Open Access

DNA analyses of a private collection of microbial green algae contribute to a better understanding of microbial diversity

BMC Research Notes20147:592

https://doi.org/10.1186/1756-0500-7-592

Received: 11 April 2014

Accepted: 27 August 2014

Published: 2 September 2014

Abstract

Background

DNA comparison is becoming the leading approach to the analysis of microbial diversity. For eukaryotes, the internal transcribed spacer 2 (ITS2) has emerged as a conspicuous molecule that is useful for distinguishing between species. Because of the small number of usable ITS data in GenBank, ITS2 sequence comparisons have only been used for limited taxa. However, major institutions with planktonic algal culture collections have now released small subunit (SSU) to ITS rDNA sequence data for their collections. This development has uplifted the level of molecular systematics for these algae.

Results

Forty-three strains of green algae isolated from German inland waters were investigated by using SSU-ITS rDNA sequencing. The strains were isolated through the direct plating method. Many of the strains went extinct during the years of culture. Thus, it could be expected that the surviving strains would be common, vigorous species. Nevertheless, 12 strains did not match any known species for which rDNA sequences had been determined. Furthermore, the identity of one strain was uncertain even at the genus level.

Conclusions

The aforementioned results show that long-forgotten and neglected collections may be of great significance in understanding microbial diversity, and that much work still needs to be done before the diversity of freshwater green algae can be fully described.

Keywords

Green algae Internal transcribed spacer 2 (ITS2) Microbial diversity Private collection Species delimitation

Background

With the current rates of biodiversity loss, biodiversity conservation is receiving much attention. In many parts of the world, various animals and plants are being intensively studied, and conservation efforts for many species are well underway. Knowledge of microbial diversity, however, remains rudimentary. The current number of known microorganism species is a fraction of the diverse population of microorganisms [1, 2]. Much research, particularly the identification and description of species, is still required before microorganism diversity can be accurately quantified.

The development of the microscope made it possible to observe organisms smaller than dust; and with modern technological advances, the field of microbiology, especially microorganism systematics, has blossomed. DNA research methods have now facilitated the development of an entirely new level of systematics, thereby redefining the species concept. In particular, studies on various types of microorganisms have led to the development of their systematics. New techniques have revealed that many separate species were originally mistakenly grouped together as single morphospecies. A well-known example of this is Chlorella, which are common algae in natural water bodies. They are composed of simple, green, spherical cells containing a minimal set of organelles: one nucleus, one chloroplast, and one mitochondrion. These algae were originally considered one species, but small subunit (SSU) rDNA phylogenies have since shown that the group is in fact polyphyletic, consisting of morphospecies in more than one class [3, 4]. Based on the initial phylogenetic studies, Huss et al. [4] divided “true” Chlorella to four monophyletic species, including the type species Chlorella vulgaris. However, later studies identified some species that are morphologically different and draw their phylogenetic origins from the “true” Chlorella group e.g. [59].

The internal transcribed spacer 2 (ITS2) has emerged as a useful and conspicuous molecule for distinguishing between species. The ITS2 is located between 5.8S rRNA and LSU rRNA, which plays an important role in processing events during rRNA maturation e.g. [10, 11], and the secondary structures of ITS2 are highly conserved throughout the eukaryota [12]. The primary sequence of ITS2 region is highly conserved within species, but is highly divergent between species e.g. [13]. In general, the diversity of ITS2 sequence comparisons are either less than 2% or more than 10% (gaps are counted as a fifth character). This characteristic strongly encourages a species concept based on ITS2 sequence differences. Compensatory base changes (CBCs) in the ITS2 secondary structure also encourage species separation e.g. [14, 15]. The presence of at least one CBC has been correlated with the separation of two species, which is classified here by using the biological species concept based on the production of fertile offspring [14]. This hypothesis has been supported by data on various eukaryotic groups, including plants, fungi, and animals ([15] and references therein). Species concepts based on ITS2 differences in chlorophytes have gained wide acceptance. They also serve as a key to identifying and describing true microorganism diversity. This development also reveals the possible pitfalls of classification based on morphological characteristics.

In recent years, molecular phylogeny has been increasingly applied to the floristic study of planktonic green algae e.g. [1619]. However, these studies analyzed SSU rDNA only and avoided species-level designation. This may have been largely because of the small amount of usable ITS data available in GenBank. However, major institutions holding algal culture collections, the Culture Collection of Algae and Protozoa (CCAP, UK), and the National Institute for Environmental Studies (NIES, Japan), have now released SSU to ITS rDNA sequence data for their collections. This has led to unprecedented developments in molecular systematics. Making use of these data, I have investigated the laboratory’s historical stocks of green algae, many of which have been neglected and forgotten about, which were isolated from German inland waters. Based on my findings, I discuss the potential of such archival lab stocks in supplementing our knowledge of biodiversity.

Methods

Cultures

Strains of green algae were established through simple methods. Water samples of several hundred microliters each were spread onto 1% agar plates containing 20% Gamborg’s B5 basal medium with mineral organics (Sigma-Aldrich, MO), to which was added 1/10 volume of lettuce juice medium [20] at pH 7.5. (The resulting medium is hereafter referred to as 1/5 GL medium.) The plates were incubated for two or three weeks under illumination by a light-emitting diode (LED) lamp (12 h/12 h light/dark cycle) at 15°C. Small single colonies (ca. 300 μm) that emerged were dissolved in water, and then spread onto a new plate under the same conditions. Single colonies that emerged from these cultures were transferred to liquid 1/5 GL medium (Table 1).
Table 1

Strains sequenced in the study and their taxonomic designation

Strain

rDNA sequence

Taxonomic designation

Acc. no.

Coverage

SSU intron*

 

GB1a

AB917097

SSU–ITS2

 

Desmodesmus sp.

GB1c

AB917098

SSU–ITS2

 

‘Ankistrodesmus’ gracilis

GB1d

AB917099

SSU–ITS2

 

Pectinodesmus sp.

GB1e

AB917100

SSU–ITS2

 

Acutodesmus obliquus

GB1g

AB917101

SSU–ITS2

 

Acutodesmus obliquus

GB1h

AB917102

SSU–ITS2

 

Pectinodesmus sp.

GB1j

AB917103

SSU–5′LSU

516

Pectinodesmus sp.

GB1k

AB917104, -105

SSU–5′LSU

 

Micractinium sp.

GS2i

AB917106

SSU–ITS2

516, 943, 1512

Desmodesmus brasiliensis

GS2j

AB917107

SSU–ITS2

516

Desmodesmus opoliensis

GS2k

AB917108

3′SSU–ITS2

Desmodesmus opoliensis

GS2L

AB917109

SSU–ITS2

516, 943, 1512

Desmodesmus brasiliensis

GS2m

AB917110

SSU–ITS2

516

Desmodesmus opoliensis

GS2n

AB917111

SSU–ITS2

516, 943, 1512

Desmodesmus brasiliensis

GS2o

AB917112

SSU–ITS2

 

Desmodesmus armatus

GS2p

AB917113

SSU–ITS2

 

Desmodesmus armatus

GS3a

AB917114

SSU–ITS2

 

Acutodesmus obliquus

GS3b

AB917115

3′SSU–5′LSU

Acutodesmus obliquus

GS3c

AB917116

SSU–ITS2

943

Tetranephris brasiliensis

GS3d

AB917117

3′SSU–5′LSU

Acutodesmus obliquus

GS3e

AB917118

SSU–ITS2

 

Acutodesmus obliquus

GS3f

AB917119

SSU–ITS2

943

Tetranephris brasiliensis

GS3g

AB917120

3′SSU–5′LSU

Acutodesmus obliquus

GS3h

AB917121

3′SSU–5′LSU

Acutodesmus obliquus

GS3i

AB917122

SSU–ITS2

 

Acutodesmus obliquus

GS3j

AB917123

3′SSU–ITS2

Tetranephris brasiliensis

GS3k

AB917124

3′SSU–5′LSU

Acutodesmus obliquus

GS3m

AB917125

SSU–ITS2

 

Acutodesmus obliquus

GS3n

AB917126

SSU–ITS2

 

Acutodesmus obliquus

GS3p

AB917127

SSU–ITS2

 

Acutodesmus obliquus

GM4a

AB917128

SSU–5′LSU

 

Desmodesmus sp.

GM4b

AB917129

SSU–5′LSU

156, 943, 1046, 1139, 1512

Selenastraceae sp.

GM4c

AB917130

SSU–ITS2

516, 1512

Desmodesmus sp.

GM4d

AB917131

SSU–5′LSU

516, 943, 1046

Nephrochlamys subsolitaria

GM4e

AB917132

SSU–5′LSU

40, 156, 516, 1046, 1139, 1512

Neochloris sp.

GM4f

AB917133

SSU–ITS2

 

Desmodesmus armatus

GM4g

AB917134

SSU–ITS2

516, 943, 1512

Desmodesmus bicellularis

GM4h

AB917135

SSU–ITS2

516, 943

Desmodesmus armatus

GM4i

AB917136

SSU–5′LSU

516

Desmodesmus sp.

GM4j

AB917137

SSU–5′LSU

516, 1512

Desmodesmus sp.

GM4k

AB917138

SSU–5′LSU

 

Desmodesmus armatus

GM4n

AB917139

SSU–5′LSU

 

Desmodesmus pannonicus

GA5a

AB917140

SSU–5′LSU

 

Coccomyxa sp.

*Insertion position corresponding to Escherichia coli rRNA gene.

Strain names indicate the water source in Germany from which the strain originated. GB1: a pond in the Botanischer Garten, Berlin, July 28, 2011; GS2: an artificial pond in the Mittlerer Schlossgarten, Stuttgart, July 31; GS3: a fountain in the Oberer Schlossgarten, Stuttgart, July 31; GM4: an artificial pond in Denninger Anger Park, Munich, Aug. 1; GA5: Alpsee Lake in the Ostallgäu district of Bavaria, Aug. 2.

The established colonies were cultured on two different media: first 1/5 GL, C medium [21], and then 0.1% Hyponex (Hyponex, Osaka). They were maintained under LED illumination (12 h/12 h light/dark cycle) at 10°C. The strains are available from the author upon request.

The culture stocks were observed under an Olympus BX60 light microscope (Olympus, Tokyo), and images were obtained with an Olympus DP72 digital camera.

DNA extraction, amplification, and sequencing

DNA extractions using the DNeasy Plant Mini Kit (Qiagen, Düsseldorf, Germany) were performed. Polymerase chain reaction (PCR) was carried out to amplify SSU to ITS rDNA by using the primer pairs SR-1/SR-9 [22], SR-6 [22]/SR-12 k [23], INT-4 F [24]/ITS4 [25] and INT-4 F/HLR-3R [23]. PCR products were purified with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany), and were directly sequenced by using the Operon DNA sequencing service (Operon Biotechnology, Tokyo). Some strains were amplified and sequenced only their ITS rDNA locus.

Phylogenetic analyses

The SSU rDNA sequences were first checked for group I introns insertions (methods for group I intron ascertainment [26]). The joined exons were then submitted to the Basic Local Alignment Search Tool for Nucleotides (BLASTN, National Center for Biotechnology Information) for comparisons between sequences. Phylogenetic analyses of Chlorophyceae and Trebouxiophyceae were conducted separately. Taxon samplings for tree analyses were mainly based on recent papers [2729]. The closest organisms for each strain that had been identified through BLASTN search were subsequently added. The SSU rDNA sequences were initially aligned automatically by using Clustal X2 software [30] and then aligned manually, taking into account secondary structure models for C. vulgaris[3] and Heterochlorella luteoviridis[31]. The 5′ and 3′ terminal regions were removed.

Two phylogenetic trees were constructed through the maximum likelihood (ML) method in PAUP 4.0b10 (Sinauer Associates, MA) or MEGA5 [32], and the neighbor-joining (NJ) method in the Saitou and Nei model in Clustal X2. The best-fit evolutionary models were identified by using Modeltest 3.7 [33] or MEGA5. By utilizing the results to derive the settings, a heuristic search using the NJ tree as the starting tree and a nearest-neighbor interchange swapping algorithm was performed. Bootstrap probabilities were computed for 100 (ML) and 1000 (NJ) replicates.

For advanced analyses at the genus or species level, phylogenetic analyses using both NJ and ML methods were conducted by examining ITS2 or SSU-ITS rDNA in conjunction with the results of recent studies of several different groups of species (Scenedesmus-related species: [34], Desmodesmus: [35], Chlorellaceae: [36]). ITS2 sequences were folded by using Mfold [37], and the secondary structures were used to aid manual alignment of those sequences and to check the presence/absence of CBC. For each tree, identical sequences were treated as one operational taxonomic unit.

Results and Discussion

BLASTN analyses indicated that all strains analyzed in this study belonged to either Chlorophyceae or Trebouxiophyceae. These two classes were therefore treated separately.

Chlorophyceans

The SSU rDNA tree for members of Chlorophyceae separated them into three broad groups: Selenastraceae, the Neochloris-Hydrodictyotaceae clade, and Scenedesmaceae (Figure 1).
Figure 1

Unrooted phylogenetic tree for Chlorophyceae based on SSU rDNA gene sequences (length 1605 nt). Algal strains analyzed in this study were indicated by alphanumeric codes in bold. This tree was constructed by using the maximum likelihood (ML) method under the GTR + I + G evolutionary model. Numbers at each node represent bootstrap probabilities from ML/NJ analyses; only values above 50% are shown.

Five strains (GB1c, GS3c and -f, and GM4b and -d) were included in the Selenastraceae clade (Figure 1). Phylogenetic relationships for this group were constructed based on SSU rDNA analysis [28, 38, 39], which indicated that some genera were polyphyletic. However, many strains remained inadequately treated. Lacking data for more rapidly evolving molecules for this group, such as ITS, necessitated the use of the SSU rDNA phylogeny to determine the relationships between the cultured strains and previously analyzed members of this group.

GS3c and -f were grouped with Tetranephris brasiliensis, the type species for Tetranephris. There are three Tetranephris SSU rDNA sequences in GenBank (HM483517, HM565927, and HM565929), all of which are from T. brasiliensis. GS3c and -f are closest to the sequence HM565927, and have only one transition in 1601 aligned sites (data not shown). Light microscopy revealed solitary, crescent-shaped cells containing some granules (Figure 2A and B), which agrees with the morphological characteristic of T. brasiliensis and thus confirms that GS3c and -f are T. brasiliensis strains. Furthermore, the strain GS3j, which was analyzed by ITS sequencing only, had an ITS sequence and morphology identical to that of GS3c and -f (Table 1).
Figure 2

Light microscopy images of algal strains identified as members of Selenastraceae from a private collection. Strains were collected from German inland waters and were named according to their source (Table 1). A: GS3c, B: GS3f, C: GM4b, D: GB1c, E: GM4d.

GM4b was grouped with Monoraphidium dybowskii in the ML tree (Figure 1), but this was not supported by the NJ analyses. Of note, however, was that the type species of Monoraphidium, Monoraphidium griffithii, was placed in a different group in this family. GM4b cells ranged from horseshoe- to donut-shaped (M. dybowskii cells are rhomboidal to labiate), with the diameters of the thickest parts between 2.5 and 4.0 μm (Figure 2C). Therefore, GM4b could not be identified even to genus level at this stage.

GB1c was clustered with Ankistrodesmus gracilis (Figure 1). Notably, the type species of Ankistrodesmus, Ankistrodesmus fusiformis, was placed in a different group in this family. GB1c cells were mainly crescent-shaped, 16–32 μm long, and 4–9 μm wide (Figure 2D). These morphocharacters are slightly different from that typical of A. gracilis (colony formation of 8–32 narrower crescent cells). As only one transition out of 1682 aligned sites between GB1c and A. gracilis was observed, GB1c was tentatively designated as a strain of A. gracilis.

GM4d was clustered with Nephrochlamys subsolitaria and Monoraphidium minutum (Figure 1). The GM4d sequence differed from that of N. subsolitaria by only two indels, and from that of M. minutum by five indels and four or five substitutions. The shape of GM4d cells resembled beans or curved cylinders (Figure 2E), similar to those of N. subsolitaria and M. minutum. Despite the pending taxonomic status of M. minutum, GM4d could be tentatively designated as a strain of N. subsolitaria.

GM4e was grouped with the Neochloris species, and the Pediastrum-Hydrodictyon clade (Hydrodictyotaceae) was identified as a sister group to this clade (Figure 1). A close relationship between Neochloris and the Hydrodictyotaceae has been reported many times e.g. [40], and they have been classified as Sphaeropleales. This group includes Scenedesmaceae [41]. Neochloris aquatica, the type species of this genus, has been described based on UTEX 138 (the culture held at The Culture Collection of Algae, University of Texas) and on CCAP 254/5. However, their SSU rDNA sequences differ considerably from each other. When the ITS2 sequence of GM4e was submitted to BLASTN, it showed almost 90% similarity to the UTEX 138 strain of N. aquatica, AY577764, which includes 5.8S-ITS2 rDNA [42]. However, it was completely different from the CCAP 254/5 sequence (FR865697, including SSU-ITS2 rDNA). Preliminary analysis of the ITS2 secondary structure showed that both GM4e and N. aquatica UTEX 138 have a Y-shaped helix I (Figure 3), similar to that of Sphaeropleales [4345]. When the CCAP 254/5 sequence was submitted to BLASTN, it showed more than 99% similarities (including ITSs) to several strains of Chlorella vulgaris (Trebouxiophyceae). It is possible, therefore, that the CCAP 254/5 strain had been confused with another strain.
Figure 3

ITS2 secondary structure that features a Y-shaped helix I (shown in blue).

Light microscopy revealed small to large spherical cells (3.2–32 μm) with a parietal chloroplast including one or more pronounced granules (Figure 4). Some cells contained segmentalized aggregations, which could be the development stage of zoospores. There is therefore no reason to exclude GM4e from Neochloris. AlgaeBase (http://www.algaebase.org) lists Neochloris as the taxonomically accepted genus name for eight species. Therefore, GM4e was designated as a species of Neochloris, although further comparative research with the other Neochloris species is required before this can be confirmed.
Figure 4

Light microscopy image of cells from strain GM4e of a private collection. This strain was collected from German inland waters and was named according to its source (Table 1).

GM4e was particularly intron-rich; it contained six group I introns at S40, S156, S516, S1046, S1139, and S1512 (Table 1), which elongated its SSU rDNA to 4.3 kb. This ties with Selenastrum capricornutum[46] for the most intron insertions in the SSU rDNA. In contrast, previously described Neochloris species do not have any introns. Descriptions of these introns will be published elsewhere.

The remaining strains identified as Chrolophyceans all belonged to Scenedesmaceae, within which they were clearly separated into two clades, namely, Coelastroidea-Scenedesmoideae and Desmodesmus[47] (Figure 1).

The Scenedesmus-related species were recently re-analyzed by using molecular phylogenetic techniques and electron microscopy e.g. [34, 47], which generated several new genera and many new species. Strains GB1j, -d, and -h were grouped with one of the new genera, Pectinodesmus, in the ITS2 analyses (Figure 5). All three strains formed four-cell coenobia comprising spindle cells (Figure 6A-C). This morphology coincides with that of Pectinodesmus, but it is insufficient for clear delineation of species in this genus [34]. Pectinodesmus regularis has been recognized as an independent species, but its genetic differences from other species are tenuous [34]. Furthermore, many genetically distinct strains dispersed throughout this clade are tentatively treated as Pectinodesmus pectinatus (Figure 5), obfuscating the distinction between species. Comparison of the ITS2 structures reveal that GB1j has one CBC and two hemi-CBCs (a compensatory base change on only one side of a pairing, the next-best “proof” of a CBC), and that GB1d and -h have one CBC when compared with P. pectinatus sensu stricto. Identification of strains GB1j, -d, and -h at species level must therefore be suspended until the phylogeny of the Pectinodesmus species becomes clear.
Figure 5

Neighbor-joining tree inferred from ITS2 sequence comparisons (length 283 nt). The Coelastrum species were used to root this tree. Numbers at each node represent bootstrap probabilities from NJ/ML (GTR + G model) analyses; only values above 50% are shown.

Figure 6

Light microscope images of algal strains identified as members of Scenedesmus sensu lato. Strains were collected from German inland waters and were named according to their source (Table 1). A: GB1j, B: GB1d, C: GB1h, D: GB1g, E: GS3g, F: GS3k, G: GB1e, H: GS3a, I: GS3b, J: GS3d, K: GS3h.

Thirteen strains (GB1e, -g, GS3a, -b, -d, -e, -g, -h, -i, -k, -m, -n, and -p) were clustered with known Acutodesmus obliquus strains (Figure 5). These culture strains had single- to eight-cell coenobia, and most cells were shorter and wider than those of typical A. obliquus strains (Figure 6D-K). This morphology could be related to the medium in which they were cultured. As CBC of these strains has not been found upon comparison with A. obliquus strains, they could be identified as A. obliquus.

Fawley et al. [16] suggested that a difference of even a single nucleotide in the SSU rDNA sequence could distinguish between species. However, this does not appear to be the case. Some strains with different SSU rDNA sequences had identical ITS sequences. For instance, there was one transition between the SSU rDNA sequences of GB1e and GS3e, but no substitutions in their ITS sequences.

Seventeen strains were included in the Desmodesmus genus (Figure 1). Most of these strains had typical Desmodesmus forms, i.e., four- or eight-celled coenobia with or without spines on the terminal cells (Figure 7). Species of this genus have been characterized based on cell-shape, spines, and cell-wall appendages. However, several species could not be unambiguously distinguished even by scanning electron microscopy. As a result, recent studies have used molecular comparisons e.g. [4850].
Figure 7

Light microscope images of algal strains identified as Desmodesmus species from a private collection. Strains were collected from German inland waters and were named according to their source (Table 1). A: GM4g, B: GM4i, C: GS2o, D: GM4h, E: GS2p, F: GM4k, G: GS2j, H: GS2k, I: GS2m, J: GM4a, K: GB1a, L: GM4c, M: GM4j, N: GS2i, O: GS2L, P: GM4n.

Based on the ITS2 analyses, GM4g was clustered with Desmodesmus bicellularis strains (Figure 8). However, GM4g comprised single ellipsoids 5.1–15 μm long without any spines (Figure 7A), whereas D. bicellularis formed two- to eight-celled coenobia. Furthermore, Johnson et al. [48] observed irregularly shaped D. bicellularis cells with spine-like appendages (DQ417558). Because the GM4g ITS2 sequence was 100% identical to that of D. bicellularis strains, GM4g is probably a different morphological form of D. bicellularis.
Figure 8

Neighbor-joining tree inferred from ITS2 sequence comparisons (length, 276 nt). Desmodesmus serratus, D. lunatus, D. costato-granulatus, D. ultrasquamata, and D. elegans were used to root this tree. Numbers at each node represent bootstrap probabilities from NJ/ML (SYM + I + G model) analyses; only values above 50% are shown.

GM4i formed four- or eight-cell coenobia. Although this morphology is not clearly shown in Figure 7B, each cell had a few small spines at the longitudinal ends. GM4i was clustered with Desmodesmus multivariabilis strains (Figure 8); however, it had a CBC at helix Ib when compared with these strains. Species-level identification is therefore not yet possible.

The strains GS2o, -p, GM4f, -h and -k were clustered with Desmodesmus armatus strains (Figure 8). They formed four- or eight-cell coenobia with long spines at the apices of the terminal cells, but the spines of GM4h were less obvious (Figure 7C-F). Based on the ITS2 phylogeny and the absence of CBC between these five and the previously identified D. armatus strains, they could be designated as D. armatus strains.

GS2j, -k, and -m were clustered with Desmodesmus opoliensis strains (Figure 8). In this study, the three D. opoliensis strains were employed from GenBank. Although these D. opoliensis ITS2 discrepancies are minor (5–7 changes out of 253 aligned sites), there is a CBC at helix IV and hemi-CBCs at helices Ic, II, and IV among them. Each of the variable sites found in the GS2j, -k, and -m strains applies to any of such variation. Their morphologies (two- or four-cell coenobia with long spines at the apices of terminal cells; Figure 7G-I) match those of the previously identified D. opoliensis strains. Therefore, they were tentatively identified as D. opoliensis strains.

GB1a and GM4a were placed in a clade comprising as-yet unidentified species, which is somewhat independent from previously described species of a sister clade to Desmodesmus asymmetricus (Figure 8). There was no CBC between GB1a, GM4a, and the two nameless strains Tow 10/11 T-2 W and Mary 6/3 T-2d, which were collected from Minnesota, USA [16]. They were all clearly separated from MAT-2008c by two CBCs and two hemi-CBCs. GB1a and GM4a formed two-, four- or eight-celled coenobia, of which each cell had a few spines (Figure 7J and K). Cells were comparatively small (3–6 × 8–13 μm).

Based on the ITS2 sequences, GM4c and -j were separated from the other Desmodesmus species. Tree analyses showed that their closest taxon was Desmodesmus sp. AKS-13 (Figure 8), but they were distinguished from each other by two hemi-CBCs at helices II and III. GM4c and -j cells were nearly spherical (3.5–10.5 μm), but some were ellipsoidal (up to 5.5 × 8 μm; Figure 7L and M).

GS2i, -L, and -n formed two- or four-cell coenobia (Figure 7N and O). Each cell had a few short spines, and some cells had granulated protoplasm. They were clustered with Desmodesmus brasiliensis CCAP 258/39 (Figure 8), with which there was no variation except for an N residue in the CCAP 258/39 sequence. CCAP has released five sequences (all including ITS2) for D. brasiliensis cultures. The ITS2 sequences of GS2i, -L, and -n closely matched the CCAP 258/39 sequence, but did not match the other four strains as closely (CCAP 258/40, 258/42, 258/43, and 258/44; these sequences were not used in this study). D. brasiliensis therefore contains several genetically distinct species. Because the authentic strain of D. brasiliensis has not yet been determined, I tentatively designate these three as strains of D. brasiliensis.

GM4n formed two- or four-cell coenobia or occurred as single cells (Figure 7P). Each cell had some spines, which projected in various directions. The ITS2 sequence for this strain was completely consistent with those of the D. pannonicus strains (Figure 8), so I designate GM4n as a strain of this species.

Trebouxiophyceans

Of the 43 strains of green algae that were analyzed in this study, only two were from Trebouxiophyceae.

Strain GA5a was clustered with Coccomyxa and Paradoxia species (Figure 9). These genera have been treated incertae sedis within the Trebouxiophyceae [51], and have not yet been revisited in detail by using molecular phylogenetic techniques. GA5a cells were simple ellipsoids, 7.0–11 μm long and 4.2–8.0 μm wide (Figure 10). They contained a girdle-shaped chloroplast without a visible pyrenoid. This morphology matches that of Coccomyxa or of the species classified hitherto as Pseudococcomyxa (shown as Coccomyxa simplex in Figures 9 and 11). BLASTN search for the GA5a ITS2 sequence found matches with several sequences. The GA5a sequence was identical to that of Choricystis sp. GSE4G (HE586518) (Figure 11). This was the case for both the ITS2 and the SSU-ITS rDNA. HE586518 was identified not as Coccomyxa or Paradoxia but as Choricystis. The morphology of Paradoxia was completely dissimilar to that of GA5a. In contrast, Coccomyxa and Choricystis have somewhat similar ellipsoidal single cells. However, Choricystis is a phylogenetically distinct genus, separate from Coccomyxa (Figure 9). Unfortunately, the authors who registered HE586518 have not yet published any information about this strain. For this reason, although GA5a is probably the same species as Choricystis sp. GSE4G, it may have to be designated as Coccomyxa sp. until publication of its further details.
Figure 9

Phylogenetic tree for Trebouxiophyceae inferred based on SSU rDNA gene sequences (length 1624 nt). Algal strains analyzed in this study were indicated by alphanumeric codes in bold. This tree was rooted with Makinoella, Oocystis, and Tetrachlorella and was constructed by using the maximum likelihood (ML) method under the TN93 + I + G evolutionary model. Numbers at each node represent bootstrap probabilities from ML/NJ analyses; only values above 50% are shown.

Figure 10

Light microscopy image of the GA5a algal strain from a private collection. This strain was collected from German inland waters and was named according to its source (Table 1).

Figure 11

Neighbor-joining tree inferred from ITS2 (helices I - III) sequence comparisons (length 194 nt). Numbers at each node represent bootstrap probabilities from NJ/ML (GTR + I + G model) analyses; only values above 50% are shown.

Strain GB1k was included in the so-called “Chlorella clade” in Chlorellaceae (Figure 9). Members of this clade have been well studied, and recent studies have laid out guidelines for its genera and species delimitations [7, 36, 5257]. GB1k was grouped with Micractinium in the SSU-ITS rDNA tree (Figure 12). This clade was independent of other clades, and had a long node and overall higher bootstraps. GB1k comprised spherical cells (4.5–8.5 μm) with a cup-shaped chloroplast containing a pyrenoid. Single cells were rare, and most cells formed coenobia (Figure 13). Micractinium is essentially a colonial species with several spines. However, recent studies have suggested that such morphological characteristics are unsuitable for defining genera within Chlorellaceae e.g. [52, 56]. In this regard, Luo et al. [56] instead examined the non-homoplasious synapomorphic molecular signature of the SSU rDNA or ITS2 sequence for the genus as well as its phylogenetic relationship. The only intelligible signature of Micractinium is C-G pairing at the tip of ITS2 helix III, which was found in GB1k (Figure 14). In ITS2 sequence comparisons, differences between GB1k and any other Micractinium species reached at least 25% (with gaps counted as fifth character). Although these large differences affect lengths or structures of helices and prevent accurate counting of CBC, there were at least two CBCs between GB1k and any other Micractinium species. Because the genus Micractinium is characterized by a colonial species with several spines, only two Micractinium species are described as having spherical cells (Micractinium reisseri and Micractinium inermum). GB1k is therefore probably a nondescript species of Micractinium.
Figure 12

Maximum likelihood tree constructed from SSU-ITS rDNA sequences (length 2487 nt). Hegewaldia parvula was used to root this tree, which was constructed by using the GTR + I + G evolutionary model. Numbers at each node represent bootstrap probabilities from ML/NJ analyses; only values above 50% are shown.

Figure 13

Light microscopy image of the GB1k algal strain from a private collection. This strain was collected from German inland waters and was named according to its source (Table 1).

Figure 14

Predicted ITS2 secondary structure diagram for GB1k. The synapomorphic signature of genus Micractinium[56] is highlighted. The signature (+UU) at the terminal loop of helix I indicates the polymorphism (indels) in the GB1k genome.

Conclusions

Many microbiology laboratories have vast microbial stocks isolated from natural environments. Some may have been collected as part of a search for new enzymes, antibiotics, or other applications. However, these stocks are often carelessly treated after the study has been completed, and are generally lost thereafter. My laboratory similarly owns large green algal stocks collected from natural water sources in various places. However, the number of culture stocks has been decreasing over time. There are numerous probable causes for this, including changes in the culture medium or other culture conditions, unexpected desiccation, and changes in culture management. If these stocks are likely to contain some interesting species, then it may be better to donate the strains to culture collection institutions, rather than to keep them for no reason. The present study focused on a private collection isolated from German inland waters, and examined their distinctiveness as a preliminary step toward donating the strains to a public collection. The method of establishing strains was very simple (q.v. Methods). This method may have selected only the species with the ability to grow faster and resistance to minor desiccation. Species that grow slowly or are unable to colonize an agar plate would be excluded from the selection. In addition, culture stocks have gone through a change in culture media, and some morphologically characteristic strains have already died out. That is, the culture stocks considered here are likely to be common and very vigorous species. Nevertheless, 12 strains out of 43 did not match any known species of which rDNA sequences have been determined (Table 1). One strain was nondescript even at the genus level. This fact suggests that the true diversity of freshwater green algae is still a long way from being fully described, and that even small laboratory stocks can contribute to our understanding of the diversity of microorganisms. I sincerely hope that this study prompts all researchers around the world to examine private microbial stocks and to publish their results.

There is intense debate about the diversity and distribution of eukaryotic microorganisms. One argument suggests that there is a limited number of species and that most of them are global in distribution e.g. [58]. The opposite argument states that large numbers of species exist although many species have possibly adapted locally; however, their simple morphology prevents discrimination between species e.g. [16, 59]. Now that microbes can be identified to species level by using DNA comparisons, as shown for some strains in this study, it is becoming apparent that a large number of freshwater green algal species exists in the world. Some strains in this study matched “brasiliensis” species (Tetranephris, Desmodesmus), and one (GB1a) matched a Fawley’s strain collected from the USA. There are therefore still many inconsistencies in patterns of species localization or ubiquity.

Abbreviations

BLASTN: 

Basic local alignment search tool for nucleotides

CCAP: 

Culture collection of algae and protozoa

ITS: 

Internal transcribed spacer

NIES: 

National institute for environmental studies (Japan)

SAG: 

The culture collection of algae at Göttingen University

SSU: 

Small subunit

UTEX: 

The culture collection of algae, University of Texas.

Declarations

Acknowledgments

This work was supported by The Sumitomo Foundation Environmental Research Grant (103180), The Institute for Fermentation, Osaka, and JSPS KAKENHI (25840141).

Authors’ Affiliations

(1)
Department of Bioscience, Nagahama Institute of Bio-Science and Technology

References

  1. Adl SM, Leander BS, Simpson AGB, Archibald JM, Anderson OR, Bass D, Bowser SS, Brugerolle G, Farmer MA, Karpov S, Kolisko M, Lane CE, Lodge DJ, Mann DG, Meisterfeld R, Mendoza L, Moestrup Ø, Mozley-Standridge SE, Smirnov AV, Spiegel F: Diversity, nomenclature, and taxonomy of protists. Syst Biol. 2007, 56: 684-689. 10.1080/10635150701494127.PubMedView ArticleGoogle Scholar
  2. Achtman M, Wagner M: Microbial diversity and the genetic nature of microbial species. Nat Rev Microbiol. 2008, 6: 431-40.PubMedGoogle Scholar
  3. Huss VAR, Sogin ML: Phylogenetic position of some Chlorella species within the chlorococcales based upon complete small-subunit ribosomal RNA sequences. J Mol Evol. 1990, 31: 432-442. 10.1007/BF02106057.PubMedView ArticleGoogle Scholar
  4. Huss VAR, Frank C, Hartmann EC, Hirmer M, Kloboucek A, Seidel BM, Wenzeler P, Kessler E: Biochemical taxonomy and molecular phylogeny of the genus Chlorella sensu lato (Chlorophyta). J Phycol. 1999, 35: 587-598. 10.1046/j.1529-8817.1999.3530587.x.View ArticleGoogle Scholar
  5. Ustinova I, Krienitz L, Huss VAR: Closteriopsis acicularis (G. M. Smith) Belcher et Swale is a fusiform alga closely related to Chlorella kessleri Fott et Nováková (Chlorophyta, Trebouxiophyceae). Eur J Phycol. 2001, 36: 341-351. 10.1080/09670260110001735488.View ArticleGoogle Scholar
  6. Wolf M, Krienitz L, Hepperle D: Phylogenetic position of Actinastrum hantzschii Lagerheim 1882 (Chlorophyta, Trebouxiophyceae). Algol Stud. 2002, 104: 59-67.Google Scholar
  7. Krienitz L, Hegewald E, Hepperle D, Huss VAR, Rohr T, Wolf M: Phylogenetic relationship of Chlorella and Parachlorella gen. nov. (Chlorophyta, Trebouxiophyceae). Phycologia. 2004, 43: 529-542. 10.2216/i0031-8884-43-5-529.1.View ArticleGoogle Scholar
  8. Pröschold T, Bock C, Luo W, Krienitz L: Polyphyletic distribution of bristle formation in Chlorellaceae: Micractinium, Diacanthos, Didymogenes and Hegewaldia gen. nov. (Trebouxiophyceae, Chlorophyta). Phycol Res. 2010, 58: 1-8. 10.1111/j.1440-1835.2009.00552.x.View ArticleGoogle Scholar
  9. Krienitz L, Bock C, Kotut K, Pröschold T: Genotypic diversity of Dictyosphaerium–morphospecies (Chlorellaceae, Trebouxiophyceae) in African inland waters, including the description of four new genera. Fottea. 2012, 12: 231-253.View ArticleGoogle Scholar
  10. Musters W, Boon K, van der Sande CA, van Heerikhuizen H, Planta RJ: Functional analysis of transcribed spacers of yeast ribosomal DNA. EMBO J. 1990, 9: 2989-2996.Google Scholar
  11. Hadjiolova KV, Normann A, Cavaillé J, Soupéne E, Mazan S, Hadjiolov AA, Bachellerie J-P: Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells. Mol Cell Biol. 1994, 14: 4044-4056.PubMedPubMed CentralView ArticleGoogle Scholar
  12. Schultz J, Maisel S, Gerlach D, Muller T, Wolf M: A common core of secondary structure of the internal transcribed spacer 2 (ITS2) throughout the Eukaryota. RNA. 2005, 11: 361-364. 10.1261/rna.7204505.PubMedPubMed CentralView ArticleGoogle Scholar
  13. Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K, Li Y, Li X, Jia X, Lin Y, Leon C: Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One. 2010, 5: e8613-10.1371/journal.pone.0008613.PubMedPubMed CentralView ArticleGoogle Scholar
  14. Coleman AW: The significance of a coincidence between evolutionary landmarks found in mating affinity and a DNA sequence. Protist. 2000, 151: 1-9. 10.1078/1434-4610-00002.PubMedView ArticleGoogle Scholar
  15. Müller T, Philippi N, Dandekar T, Schultz J, Wolf M: Distinguishing species. RNA. 2007, 13: 1469-1472. 10.1261/rna.617107.PubMedPubMed CentralView ArticleGoogle Scholar
  16. Fawley MW, Fawley KP, Buchheim MA: Molecular diversity among communities of freshwater microchlorophytes. Microbial Ecol. 2004, 48: 489-499. 10.1007/s00248-004-0214-4.View ArticleGoogle Scholar
  17. Fawley MW, Fawley KP, Owen HA: Diversity and ecology of small coccoid green algae from Lake Itasca, Minnesota, USA, including Meyerella planktonica, gen. et sp. nov. Phycologia. 2005, 44: 35-48. 10.2216/0031-8884(2005)44[35:DAEOSC]2.0.CO;2.View ArticleGoogle Scholar
  18. Lewis LA, Lewis PO: Unearthing the molecular phylodiversity of desert soil green algae (Chlorophyta). Syst Biol. 2005, 54: 936-947. 10.1080/10635150500354852.PubMedView ArticleGoogle Scholar
  19. De Wever A, Leliaert F, Verleyen E, Vanormelingen P, Van der Gucht K, Hodgson DA, Sabbe K, Vyverman W: Hidden levels of phylodiversity in Antarctic green algae: further evidence for the existence of glacial refugia. Proc R Soc B. 2009, 276: 3591-3599. 10.1098/rspb.2009.0994.PubMedPubMed CentralView ArticleGoogle Scholar
  20. Hosoya H, Kimura K, Matsuda S, Kitaura M, Takahashi T, Kosaka T: Symbiotic algae-free strains of the green paramecium Paramecium bursaria produced by herbicide paraquat. Zool Sci. 1995, 12: 807-810. 10.2108/zsj.12.807.View ArticleGoogle Scholar
  21. Ichimura T: Sexual cell division and conjugation-papilla formation in sexual reproduction of Closterium strigosum. Proceedings of the Seventh International Seaweed Symposium: 1971; Tokyo. Edited by: Nishizawa K, Arasaki S, Chihara M, Hirose H, Nakamura V, Tsuchiya Y. 1971, 208-214.Google Scholar
  22. Nakayama T, Watanabe S, Mitsui K, Uchida H, Inouye I: The phylogenetic relationship between the Chlamydomonadales and Chlorococcales inferred from 18S rDNA sequence data. Phycol Res. 1996, 44: 47-55. 10.1111/j.1440-1835.1996.tb00037.x.View ArticleGoogle Scholar
  23. Hoshina R, Kato Y, Kamako S, Imamura N: Genetic evidence of “American” and “European” type symbiotic algae of Paramecium bursaria Ehrenberg. Plant Biol. 2005, 7: 526-532. 10.1055/s-2005-865912.PubMedView ArticleGoogle Scholar
  24. Hoshina R, Kamako S, Imamura N: Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan. Plant Biol. 2004, 6: 447-453. 10.1055/s-2004-820888.PubMedView ArticleGoogle Scholar
  25. White TJ, Bruns T, Lee S, Taylor J: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and application. Edited by: Innis M, Gelfand D, Sninsky J, White T. 1990, San Diego: Academic Press, 315-322.Google Scholar
  26. Hoshina R: Analysis of an intron intervening the SSU rDNA of Chlorella sp. T-24-5, a photobiont of Paramecium bursaria. Jpn J Protozool. 2012, 45: 17-27.Google Scholar
  27. Hoshina R, Imamura N: Multiple origins of the symbioses in Paramecium bursaria. Protist. 2008, 159: 53-63. 10.1016/j.protis.2007.08.002.PubMedView ArticleGoogle Scholar
  28. Krienitz L, Bock C, Nozaki H, Wolf M: SSU rRNA gene phylogeny of morphospecies affiliated to the bioassay alga “Selenastrum capricornutum” recovered the polyphyletic origin of crescent-shaped Chlorophyta. J Phycol. 2011, 47: 880-893. 10.1111/j.1529-8817.2011.01010.x.View ArticleGoogle Scholar
  29. Marin B: Nested in the Chlorellales or independent class? Phylogeny and classification of the Pedinophyceae (Viridiplantae) revealed by molecular phylogenetic analyses of complete nuclear and plastid-encoded rRNA operons. Protist. 2012, 163: 778-805. 10.1016/j.protis.2011.11.004.PubMedView ArticleGoogle Scholar
  30. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace M, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0. Bioinformatics. 2007, 23: 2947-2948. 10.1093/bioinformatics/btm404.PubMedView ArticleGoogle Scholar
  31. Cannone JJ, Subramanian S, Schnare MN, Collett JR, D'Souza LM, Du Y, Feng B, Lin N, Madabusi LV, Müller KM, Pande N, Shang Z, Yu N, Gutell RR: The Comparative RNA Web (CRW) Site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics. 2002, 3: 2-10.1186/1471-2105-3-2.PubMedPubMed CentralView ArticleGoogle Scholar
  32. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28: 2731-2739. 10.1093/molbev/msr121.PubMedPubMed CentralView ArticleGoogle Scholar
  33. Posada D, Crandall KA: MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998, 14: 817-818. 10.1093/bioinformatics/14.9.817.PubMedView ArticleGoogle Scholar
  34. Hegewald E, Bock C, Krienitz L: A phylogenetic study on Scenedesmaceae with the description of a new species of Pectinodesmus and the new genera Verrucodesmus and Chodatodesmus (Chlorophyta, Chlorophyceae). Fottea. 2013, 13: 149-164.View ArticleGoogle Scholar
  35. Eliáš M, Němcová Y, Škaloud P, Neustupa J, Kaufnerová V, Šejnohová L: Hylodesmus singaporensis gen. et sp. nov., a new autosporic subaerial green alga (Scenedesmaceae, Chlorophyta) from Singapore. Int J Syst Evol Microbiol. 2010, 60: 1224-1235. 10.1099/ijs.0.012963-0.PubMedView ArticleGoogle Scholar
  36. Hoshina R, Fujiwara Y: Molecular characterization of Chlorella cultures of the NIES culture collection with description of Micractinium inermum sp. nov., Didymogenes sphaerica sp. nov., and Didymogenes soliella sp. nov. (Chlorellaceae, Trebouxiophyceae). Phycol Res. 2013, 61: 124-132. 10.1111/pre.12010.View ArticleGoogle Scholar
  37. Zuker M: Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003, 31: 3406-3415. 10.1093/nar/gkg595.PubMedPubMed CentralView ArticleGoogle Scholar
  38. Krienitz L, Ustinova I, Friedl T, Huss VAR: Traditional generic concepts versus 18S rRNA gene phylogeny in the green algal family Selenastraceae (Chlorophyceae, Chlorophyta). J Phycol. 2001, 37: 852-865. 10.1046/j.1529-8817.2001.01004.x.View ArticleGoogle Scholar
  39. Fawley MW, Dean ML, Dimmer SK, Fawley KP: Evaluating the morphospecies concept in the Selenastraceae (Chlorophyceae, Chlorophyta). J Phycol. 2005, 42: 142-154.View ArticleGoogle Scholar
  40. Friedl T, O’Kelly CJ: Phylogenetic relationships of green algae assigned to the genus Planophila (Chlorophyta): evidence from 18S rDNA sequence data and ultrastructure. Eur J Phycol. 2002, 37: 373-384. 10.1017/S0967026202003712.View ArticleGoogle Scholar
  41. Deason TR, Silva PC, Watanabe S, Floyd GL: Taxonomic status of the species of the green algal genus Neochloris. Pl Syst Evol. 1991, 177: 213-219. 10.1007/BF00937958.View ArticleGoogle Scholar
  42. McManus HA, Lewis LA: Molecular phylogenetics, morphological variation and colony-form evolution in the family Hydrodictyaceae (Sphaeropleales, Chlorophyta). Phycologia. 2005, 44: 582-595. 10.2216/0031-8884(2005)44[582:MPMVAC]2.0.CO;2.View ArticleGoogle Scholar
  43. Keller A, Schleicher T, Förster F, Ruderisch B, Dandekar T, Müller T, Wolf M: ITS2 data corroborate a monophyletic chlorophycean DO-group (Sphaeropleales). BMC Evol Biol. 2008, 8: 218-10.1186/1471-2148-8-218.PubMedPubMed CentralView ArticleGoogle Scholar
  44. Fucikova K, Lewis LA: Intersection of Chlorella, Muriella and Bracteacoccus: resurrecting the genus Chromochloris KOL et CHODAT (Chlorophyceae, Chlorophyta). Fottea. 2012, 12: 83-93.View ArticleGoogle Scholar
  45. Markert SM, Müller T, Koetschan C, Friedl T, Wolf M: ‘Y’ Scenedesmus (Chlorophyta, Chlorophyceae): the internal transcribed spacer 2 rRNA secondary structure re-revisited. Plant Biol. 2012, 14: 987-996. 10.1111/j.1438-8677.2012.00576.x.PubMedView ArticleGoogle Scholar
  46. Booton GC, Floyd GL, Fuerst PA: Multiple group I introns detected in the nuclear small subunit rDNA of the autosporic green alga Selenastrum capricornutum. Curr Genet. 2004, 46: 228-234. 10.1007/s00294-004-0525-5.PubMedView ArticleGoogle Scholar
  47. Hegewald E, Wolf M, Keller A, Friedl T, Krienitz L: ITS2 sequence–structure phylogeny in the Scenedesmaceae with special reference to Coelastrum (Chlorophyta, Chlorophyceae), including the new genera Comasiella and Pectinodesmus. Phycologia. 2010, 49: 325-335. 10.2216/09-61.1.View ArticleGoogle Scholar
  48. Johnson JL, Fawley MW, Fawley KP: The diversity of Scenedesmus and Desmodesmus (Chlorophyceae) in Itasca State Park, Minnesota, USA. Phycologia. 2007, 46: 214-229. 10.2216/05-69.1.View ArticleGoogle Scholar
  49. Fawley MW, Fawley KP, Hegewald E: Taxonomy of Desmodesmus serratus (Chlorophyceae, Chlorophyta) and related taxa on the basis of morphological and DNA sequence data. Phycologia. 2011, 50: 23-56. 10.2216/10-16.1.View ArticleGoogle Scholar
  50. Fawley MW, Fawley KP, Hegewald E: Desmodesmus baconii (Chlorophyta), a new species with double rows of arcuate spines. Phycologia. 2013, 52: 565-572. 10.2216/12-116.1.View ArticleGoogle Scholar
  51. Krienitz L, Bock C: Present state of the systematics of planktonic coccoid green algae of inland waters. Hydrobiologia. 2012, 698: 295-326. 10.1007/s10750-012-1079-z.View ArticleGoogle Scholar
  52. Luo W, Pflugmacher S, Pröschold T, Walz N, Krienitz L: Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae). Protist. 2006, 157: 315-333. 10.1016/j.protis.2006.05.006.PubMedView ArticleGoogle Scholar
  53. Bock C, Pröschold T, Krienitz L: Two new Dictyosphaerium-morphotype lineages of the Chlorellaceae (Trebouxiophyceae): Heynigia gen. nov. and Hindakia gen. nov. Eur J Phycol. 2010, 45: 267-277. 10.1080/09670262.2010.487920.View ArticleGoogle Scholar
  54. Bock C, Krienitz L, Pröschold T: Taxonomic reassessment of the genus Chlorella (Trebouxiophyceae) using molecular signatures (barcodes), including description of seven new species. Fottea. 2011, 11: 293-312.View ArticleGoogle Scholar
  55. Bock C, Pröschold T, Krienitz L: Updating the genus Dictyosphaerium and description of Mucidosphaerium gen. nov. (Trebouxiophyceae) based on morphological and molecular data. J Phycol. 2011, 47: 638-652. 10.1111/j.1529-8817.2011.00989.x.View ArticleGoogle Scholar
  56. Luo W, Pröschold T, Bock C, Krienitz L: Generic concept in Chlorella-related coccoid green algae (Chlorophyta, Trebouxiophyceae). Plant Biol. 2010, 12: 545-553. 10.1111/j.1438-8677.2009.00221.x.PubMedView ArticleGoogle Scholar
  57. Hoshina R, Iwataki M, Imamura N: Chlorella variabilis and Micractinium reisseri sp. nov. (Chlorellaceae, Trebouxiophyceae): Redescription of the endosymbiotic green algae of Paramecium bursaria (Peniculia, Oligohymenophorea) in the 120th year. Phycol Res. 2010, 58: 188-201. 10.1111/j.1440-1835.2010.00579.x.View ArticleGoogle Scholar
  58. Finlay BJ: Global dispersal of free-living microbial eukaryote species. Science. 2002, 296: 1061-1063. 10.1126/science.1070710.PubMedView ArticleGoogle Scholar
  59. Coleman AW: Microbial eukaryote species. Science. 2002, 297: 337-PubMedView ArticleGoogle Scholar

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