Chlorella variabilis and Micractinium reisseri sp. nov. - Algaebase.org

Phycological Research 2010; 58: 188–201

Chlorella variabilis and Micractinium reisseri sp. nov. (Chlorellaceae, Trebouxiophyceae): Redescription of the endosymbiotic green algae of Paramecium bursaria (Peniculia, Oligohymenophorea) in the 120th year pre_579

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Ryo Hoshina,1 Mitsunori Iwataki3 and Nobutaka Imamura2* 1

Department of Biomedical Science, College of Life Sciences, 2Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, and 3Institute for East China Sea Research, Nagasaki University, Nagasaki, Japan

SUMMARY Symbiotic algae of the ciliate Paramecium bursaria (Ehrenberg) Focker are key species in the fields of virology and molecular evolutionary biology as well as in the biology of symbiotic relationships. These symbiotic algae were once identified as Zoochlorella conductrix Brandt by the Dutch microbiologist, Beijerinck 120 years ago. However, after many twists and turns, the algae are today treated as nameless organisms. Recent molecular analyses have revealed several different algal partners depending on P. bursaria strains, but nearly all P. bursaria contains a symbiont belonging to either the so-called ‘American’ or ‘European’ group. The absence of proper names for these algae is beginning to provoke ill effects in the above-mentioned study areas. In the present study, we confirmed the genetic autonomy of the ‘American’ and ‘European’ groups and described the symbionts as Chlorella variabilis Shihira et Krauss and Micractinium reisseri Hoshina, Iwataki et Imamura sp. nov., respectively (Chlorellaceae, Trebouxiophyceae). Key words: Chlorella variabilis, ITS2, Micractinium reisseri, Paramecium bursaria, species concept.

INTRODUCTION The unicellular ciliate Paramecium O. F. Müller (Peniculia, Oligohymenophorea) is one of the most studied protists (Fokin et al. 2004). Within this genus, P. bursaria (Ehrenberg) Focker is an invaluable asset. Paramecium bursaria is a single-celled protozoan that maintains several hundred algal cells within its own cytoplasm, lending it a green color (Karakashian et al. 1968). Thus, P. bursaria is known as the green Paramecium. The alga lives inside the ciliate, providing it with photosynthate, while the ciliate provides the alga

with protection from other protozoans or viruses and chauffeurs it to brightly lit areas for optimal photosynthesis (Hoshina & Imamura 2009a). Therefore, P. bursaria is a ciliate that, like corals and lichens, has established a mutualistic relationship with an algal species. Unlike in corals or lichens, even though P. bursaria is intrinsically a heterotrophic protist, the daughter cells inherit the same symbionts that were retained by the mother cell (Siegel 1960). Although the systematics of the chlorophytes remains somewhat disorganized, researchers have determined the progenitor, identified the known species, and described new species for the Paramecium symbiont. The genus Zoochlorella Brandt was introduced in 1882 by Brandt, who so designated algae isolated from the green coelenterate Hydra viridis Linnaeus (Z. conductrix Brandt) and sponges (Z. parasitica Brandt). The P. bursaria symbiotic alga was later assigned to Z. conductrix by Beijerinck (Beijerinck 1890), although Brandt (1882) appeared to have regarded the symbiont as the same as that possessed by hydra. Today, the first-described Zoochlorella is considered synonymous with the well-known genus Chlorella Beijerinck (Silva 1999). Although Zoochlorella is an older name than Chlorella, the genus Chlorella was determined to be conserved with the type species C. vulgaris Beijerinck, and consequently, Zoochlorella was rejected (Appendix IIIA, Greuter et al. 2000). The symbiotic alga was later renamed as an independent species, ‘C. paramecii’ nom. nud., by Loefer. However, he did not publish a description for this nomenclature, for which reason Shihira and Krauss (1965) later rejected ‘C. paramecii’ and instead

*To whom correspondence should be addressed. Email: [email protected] Communicating editor: M. Hoppenrath. Received 1 September 2009; accepted 25 February 2010. doi: 10.1111/j.1440-1835.2010.00579.x

© 2010 Japanese Society of Phycology

Taxonomy of the photobionts of Paramecium

described a new species, C. variabilis Shihira et Krauss, based on authentic strains no. 130 from the Indiana Algal Culture Collection (which was later moved to the Culture Collection of Algae at the University of Texas [UTEX]; the strain is currently not available) and no. 211/6 from the Cambridge Collection (which was later moved to the Culture Collection of Algae and Protozoa [CCAP], UK; this strain is also currently not available), which were claimed to be Loefer’s ‘C. paramecii’. However, strain 211/6 showed distinct biochemical and physiological characteristics compared with the other strains of P. bursaria symbionts (later identified as Auxenochlorella protothecoides [Krüger] Kalina et Puncˇochárˇová, Douglas & Huss 1986; Kessler & Huss 1990), raising questions as to whether it was a symbiont at all. Consequently, the species name C. variabilis is no longer in use. A duplicate of 211/6 was transferred to the Culture Collection of Algae at the University of Göttingen (SAG), Germany, as strain 211-6. This strain, however, showed symbiotic characteristics (Kessler & Huss 1990). Modern observations of the symbiotic algae are represented by the studies conducted by German researchers (Professors E. Kessler, W. Reisser, V.A.R. Huss, and their coworkers). These researchers have examined algal strains isolated from P. bursaria collected in Germany and the United States, strains Pbi and NC64A, respectively (NC64A(M) and NC64A(P) appear in some literature; the former is regarded as the true NC64A, and the latter is unlikely to be of symbiotic origin; Douglas and Huss 1986). Their studies demonstrated that the ‘American’ and ‘European’ algal strains each possess certain distinct characteristics. The general conclusion of these taxonomic studies was that no reason exists to distinguish the symbiotic algae from the genus Chlorella. Rather, one may reasonably assume that these symbionts are derived from free-living Chlorella spp. but with some evolved characteristics. A series of symbiont analyses resulted in the discovery of the Chlorella virus, which specifically infects the symbiotic alga NC64A and other ‘American’ symbionts but does not infect ‘European’ symbionts or free-living Chlorella; likewise, a distinct virus specifically infects Pbi and other ‘European’ symbionts (Reisser et al. 1988). Thus, these viruses were designated the NC64A and Pbi viruses, respectively. Reisser et al. (1990) confirmed that each virus is able to identify its host species based on a key factor present in the algal cell wall. Takeda (1995) analyzed the cell wall sugar composition of symbiotic algae. He noted that the characteristics of the symbiont cell wall indicated a relationship with Chlorella species (especially C. kessleri Fott et Nováková); however, the distinct proportion of compositional sugars indicated that the symbiotic algae belong to a new species. Thus, the question of whether symbiotic © 2010 Japanese Society of Phycology

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algae represent an independent species has become an increasingly important focus in more recent studies. Isozyme distribution patterns for several enzymes have demonstrated uniformity within the ‘American’ vs. ‘European’ algae and multiplicity among different groups (i.e. ‘American’, ‘European’, and free-living Chlorella spp.; Linz et al. 1999). Similar results were also obtained through the analysis of universal primer polymerase chain reaction (UPPCR) fragmentation patterns (Kvitko et al. 2001). Recent molecular analyses have revealed many facts about P. bursaria symbiotic algae: (i) P. bursaria almost always contains single cloned algae as naturally occurring photobionts; (ii) in most cases, the symbionts are either ‘American’ or ‘European’, but in two exceptional cases, Chlorella vulgaris and Coccomyxa sp. have been identified; (iii) the ‘American’ and ‘European’ groups are characterized by different length small subunit (SSU) rDNA due to the varying number of group I intron insertions (three-intron or single-intron); (iv) each group (‘American’ or ‘European’) of algae has a highly uniform SSU rDNA (including introns)-ITS1-5.8S rDNA-ITS2 sequence; (v) ‘American’ and ‘European’ sequences differ by only seven or eight nucleotides in the SSU rDNA (exon), whereas the ITS sequences exhibit significant differences (approximately 20%); (vi) both the ‘American’ and ‘European’ groups belong to the Chlorella clade (sensu Krienitz et al. 2004); (vii) each group is equivalent to a species discrete from any known free-living species; (viii) ancestral P. bursaria may have obtained the ‘American’ and ‘European’ algae separately; and (ix) the affiliation of SAG 211-6 to the ‘American’ type has also become apparent (Hoshina et al. 2004, 2005; Gaponova et al. 2007; Summerer et al. 2008; Hoshina & Imamura 2008a; Luo et al. 2010). Both the ‘American’ and ‘European’ groups have attracted attention because of their particularly evolved group I introns (Hoshina & Imamura 2008b, 2009b). Johansen and Haugen (2001) proposed a nomenclature system for the rDNA group I introns based on the host species name (one-letter abbreviation of the genus name and two-letter abbreviation of the specific epithet) and insertion site; this system has been well accepted in the arena of intron study. However, because of the lack of a species name for the symbionts, this rule cannot be applied to their introns. In virology, the Chlorella virus has developed into a very important taxon possessing a large dsDNA genome that encodes many proteins with some unique features. The virus led to the establishment of the viral genus Chlorovirus (family Phycodnaviridae) based on three criteria: viruses that infect P. bursaria ‘American’ symbionts, viruses that infect P. bursaria ‘European’ symbionts, and viruses that infect the green hydra symbionts (for a review on the Chlorella virus, see Van Etten 2003). The

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namelessness of these algal symbionts has coerced virologists into using strange terminologies, such as the NC64A virus and the Pbi virus. The greatest harm associated with the anonymity is the irresponsible species identifications that subsequently generate a chain reaction of misidentification and further disorder. In fact, three sequence entries of ‘American’ symbionts are registered as ‘Chlorella vulgaris’ (AB191205-207), and according to these misidentified entries, further misidentifications of the symbionts have already occurred (Kodama et al. 2007; Kodama & Fujishima 2008, 2009a,b). Albeit an exceptional case, true C. vulgaris has been found from one P. bursaria strain (Hoshina & Imamura 2008a), which further complicates matters. The underlying problem is clear. Each ‘American’ and ‘European’ symbiont must be assigned a species name as soon as possible to avoid further taxonomical confusion. The P. bursaria symbionts will fascinate protozoologists in the decades ahead and will remain important study species for molecular evolutionary biologists and virologists. The current International Code of Botanical Nomenclature notes that ‘The purpose of giving a name to a taxonomic group is not to indicate its characters or history, but to supply a means of referring to it and to indicate its taxonomic rank’ (Preamble, McNeill et al. 2006). Today, most, if not all, of the endosymbiotic algae of P. bursaria seem to be autonomous from other known species. Therefore, today, 120 years after Beijerinck identified a symbiont alga from P. bursaria, we redescribe the ‘American’ and ‘European’ algae to provide a means of referencing them correctly.

MATERIALS AND METHODS Culture Cells of P. bursaria symbiont strains ATCC 50258 (NC64A) and CCAP 211/83 (Pbi) were cultured in C medium (Ichimura 1971) with 2.3 mM casamino acids. Cells were maintained under fluorescent illumination (16 : 8 h light : dark (LD), 50 mmol photons m-2 s-1) at 25°C.

Microscopy Cells of symbiotic algae were observed under light microscopy (BX51; Olympus, Tokyo, Japan), and photos were taken with an Olympus model DP50 digital camera. For transmission electron microscopy, the cultured cells were prepared and investigated according to Iwataki et al. (2002) and were examined using a JEM 1010 (JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV.

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DNA extraction, amplification, and sequencing For DNA extraction, we used a DNeasy plant mini kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s instructions. We designed two reversedirection primers, Ls3Ce2 (5′-CGA ACC ACG GCT GAA TCT-3′) and Ls3Ce3 (5′-CGA ACC ACG GCT GAA TCT C-3′); both anneal to the 3′ side of the H2836 helix (named large subunit (LSU) rRNA in Escherichia coli). The Pbi rRNA cistron was amplified using PCR with the following eight primer pairs: SR-1/SR-5, SR-4/SR-9, SR-8/SR12, INT4F/HLR3R, HLR0F/LR5, HLR5F/LR8, HLR7F/HLR9R, and HLR9F/Ls3Ce3. The primers used for PCR and sequencing are described in Hoshina et al. (2004) and Hoshina and Imamura (2008b). The LSU rDNA 3′ end of NC64A was amplified with the primer pair HLR10Fk/Ls3Ce2. The PCR products were confirmed using agarose gel electrophoresis, purified via polyethylene glycol precipitation, and then sequenced directly. A fragment of the HLR9F/Ls3Ce3 (Pbi) product could not be read by direct sequencing; thus, this region was re-amplified with the primer pair HLR10F/LR12k, which was subcloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA). The subcloning procedure defined polymorphisms with one nucleotide indels within the L2449 intron, indicating that the sequence was made unreadable by one of the nucleotide indels.

SSU rDNA + ITS2 phylogeny We collected chlorellacean strains with published SSU rDNA and ITS2 sequences from GenBank in May 2009. Redundant strains of some species, for example, data from more than 10 strains of ‘American’ symbionts, were omitted. We used two sequence datasets of X74001 (SSU rDNA) with AY323463 (ITS2), and FM205860 (contains both SSU rDNA and ITS2) for strain 260 of the Culture Collection of Autotrophic Organisms (CCALA), Czech Republic. The SSU rDNA sequences were initially aligned using Clustal X version 2.0.10 (Larkin et al. 2007) and then manually aligned taking into consideration secondary structure models for Chlorella vulgaris (Huss & Sogin 1990). Introns and 5′ and 3′ terminal regions were removed; thereafter, 1752 aligned sites remained (these alignment data are available at our website, http://www.ritsumei.ac.jp/pharmacy/imamura/chlorella. html/). The ITS2 sequences were folded using Mfold 3.2 (Mathews et al. 1999; Zuker 2003), and the resulting secondary structures were used to assist in the manual alignment of the ITS2 sequences. We excluded rapidly evolving helices I and IV as well as the top half of helix II and the top of helix III because of unreliable alignment. Consequently, 136 sites remained; this © 2010 Japanese Society of Phycology


ITS2 alignment is shown in Supplementary Fig. S1. Further analyses were conducted using the combined data from the SSU rDNA and ITS2 alignments (number of parsimony-informative characters = 100, and 1739 characters were constant). The phylogenetic analyses were based on maximum likelihood (ML) methods in PAUP 4.0b10 (Swofford 2003). Based on the Akaike’s Information Criterion, the best-fit evolutionary models for ML analysis were determined using Modeltest 3.7 (Posada & Crandall 1998), which selected the TrNef + G + I evolutionary model with the following parameters: substitution-rate matrix of AC = 1, AG = 3.1455, AT = 1, CG = 1, CT = 7.5961, and GT = 1; proportion of sites assumed to be invariable = 0.8376; rates for variable sites assumed to follow a gamma distribution with shape parameter = 0.9101; and number of rate categories = 4. With these settings, a heuristic search was performed using the neighbor-joining tree as the starting tree and a nearest-neighbor interchange swapping algorithm. Bootstrap probabilities were computed for 100 replicates with these settings. Further bootstrap analyses (100 replicates each) were performed using the neighbor-joining method of Jukes and Cantor, minimum evolution of maximum composite likelihood with gamma parameter = 0.8, and maximum parsimony in MEGA version 4.1 (Tamura et al. 2007). All trees are shown in Supplementary Fig. S2.

Pairwise analyses among Chlorella-related species Small subunit rDNA (exon only) and ITS2 sequences of selected species of Chlorella, Micractinium Fresinius, and Meyerella Fawley et K. Fawley were compared. SSU rDNA sequences of 12 members were initially aligned using Clustal X and then manually aligned by eye. The 5′ and 3′ terminal regions were removed, and 1742 sites were used. ITS2 sequences were also aligned manually. Unreliable areas of helices I, II, and IV were removed, and 193 aligned sites remained (alignment data are shown in Supplementary Fig. S3).

RESULTS Chlorella variabilis Shihira et Krauss 1965 Figs 1–3 Solitary cells without mucilaginous covering, planktonic, spherical or ovoid, 2.3–5.8 ¥ 2.5–6.6 mm. Chloroplast single cup- or girdle-shaped, with an ellipsoidal pyrenoid covered by grains of starch. Thylakoid lamellae penetrating pyrenoid matrix. Asexual reproduction by autospores. Differs from other species of the genus by the order of the nucleotides in the SSU rRNA and ITS2. © 2010 Japanese Society of Phycology

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Holotype: figures 3 and 4 in Shihira and Krauss 1965 (page 43) Synonym: ‘Chlorella paramecii’ Loefer nom. nud. Type locality: Endocyte of P. bursaria collected from USA Distribution: Only known endocyte of P. bursaria collected from the eastern United States (and unspecified regions), Japan, Shanghai (China), and Melbourne (Australia)

Micractinium reisseri Hoshina, Iwataki et Imamura sp. nov. Figs 4–6 Cellulae solitariae, sine tegumento gelatinoso, planctonicae, sphericae, 3.7–8.0 mm in diametro. Chloroplastus unicus parietalis patera vel poculum pyrenoide ellipsoidea granis amyli tecto. Matrix pyrenoidis penetrantibus lamelli thylacoidum. Propagatio asexualis per autosporas. Speciebus ceteris generis ordine nucleotidorum in SSU rRNA et ITS2 differt. Solitary cells without mucilaginous covering, planktonic, spherical, 3.7–8.0 mm in diameter. Chloroplast single parietal, saucer- or cup-shaped with an ellipsoidal pyrenoid covered by grains of starch. Thylakoid lamellae penetrating pyrenoid matrix. Asexual reproduction by autospores. Differs from other species of the genus by the order of the nucleotides in the SSU rRNA and ITS2. Holotype: TNS-AL-56965 in TNS (Department of Botany, National Museum of Nature and Science, Tokyo), resin embedded CCAP 211/83 (Pbi) Type locality: Endocyte of P. bursaria collected from Göttingen, Germany. Distribution: Only known endocyte of P. bursaria collected from western European areas: England, Germany, Austria, and northern Europe, Karelia region (Russia). Etymology: Named in honor of the work of Professor Werner Reisser on symbiotic algae. As described below, SSU rDNA data of hydra symbionts (primary owner of ‘Z. conductrix’) differ from those of P. bursaria symbionts. Thus, ‘Z. conductrix’ cannot be used to refer to P. bursaria symbionts. ‘C. paramecii’ obviously violates current Botanical Code. In terms of the problematic ‘C. variabilis’, although CCAP 211/6 cannot be confirmed, the duplicate strain SAG 211-6 matches the other ‘American’ symbionts in its rDNA-ITS level. Apparently, at one of the two collections, the strain was mislabeled at some point after the transfer to Göttingen. The assumption that the SAG strain is the Loefer’s isolate is much more reasonable, as its sequence is identical to that of the ‘American’ symbionts. Thus, we have confirmed SAG 211-6 as the true authentic strain and the validity of the species name ‘C. variabilis’. The ‘European’ algae should be considered a new species of the genus Micractinium

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Fig. 1–6. Morphology and cytology of Chlorella variabilis NC64A and Micractinium reisseri Pbi. Scale bars are 10 mm for light micrographs (Figs 1,4) and 1 mm for transmission electron micrographs (Figs 2, 3, 5 and 6). 1. Chlorella variabilis, general morphology. 2. Chlorella variabilis, vegetative cell. Arrowhead indicates the thylakoid membranes penetrating the pyrenoid matrix. 3. Chlorella variabilis, cell during division. 4. Micractinium reisseri, general morphology. 5. Micractinium reisseri, arrowhead indicates the thylakoid membranes penetrating the pyrenoid matrix. 6. Magnified view of M. reisseri pyrenoid. Arrowhead indicates the double-layered thylakoid.



(see below), and we have named it M. reisseri. Pbi and NC64A are the most widely used strains, and we chose strain Pbi as the authentic strain for M. reisseri. Pbi was isolated in 1974 by Dr Werner Reisser from P. bursaria collected from a pond in the Old Botanical Garden of Göttingen University, Germany, and the name was derived from, simply, ‘Paramecium bursaria isolate’. This strain is available from CCAP. NC64A first appeared in a report by Karakashian and Karakashian in 1965. This strain was isolated from P. bursaria syngen 1 collected in North Carolina, USA, and resides at the ATCC and CCAP. Known strains with affiliations that were confirmed by DNA sequence analysis are listed in Table 1.

Morphological and cytological observations The vegetative cells of C. variabilis in culture were spherical, ellipsoidal, or ovoid, and 4.0–6.5 mm in diameter (Fig. 1). Each cell contained a cup- to girdleshaped chloroplast. A pyrenoid was usually seen within the chloroplast. The mother cells produced four autospores. The vegetative cells of M. reisseri in culture were almost spherical and 5–8 mm in diameter (Fig. 4), somewhat larger than C. variabilis. A single saucer- or cup-shaped chloroplast per cell was present. A pyrenoid was usually seen within the chloroplast. The mother cells produced four autospores. Outer structures, for example, mucilage, spines, or threads, have never been observed in either C. variabilis or M. reisseri. A single-layered cell wall and pyrenoid structure cleaved by the thylakoid membranes were commonly observed in C. variabilis and M. reisseri in electron microscopic observations (Figs 2,3 and 5). The pyrenoid was surrounded by thinner starch grains in each species in this study, although several studies have reported a pyrenoid with thick starch grains when symbionts inhabit P. bursaria (e.g. Reisser 1987, 1988); this thickness seems to vary according to culture conditions. Observed C. variabilis maintains consistency with the original description (Shihira & Krauss 1965), although these morphological and cytological characters are common in most chlorellacean taxa.

Sequence of the rRNA cistron We sequenced the rRNA cistron of the strain Pbi. Between the primers SR-1 (SSU rDNA 5′ end) and Ls3Ce3 (LSU rDNA 3′ end), sequences reached 6460 or 6461 bases (due to polymorphism including indels) including the primer sequences (AB506070/ AB506071). These sequences included two group I introns at SSU rRNA 651 and LSU rRNA 2449 (the numbering reflects their homologous positions in the E. coli rRNA gene; Fig. 7). SSU rDNA (with S651 intron), © 2010 Japanese Society of Phycology

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ITS1, 5.8S rDNA, and ITS2 sequences were completely identical to those we previously introduced as the ‘European’ algae, SW1-ZK, and the algal sequence data directly amplified from whole P. bursaria (strains CCAP 1660/11, 1660/12) extracts (Table 1). The exonic region of Pbi LSU rDNA differed at three or four (due to polymorphisms of SW1-ZK) nucleotide sites compared with SW1-ZK. The L2449 intronic sequence of Pbi differed by one to three (due to polymorphisms of Pbi) nucleotide sites compared with SW1-ZK. Concomitantly with the sequencing of Pbi, we further sequenced NC64A LSU rDNA with the newly designed 3′ end primer Ls3Ce2 and added another 270 bp (AB506072) to our previous NC64A sequences (AB206549 (SSU rDNA–ITS2) and AB236862 (LSU rDNA)). Comparisons of the LSU rDNA sequences revealed a total of 70 nucleotide substitutions (0.0227 substitutions per site) between Pbi and NC64A, 85 substitutions (0.0276) between Pbi and C. vulgaris NIES-227 (AB237642), and 59 substitutions (0.0191) between NC64A and C. vulgaris. For the first 800 bp of the LSU rDNA sequences (expected to be a mutation-intensive region, see Hoshina & Imamura 2008b), we found 41 substitutions (0.0513) between Pbi and NC64A, 49 substitutions (0.0613) between Pbi and C. vulgaris, and 39 substitutions (0.0488) between NC64A and C. vulgaris.

Internal transcribed spacer 2 secondary structure Secondary structure diagrams of the C. variabilis SAG 211-6 and M. reisseri Pbi ITS2s are shown in Fig. 8. Both structures possess ITS2 motifs conserved among the green algae (Mai & Coleman 1997), i.e. fourfingered hand (four helices), pyrimidine-pyrimidine bulge on helix II, and the conserved sequence TGGT (UGGU) on the 5′ side of helix III. Of special note is the difference in paired nucleotides at the tip of helix III. Chlorella variabilis exhibits G-C pairing, whereas M. reisseri exhibits C-G pairing. This compensatory base change (CBC), i.e. a G-C to C-G change, is the synapomorphic signature of Micractinium (Luo et al. 2006, 2010). We found distinct characteristics in the M. reisseri ITS2 structure. In all members of the Chlorella clade, helix II is composed of two double-stranded regions articulated by an elbow-like bulge. Micractinium reisseri has a significantly large elbow with 10 ‘bachelor’ nucleotides, although the other species have three to six bachelor nucleotides (also refer to alignment data in Supplementary Figs S1 and S3).

Phylogenetic placement Phylogenetic analyses with the combined dataset of the SSU rDNA and ITS2 were performed, and an ML tree is

P. bursaria strain

Micractinium reisseri Algal strain Göttingen, Germany Schwarzwald, Germany Göttingen, Germany Cambridge, UK Cambridge, UK Wildbichl, Austria Piburger See, Austria Russia Karelia, Russia Karelia, Russia Karelia, Russia

SSU-ITS1-5.8S-ITS2-LSU SSU-ITS1-5.8S-ITS2-LSU SSU-ITS1-5.8S-ITS2 SSU-ITS1-5.8S-ITS2 SSU-ITS1-5.8S-ITS2 SSU (partial), ITS1 SSU (partial), ITS1 SSU (partial), ITS1 SSU (partial), ITS1 SSU (partial) SSU (partial)

Available region

SSU-ITS1-5.8S-ITS2 SSU (partial) SSU-ITS1-5.8S-ITS2-LSU SSU-ITS1-5.8S-ITS2 SSU-ITS1-5.8S-ITS2 SSU-ITS1-5.8S-ITS2 SSU-ITS1-5.8S-ITS2 SSU-ITS1-5.8S-ITS2 SSU-ITS1 SSU-ITS1 SSU-ITS1 SSU-ITS1-5.8S-ITS2 SSU-ITS1-5.8S-ITS2

Ohio, USA USA Aichi, Japan Nagano, Japan (cross breed, Japan-Japan) Shimane, Japan Ibaraki, Japan Hiroshima, Japan Hiroshima, Japan Miyazaki, Japan Oita, Japan Shanghai, China Melbourne, Australia Collection site

SSU, ITS2 SSU-ITS1-5.8S-ITS2-LSU

Available region

USA North Carolina, USA

P. bursaria collection site

†Algal DNA sequence directly obtained from whole Paramecium extract (Hoshina et al. 2005).

CCAP 1660/11 CCAP 1660/12

SW1

OK1 So13 F36 KM2 Dd1 Bnd1 HB2-2 shiP-7 takaP-3 Cs2 MRBG1

SAG 211-6 ATCC 50258/CCAP 211/84 (NC64A) ATCC 30562 N-1-A NIES-2541 (OK1-ZK) So13-ZK NIES-2540 (F36-ZK) KM2-ZK/pbKM2 Dd1-ZK Bnd1-ZK HB2-2-1 shiP-7-A4 takaP-3-A2 (uncultured)† (uncultured)†

CCAP 211/83 (Pbi) SW1-ZK SAG 241/80 (uncultured)† (uncultured)† PbW PbPIB Pbu OCH OC-1 OC-6

P. bursaria strain

AB506070-71, FM205852 AB206547, AB437244-56 FM205851 AB206548 AB260894 EF030566, EF030583 EF030565, EF030582 EF030562, EF030579 EF030561, EF030578 AY876298 AY876299

Accession numbers (major ones)

AB206550 AY876293 AB162912, AB437257 AB162913 AB162914 AB162915, EF030567, EF030584 AB162916 AB162917 AB191205 AB191206 AB191207 AB206546 AB219527

AB260893, AB301072, FM205849 AB206549, AB236862, AB506072

Accession numbers (major ones)

Chlorella variabilis and Micractinium reisseri: sources of Paramecium bursaria and algal rDNA accession numbers

Chlorella variabilis Algal strain

Table 1.

This study; Luo et al. (2010) Hoshina et al. (2005) Luo et al. (2010) Hoshina et al. (2005) Hoshina et al. (2005) Summerer et al. (2008) Summerer et al. (2008) Summerer et al. (2008) Summerer et al. (2008) Gaponova et al. (2007) Gaponova et al. (2007)

References

Hoshina & Imamura (2008a); Luo et al. (2010) This study; Hoshina et al. (2005); Hoshina & Imamura (2008b) Hoshina et al. (2005) Gaponova et al. (2007) Hoshina et al. (2004) Hoshina et al. (2004) Hoshina et al. (2004) Hoshina et al. (2004); Summerer et al. (2008) Hoshina et al. (2004) Hoshina et al. (2004) Unpublished Unpublished Unpublished Hoshina et al. (2005) Hoshina et al. (2005)

References

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Fig. 7.

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Survey of nuclear ribosomal RNA cistrons of Chlorella variabilis and Micractinium reisseri with insertion sites of group I introns.

Subgroup IC introns are shown in normal font, and IE introns are in bold. The numbering reflects their homologous positions in the Escherichia coli rRNA gene: L, large subunit of rRNA; S, small subunit of rRNA. Figure modified from Hoshina and Imamura (2009b).

A C G G A U G G A U G G U U C G G U A A C G G G A

C G U

U

U

U C C C U A C

A C G A U G G

G C A

A U G G U U C G G U A A C G G G A

G C C

U G C C G U C G C C C

U G

U U

G A G

C

C

G C

G C

GA AG

C G

UU U A

U C

G G

C A

UG GU

G U

G C

GG

C C C

A

G

C

A C C G A U G C U A G C G C U A G C G C C G U A A A G A A U AG C C U

G

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Fig. 8. ITS2 secondary structure diagrams for Chlorella variabilis and Micractinium reisseri. Both structures possess ITS2 motifs conserved among the green algae i.e. four-fingered hand (four helices), pyrimidine-pyrimidine bulge (indicated by ‘py’) on helix II, and the conserved sequence TGGT (UGGU in boldface) on the 5′ side of helix III. The key compensatory base changes (CBCs) on the tip of helix III separating Micractinium from Chlorella are boxed. ‘E2’ indicates the elbow-like bulge connecting two double-stranded regions within helix II. Non-canonical (non-Watson-Crick) pairings (e.g. G-U pairing) are shown by dots. Models are modified from Hoshina and Imamura (2008a).

shown in Fig. 9 (all other trees in the analyses are shown in Supplementary Fig. S2). Both C. variabilis and M. reisseri were included in the Chlorella clade. We did not include the previously mentioned misidentified © 2010 Japanese Society of Phycology

‘Chlorella vulgaris’ in the phylogenetic analyses due to a lack of ITS2 data (AB191205-207, covering SSU rDNA to ITS1). However, the affiliations are obvious. ‘Chlorella vulgaris’ has three group I introns at S943,

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Fig. 9. A maximum likelihood (ML) tree constructed from a combined analysis of small subunit (SSU) rRNA and ITS2 sequences (length of 1888 bp). This tree (-ln L = 4148.15) was obtained using the TrNef + I + G evolutionary model. Bootstrap values above the internal nodes were inferred from ML (left) and minimum evolution of the maximum composite likelihood (right) analyses, whereas the neighbor-joining method of Jukes and Cantor (left) and maximum parsimony (right) are shown below the nodes; only values above 50% support are given. Species residing in Paramecium bursaria are in bold.

S1367, and S1512 (true C. vulgaris does not contain any intronic insertions), and, for example, the ‘C. vulgaris’ HB2-2-1 sequence (AB191205) is completely identical to that of C. variabilis NC64A. In contrast, the algal sequence that was directly obtained from P. bursaria CCAP 1660/10 supported its monophyly to true C. vulgaris (SAG 211-11b is the authentic culture) with 100% bootstrapping in all analyses. Chlorella variabilis claded with Meyerella planktonica Fawley et K. Fawley, but M. planktonica was more than 0.01 substitutions/ site away from the branching point. The Chlorella vulgaris – C. lobophora Andreyeva clade and the clade of C. sorokiniana – Chlorella sp. IFRPD (Institute of Food Research and Product Development at Kasetsart University, Thailand) strains were moderately to highly supported; however, the monophyly of the genus Chlorella was not supported regardless of whether C. variabilis was included. The sequence sets of strain CCALA 260 occupied two different positions: one (FM205860) in the strain C. sorokiniana – Chlorella sp. IFRPD clade and the other (X74001 + AY323463) in a clade consisting of Micractinium spp. as a sister of M. reisseri.

Evolutionary divergences among Chlorella, Micractinium, and Meyerella species Evolutionary divergences among selected Chlorella, Micractinium, and Meyerella species are shown in Table 2. The divergences between SSU rDNA sequences were very low, limited to 0.0133 substitutions per site (between C. vulgaris and Meyerella planktonica) or less. Although unreliable positions were removed in the comparisons, ITS2 divergences reached 0.1176 or more (except among C. sorokiniana and IFRPD strains). These are thought to be crucial data for the separation of each species. We compared the SSU r DNA sequences to those of the hydra symbionts. Five sets of SSU rDNA sequence data exist for the hydra symbionts, of which the three strains Esh, HvT, and Ssh (X72706, X72707, and X72854) belong to the Chlorella clade (Hoshina et al. 2005). These three sequences do not contain any group I introns, and the exonic regions differ by 0.0029–0.0040 substitutions per site (five to eight nucleotide changes) from C. variabilis and by 0.0012–0.0023 substitutions (two to © 2010 Japanese Society of Phycology


197 Asterisks indicate the strains isolated from green hydra (Huss et al. 1993/94). The number of base substitutions per site from analysis between sequences is shown. All results are based on the pairwise analyses of 14 sequences for small subunit (SSU) rDNA (lower-left) and 11 sequences for ITS2 (upper-right). Analyses were conducted using the Jukes-Cantor method in MEGA. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (pairwise deletion option). There were a total of 1742 positions for SSU rDNA and 193 for ITS2 in the final datasets.

0.0029 0.0058 0.0116

0.0087 0.0122

0.0174

0.2000 0.1641 0.2326 0.2456 0.2057 0.1896 0.2032

0.0017 0.0035 0.0075 0.0110 0.0017 0.0012 0.0029 0.0069 0.0116 0.0017 0.0023 0.0017 0.0035 0.0075 0.0122

0.0058 0.0058 0.0052 0.0046 0.0052 0.0064 0.0058 0.0064 0.0058 0.0064 0.0116 0.0116

0.0000 0.0006 0.0035 0.0006 0.0017 0.0012 0.0017 0.0012 0.0029 0.0069 0.0104

0.0006 0.0035 0.0006 0.0017 0.0012 0.0017 0.0012 0.0029 0.0069 0.0104

0.0029 0.0012 0.0023 0.0017 0.0023 0.0017 0.0023 0.0075 0.0098

0.1422 0.1752 0.1422 0.1360 0.1176 0.1941 0.1752 0.0481 0.0426 0.2284 0.1687 0.0052 0.2354 0.1752

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Chlorella vulgaris SAG 211-11b Chlorella lobophora SAG 37.88 Chlorella sorokiniana SAG 211-8k C. sorokiniana CCALA 260 Chlorella sp. IFRPD1018 Chlorella variabilis SAG 211-6 Chlorella sp. Esh* Chlorella sp. HvT* Chlorella sp. Ssh* Micractinium reisseri Pbi Micractinium sp. CCALA 260 Micractinium belenophorum SAG 42.98 Micractinium pusilum SAG 13.81 Meyerella planktonica 2/24-S-1w

0.0052 0.0040 0.0040 0.0046 0.0063 0.0035 0.0046 0.0040 0.0046 0.0040 0.0058 0.0098 0.0133

0.2158

0.0029 0.0040 0.0035 0.0040 0.0035 0.0040 0.0093 0.0081

0.0012 0.0006 0.0012 0.0006 0.0023 0.0064 0.0110

8 7 6 5 4 3 2 1

Estimates of evolutionary divergence between sequences Table 2.

0.1141

0.1784 0.1251

0.2227 0.1964 0.1557 0.1493 0.1493 0.1243 0.2963 0.2677 0.2088 0.2020 0.2298 0.2369 0.2828 0.2846 0.2032 0.1964 0.2171 0.2101 0.2341 0.2883 0.1988 0.1919 0.1784 0.1585

0.2241 0.2184 0.1697 0.1631 0.1697 0.1189

13 9

10

11

12

14


four nucleotides) from M. reisseri (ITS2 sequences of hydra symbionts were not available). Although these hydra symbiont sequences may exemplify the diversity of such entities, they can be discriminated from the P. bursaria symbionts.

DISCUSSION Because of its extremely simple morphology with an exclusively asexual reproductive system, ‘the green ball’, the genus Chlorella, has caused severe taxonomic problems. In 1999, Huss et al. restricted the true Chlorella species to C. vulgaris (type species) and three other species (C. lobophora, C. sorokiniana, and C. kessleri) due to their comparatively close relationships based on SSU rDNA phylogeny. Krienitz et al. (2004) split them into the Chlorella clade (including C. vulgaris, C. lobophora, and C. sorokiniana) and the Parachlorella clade (including C. kessleri as a member of the new genus Parachlorella Krieniz, Hegewald, Hepperle, Huss, Rohr et Wolf) based on SSU rDNA and ITS2 phylogeny. Each clade includes algae of different morphologies including colonial or coenobial life-forms; fusiform, spindle, or needle shapes; and with mucilage, bristle, spines, or threads (Ustinova et al. 2001; Wolf et al. 2002; Krienitz et al. 2004). Furthermore, Krienitz et al. (2004) designated the Chlorellaceae for the Chlorella clade and Parachlorella clade together. Another new alga, Meyerella planktonica, was described by Fawley et al. (2005). This species appears similar to the true Chlorella species in morphology and is phylogenetically included in the Chlorella clade or is basal to the clade. However, due to the lack of a pyrenoid, the authors established a new genus, Meyerella. Consequently, in consideration of the historical literature, the definition of the genus Chlorella should be automatically determined by a combination of the three criteria of morphology, cytology, and molecular phylogeny, i.e. spherical to ellipsoidal unicellular green alga lacking any motile stage (without flagella), autosporulation, possession of a single nucleus, a chloroplast with a pyrenoid whose matrix is divided by the double-layered thylakoid, a mitochondrion, and a phylogenetic affiliation to the Chlorella clade. Although it remains unclear whether all members of Chlorella have a monophyletic origin within this clade, both C. variabilis and M. reisseri meet all of these requirements (Figs 1–6 and 9). However, most recent studies have overturned such a generic concept. Luo et al. (2006) characterized the genus Micractinium, which is closely related to Chlorella but morphologically different because of the formation of bristles. However, the authors emphasized the phenotypic plasticity of bristle formation, which in some cases could only be induced by the grazing rotifer Brachionus. Micractinium and Chlorella could only be genetically differentiated by

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their variable ITS sequences, specifically by a unique (for Micractinium) CBC in helix III of ITS2 (Luo et al. 2010; see also Fig. 8 and Supplementary Fig. S3), which is a characteristic for distinguishing the genus Micractinium from other genera within the Chlorella clade. Finally, Luo et al. (2010) extended the generic concept in Chlorella-related green algae based on phylogeny and synapomorphic nucleotide changes in SSU rDNA and ITS2. Accordingly, the genus Chlorella now also contains gelatinous and colonial species such as Dictyosphaerium Nägeli, whereas the genus Micractinium contains some spherical species as well as species of the other bristled genus Diacanthos Korshikov (see Fig. 9). Didymogenes Schmidle is another genus that includes both bristle-bearing and nonbristle-bearing species (Schnepf & Hegewald 1993). The monophyly of D. anomala (G. M. Smith) Hindák (bristle-bearing) and D. palatina Schmidle (non-bristlebearing) was partially supported (Fig. 9). Our phylogenetic analyses indicate that the ‘European’ symbionts are members of Micractinium (Fig. 9). The ITS2 nucleotide-change characters of ‘European’ symbionts are also congruent (Fig. 8) to those of Micractinium indicated by Luo et al. (2006, 2010). These facts strongly indicate the affiliation of ‘European’ symbionts to the genus Micractinium. Meanwhile, we used two sequence datasets for strain CCALA 260. The SSU rDNA (X74001) sequence of CCALA 260 initially appeared in Huss et al. (1999) as ‘Prag A14’. Subsequently, Krienitz et al. (2004) released the ITS2 (AY323463) sequence and combined the two sequences (X74001 + AY323463) to use for phylogenetic analyses. Luo et al. (2010) released a string sequence of SSU-ITS1-5.8S-ITS2 (FM205860) for the same strain. Between these SSU rDNA (X74001 vs. FM205860), two nucleotide changes were present; however, divergence between these ITS2 sequences (AY323463 vs. FM205860) reached 0.1631 (data not shown). One sequence was closer to M. reisseri, but the other was C. sorokiniana. Mixed-up strains remain a serious problem plaguing algal taxonomy. ‘American’ symbionts comprise a clade with Meyerella planktonica (Fig. 9). The phylogenetic positions of Me. planktonica are fluid depending on outer and/or inner group choice for the analyses (Fawley et al. 2005, and preliminary data in this study). We believe this issue is the effect of a long branch attraction artifact (Felsenstein 1978) caused by the fact that the Me. planktonica sequence is less similar to any other sequences; therefore, the monophyly of C. variabilis and Me. planktonica is unreliable. ‘American’ symbionts can also be separated from Meyerella by the presence of pyrenoids (Figs 2,3). In the generic concepts based on nucleotide changes in SSU rDNA and ITS2, the genus Chlorella could not be distinguished by any synapomorphic character (Luo et al. 2010).

R. Hoshina et al.

Perhaps the genus Chlorella can be regarded as a paraphyletic group maintaining symplesiomorphic statuses in terms of both cytology and nucleotide lows. Considerable molecular differences still exist for Chlorella-like organisms, and we believe that the family Chlorellaceae warrants more genera. However, if one were to establish a new genus for the ‘American’ symbionts, the genus would have only one species, and few differences would exist between the generic and species concepts. Genus establishment should be postponed until the diversity and relationships become more obvious. Therefore, at present, we tentatively use Chlorella for the ‘American’ symbionts. Chlorella-related species are not distinguishable by morphology or cytology. For the identification of these species, physiological characters have often been used (Huss et al. 1999 and references therein). However, identical morphology with recondite physiology may pressure investigators to give up on the identification of Chlorella species, resulting in the generation of many unidentified strains. The National Centre for Biotechnology Information (NCBI) Taxonomy Browser, for example, contains more than 100 unidentified Chlorella strains, some of which may belong to existing Chlorella species, some of which may not belong to the genus Chlorella, and some of which may be new species of Chlorella. Of course, describing new species in this genus based on traditional means is extremely difficult. Clearly, the species concept in this genus is also, at present, facing a major turning point. Given two organisms, the more they resemble each other, the more difficult it will be to distinguish whether they belong to the same species. The internal transcribed spacer 2 is currently attracting attention as a molecular ‘barcode’ resolving such species problems (Coleman 2003, 2007; Müller et al. 2007). A remarkable feature of this molecular marker is its high divergence between species. In the ITS2 comparisons (Table 2), all species are clearly separated from each other, whereas within species, those divergences are very small. For example, more than 30 sequences of C. vulgaris have been published, for which divergence was only as high as 0.0126 substitutions per site for all 241 nucleotide position comparisons (data not shown). For M. reisseri, sequence variation has thus far not been found. Therefore, in species of the Chlorella clade, ITS2 can be regarded as a highly conserved molecule within species as well as a highly divergent molecule between species. In addition to such phylogenetic informativeness, a specific structural feature between two ITS2 secondary structures, a CBC, can also be used to distinguish two species from each other (Coleman 2000; Coleman & Vacquier 2002; Behnke et al. 2004; Young & Coleman 2004; Müller et al. 2007). When comparing ITS2 helices II and III as highly conserved regions (Coleman 2003), we always found two or more © 2010 Japanese Society of Phycology


CBCs among C. variabilis and other species (see Supplementary Fig. S3). Helix II of Micractinium species is structurally variable; for example, M. reisseri includes a large bulge (Fig. 8), and Micractinium sp. CCALA 260 has a shortened helix (see Supplementary Fig. S3). We regard such structural differences as equivalent to CBCs. Therefore, adequate data exist for definitive species-level autonomy of C. variabilis and M. reisseri. Group I intronic insertions in their rRNA genes comprise another genetic characteristic of C. variabilis and M. reisseri (Fig. 7). Although group I introns are known as continually losable mobile genetic elements, in both C. variabilis and M. reisseri, their sequences, presence or absence, and insertion positions are stable. Chlorella variabilis has eight group I introns in its nuclear ribosomal RNA cistron, the highest number in the Viridiplantae (Hoshina & Imamura 2008b). Some introns in both species insert into unique positions due to their uniquely evolved infection mechanism (Hoshina & Imamura 2009b), which will be of considerable help in identifying these species. Paramecium bursaria contains green algal symbionts, and this unicellular ciliate is a textbook example used for microscopic observation in high school science projects. However, many textbooks now in use are written as if P. bursaria temporally maintains algae that were ingested from the outside. Paramecium bursaria, as a predatory protist with quotidian phagocytotic behavior, may invite such a misunderstanding. Paramecium bursaria symbionts comprise distinct species inhabiting the cells of P. bursaria. Both C. variabilis and M. reisseri demand organic nitrogen compounds (Kamako et al. 2005) and are sensitive to Chlorella viruses, the so-called ‘NC64A virus’ and the ‘Pbi virus’, which are abundant in natural freshwater (Van Etten et al. 1991; Yamada et al. 1991). As a matter of course, they have never been collected from nature as free-living Chlorella. In the present study, we provide species names (one is a revival of an old name) for these morphologically indistinguishable algae based on DNA sequence and structural comparisons. We believe this work offers important contributions toward constructing a genetic species concept in the fields of microbial biodiversity and taxonomic analyses, as well as dispelling misunderstandings about P. bursaria symbiosis. Supplementary figures are available at our website, http://www.ritsumei.ac.jp/pharmacy/imamura/chlorella. html/.

ACKNOWLEDGEMENTS Strain Pbi was obtained from Professor James L. Van Etten (University of Nebraska) with culturing © 2010 Japanese Society of Phycology

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instructions provided by Dr James R. Gurnon (University of Nebraska).

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