Comparison Between Phylogenetic Relationships Based on 18S rDNA Sequences and Growth by Salinity of <italic>Chlorella</italic>-like Species (Chlorophyta)
Comparison Between Phylogenetic Relationships Based on 18S rDNA Sequences and Growth by Salinity of Chlorella-like Species (Chlorophyta)
Fisheries and aquatic sciences. 2012. Jun, 15(2): 125-135
Copyright ©2012, The Korean Society of Fisheries and Aquatic Science
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  • Received : March 03, 2012
  • Accepted : May 05, 2012
  • Published : June 30, 2012
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Hye Jung Lee
Sung Bum Hur
This study was carried out to understand the correlation between phylogenetic relationships based on 18S rDNA sequences and growth by salinity of Chlorella -like species. The 18S rDNA sequences of 71 Chlorella -like species which were mainly collected from Korean waters were analyzed. The 18S rDNA sequences of Chlorella -like species were divided into three groups (group A, B and C) and group B was further divided into three subgroups (subgroup B-1, B-2 and B-3). Thirty-seven Chlorella -like species in group A grew well at high salinity (32 psu) but the other groups grew well in freshwater. The sequence identities of the species in group A and B were 97.2-99.5%, but those of 6 species in group C (“ Chlorella saccharophila ), which contained group I intron sequences region were 75.0-75.4%. Two representative species of each group were cultured at different salinities (0, 16 and 32 psu) to examine the correlation between the molecular phylogenetic groups and the phenotypic characteristics on cell growth and size by different salinities. The size of cell cultured at different salinities varied according to the species of each molecular phylogenetic group. The size of “ Chlorella saccharophila in group C was bigger and more obviously elliptical rather than that of the other Chlorella -like species. Considering the results on molecular and phenotypic characteristics, the group A and B belonged to Chlorellaceae, but group C was distinctly different from them.
The genus Chlorella is unicellular green algae with sphere or elliptical shape of very small size around 2-10 μm. Since Chlorella vulgaris Beijerinck, which was a type species of the genus Chlorella was isolated (Beijerinck, 1890), a number of Chlorella were widely studied or utilized as industrial materials (Scragg et al., 2003; Yoshida et al., 2006; Rioboo et al., 2009). Most of the coccoid green algae that are very small in size and similar in shape, classification of Chlorella has been considered as one of the most difficult studies in phylogenetic classification (Krienitz et al., 2004).
Though more than 100 species of coccoid green algae were classified as members of the genus Chlorella , most of them were transferred to other genus such as Micractinium , Didymogenes and Actinastrum through taxonomic studies based on morphological characters (Fott and Nováková, 1969; Andreyeva, 1975). Because of the lack of distinct morphological characters for species for identification of Chlorella , the ultrastructure of cell walls or pyrenoids (Ikeda and Takeda, 1995; Nemcová and Kalina, 2000), comparative physiology and biochemistry (Kessler and Huss, 1992; Kadono et al., 2004), and nutrient requirements (Shihira and Krauss, 1965; Wang and Dei, 2001), were often adopted for their precise classification.
Since the first report of 18S ribosomal RNA gene (rDNA) sequence of the type species, C. vulgaris (Huss and Sogin, 1989) the polyphyly of the genus Chlorella was proposed based on 18S rDNA sequences information (Huss and Sogin, 1990; Friedl, 1997; Huss et al., 1999). After that, through accumulated biochemical data and analysis of 18S rDNA sequences, only four “true” Chlorella species ( C. kessleri , C. lobophora , C. sorokiniana , and C. vulgaris ) was proposed (Huss et al., 1999). Besides, on the basis of the phylogenetic analysis of 18S rDNA sequences, Krienitz et al. (2004) had suggested that the family Chlorellaceae is composed of Chlorella and Parachlorella clades. More recently, Luo et al. (2010) revealed that five genera of green algae ( Micractinium , Didymogenes , Actinastrum , Meyerella , and Hegewaldia ) were phylogenetically closely affiliated to the genus Chlorella inferred from nucleotide sequences of 18S rDNA and ITS regions.
Korea Marine Microalgae Culture Center (KMMCC) had collected 80 species of Chlorella -like species from coastal and inland regions in Korea or from foreign collection centers (Hur, 2008). Chlorella -like species were identified at level of genus by observation of light microscopy, but it was confusing to identify at species level because of their similar morphology. In particular, Chlorella -like species isolated from estuary water was obscure to identify its origin as marine or freshwater
Culture history of seventy-one Chlorella-like species for the study and their GenBank accession numbers for the 18S rDNA sequencesKMMCC : Korea Marine Microalgae Culture Center ; UTEX : University of Texas Culture Collection; PKNU : pond of Pukyong National University.
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Culture history of seventy-one Chlorella-like species for the study and their GenBank accession numbers for the 18S rDNA sequences KMMCC : Korea Marine Microalgae Culture Center ; UTEX : University of Texas Culture Collection; PKNU : pond of Pukyong National University.
species. Therefore, in this study, 18S rDNA sequences of 71 Chlorella -like species collected mainly from Korean waters were examined. In addition, growth and size of representative Chlorella -like species from each identified group were also tested at different salinities of culture media. And the correlation between molecular phylogenetic relationships and phenotypic characteristics on growth and size of Chlorella -like species was analyzed.
Materials and Methods
- 18S rDNA gene amplification and sequence analysis
Seventy-one Chlorella -like species used in this study were received from KMMCC in Pukyong National University. Among them, six species were obtained from abroad and the rest of the 65 species were isolated from coastal and inland regions in Korea ( Table 1 ). Forty six species of green algae which were used in the phylogenetic tree which were obtained from GenBank.
Chlorella -like species were stationary cultured in 150 mL media volume of 250 mL Erlenmeyer flask using a temperature of 25℃ with continuous illumination of 100 μmol photons m -2 s -1 for 2 weeks. F/2 medium (Guillard and Ryther, 1962) for marine Chlorella -like species and Jaworski medium (Thompson et al., 1988) for freshwater Chlorella -like species were used. Cultured microalgae were harvested and their genomic DNA was isolated using LiCl method (Hong et al., 1995) or Wizard ® Genomic DNA Purification System (Promega, Medison, WI, USA). Isolated genomic DNA was performed polymerase chain reactions (PCR) using P2F (GGC TCA TTA AAT CAG TTA TAG) / MF (ACC TGG TTG ATC CTG CCA G) forward primers and P2R (CCT TGT TAC GA(C/T) TTC TCC TTC) / RF (GTG AAC CTG C(G/A)G AAG GAT CA) reverse primers (Huss et al., 1999; Lee and Hur, 2009) for 18S rDNA gene amplification. Sequences were obtained from cloning and the sequencing process (Lee and Hur, 2009). Sequences were subjected to homology analysis by using Blast N program and aligned by using ClustalW2 program (Thomp-
continuedKMMCC : Korea Marine Microalgae Culture Center ; UTEX : University of Texas Culture Collection; PKNU : pond of Pukyong National University.
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continued KMMCC : Korea Marine Microalgae Culture Center ; UTEX : University of Texas Culture Collection; PKNU : pond of Pukyong National University.
son et al., 1994). For these species, 18S rDNA sequences were acquired and their accession numbers were registered in Gen- Bank of NCBI.
Genetic distance of sequences was calculated by Kimura 2-parameter model (Kimura, 1980) and sequence identity was carried out by Bioedit Sequence Alignment Editor version (Hall, 1999). To analyze the phylogenetic relationships of sequences, maximum likelihood (ML), maximum parsimony (MP), and neighbor joining (NJ) analysis were conducted using MEGA v.5.0 (Tamura et al., 2011). The ML analysis was constructed based on the Kimura 2-parameter with proportion of invariable sites and gamma distribution split into five categories (K2+ I + G ). This model was selected by test as best-fit substitution model of nucleotide sequence data. The MP analysis was obtained using the Close- Neighbor- Interchange algorithm (Nei and Kumar, 2000) with search level 3 in which the initial trees were obtained with the random addition of sequences (100 replicates). The NJ analysis model was used for maximum composite likelihood and 2000 bootstrap replications were carried out to support the reliability of the tree and the species with similar sequences were grouped together.
- Growth and size of representative species from each group at different salinities
Growth characteristics of the species from each group were examined and compared to each other. Two representative species showing the difference on the phylogenetic relationships of sequences from each group were selected and cultured at different salinities (0, 16, and 32 psu). The specific growth rate (s.g.r./day=3.322×log (N 1 /N 0 )/t 1 -t 0 (N 1 and N 0 : cell number at t 1 and t 0 day)) (Guillard, 1973) and size of the cell were examined. For this culture, f/2 medium (32 psu) was used for marine Chlorella -like species. For estuary and freshwater Chlorella -like species, nutrient concentrations of f/2 medium were also used and the salinity for estuary (16 psu) and freshwater (0 psu) Chlorella -like species was made with distilled water and sea water. Microalgae were cultured in 250 mL Erlenmeyer flask with 100 mL medium at 25℃ with continuous light of 100 μmol photons m -2 s -1 for 10 days. Culture experiments were replicated three times. The cell number of microalgae was counted daily using a hemacytometer. The size of 40 cells at the initial and final culture days was measured using MoticamPro 205A CCD scientific camera (Motic Instruments Inc., Richomond, BC, Canada).
Percentage of sequence identity (top right triangle) and nucleotide pairwise distance calculation (bottom left triangle) of the 18S rDNA sequences of ten representative Chlorella-like species and six species from GenbankKAS: Korean Algae from Seawater; KMMCC: Korea Marine Microalgae Culture Center; SAG: Sammlung von Algenkulturen der Universit?t G?ttingen.
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Percentage of sequence identity (top right triangle) and nucleotide pairwise distance calculation (bottom left triangle) of the 18S rDNA sequences of ten representative Chlorella-like species and six species from Genbank KAS: Korean Algae from Seawater; KMMCC: Korea Marine Microalgae Culture Center; SAG: Sammlung von Algenkulturen der Universit?t G?ttingen.
- Statistical analysis
Data were analyzed by one-way ANOVA test, and Duncan’s multiple range test (Duncan, 1955) was applied for the significance level ( P <0.05). SPSS version 10.1 (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis.
Results and Discussion
- Analysis of 18S rDNA sequences
Molecular phylogenetic analysis of 18S rDNA sequences from 71 Chlorella -like species showed three independent groups (group A, B and C) with 94-99% bootstrap value. Group B was divided into three subgroups (subgroup B-1, B-2, and B-3) ( Fig 1 .). Maximum likelihood phylogenetic analysis by using 21 representative species of the acquired Chlorella -like species sequences and 46 species of green algae sequences referred from GenBank also confirmed three groups with 97-100% bootstrap value ( Fig. 2 ). Sequence identity and genetic distance of 10 species (group A: KMMCC-234, 870; group B-1: KMMCC-9, 115; group B-2: KMMCC-132, 137; group B-3: KMMCC-6, 143; group C: KMMCC-3, 183) which were composed with two representative species from each of the five groups as well as six species obtained from GenBank were analyzed ( Table 2 ). The sequence identity of KMMCC-234 and 870 from group A was 99.5%. Sequence
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Molecular phylogenetic tree using the Neighbor-joining method inferred from 18S ribosomal DNA sequences of 71 Chlorella-like species. Tree reliability is tested by 2000 bootstraps, which indicates numbers (bold letters) at the nodes. Only values above 50% are shown.
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Maximum-likelihood tree of 18S rDNA sequences from members of the Trebouxiophyceae. Numbers above the nodes indicate support bootstrap values of maximum-likelihood (right), maximum-parsimony (middle) and neighbor-joning (left) analysis. Only values above 50% are shown. Ten representative species from each group are indicated as bold letters. Ulothrix zonata is indicated for one group.
identity compared with group A and group B showed 97.2- 97.9%, and that between KMMCC-3 and 183 from group C turned out to be same. However, group C showed low sequence identity of 75.0-75.4% with group A and B.
Group A was diverged monophyletic with bootstrap value of 99% in Fig. 2 . Thirty-seven species which corresponded to 52.1% of total were included in this group. Most of the species collected from Korean coastal waters belonged to this group. The length of 18S rDNA sequences of the species in group A was same with 1672 bp. Nine species (KMMCC-8, 86, 87, 163, 345, 434, 870, 1008, and 1009) which composed 24.3% of group A were identical in sequence and other 28 species showed sequence differences of 1-7 bp. The representative species (KMMCC-234 and 870) of group A were located in same clade with Parachlorella beijerinckii and P. kessleri and sequence identity between genus Parachlorella and species of group A was 98.7-98.9% with sequence differences of 15-21 bp. However, genus Parachlorella was closer to Closteriopsis acicula and Dicloster acuatus than group A and formed different cluster from group A. Moreover, Parachlorella was freshwater species, but most of species of group A were marine.
Chlorella sp. KAS 012 isolated from Korean sea water showed high sequence similarity over 99.5% with group A. This species exhibited very small and simple spherical morphology that was similar with the strains of Parachlorella (Aslam et al., 2007) although several egg-shaped cells of Parachlorella were observed (Krienitz et al., 2004). And Chlorella sp. KAS 012 possessed halotolerant and thermotolerant features. Aslam et al. (2007) proposed Chlorella sp. KAS 012 to genus Marinichlorella on the basis of the phenotypic and phylogenetic data. Therefore, it was judged that marine habitat species existed in Parachlorella clade. Chlorella sp. KMMCC-870 which was isolated from estuary of Geumriver was considered as a freshwater species on account of low salinity of sampling area. But this species was revised as a marine species. This misunderstanding was due to tidal currents in the estuary area.
Group B was diverged with 94-99% bootstrap value and 28 species which composed of 39.4% of total species were included in this group. Two species from UTEX (KMMCC-6 and 9) and 26 species collected from the inland area were included in this group and their sequence length was 1672- 1673 bp. Group B was divided into three subgroups. Chlorella vulgaris Beijerinck SAG 211-11b belonged to subgroup B-1. Sequence length of six species in subgroup B-1 showed same length with 1673 bp and over 99.4% identity with only 1-4 bp sequence differences ( Fig. 2 ). Five among six species in subgroup B-2 were collected from Nakdong-river and Namcheon- cheon. The sequence length of the five species was the same with 1672 bp and sequence identity was 99.5-99.9% with only 1-5 bp sequence differences. However, in case of Chlorella sp. KMMCC-132 collected from Jinhae Bay, sequence difference was 13 bp which had large difference. And sequence identity was also slightly low (98.8-99.2%). In comparison with subgroup B-1, it showed similar tendency with 98.5-98.9% of sequence similarity and 18 bp of sequence differences. Therefore, the position of KMMCC-132 in group B was not clear. The sequence length of 16 species in subgroup B-3 which were collected from freshwater was 1672 bp. The sequence identity was over 99.1% and sequence difference was 1-7 bp in subgroup B-3. In the molecular phylogentic tree with 71 Chlorella -like species ( Fig. 1 ), subgroup B-1 and B-2 were closely located, but B-3 was distantly diverged. It indicated that subgroup B-1 and B-2 were closer to each other. However, in Fig. 2 , subgroup B-1, B-2, and B-3 were located next to each other, together with Actinastrum hantzschii and Dictyospherium pulchellum .
Group C was branched with 99-100% bootstrap value and six species of “ Chlorella saccharophila were included in group C. The sequence length of them was 2075 bp and sequence identity was 99.6-100% with 1-5 bp of sequence differences. Group C included group I intron of 400 bp sequences and neighbored with Watanabea reniformis (Hanagata et al., 1998) forming a monophyletic lineage within Trebouxiophyceae. Group C was also considerably apart from group A and B in the tree. Friedl (1995) established new class Trebouxiophyceae by analysis of the ribosomal RNA sequences from coccoid green algae, which was the sister group of Chlorophyceae. Huss et al. (1999) reported four species of ‘true’ Chlorella belong to familiy Chlorellaceae in class Trebouxiophyceae. “ Chlorella saccharophila did not belong to the true Chlorella group (Huss et al., 2002).
In this study, group A and B belonged to Chlorellaceae which were diverged with 91-93% high bootstrap value in the phylogenetic tree ( Fig. 2 ), but the another group of Trebouxiophyceae was diverged with 49-69% low bootstrap value. This finding was similar to the result (45-59%) of Huss et al. (1999). This study also supported that “ Chlorella saccharophila of group C was not in ‘true’ Chlorella group. And taxonomic status of the group C species was unclear.
- Specific growth rate and size of cell by salinity
The specific growth rate by salinity of two representative Chlorella -like species from each group was shown in Fig. 3 . Group A showed the highest growth rate, whereas group C showed the lowest growth rate ( P <0.05). Group B showed intermediate tendency in growth rate. The specific growth rate of Chlorella sp. KMMCC-870 and 234 in group A was 0.75 and 0.55, respectively at 32 psu, which were significantly higher than that at 16 and 0 psu. The growth rate of both species at 0 psu was lowest with 0.46 ( P <0.05). Six species in group B showed the fastest growth rate at 0 psu with 0.24- 0.45 compared with 0.01-0.29 at 32 psu. In particular, subgroup B-3 showed the lowest growth rate with 0.01 at 32 psu as compared with that of B-1 and B-2. KMMCC-3 and 183 in group C showed growth rate of 0.13-0.18 at 0 and 16 psu, which were the lowest among the ten species. These species
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Specific growth rate of ten representative Chlorella-like species cultured at different salinities (■ : 0 psu; ׈ : 16 psu; ⌨ : 32 psu). The different letters on the bar mean a significant difference (P<0.05). KMMCC: Korea Marine Microalgae Culture Center.
showed significant high growth rate at 0, 16 psu rather than 32 psu ( P <0.05).
With regards to cell size after 10 days of culture at different salinities ( Fig. 4 ), the size of KMMCC-234 and 870 in group A were 3.12 μm and 3.60 μm, respectively at 32 psu. However, the cell sizes of two species at 16 psu were 3.63 μm and 3.94 μm, and those were 3.94 μm and 4.36 μm at 0 psu, respectively. The cell size of KMMCC-234 and 870 was significantly larger in lower salinity ( P <0.05). The size at the final day showed a decreased tendency compared to that at inoculation day at 32 psu. In the case of KMMCC-870 which was collected at the Geum-river, the cell size was larger as with its lower salinity. This result was also in conformity with molecular phylogenetic analysis and cell growth rate as salinity, suggesting that it was originally a marine species.
The cell size of six species (KMMCC-6, 9, 115, 132, 137, and 143) in group B varied from 3.49 μm to 4.93 μm at 0 psu after 10 days of culture. The cell size was 3.91-5.43 μm at 16 psu and 4.59-7.70 μm at 32 psu. Especially the cell size of C. vulgaris KMMCC-6 and 143 in subgroup B-3 was 3.77 μm and 3.49 μm, respectively at 0 psu. The final size of the species was not significantly different compared with initial size. But the cell sizes of them were 4.96 μm and 5.18 μm, respectively at 16 psu, and 5.85 μm and 7.25 μm, respectively at 32 psu. The cell sizes were larger at higher salinity ( P <0.05). Among these, the difference in cell size of KMMCC-143 between 0 psu and 32 psu was more than two times. The cell size of the species in group A and B showed significantly larger as the conditions of salinity when compared to the optimum salinity for growth rate.
Group C showed no significant difference in cell size by sa-linity, which was contrary to that of both group A and B. This feature also corresponded with molecular phylogenetic analysis. The cell size of microalgae increased under unsuitable salinity conditions due to effect of proline (Szekely, 2004). Proline increased the size of the cytoplasm and free water contents along with glycine betain (Record et al., 1998) and engaged in increased salt resistance of microalgae (Hiremath and Matad, 2010).
Elliptical “ Chlorella saccharophila were characterized by unequal size of autospores during sporogenesis (Fott and Nováková, 1969). This feature has the advantage of survival under unsuitable environments (Darienko et al., 2010). Bigger autospores increased proliferation rate than smaller ones, while smaller ones were widely spread by the wind. So, “ Chlorella saccharophila were found in a wide range of extreme environmental conditions such as exposure to trees, rocks, and acidic soil or high temperature soil (Kessler, 1965; Huss et al., 2002; Mikhailyuk et al., 2003; Gray et al., 2007). Because of the morphological and taxonomical differentiation from ‘true’ Chlorella , Darienko et al. (2010) proposed that “ Chlorella saccharophila be transferred to the Chloroidium saccharophilum .
In conclusion, the three groups of Chlorella -like species based on the sequence analysis of 18S rDNA showed corresponding tendency with growth and size variation of the cell cultured at different salinities. Thus, the comparison between phylogenetic relationships based on 18S rDNA sequences and phenotypic growth characteristics on salinity in the culture experiment can be considered as an useful method for systematic classification of Chlorella -like species which are difficult to identify.
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Size variations of Chlorella-like species cultured at different salinities during ten days ( I : size at the inoculation day; ■ : 0 psu; ὎ : 16 psu; ⌨ : 32 psu). The different letters on the bar mean a significant difference (P<0.05). KMMCC: Korea Marine Microalgae Culture Center.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2011-0027480) and the Marine Biomaterials Research Center grant from Marine Biotechnology Program funded by the Ministry of Land, Transport and Maritime Affairs, Korea.
Andreyeva VM. 1975 Rod Chlorella: morfologya, sistematika, printsipy klassifikatsii. Nauka Leningrad 88 -
Aslam Z , Shin W , Kim MK , Im WT , Lee ST. 2007 Marinichlorella kaistiae gen. et sp. nov. (Trebouxiophyceae, Chlorophyta) based on polyphasic taxonomy. J Phycol 43 576 - 584
Beijerinck MW. 1890 Culturversuche mit zoochlorellen, lichenengo-parsiniden und anderen niederen algen I-III. Botanische Zeitung 48 726 - 740
Darienko T , Gustavs L , Mudimu O , Menendez CR , Schumann R , Karsten U , Friedl T , Pröschold T. 2010 Chloroidium, a common terrestrial coccoid green alga previously assigned to Chlorella (Trebouxiophyceae, Chlorophyta). Eur J Phycol 45 79 - 95
Duncan DB. 1955 Multiple range and multiple F tests. Biometrics 11 1 - 42
Fott B , Nováková M. 1969 A monograph of the genus Chlorella. The fresh water species. In: Studies in phycology. Fott B, ed. Academia Prague 10 - 74
Friedl T. 1995 Inferring taxonomic positions and testing genus level assignments in coccoid green algae: a phylogenetic analysis of 18S ribosomal RNA sequences from Dictyochloropsis reticulata and from members of the genus Myrmecia (Chlorophyta, Trebouxiophyceae cl. nov.). J phycol 31 632 - 639
Friedl T. 1997 The evolution of the green algae. Plant Syst Evol Suppl 11 87 - 101
Gray DW , Lewis LA , Cardon ZG. 2007 Photosynthetic recovery following desiccation of desert green algae (Chlorophyta) and their aquatic relatives. Plant Cell Environ 30 1240 - 1255
Guillard RRL. 1973 Division rates. In: Handbook of Phycological Methods: Culture Methods and Growth Measurements. Stein JR ed. Cambridge University Press London, U.K. 289 - 311
Guillard RRL , Ryther JH. 1962 Studies on marine planktonic diatoms. Cyclotella nana Hustedt and Detonula confervacea (Clece) Gran. Can J Microbiol 3 229 - 239
Hanagata N , Karube I , Chihara M , Silva PC. 1998 Reconsideration of the taxonomy of ellipsoidal species of Chlorella (Trebouxiophyceae, Chlorophyta), with establishment of Watanabea gen. nov. Phycol Res 46 221 - 229
Hall TA. 1999 Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41 95 - 98
Hiremath S , Mathad P. 2010 Impact of salinity on the physiological and biochemical traits of Chlorella vulgaris Beijerinck. J Algal Biomass Utln 1 51 - 59
Hong YK , Kim SD , Polne-Fuller M , Gibor A. 1995 DNA extraction conditions from Porphyra perforata using LiCl. J Appl Phycol 7 101 - 107
Hur SB. 2008 Korea Marine Microalgae Culture Center-List of strains. Algae 23 1 - 68
Huss VAR , Ciniglia C , Cennamo P , Cozzolino S , Pinto G , Pollio A 2002 Phylogenetic relationships and taxonomic position of Chlorella-like isolates from low pH environments (pH<3.0). BMC Evol Biol 26 2 - 13
Huss VAR , Frank C , Hartmann EC , Hirmer M , Kloboucek A , Seidel BM , Wenzeler P , Kessler E. 1999 Biochemical taxonomy and molecular phylogeny of the genus Chlorella sensu lato (Chlorophyta). J Phycol 35 587 - 598
Huss VAR , Sogin ML. 1989 Primary structure of the Chlorella vulgaris small subunit ribosomal RNA coding region. Nucl Acids Res 17 1255 -
Huss VAR , Sogin ML. 1990 Phylogenetical position of some Chlorella species within the Chlorococcales based upon complete small-subunit ribosomal RNA sequences. J Mol Evol 31 431 - 442
Ikeda T , Takeda H. 1995 Species-specific differences of pyrenoids in Chlorella (Chlorophyta). J Phycol 35 587 - 598
Kadono T , Kawano T , Hosoya H , Kosaka T. 2004 Flow cytometric studies of the host-regulated cell cycle in algae symbiotic with green paramecium. Protoplasma 223 133 - 141
Kessler E. 1965 Physiologische und biochemische beiträge zur taxonomie der gattung Chlorella. I. S?ureresistenz als taxonomisches merkmal. Arch Mikrobiol 52 291 - 296
Kessler E , Huss VAR. 1992 Comparative physiology and biochemistry and taxonomic assignment of the Chlorella (Chlorophyceae) strains of the culture collection of the university of Texas at Austin. J Phycol 28 550 - 553
Kimura M. 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16 111 - 120
Krienitz L , Hegewald EH , Hepperle D , Huss VAR , Rohr T , Wolf M. 2004 Phylogenetic relationship of Chlorella and Parachlorella gen. nov. (Chlorophyta, Trebouxiophyceae). Phycologia 43 529 - 542
Lee HJ , Hur SB. 2009 Genetic relationships among multiple strains of the genus Tetraselmis based on partial 18S rDNA sequences. Algae 24 205 - 212
Luo W , Pröschold T , Bock C , Krienitz L. 2010 Generic concept in Chlorella-related coccoid green algae (Chlorophyta, Trebouxiophyceae). Plant Biol 12 545 - 223
Mikhailyuk TI , Demchenko EM , Kondratyk SY. 2003 Algae of granite outcrops from the left bank of Pivdennyi bug river (Ukraine). Biologia 58 589 - 601
Nei M , Kumar S. 2000 Molecular Evolution and Phylogenetics. Oxford University Press New York 115 - 140
Nemcová Y , Kalina T. 2000 Cell wall development, microfibril and pyrenoid structure in type strains of Chlorella vulgaris, C. kessleri, C. sorokiniana compared with C. luteoviridis (Trebouxiophyceae, Chlorophyta). Algological Studies 100 95 - 105
Record MT Jr , Couternay ES , Cayley S , Guttman JH. 1998 Biophysical compensation mechanism buffering E. coli proteins, nucleic acid interactions against changing environment. Trend Biochem Sci 23 190 - 194
Rioboo C , O’Connor JE , Prado R , Herrero C , Cid A. 2009 Cell proliferation alterations in Chlorella cells under stress conditions. Aquatic Toxicol 94 229 - 237
Scragg AH , Morrison J , Shales SW. 2003 The use of a fuel containing Chlorella vulgaris in a diesel engine. Enzyme Microb Technol 33 884 - 889
Shihira J , Krauss RW. 1965 Chlorella. Physiology and taxonomy of forty-one isolates. University of Maryland Maryland , USA. MD. Thesis
Szekely G. 2004 The role of proline in Arabidopsis thaliana osmotic stress response. Acta Biol Szeged 48 81 -
Tamura K , Peterson D , Peterson N , Stecher G , Nei M , Kumar S. 2011 MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28 2731 - 2739
Thompson A , Rhodes J , Pettman I. 1988 Culture collection of algae and protozoa catalogue of strains. Natural Environment Research Council Swindon 164 -
Thompson JD , Higgins DG , Gibson TJ. 1994 Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22 4673 - 4680
Wang WX , Dei RC. 2001 Effects of major nutrient additions on metal uptake in phytoplankton. Environ Pollut 111 233 - 240
Yosida N , Ikeda R , Okuno T. 2006 Identification and characterization of heavy metal-resistant unicellular alga isolated from soil and its potential for phytoremediation. Bioresour Technol 97 1843 - 1849