, ITS-2 regions usingthe software SeqAssam (Hepperle 2004). Overall, 29 strains werenewly sequenced during this study. Part of the genomic DNA wasstored at the BGBM DNA Network (Gemeinholzer et al. 2009).

Phylogenetic analyses. Phylogenetic analyses were performedseparately on the SSU rRNA genes; on the combined ITS-1,5.8S, and ITS-2 rRNA genes; and on a concatenated set of SSU,5.8S, ITS-1, and ITS-2 gene sequences. The data set wasconstructed by adding members of the different genera of theChlorella and Parachlorella clades (Aslam et al. 2007, Bock et al.2010, Luo et al. 2010) to the newly sequenced strains. Inaddition, sequences of strains with the Dictyosphaerium mor-phology published by Krienitz et al. (2010) were included.Marinichlorella was excluded for the combined analyses since nopublished ITS sequences are currently available for this genus.Catena viridis (SAG 65.94) was chosen as outgroup for allanalyses since previous publications (Krienitz et al. 2003, Bocket al. 2010) showed its suitability as outgroup for the Chlorellaand Parachlorella clades. The sequences were manually alignedwith the SequentiX Alignment Editor (Hepperle 2004) basedon the rRNA secondary structure of Micractinium pusillumFresenius as reference (figs. S1 and S2 in Luo et al. 2006).

UPDATING GENUS DICTYOSPHAERIUM AND DESCRIPTION OF MUCIDOSPHAERIUM GEN. NOV. 639

Introns were excluded. The complete alignment of the regionsconsidered is available at Treebase (http://www.treebase.org).Each data set was analyzed by distance (neighbor joining [NJ])and maximum parsimony (MP) using PAUP* (portable version4.0b10) (Swofford 2002). The MP analyses were performedwith heuristic search options based on simple taxon addition,tree-bisection-reconnection (TBR) branch-swapping algorithmand Multrees options enabled. Maximum-likelihood (ML)analyses were performed using Treefinder (Jobb 2008). Forthe combined analyses, the data sets were partitioned, applyingdifferent models to each partition. The partitions, models, andparameters proposed by Treefinder under AICc criteria were asfollows: SSU (1,701 bp, J2+I+G model), ITS-1 (384 bp, J1+Gmodel), 5.8S (139 bp, HKY model), ITS-2 (321 bp, J2+Gmodel). The GenBank accession numbers for all the newsequences produced in the study are included in Table S1. Totest the confidence of the trees topologies, bootstrap analyseswere performed for NJ (1,000 replicates), MP (1,000 repli-cates), and ML (1,000 replicates). Bayesian analyses wereperformed using MrBayes version 3.1 (Huelsenbeck andRonquist 2001). Two runs with four chains of Markov chainMonte Carlo iterations were performed for 2,000,000 genera-tions with tree sampling every 100 generations. For thepartitioned data set, a general time reversible model withgamma shape parameter and proportion of invariable sites(GTR+I+G) was applied to each partition. The parameters wereunlinked and allowed to vary across the partitions. The SSUrRNA analyses were also performed using the GTR+I+G. Thestationary distribution was assumed when the average standarddeviations of split frequencies between two runs was lower than0.01. The first 25% of the calculated trees was discarded asburn-in. A 50% majority-rule consensus tree was calculated forposterior probabilities (PP).

Analyses of the secondary structure. We compared the SSU ofall sequences manually with the secondary structure model ofM. pusillum (CCAP 248 ⁄ 5) (figs. S1 and S2 in Luo et al. 2006)to locate nonhomoplasious synapomorphies (NHS), hemicom-pensatory base changes (h-CBCs), and compensatory basechanges (CBCs) according to Marin et al. (2003) and Coleman(2003, 2007). We constructed the secondary structure of theITS-1 and ITS-2 with the help of mfold (Zuker 2003). To locateNHS, h-CBCs, and CBC in the ITS-1 and ITS-2 of the strains,the program 4SALE was used (Seibel et al. 2006, 2008, Schultzand Wolf 2009). Structures were drawn by PseudoViewer (Byunand Han 2006).

RESULTS

Genera and species descriptions.Dictyosphaerium Nageli 1849.Emended description: Thallus colonial or single-

celled, planktonic, adult cells oval or broadly ellip-

soid to spherical. Chloroplast single, parietal, cupor saucer shaped with ellipsoid to spherical pyre-noid, sometimes indistinct. In some species, twostarch envelopes occur on the surface of the pyre-noid. Cells attached to remnants of the mother cellwall, forming pseudotetrachotomously or pseudodi-chotomously branched colonies. Cell wall smooth orgranular. Colonies surrounded by a mucilaginousenvelope, except in D. coacervatum. Reproduction by2–4 autospores. Oogamy only observed in one spe-cies (D. indicum).

Type species: Dictyosphaerium ehrenbergianum Nageli1849, Gatt. einzell. Algen p. 72, pl. II E.

Dictyosphaerium ehrenbergianum Nageli 1849.Cells in colonies or single, planktonic; adult cells

oval, 6–9 · 4–6 lm, with mucilaginous envelope.Young cells oval, closely appressed, 4–5.5 · 2–3 lm.Release of the autospores by rupture of the mothercell wall that takes place obliquely or horizontally.Colonies 4–32 celled, connected via mucilaginousstalks. The connection between the stalks and thecells occurs on the longer side of the cells. Diameterof colonies up to 80 lm. Chloroplast single, parie-tal, cup or saucer shaped with ellipsoid to sphericalpyrenoid, on which two starch grains occur. It dif-fers from other species of the genus in the 18SrRNA and ITS rRNA sequences.

Holotype: Nageli 1849, Gatt. einzell. Algen p. 72,pl. II E.

Epitype: Strain CCAP 222 ⁄ 1A, cryopreserved at theCulture Collection of Algae and Protozoa, Oban,Scotland; Krienitz et al. 2010, J. Phycol. 46: 560.

Dictyosphaerium libertatis C. Bock, Proschold etKrienitz sp. nov. (Figs. 1a and 6, a–c).

Diagnosis: Cellulae in coloniis vel solitariae, planc-tonicae, tegumento mucilaginoso vestitae. Cellulaeadultae ovales 4–5 (7) · 3–4.5 lm. Propagatio perautosporas ruptura cellularum maternarum obliquevel horizontaliter. Autosporae ovales vel ovoides,arte confines, 4–5 · 3–4.5 lm. Cellulae in coloniis4–16 (raro 32–64), funibus subtilibus mucilaginosisiunctae. Coloniae ad 50 lm latae. Chloroplastus uni-cus, parietalis, poculiformis patelliformisve; pyreno-ides ellipticum vel sphaericum, stratis duobus amylitectum. A speciebus ceteri generis ordine nucleoti-dorum in 18S rRNA et ITS rRNA differt.

TABLE 1. Primers used in this study.

Primer Sequence (5¢–3¢) Target Reference

18SF AACCTGGTTGATCCTGCCAGT SSU Katana et al. (2001)18SR TTGATCCTTCTGCAGGTTCACC SSU Katana et al. (2001)NS7m GGCAATAACAGGTCTGT SSU-ITS An et al. (1999)LR1850 CCTCACGGTACTTGTTC ITS An et al. (1999)528F TGCCAGCAGCYGCGGTAATTCCAGC SSU Marin et al. (1998)536R GWATTACCGCGGCKGCTG SSU Marin et al. (1998)920F GAAACTTAAAKGAATTG SSU Marin et al. (1998)920R TTCCGTCAAT TCCTTTRAGTTTC SSU Modified after Marin et al. (1998)1400F CTGCCCTTTGTACACACCGCCCGTC SSU-ITS Modified after Oliveira and Ragan (1994)GR GGGATCCATATGCTTAAGTTCAGCGGGT ITS Coleman et al. (1994)

ITS, internally transcribed spacer region.

640 CHRISTINA BOCK ET AL.

Cells in colonies or single, planktonic, with muci-laginous envelope. Adult cells oval, 4–5 (7) · 3–4.5lm. Propagation by release of the autospores afterrupture of the mother cell wall obliquely or hori-zontally. Autospores oval or ovoid, closely appressed,4–5 · 3–4.5 lm. Cells in colonies 4–16, rarely 32–64,connected via mucilaginous stalks. Colonies up to50 lm wide. Chloroplast single, parietal, cup or sau-cer shaped; pyrenoid ellipsoid to spherical, coveredby two starch envelopes. It differs from the otherspecies of the genus in the 18S rRNA and ITS rRNAsequences.

Holotype: Material of the authentic strain CCAP222 ⁄ 92 is cryopreserved at the Culture Collection ofalgae and Protozoa, Oban, Scotland.

Isotype: An air-dried and a formaldehyde-fixedsample of the strain CCAP 222 ⁄ 92 were deposited atthe Botanical Museum at Berlin-Dahlem under thedesignation B40 0040720.

Type locality: Artificial pond in Uhuru-park,Nairobi, Kenya (1�17.369¢ S, 36�48.995¢ E).

Etymology: From the Latin libertas (= liberty), refersto type locality Uhuru (= liberty in Swahili).

Dictyosphaerium lacustre C. Bock, Proschold etKrienitz sp. nov. (Figs. 1b and 6, d–f).

Diagnosis: Cellulae in coloniis vel solitariae, planc-tonicae, tegumento mucilaginoso vestitae. Cellulaeadultae globosae vel ovales, 6–8.5 · 4.5–6.5 lm.Propagatio per autosporas ruptura cellularum mater-narum oblique vel horizontaliter. Autosporae ovalesvel reniformes, arte confines, 5.4–6.8 · 3–4.5 lm.Cellulae in coloniae 16–32 (raro 64), funibus subtilismucilaginosis ad latus longum cellularum iunctae.Coloniae ad 45 lm latae. Chloroplastus unicus,parietalis, poculiformis patelliformisve; pyrenoidesstratis duobus amyli tectum. A speciebus ceteri gene-ris ordine nucleotidorum in 18S rRNA et ITS rRNAdiffert.

Cells colonial or single, planktonic, with mucilagi-nous envelope. Adult cells spherical to oval, 6–8.5 ·4.5–6.5 lm. Propagation by release of the autosporesby rupture of the mother cell wall obliquely orhorizontally. Autospores oval to reniform, closelyappressed, 5.4–6.8 · 3–4.5 lm. Cells in colonies 16–32, rarely up to 64, connected via mucilaginousstalks attached at the long side of the cells. Coloniesup to 45 lm wide. Chloroplast single, parietal, cupor saucer shaped; pyrenoid ellipsoid to sphericalpyrenoid, covered by two starch envelopes. It differsfrom other species of the genus by the order of thenucleotides in 18S rRNA and ITS rRNA.


Isotype: An air-dried and a formaldehyde-fixedsample of the strain CCAP 222 ⁄ 62 were deposited atthe Botanical Museum at Berlin-Dahlem under thedesignation B40 0040721.

Type locality: Lake Jabeler See, Mecklenburg-Western Pomerania, Germany (53�31¢36¢¢ N, 12�32¢25¢¢ E).

Etymology: From the Latin lacustre (= living inlakes).

Mucidosphaerium C. Bock, Proschold et Krienitzgen. nov.

Diagnosis: Microalgae virides, sphaericae, plank-tonicae. Chloroplastus unicus, parietalis, poculifor-mis; pyrenoides unicum, amylo tectum. Reproductioasexualis autosporarum liberatione (4 autosporaeper sporangium), reproductio sexualis ignota. Cellu-lae solitariae vel in coloniis 4–64 cellularibus, funi-bus subtilis iunctae, interdum tegumento gelatinosovestitae. A generibus ceteris familiae ordine nucleot-idorum in 18S rRNA et ITS rRNA differt.

Green, spherical, planktonic microalgae. Chloro-plast single, parietal, cup shaped, containing astarch-covered single pyrenoid. Asexual reproduc-tion by autosporulation (four autospores per spo-rangium), sexual reproduction not known. Cells stayattached to the remnants of the mother cell wallafter rupture, forming large colonies with up to 64cells. Cells often single celled in culture owing to

FIG. 1. Illustration of light microscopical characters of the newDictyosphaerium and Mucidosphaerium species. Scale bars, 10 lm(applies to all figures in plate). (a) Dictyosphaerium libertatis fromthe authentic strain CCAP 222 ⁄ 92. Iconotype. (b) Dictyosphaeriumlacustre from the authentic strain CCAP 222 ⁄ 62. Iconotype. (c)Mucidosphaerium palustre from the authentic strain CCAP 222 ⁄ 96.Iconotype. (d) Mucidosphaerium planctonicum from the authenticstrain ACOI 1719. Iconotype.


loss of mucilaginous stalks. Cells single or con-nected by fine stalks to 4- to 64-celled colonies,which are covered by mucilaginous envelopes.Genus differs from other genera of the family bythe order of the nucleotides in the 18S rRNA andITS rRNA sequences.

Typus generis: Mucidosphaerium sphagnale (Hindak)C. Bock, Proschold et Krienitz comb. nov.

Etymology: From the Latin mucidus (= mucous),sphaera (= sphere).

Mucidosphaerium palustre C. Bock, Proschold etKrienitz sp. nov. (Figs. 1c and 6, j and k).

Diagnosis: Cellulae in coloniis vel solitariae, planc-tonicae, tegumento mucilaginoso vestitae. Cellulaeadultae sphaericae, 5–8 lm in diametro. Propagatioper autosporas ruptura cellularum maternarum hor-izontaliter. Autosporae ovales vel reniformes, arteconfines, 4.4–5.7 · 3.2–4.2 lm, funibus subtilibusi-unctae ad apices angustis plus minusve obliqued.Cellulae in coloniis 16–32 (raro 64) cellularibus. Co-loniae usque ad 55 lm in diametro. Chloroplastusunicus, parietalis, poculiformis patelliformisve; py-renoides stratis duobus amyli tectum. A speciebusceteris generis ordine nucleotidorum in 18S rRNAet ITS rRNA differt.

Cells colonial or single, planktonic, with mucilagi-nous envelope. Adult cells spherical, 5–8 lm indiameter. Release of the autospores after rupture ofmother cell wall horizontally. Autospores oval, nar-row oval to asymmetric reniform, closely together,4.4–5.7 · 3.2–4.2 lm, connected via mucilaginousstalks at their narrow end, slightly tilted. Cells in16- to 32-celled (rarely 64) colonies. Diameter ofcolonies up to 55 lm. Chloroplast single, parietal,cup or saucer shaped with ellipsoid to sphericalpyrenoid, covered by two starch envelopes. It differsfrom other species of the genus by the order of thenucleotides in 18S rRNA and ITS rRNA.


Isotype: An air-dried and a formaldehyde-fixedsample of strain CCAP 222 ⁄ 96 were deposited at theBotanical Museum at Berlin-Dahlem under the des-ignation B40 0040722.

Type locality: Fish pond Klimpsmoorsheide Lune-stedt, Lower Saxony, Germany (53�26¢20.03¢¢ N.8�46¢47.58¢¢ E).

Etymology: From the Latin palustre (= living inswamps).

Mucidosphaerium planctonicum C. Bock, Proscholdet Krienitz sp. nov. (Figs. 1b and 6l).

Diagnosis: Cellulae in coloniis vel solitariae, planc-tonicae, tegumento mucilaginoso vestitae. Cellulaeadultae sphaericae, 5.2–7 lm in diametro. Propaga-tio per autosporas ruptura cellularum maternarumoblique vel horizontaliter. Autosporae ovales velelongatae, 5.3–6.5 · 3.5–4.5 lm, funibus subtilibusiunctae ad apices angustis plus minusve oblique.Cellulae in coloniis, 4–16 (raro 64) cellularibus.

Coloniae usque ad 55 lm latae. Chloroplastus uni-cus, parietalis, poculiformis patelliformisve; pyreno-ides stratis duobus amyli tectum. A speciebus ceterisgeneris ordine nucleotidorum in 18S rRNA et ITSrRNA differt.

Cells colonial or single, planktonic, with mucilagi-nous envelope. Adult cells spherical, 5.2–7 lm.Release of the autospores after rupture of mothercell wall obliquely or horizontally. Autospores ovalto elongate, 5.3–6.5 · 3.5–4.5 lm, connected viamucilaginous stalks at their narrow end, slightlytilted. Cells in 4- to 16-celled (rarely 64) colonies.Diameter of colonies up to 55 lm. Chloroplast sin-gle, parietal, cup or saucer shaped, ellipsoid tospherical, pyrenoid covered by two starch grains. Itdiffers from other species of the genus by the orderof the nucleotides in 18S rRNA and ITS rRNA.

Holotype: An air-dried and a formaldehyde-fixedsample of the strain ACOI 1719 were deposited atthe Botanical Museum at Berlin-Dahlem under thedesignation B40 0040723.

Isotype: Material of the authentic strain ACOI1719 is maintained at the Coimbra Collection ofAlgae, Portugal.

Type locality: Abrantes, Campo Militar de Sta Mar-garida, Barragem do Monte Novo.

Etymology: From the Latin plancton (= plankton).Mucidosphaerium sphagnale (Hindak) C. Bock,

Proschold et Krienitz comb. nov.Cells colonial or single, planktonic, adult cells

spherical to widely oval, 4–6 (7.5) lm, with mucilagi-nous envelope. Young cells elongate to semilunate,4–5.8 · 2.5–3.7 lm. The autospores slant after rup-ture of mother cell wall in 180�, connected at theirnarrow ends to the mucilagenous stalks, slightlytilted. Colonies 4–16 celled. Diameter of coloniesup to 50 lm. Chloroplast single, parietal, cup orsaucer shaped, ellipsoid to spherical, pyrenoid cov-ered by two starch grains. It differs from other spe-cies of the genus by the order of the nucleotides in18S rRNA and ITS.

Basionym: Dictyosphaerium sphagnale Hindak 1980,Biol. Prace 26(6): 54.

Synonym: Dictyosphaerium elegans Bachm. sensuSkuja 1956 et Hindak 1977.

Holotype: Fig. 105:1 in Hindak 1980, Biol. Prace26(6): 54.

Epitype (designated here): Material of the authen-tic strain CCAP 222 ⁄ 93 is cryopreserved at theCulture Collection of Algae and Protozoa, Oban,Scotland.

Mucidosphaerium pulchellum (H. C. Wood) C. Bock,Proschold et Krienitz comb. nov.

Cells colonial or single, planktonic, adult cellsspherical, 6–8 lm, with mucilaginous envelope.Cells first distance from each other, later closelytogether. Young cells asymmetric oval to ovoid,(2.8)3.1–5.8()7) · 3.0–4.5()6) lm. Release of theautospores after rupture of mother cell wall hori-zontally. Colonies 16–32 celled, rarely up to 64


celled, connected via mucilaginous stalks. Youngcells connected at their narrow end, adult cells atthe side of location of the chloroplast containingthe pyrenoid. Diameter of colonies up to 80 lm.Chloroplast single, parietal, cup or saucer shaped,ellipsoid to spherical, pyrenoid covered by twostarch grains. It differs from other species of thegenus by the order of the nucleotides in 18S rRNAand ITS rRNA.

Basionym: Dictyosphaerium pulchellum H. C. Wood1872, Smiths. Contr. Knowledge 19(241): 84.

Holotype: Tab. 10:4 in Wood 1872, Smiths. Contr.Knowledge 19(241): 84.

Epitype (designated here): Material of the strainUTEX 731 is deposited at the Culture Collection ofAlgae at the University of Texas at Austin.

Phylogenetic analyses. The SSU rRNA tree showedthe separation of the Chlorellaceae in the Chlorellaand Parachlorella clades (Fig. S1 in the supplemen-tary material). The different genera within these twoclades were not resolved, except for Marinichlorella,which formed an independent lineage at the base ofthe Parachlorella clade. The topologies recoveredfrom the analyses of the partitioned data set ITS-1,5.8S, ITS-2 (Fig. S2 in the supplementary material)and the concatenated data set SSU, ITS-1, 5.8S,ITS-2 (Fig. 2) were in general congruent. Discrepan-cies were only observed in the placement ofsubclades with weak or no statistical support. Withinthe phylogenetic tree of the combined SSU and ITSrRNA sequences (Fig. 2), species with the Dictyosphae-rium morphotype occurred in three differentsubclades: the Dictyosphaerium subclade, the Mucidosp-haerium subclade, and an independent subcladeof only two strains (CCAP 222 ⁄ 1C, ACOI 1988). Thebranching order of these subclades and of the genusParachlorella was not resolved. The Dictyosphaeriumsubclade was moderately to highly supported (0.99PP ⁄ 61–98 bootstrap percentages [BP]), andincluded two new independent lineages correspond-ing to the new species D. libertatis and D. lacustre.

The new Mucidosphaerium subclade consisted ofseveral strains from Europe and one from Canada.This new subclade was moderately to highly sup-ported (1.0 PP ⁄ 73–88 BP). Within this subclade,four lineages could be detected, corresponding tothe four different species M. sphagnale, M. palustre,M. pulchellum, and M. planctonicum. M. palustre wassister to M. sphagnale with high statistical support inall analyses (1.0 PP ⁄ 97–100 BP). The branchingorder of the remaining species was not resolved inthese analyses.

Secondary structure. The secondary structure ofthe ITS-1 and ITS-2 of Dictyosphaerium and Mucido-sphaerium showed the typical branching pattern gen-erally found in the Chlorellaceae with four heliceseach. D. ehrenbergianum (CCAP 222 ⁄ 1A) andM. sphagnale (CCAP 222 ⁄ 93) differ in the ITS-1 ineight CBCs and one h-CBC; in the ITS-2, in 17CBCs and three h-CBCs. Comparison of the struc-

ture between the authentic strains of the Dictyosphae-rium species (CCAP 222 ⁄ 1A, CCAP 222 ⁄ 92, CCAP222 ⁄ 62) and Mucidosphaerium species (CCAP222 ⁄ 93, CCAP 222 ⁄ 96, UTEX 731, ACOI 1719)revealed several h-CBCs and CBCs. Interspecies basechanges in Dictyosphaerium range from three to eightCBCs and one to two h-CBCs in the ITS-1, and fourto 10 CBCs and three to five h-CBCs in the ITS-2(Fig. 3). Base changes in Mucidosphaerium ranged,respectively, from 6 to 14 CBCs and 3 to 6 h-CBCswithin the ITS-1, and 5 to 13 CBCs and 2 to 3 h-CBCs in the ITS-2 (Fig. 4).

To test if we could discover generic signaturesaccording to Luo et al. (2010) among different lin-eages within the Parachlorella clade, we comparedthe secondary structure of the SSU and 5.8S. Weexcluded the lineage of strain CCAP 222 ⁄ 25, whichrequires further sampling until an unambiguousplacement at the genus level may be proposed. Theanalyses of the different lineages revealed specificchanges that we consider indicative of separation atgenus level; these are summarized in Figure 5. Ele-ven positions could be identified as unique for someof the genera. The combination of the base changesat each position can be used as marker for thedifferent lineages. For example, the genus Dictyo-sphaerium can be distinguished from Mucidosphaeriumat the position 7, where only Mucidosphaerium showsa C instead of an A. A similar pattern can be foundfor any lineage within the Parachlorella clade, includ-ing Marinichlorella, for which we could only analyzethe SSU.

Morphological observation by LM. The connectionof the cells to mucilaginous stalks emerging fromthe center of the colonies and the presence of a sur-rounding mucilaginous envelope were featuresshared by Dictyosphaerium and Mucidosphaerium. Theoval cell form of adult cells and the connectionto the stalks at the broader side of the cellsdistinguished members of Dictyosphaerium from thespherical adult cells of Mucidosphaerium. Themorphological characteristics of the different spe-cies of Dictyosphaerium and Mucidosphaerium are sum-marized in Table 2.

The adult cells of D. libertatis (CCAP 222 ⁄ 92)showed the smallest size (4–5 [7] · 3–4.5 lm)(Fig. 6, a–c) compared with the two other Dictyo-sphaerium species (D. lacustre 6–8.5 · 4.5–6.5 lm;D. ehrenbergianum 6–9 · 4–6 lm). The broadly ovalcells of D. lacustre became almost spherical duringaging, distinguishing this species from the others(Fig. 6, d–f). The young cells were oval to subovoidin D. libertatis, whereas D. ehrenbergianum showedmainly oval cells and D. lacustre oval or sometimesreniform to broadly ellipsoid cells. The number ofcells per colony ranged for all species from four to32, except for D. libertatis (for which it was rare toobserve >16 cells per colony).

The adult cells of M. sphagnale (Fig. 6g) hada smaller cell size (4–6 lm) than the other


Mucidosphaerium species (M. pulchellum, 6–8 lm; M.palustre, 5–8 lm; M. planctonicum, 5.2–7 lm). Themain character distinguishing this species was themode of release of the autospores. The autosporesslanted from the mother cell at an angle of 180�and joined the mucilaginous stalks at their narrowend, as it was already reported in the originaldescription of this species. In our observations, the

connection was in most cases slightly tilted to thelonger side of the young cells. The autospores ofall other Mucidosphaerium strains slid more or lesshorizontally or obliquely out of the remnants ofthe mother cell wall. The young cells of M. sphagnaleshowed in our strains the typical elongated tosemilunate cell form. They were larger thanpreviously reported (4–5.8 · 2.5–3.7 lm instead of

FIG. 2. Maximum-likelihood (ML) phylogenetic tree of the Chlorellaceae as inferred from concatenated rRNA gene sequence set ofSSU, ITS-1, 5.8S, and ITS-2. Support values correspond to Bayesian PP, ML BP, maximum-parsimony BP, and neighbor-joining BP.Hyphens correspond to values <50% for BP and <0.95 for PP. Branch lengths represent substitutions per site. BP, bootstrap percentages;ITS, internally transcribed spacer region; PP, posterior probability.


FIG. 3. Comparison of the secondary structure of the ITS-1 and ITS-2 according to Coleman (2003) of the authentic strains of Dictyo-sphaerium (CCAP 222 ⁄ 1A = D. ehrenbergianum, CCAP 222 ⁄ 92 = D. libertatis, CCAP 222 ⁄ 62 = D. lacustre). Base changes are marked in blackboxes, and additional base pairs in white boxes. ITS, internally transcribed spacer region.


FIG. 4. Comparison of the secondary structure of the ITS-1 and ITS-2 according to Coleman (2003) of the authentic strains of Mucido-sphaerium (CCAP 222 ⁄ 93 = M. sphagnale, CCAP 222 ⁄ 96 = M. palustre, UTEX 731 = M. pulchellum, ACOI 1719 = M. planctonicum). Basechanges are marked in black boxes, and additional base pairs in white boxes. ITS, internally transcribed spacer region.


2–4 · 1.6–2.4 lm). Typical for this species was thatthe colony consisted mainly of 4–16 cells. The ori-ginal description of D. pulchellum from Wood(1872) was very vague in terms of cell size andshape of adult and young cells. It was redefined byKomarek and Perman (1978), who reported forthe adult cells a size of 6–8 lm and for the youngcells of (2.8–) 4–6 · (2.5–) 4–5.5 lm. In our obser-vations, M. pulchellum matched the cell size of theadult cells, whereas the young cells were irregularoval to asymmetric ovoid, 3–5.8 · 3–4.4 lm (Fig. 6,

h and i). The young cells were attached to thestalks at their narrow end and subsequently on theside of the chloroplast containing the pyrenoid. M.palustre differed from M. pulchellum in the slightlysmaller cell size; young cells were oval to reniformand connected to the stalks slightly tilted at thebroader side of the cells (Fig. 6, j and k). M. planctoni-cum showed fine gelatinous stalks in the older coloniesand oval to elongated young cells, which were largerthan in the other species (5.3–6.5 · 3.5–4.5 lm)(Fig. 6l).

FIG. 5. Comparison of the secondary structure of the SSU, 5.8S, and part of the LSU of the Parachlorella clade. Dictyosphaerium (CCAP222 ⁄ 1A) was chosen as core structure. NHS, h-CBCs, and CBCs are marked in circles. CBC, compensatory base change; h-CBCs, hemicom-pensatory base changes; NHS, nonhomoplasious synapomorphies.


DISCUSSION

Our molecular analyses combined with the mor-phological comparison of taxa with Dictyosphaerium-like morphology led to the erection of the newgenus Mucidosphaerium within the Parachlorella cladeof the Chlorellaceae. The phylogenetic position ofthis new genus cannot be resolved in our molecularanalyses due to lack of statistical support at thecorresponding nodes. Nevertheless, by comparingD. ehrenbergianum (strain CCAP 222 ⁄ 1A) withM. sphagnale (CCAP 222 ⁄ 93), we found nine CBCs(including h-CBCs) within the ITS-1 and 20 CBCs(including h-CBCs) within the ITS-2. Krienitz et al.(2010) detected a similar number of CBCs by com-paring Parachlorella beijerinckii (strain SAG 2046) withD. ehrenbergianum (strain CCAP 222 ⁄ 1A) (nine CBCsin the ITS-1; 18 CBCs in the ITS-2) and used this asan additional criterion to separate Dictyosphaeriumfrom Parachlorella. The morphology of this newgenus is characterized by spherical cells, colonial lifeform, and a surrounding mucilaginous envelope; itis easily distinguished from Dictyosphaerium for thedifferent shape of the cells. The species D. pulchel-lum and D. sphagnale are transferred to this genus.Members of the genuine Dictyosphaerium show onlyoval adult cells with the connection to the mucilagi-nous stalks at their broader side, as it is typical forthe type species D. ehrenbergianum. Several new spe-cies could be observed within both genera.

In a first attempt to revise the genus Dictyosphaeriumbased on molecular data, the polyphyletic origin ofthe Dictyosphaerium morphotype was demonstrated,and a new epitype for the type species D. ehrenbergia-num within the Parachlorella clade was established(Krienitz et al. 2010). Our analyses revealed twonew lineages in close relationship with the authenticstrain of D. ehrenbergianum (CCAP 222 ⁄ 1A). The cor-responding strains show differences in cell size andshape, and we observed considerable differences inthe secondary structure of the ITS-1 and ITS-2among the lineages. As demonstrated in previousstudies, one CBC in a conserved region of the ITS-2can be used for species delineation (Coleman 2003,2009, Seibel et al. 2008, Ruhl et al. 2009). It is fur-ther shown that the absence or presence of CBCs inthe ITS-2 predicts in some cases sexual compatibil-ity, for example, in some microalgae or the proto-zoan Paramecium aurelia complex (Coleman 2003,2005, Muller et al. 2007). On the basis of morpho-logical differences and the CBC content in theITS-1 and ITS-2 between the three lineages, wedescribe the two new species D. libertatis sp. nov.and D. lacustre sp. nov.

Species with the Dictyosphaerium morphotype con-taining pyrenoids and spherical cells have been iden-tified either as D. pulchellum or D. sphagnale (Hindak1977, Komarek and Fott 1983). Since they differ onlyslightly in the morphology of the young cells, habitatpreferences have been used for distinction of these

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species. Morphotypes occurring in bogs were mostlyreferred to as D. sphagnale, whereas morphotypesfound in others habitats were referred to D. pulchel-lum. We analyzed 14 different strains of the sphericalmorphotype. Our results demonstrated that theybelong to the Parachlorella clade but are not membersof the genus Dictyosphaerium. The new genus Muci-dosphaerium is established next to Dictyosphaerium andParachlorella. Five strains from different bog lakes

match the original description of D. sphagnale withsmall differences in the cell size of the young cells.D. sphagnale is transferred here to Mucidosphaerium.Wood (1872) described D. pulchellum, providing aniconotype and a short description of the morphology.Subsequently, the species was better characterizedby Komarek and Perman (1978). On the basis ofdescription of Komarek and Perman, we identifiedfour strains from culture collections as D. pulchellum,

FIG. 6. Microphotographs of selected species of the genera Dictyosphaerium and Mucidosphaerium. Scale bars, 10 lm (applies to all fig-ures in plate). (a, b) D. libertatis (strain CCAP 222 ⁄ 92). (c) D. libertatis (strain CCAP 222 ⁄ 92). The culture is stained with drawing ink todemonstrate the mucilaginous envelope surrounding the colony. (d) D. lacustre (strain CCAP 222 ⁄ 85). (e, f) D. lacustre (strain CCAP222 ⁄ 62). (g) M. sphagnale (strain KR 2009 ⁄ 1). (h, i) M. pulchellum (strain UTEX 731). (j, k) M. palustre (strain CCAP 222 ⁄ 96). (l) M. planc-tonicum (strain ACOI 1719).


and we transfer this species to Mucidosphaerium. Inaddition to these two new combinations, two strainsrepresentative of new lineages belong to Mucido-sphaerium. Both show spherical cells and differ intheir morphology from the above-mentioned species(Table 2). We detected CBCs and h-CBCs in theITS-1 and ITS-2 between these lineages to furthersupport their delineation as new species, M. palustresp. nov. and M. planctonicum sp. nov.

The spherical morphotype of Dictyosphaerium hasalso been observed in the genus Heynigia (Bocket al. 2010) and in some species of Chlorella (Krie-nitz et al. 2004, Luo et al. 2010). This polyphyly isstrongly supported by our results. Species identifica-tion based only on light microscopic observation ishighly difficult in this morphotype because a mor-phological character separating these taxa unam-biguously is lacking.

The genus Dictyosphaerium formerly consisted of16 species (Komarek and Fott 1983, Hindak 1984,Comas 1996, Ling and Seppelt 1998). The specieswithout pyrenoids (D. botrytella, D. densum, D. anom-alum, D. elegans) were transferred by Hindak (1988)to the genus Pseudodictyosphaerium. Recent molecularstudies confirmed this separation but revealed thatspecies of Pseudodictyosphaerium are nested withinthe genus Mychonastes P. D. Simpson et Van Valken-burg (Krienitz et al. 2011). Studies focusing onD. tetrachotomum (spherical cells, attachments at theirnarrow side), which is one of the most common spe-cies in inland waters, revealed that this species isnot closely related to D. ehrenbergianum. Strains withthe morphology typical of D. tetrachotomum form amonophytletic group within the Chlorella clade, cor-responding to the genus Hindakia (Bock et al.2010). The species D. chlorelloides and D. elongatumare nested within the genus Chlorella (Bock et al.2011). The phylogenetic positions of D. indicum, D.subsolitarium, and D. granulatum remain unresolved.D. indicum is the only species of Dictyosphaerium forwhich an oogamic stadium was observed in additionto the vegetative cells (Iyengar and Ramanathan1949). Unfortunately, there are no authentic strainsavailable for this species. D. subsolitarium has smallcells (1.5–3 lm) and a barely visible pyrenoid(Komarek and Fott 1983). We could not identify thismorphology in any strain examined. D. granulatumis characterized by granules on the cell walls; thischaracter was not observed in any strain. Ling andSeppelt (1998) described a new species of Dictyo-sphaerium from the Antarctic region, D. dichotomum.Unfortunately, the culture was lost during the jour-ney due to saltwater inflow, and only a drawing ofthis species exists. Comas (1996) described a newspecies from Cuba, D. coacervatum. This species hasbroad stalks and lacks the typical mucilaginousenvelope. New collections of these species will benecessary to clarify their phylogenetic positions.

On the basis of molecular and morphologicaldata, we have established the new genus Mucido-

sphaerium. The spherical-celled morphotype is ofpolyphyletic origin within the Chlorellaceae. Lightmicroscopic observations on algae exhibiting thismorphology are difficult and bear a high risk ofmisidentification. Further studies considering othercharacters (for example, the ultrastructure of thecell wall) are needed to reveal any morphologicaldifferences between the genera and species with thismorphological habit.

This research was supported by the German Science Founda-tion (DFG) (KR 1262 ⁄ 11-1, 2) and NERC Oceans 2025 andNERC MGF 154 sequencing grant. We thank E. Novelo forproviding two strains, and M. Degebrodt and M. Papke fortechnical assistance. We thank T. Mehner and the partici-pants of the workshop ‘‘Scientific Writing’’ for helpful discus-sions on an early draft of this article. We are grateful toFabio Rindi and an unknown reviewer for the constructivecomments.

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Supplementary Material

The following supplementary material is avail-able for this article:

Figure S1. Maximum-likelihood (ML) phyloge-netic tree (50% majority rule consensus) of theChlorellaceae as inferred from SSU genesequences.

Figure S2. Maximum-likelihood (ML) phyloge-netic tree (50% majority rule consensus) of theChlorellaceae as inferred from concatenatedrRNA gene sequence set of ITS-1, 5.8S, and ITS-2.

Table S1. List of strains used in this study.

This material is available as part of the onlinearticle.

Please note: Wiley-Blackwell are not responsi-ble for the content or functionality of any sup-porting materials supplied by the authors. Anyqueries (other than missing material) should bedirected to the corresponding author for thearticle.


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