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Molecular evidence identifies bloom-forming Phaeocystis (Prymnesiophyta) from coastal waters of southeast China as Phaeocystis globosa
Biochemical Systematics and Ecology 30 (2002) 15–22
Molecular evidence identifies bloom-forming Phaeocystis (Prymnesiophyta) from coastal waters of southeast China as Phaeocystis globosa Yue-Qin Chen*, Ning Wang, Peng Zhang, Hui Zhou, Liang-Hu Qu Key Laboratory of Gene Engineering of Education Ministry, Biotechnology Research Center, Zhongshan Universtiy, Guangzhou 510275, PR China Received 12 July 2000; accepted 23 April 2001
Abstract Sequence data from the 18S small subunit ribosomal RNA gene have been used to identify the species of a Phaeocystis (Prymnesiophyta) that caused harmful algae blooms in the coastal waters of southeast China. This Phaeocystis has morphological and physiological features that differ from those previously described for either P. globosa Scherffel or P. pouchetii (Hariot) Lagerheim. However, the sequence comparison of the Phaeocystis 18S rDNA clearly showed that it was remarkably similar to several isolates of P. globosa. Thus, the species isolated from the southeast coast of China is identified as P. globosa rather than P. cf. pouchetii or another species. Our results also demonstrate that phenotypes of different members of the genus Phaeocystis are variable, apparently changing in response to environmental conditions. It is concluded that, on the basis of this phylogenetic analysis, the bloom forming southeast China coast species of Phaeocystis most likely originated from an endemic warm-water, rather than a foreign source. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Phaeocystis species; 18S rRNA gene; Molecular identification; Dispersal mechanism
1. Introduction A dense bloom of a toxic species of Phaeocystis first occurred in the coastal waters of southeast China in the autumn of 1997 and reoccurred in the summer of 1999. These episodes caused great economic losses to the local aquaculture industry. The *Corresponding author. Tel.: +86-20-84112399; fax: +86-20-84036551. E-mail address: [email protected] (Yue-Qin Chen). 0305-1978/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 1 ) 0 0 0 5 4 - 0
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Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
causative agent of these toxic blooms has a polymorphic life cycle involving both colonial and flagellated cells. Colonies have a spherical shape and numerous derived forms with 103–104 cells disposed evenly along the periphery and covered by a gelatinous coat. Colonies are 2–30 mm in diameter, often an air bubble is visible in large colonies. The flagellated cells are motile with 3–8 mm, each cell possessing two almost equal flagella and a short haptonema, occasionally, a trichocyst structure was observed under a light microscope. This species was initially identified as Phaeocystis cf. pouchetii, thus belonging to the Prymnesiophytes (He et al., 1999). However, the colony morphology and physiology of the species is different from that of Phaeocystis species, such as P. globosa Scherffel and P. pouchetii (Hariot) Lagerheim that had been described previously (Larson et al., 1992; Baumann et al., 1994). For example, the Phaeocystis species from coastal regions of southeast China can form a colony with a 30 mm diameter, much larger than the size observed with either P. globosa or P. pouchetii. The identification of this species is still usually performed on the basis of its phenotypic characteristics. Recently, the geographic range of Phaeocystis species seems to have increased at both the regional and the global scale causing significant public health and economic problems. The study of the spread of these species has become a very important theme in ‘‘red-tide’’ research. However, taxonomic uncertainties have caused major difficulties in interpreting the significance of the ‘‘dispersal’’ of Phaeocystis species. The genus Phaeocystis was created by Lagerheim in 1893 to accommodate the colonial state of an alga originally described as Tetraspora pouchetii by Hariot in Pouchet. The criteria used to distinguish the Phaeocystis species have long been questioned because of the phenotypic plasticity of these organisms and the difficulty in assigning a species to the different form. One of the most striking characteristics of Phaeocystis is their complex life cycle, with different flagellate stages alternating with non-motile, single-celled and colonial stages in a series of stages not yet fully understood (Rousseau et al., 1994). This feature, coupled with the morphological similarity among Phaeocystis species, has resulted in much confusion about the different taxa within the genus (Sournia, 1988; Jahnke and Baumann, 1987). To avoid the problems associated with the phenotypic plasticity of these organisms, it is necessary to identify them on the basis of their genetic information. Ribosomal DNA contains regions of highly conserved sequence interspersed with variable regions, making it eminently appropriate for use in molecular phylogenetic Table 1 The isolates used in this study, their origin and Genbank accession number Species
Strains
Origin
Abbreviation
Genbank accession
Phaeocystis sp. P. globosa P. globosa P. pouchetii P. pouchetii P. jahnii P. cordata
Santou 97 CCMP 627 SK35 P361 SK34 Unknown Unknown
Southeast China sea Mexico German bight Svalbard (Norway) Greenland sea Mediterranean sea Mediterranean sea
Santou Pgl627 Pglsk35 Ppo361 Ppo34 Pja Pco
AJ279499 AJ278035 X77476 AJ278036 X77475 AF163148 AF163147
Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
17
analysis (Medlin et al., 1994). In this study, we sequenced the 18S rDNA of Phaeocystis species from the coastal regions of southeast China and in three strains of P. globosa and P. pouchetii. The rRNA sequence comparison of several colonyforming Phaeocystis isolates from coastal regions of southeast China allowed us to determine their phylogenetic placement among the known Phaeocystis sp. 2. Materials and methods 2.1. Cultures The sources of Phaeocystis species used in this study are indicated in Table 1. Isolates of P. globosa CCMP627 and P. pouchetii P361 were kindly donated by Dr. L.K. Medlin at Alfred Wegener Institute of Polar and Marine Research, Germany. Isolates of Phaeocystis sp. Santou 97, the causative agents of the southeast China coast blooms in 1997 and 1999, were kindly provided by Dr. S.H. Lu at Institute of Hydrobiology, Jinan University, Guangzhou. 2.2. Oligonucleotide primers, DNA isolation and 18S rDNA amplification The primers used for amplification and sequencing of 18S rDNA were: 18N1: 50 CTATCTGGTTGATCCTGCCAG-30 , 18N5: 50 -TGGTGCCAGCAGCCGCGGTA-30 and 18N11R: 50 -TGATCCTTCCGCAGGTTCACC-30 corresponding respectively, to positions 1–21bp, 812–830bp and 1775–1796bp of eukaryotic 18S rDNA, (Sogin, 1990). These oligonucleotides were synthesized for us by the Shanhai Biochemistry Institute (Academy of Sciences, People’s Republic of China). Cultures were harvested by centrifugation. They were immediately suspended in extraction buffer (1% SDS; 10 mm EDTA (pH 8.0); 10 mm Tris-HCl (pH 7.5); 10 mm NaCl). The DNA was extracted by following the procedure of Chen et al. (1997). The primers used for amplification were: 18N1 and 18N11R; The PCR amplification consisted of an initial denaturation step of 941 for 5 min, followed by 30 cycles of 941C for 1 min, 551C for 0.5 min and 721C for 1 min, and a final extension step of 721C for 10 min. The amplification products were purified with Qiaquick PCR Purification Kit (QIAGEN, Germany), and then used for direct sequencing. 2.3. Sequencing and molecular character analysis PCR products of 18S rDNA were sequenced directly. Primers used for sequencing were: 18N1, 18N5 and 18N11R. Sequence management and editing were carried out using the PCgene 6.0 Package. Sequence alignment was improved by hand according to the four criteria proposed by Barriel (1994). The aligned sequences were also converted to a distance matrix with Kimura models of nucleotide substitution (Kimura, 1980) using the DNA dist program of the Phylip package (Version 3.5c from J. Felsenstein, University of Washington). Phylogenetic trees were constructed by the neighbor–joining method and parsimony method. Bootstrap analyses for 100 replicates were performed to provide confidence estimates for tree topologies.
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Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
3. Results and discussion Complete 18S rRNA sequences were determined for the Phaeocystis sp. strain Santou 97, P. globosa strain CCMP 627 and P. pouchetii strain P361, and deposited in Genbank (accession number AJ279499 for Phaeocystis sp. strain Santou 97, AJ278035 for P. globosa strain CCMP 627 and AJ278036 for P. pouchetii strain P361). These sequences with four other homologous sequences from P. globosa strain SK31, P. pouchetii strain SK34, P. jahnii and P. cordata were unambiguously aligned (data not shown), using Pavlova gyrans as the outgroup. The relationship among the seven Phaeocystis strains was analyzed with both distance and parsimony methods using the complete 18S rRNA sequences. Similarity values among the strains of Phaeocystis and P. gyrans, range from 87% to 90% (Table 2). The number of nucleotides separating well-established species in this study suggests that there were sufficient nucleotide differences to accurately analyze the colony-forming species. The Phaeocystis strains could be arranged into four distinctive groups according to the variations in their 18S rDNA (Fig 1.). Phaeocystis globosa and Phaeocystis sp. Santou 97 varied from 0 to 4 nucleotides. Two isolates of Phaeocystis globosa (CCMP627 and SK35) and Phaeocystis sp. (strain Santou 97), despite originating from different sites, present a single clade (Clade I). P. pouchetii (strains P361 and SK34) deviated from Clade I, and formed Clade II. P. cordata and P. jahnii have morphological features that distinguish them from the Phaeocystis species mentioned above, and they formed another two Clades (III and IV). P. cordata does not form colonies and only produces motile, flagellated cells; in contrast P. jahnii has nonmotile cells and mucilaginous colonies, consisting of aggregates of only 4–8 cells (Zingone et al., 1999). The genetic diversity of isolates are congruent with their morphological disparity. Previously published descriptions of the morphology and physiology of Phaeocystis cells are undoubtedly confusing because of taxonomic difficulties in identifying the colony-forming stage of P. globosa and P. pouchetii. P. globosa from temperate waters has been described by Scherffel (1990), as forming spherical colonies with cells homogeneously arranged within a gelatinous matrix, the older stages can assume distorted pear-shapes (Jahnke and Baumann, 1986). Phaeocystis Table 2 Distance values between 18S rDNA sequences of Phaeocystis species
1 2 3 4 5 6 7 8
Ppo361 Ppo34 Pgl627 Santou Pglsk35 Pco Pja Pgy
1
2
3
4
5
6
7
8
0.0000
0.0031 0.0000
0.0166 0.0159 0.0000
0.0120 0.0098 0.0052 0.0000
0.0127 0.0105 0.0065 0.0013 0.0000
0.0180 0.0147 0.0251 0.0179 0.0192 0.0000
0.0343 0.0309 0.0389 0.0316 0.0329 0.0238 0.0000
0.1365 0.1324 0.1459 0.1370 0.1386 0.1264 0.1308 0.0000
Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
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Fig. 1. Molecular phylogenetic tree inferred from the 18S rDNA sequences of Phaeocystis species using the neighbor–joining method, numbers above each branch represent percentages of 100 bootstrap repetitions. Topology of the parsimony tree is almost exactly the same as this NJ tree. The numbers below each branch denote the bootstrap values obtained by the parsimony methods.
pouchetii occurs in cold waters and forms lobed, globular colonies with cells arranged in patches (B.atje and Michaelis, 1986). Most early workers distinguished P. globosa and P. pouchetii on the basis of the differences in their cell distribution and colony morphology. However, Kornmann (1955) expressed doubts about this differentiation into two species, he believed that ‘‘P. globosa’’ cells were actually a juvenile form of P. pouchetii. However, the subsequently revealed degree of genetic divergence between the two suggests that they are separate species instead of different stages of the life cycle of same species. For the strains of Phaeocystis sp. from coastal regions of southeast China, no motile cells have yet been observed and without a clear picture of the entire life cycle, we are unable to classify the stages of the life cycle in the fashion suggested by Kornmann (1955). The features of these strains are quite different from those of P. globosa and P. poutchii. The diameter of this strain’s colonies can attain 30 mm, much greater than either P. globosa or P. poutchii, and they grow at temperatures up to 301C (Table 3). However, 18S rDNA sequence analysis clearly showed that isolates of Phaeocystis sp. strain Santou 97 and P. globosa were remarkably similar and can be differentiated from P. cf pouchetii or other species. In addition, the rDNA ITS sequences recently obtained from several Phaeocystis species also demonstrated that Phaeocystis sp. and Phaeocystis globosa are also identical (unpublished results).
20
Species
Colony morphology
Physiology
Maximum size
Shape
Cell distribution
Growth optimum
Temperature tolerance
P. globasaa P. pouchetiia
8–9 mm 1.5–2 mm
Spherical and numerous derived forms Spherical up to a colony diameter of 0.1 mm, lobed above 0.3 mm
161C 81C
0.6–221C o2–141C
P. sp (Santou)b
2–30 mm
Same as P. globosa
Evenly along the periphery Only in the curves of the lobes of larger colonies, and mostly regular; four cells form a square, cell free mucilage in between Same as P. globosa
201C
? to 301C
a b
Jahnke and Baumann (1987). He et al. (1999).
Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
Table 3 Colony features in morphology and physiology of Phaeocystis
Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
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There is circumstantial evidence that colony formation is dependent on environmental conditions and may vary with growth stage although a clear relationship has not yet been established. Several explanations have been put forward to explain the mechanism of Phaeocystis related blooms on the coast of China, including the increases in the level of pollution of Chinese coastal waters, global warming etc. (Liang et al., 1999), but the origin of causative Phaeocystis species remained unknown. Is the ‘‘red-tide’’ forming species a previously unnoticed endemic species or an introduced flora? To distinguish between endemic and introduced flora, historical records of species abundance in the region are required. The absence of such data is difficult to exclude the possibility that Phaeocystis species were already present in a given area. To our knowledge, P. globosa has never been reported in Chinese coastal waters, thus dispersal of an endemic organism must be inferred from other data. On the basis of an earlier phylogenetic analysis (Medlin et al., 1994), it was concluded that Phaeocystis most probably originated as a warm-water genus with a global distribution. Medlin et al. (1994) estimated that the separation of the cold-water species, such as P. pouchetii and the warm-water Phaeocystis species, such as P. globosa most likely occurred about 50 million years ago (mya). At this time when the world’s oceans were thermodynamically more homogeneous than today and the climate was generally very warm (Frakes et al., 1992), consistent with the hypothesis that Phaeocystis originated as a warm-water cosmopolitan genus, which means P. globosa is a cosmopolitan species too. According to this hypothesis, the Phaeocystis species (P. globosa) from southeast coast of China would be endemic rather than a newly introduced species. This implies that the outbreaks of Phaeocystis blooms in the southeast Chinese coast might have a direct connection with the increase in pollution of Chinese coastal waters and the prevalence of a previously unnoticed endemic species which can propagate rapidly and reach high cell densities in these polluted waters. The recent alarming increase in algal blooms in Chinese waters could be due to the growing pollution that can make these endemic but normally low level populations evenmore dangerous in the future. In conclusion, our results clearly showed that phenotypes of the genus Phaeocystis may vary in response to changing environmental conditions as well as growth stage. Because of this phenotypic plasticity, it is critical to develop DNA probes to distinguish between the different Phaeocystis species and to facilitate their identification in field samples. It was further demonstrated that molecular phylogeny data could be used not only to assess ecological/taxonomic problems, but also to test theories of geographical distribution of taxa. Such an approach would be extremely useful to explain the dispersal mechanism of ‘‘red-tide’’ species.
Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 39770058 and 39970063) and Funds for Young Scholar from the Education
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Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
Ministry of China. We are grateful to Dr. L.K. Medlin at Alfred Wegener Institute of Polar and Marine Research, Germany for providing isolates of Phaeocystis globosa (strain CCMP627) and Phaeocystis pouchetii (strain P361), and Dr. Lu SongHui at The Institute of Hydrobiology, Jinan University, Guangzhou for providing isolates of Phaeocystis sp. (strain Santou 97) from the coastal region of southeast China. Special thanks go to Dr. H.M. Krisch of the LMGM CNRS, Toulouse, France for revising the text of the manuscript.
References Barriel, V., 1994. Phylog!enies moleculaires et insertions-deletion de nucleotides CR. Acid Sci. Paris Sci. de la vie life sci. 317, 693–701. B.atje, M., Michaelis, H., 1986. Phaeocystis pouchetii blooms in the east Frisian coastal waters (German bight, North sea). Mar. Biol. 93, 21–27. Baumann, M.E.M., Lancelot, C., Brandini, F.P, Sakshang, E., John, D.M., 1994. The taxonomic identity of the cosmopolitan prymnesiophyte phaeocystis: a morphological and ecophysiological approach. J. Mar. Sys. 5, 5–22. Chen, Y.Q., Qu, L.H., Qiu, X.Z., Zeng, L.M., Qi, Y.Z., 1997. Method for PCR amplification of dinoflagellate rDNA with individual cells and its amplification in the identification of algae species. Acta Sci.. Nat. Univ. Sunyatseni. 36 (4), 66–69. Frakes, L.A., Francis, J.E., Syktus, J.I., 1992. Climate Modes of the Phanerozoic. Cambridge University Press, Cambridge, p. 274. He, J.W., Shi, Z.X., Zhang, Y.H., Liu, Y.D., Jiang, T.J., Yi, Y.W., Qi, Y.Z., 1999. Morphological characteristics and toxins of Phaeocystis cf. pouchetii (Prymnesiophyceae). Ocean et Limn. Sin. 30 (2), 172–179. Jahnke, J., Baumann, M., 1986. Die marine planktonalgae Phaeocystis globosa: eine Massenform unserer Ku. stengew.asser. Mikrokosmos 75, 357–359. Jahnke, J., Baumann, M.E.M., 1987. Differentiation between Phaeocystis pouchetii (Har.) Lagerheim and Phaeocystis globosa Scherffel I. colony shapes and temperature tolerances. Hydro. Biol. Bull. 21, 141–147. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitution through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. Kornmann, P., 1955. Beobachtungen an Phaeocystis-kulturen. Helgol. Wiss. Meeresunters. 5, 218–233. Larson, A., Kirk, M.M., Kirk, D.L., 1992. Molecular phylogeny of the volvocine flagellates. Mol. Biol. Evol. 9, 85–105. Liang, S., Qian, H.L., Xu, Sh.G., 1999. Collected Works of the South China Sea Resources and Environment Research. ZhongShan University Press, Guangzhou, pp. 229–235. Medlin, L.K., Lange, M., Baumann, M., 1994. Genetic differentiation among three colony-forming species of Phaeocystis: further evidence for the phylogeny of the prymnesiophyta. Phycologia 33 (3), 199–212. Rousseau, V., Vaulot, D., Casotti, R., Cariou, .V., Lenz, .J., Gunkel, .J., Baumann, M., 1994. The life cycle of Phaeocystis (Prymnesiophyceae): evidence and hypotheses. J. Mar. Syst. 5, 23–39. Scherffel, A., 1990. Phaeocystis globosa nov.spec.nebst einigen betrachtungen u. ber die phylogenie niederer. insbesondere brauner organismen. Wissenschaftliche, Meeresuntersuchungen, Neue Folge Abteilung Helgoland 4, 1–28. Sogin, M.L., 1990. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, pp. 95–102. Sournia, A., 1988. Phaeocystis (Prymnesiophyceae): how many species. Nova Hedwigia 47, 211–217. Zingone, A., Chretiennot–Dinet, M.J., Lange, M., Medlin, L., 1999. Morphological and genetic characterization of Phaeocystis cordata and P. Jahnii (Prymnesiophyceae), two new species from the Mediterranean sea. J. Phycol. 35, 1322–1337.
Molecular evidence identifies bloom-forming Phaeocystis (Prymnesiophyta) from coastal waters of southeast China as Phaeocystis globosa Yue-Qin Chen*, Ning Wang, Peng Zhang, Hui Zhou, Liang-Hu Qu Key Laboratory of Gene Engineering of Education Ministry, Biotechnology Research Center, Zhongshan Universtiy, Guangzhou 510275, PR China Received 12 July 2000; accepted 23 April 2001
Abstract Sequence data from the 18S small subunit ribosomal RNA gene have been used to identify the species of a Phaeocystis (Prymnesiophyta) that caused harmful algae blooms in the coastal waters of southeast China. This Phaeocystis has morphological and physiological features that differ from those previously described for either P. globosa Scherffel or P. pouchetii (Hariot) Lagerheim. However, the sequence comparison of the Phaeocystis 18S rDNA clearly showed that it was remarkably similar to several isolates of P. globosa. Thus, the species isolated from the southeast coast of China is identified as P. globosa rather than P. cf. pouchetii or another species. Our results also demonstrate that phenotypes of different members of the genus Phaeocystis are variable, apparently changing in response to environmental conditions. It is concluded that, on the basis of this phylogenetic analysis, the bloom forming southeast China coast species of Phaeocystis most likely originated from an endemic warm-water, rather than a foreign source. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Phaeocystis species; 18S rRNA gene; Molecular identification; Dispersal mechanism
1. Introduction A dense bloom of a toxic species of Phaeocystis first occurred in the coastal waters of southeast China in the autumn of 1997 and reoccurred in the summer of 1999. These episodes caused great economic losses to the local aquaculture industry. The *Corresponding author. Tel.: +86-20-84112399; fax: +86-20-84036551. E-mail address: [email protected] (Yue-Qin Chen). 0305-1978/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 1 ) 0 0 0 5 4 - 0
16
Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
causative agent of these toxic blooms has a polymorphic life cycle involving both colonial and flagellated cells. Colonies have a spherical shape and numerous derived forms with 103–104 cells disposed evenly along the periphery and covered by a gelatinous coat. Colonies are 2–30 mm in diameter, often an air bubble is visible in large colonies. The flagellated cells are motile with 3–8 mm, each cell possessing two almost equal flagella and a short haptonema, occasionally, a trichocyst structure was observed under a light microscope. This species was initially identified as Phaeocystis cf. pouchetii, thus belonging to the Prymnesiophytes (He et al., 1999). However, the colony morphology and physiology of the species is different from that of Phaeocystis species, such as P. globosa Scherffel and P. pouchetii (Hariot) Lagerheim that had been described previously (Larson et al., 1992; Baumann et al., 1994). For example, the Phaeocystis species from coastal regions of southeast China can form a colony with a 30 mm diameter, much larger than the size observed with either P. globosa or P. pouchetii. The identification of this species is still usually performed on the basis of its phenotypic characteristics. Recently, the geographic range of Phaeocystis species seems to have increased at both the regional and the global scale causing significant public health and economic problems. The study of the spread of these species has become a very important theme in ‘‘red-tide’’ research. However, taxonomic uncertainties have caused major difficulties in interpreting the significance of the ‘‘dispersal’’ of Phaeocystis species. The genus Phaeocystis was created by Lagerheim in 1893 to accommodate the colonial state of an alga originally described as Tetraspora pouchetii by Hariot in Pouchet. The criteria used to distinguish the Phaeocystis species have long been questioned because of the phenotypic plasticity of these organisms and the difficulty in assigning a species to the different form. One of the most striking characteristics of Phaeocystis is their complex life cycle, with different flagellate stages alternating with non-motile, single-celled and colonial stages in a series of stages not yet fully understood (Rousseau et al., 1994). This feature, coupled with the morphological similarity among Phaeocystis species, has resulted in much confusion about the different taxa within the genus (Sournia, 1988; Jahnke and Baumann, 1987). To avoid the problems associated with the phenotypic plasticity of these organisms, it is necessary to identify them on the basis of their genetic information. Ribosomal DNA contains regions of highly conserved sequence interspersed with variable regions, making it eminently appropriate for use in molecular phylogenetic Table 1 The isolates used in this study, their origin and Genbank accession number Species
Strains
Origin
Abbreviation
Genbank accession
Phaeocystis sp. P. globosa P. globosa P. pouchetii P. pouchetii P. jahnii P. cordata
Santou 97 CCMP 627 SK35 P361 SK34 Unknown Unknown
Southeast China sea Mexico German bight Svalbard (Norway) Greenland sea Mediterranean sea Mediterranean sea
Santou Pgl627 Pglsk35 Ppo361 Ppo34 Pja Pco
AJ279499 AJ278035 X77476 AJ278036 X77475 AF163148 AF163147
Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
17
analysis (Medlin et al., 1994). In this study, we sequenced the 18S rDNA of Phaeocystis species from the coastal regions of southeast China and in three strains of P. globosa and P. pouchetii. The rRNA sequence comparison of several colonyforming Phaeocystis isolates from coastal regions of southeast China allowed us to determine their phylogenetic placement among the known Phaeocystis sp. 2. Materials and methods 2.1. Cultures The sources of Phaeocystis species used in this study are indicated in Table 1. Isolates of P. globosa CCMP627 and P. pouchetii P361 were kindly donated by Dr. L.K. Medlin at Alfred Wegener Institute of Polar and Marine Research, Germany. Isolates of Phaeocystis sp. Santou 97, the causative agents of the southeast China coast blooms in 1997 and 1999, were kindly provided by Dr. S.H. Lu at Institute of Hydrobiology, Jinan University, Guangzhou. 2.2. Oligonucleotide primers, DNA isolation and 18S rDNA amplification The primers used for amplification and sequencing of 18S rDNA were: 18N1: 50 CTATCTGGTTGATCCTGCCAG-30 , 18N5: 50 -TGGTGCCAGCAGCCGCGGTA-30 and 18N11R: 50 -TGATCCTTCCGCAGGTTCACC-30 corresponding respectively, to positions 1–21bp, 812–830bp and 1775–1796bp of eukaryotic 18S rDNA, (Sogin, 1990). These oligonucleotides were synthesized for us by the Shanhai Biochemistry Institute (Academy of Sciences, People’s Republic of China). Cultures were harvested by centrifugation. They were immediately suspended in extraction buffer (1% SDS; 10 mm EDTA (pH 8.0); 10 mm Tris-HCl (pH 7.5); 10 mm NaCl). The DNA was extracted by following the procedure of Chen et al. (1997). The primers used for amplification were: 18N1 and 18N11R; The PCR amplification consisted of an initial denaturation step of 941 for 5 min, followed by 30 cycles of 941C for 1 min, 551C for 0.5 min and 721C for 1 min, and a final extension step of 721C for 10 min. The amplification products were purified with Qiaquick PCR Purification Kit (QIAGEN, Germany), and then used for direct sequencing. 2.3. Sequencing and molecular character analysis PCR products of 18S rDNA were sequenced directly. Primers used for sequencing were: 18N1, 18N5 and 18N11R. Sequence management and editing were carried out using the PCgene 6.0 Package. Sequence alignment was improved by hand according to the four criteria proposed by Barriel (1994). The aligned sequences were also converted to a distance matrix with Kimura models of nucleotide substitution (Kimura, 1980) using the DNA dist program of the Phylip package (Version 3.5c from J. Felsenstein, University of Washington). Phylogenetic trees were constructed by the neighbor–joining method and parsimony method. Bootstrap analyses for 100 replicates were performed to provide confidence estimates for tree topologies.
18
Yue-Qin Chen et al. / Biochemical Systematics and Ecology 30 (2002) 15–22
3. Results and discussion Complete 18S rRNA sequences were determined for the Phaeocystis sp. strain Santou 97, P. globosa strain CCMP 627 and P. pouchetii strain P361, and deposited in Genbank (accession number AJ279499 for Phaeocystis sp. strain Santou 97, AJ278035 for P. globosa strain CCMP 627 and AJ278036 for P. pouchetii strain P361). These sequences with four other homologous sequences from P. globosa strain SK31, P. pouchetii strain SK34, P. jahnii and P. cordata were unambiguously aligned (data not shown), using Pavlova gyrans as the outgroup. The relationship among the seven Phaeocystis strains was analyzed with both distance and parsimony methods using the complete 18S rRNA sequences. Similarity values among the strains of Phaeocystis and P. gyrans, range from 87% to 90% (Table 2). The number of nucleotides separating well-established species in this study suggests that there were sufficient nucleotide differences to accurately analyze the colony-forming species. The Phaeocystis strains could be arranged into four distinctive groups according to the variations in their 18S rDNA (Fig 1.). Phaeocystis globosa and Phaeocystis sp. Santou 97 varied from 0 to 4 nucleotides. Two isolates of Phaeocystis globosa (CCMP627 and SK35) and Phaeocystis sp. (strain Santou 97), despite originating from different sites, present a single clade (Clade I). P. pouchetii (strains P361 and SK34) deviated from Clade I, and formed Clade II. P. cordata and P. jahnii have morphological features that distinguish them from the Phaeocystis species mentioned above, and they formed another two Clades (III and IV). P. cordata does not form colonies and only produces motile, flagellated cells; in contrast P. jahnii has nonmotile cells and mucilaginous colonies, consisting of aggregates of only 4–8 cells (Zingone et al., 1999). The genetic diversity of isolates are congruent with their morphological disparity. Previously published descriptions of the morphology and physiology of Phaeocystis cells are undoubtedly confusing because of taxonomic difficulties in identifying the colony-forming stage of P. globosa and P. pouchetii. P. globosa from temperate waters has been described by Scherffel (1990), as forming spherical colonies with cells homogeneously arranged within a gelatinous matrix, the older stages can assume distorted pear-shapes (Jahnke and Baumann, 1986). Phaeocystis Table 2 Distance values between 18S rDNA sequences of Phaeocystis species
1 2 3 4 5 6 7 8
Ppo361 Ppo34 Pgl627 Santou Pglsk35 Pco Pja Pgy
1
2
3
4
5
6
7
8
0.0000
0.0031 0.0000
0.0166 0.0159 0.0000
0.0120 0.0098 0.0052 0.0000
0.0127 0.0105 0.0065 0.0013 0.0000
0.0180 0.0147 0.0251 0.0179 0.0192 0.0000
0.0343 0.0309 0.0389 0.0316 0.0329 0.0238 0.0000
0.1365 0.1324 0.1459 0.1370 0.1386 0.1264 0.1308 0.0000
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Fig. 1. Molecular phylogenetic tree inferred from the 18S rDNA sequences of Phaeocystis species using the neighbor–joining method, numbers above each branch represent percentages of 100 bootstrap repetitions. Topology of the parsimony tree is almost exactly the same as this NJ tree. The numbers below each branch denote the bootstrap values obtained by the parsimony methods.
pouchetii occurs in cold waters and forms lobed, globular colonies with cells arranged in patches (B.atje and Michaelis, 1986). Most early workers distinguished P. globosa and P. pouchetii on the basis of the differences in their cell distribution and colony morphology. However, Kornmann (1955) expressed doubts about this differentiation into two species, he believed that ‘‘P. globosa’’ cells were actually a juvenile form of P. pouchetii. However, the subsequently revealed degree of genetic divergence between the two suggests that they are separate species instead of different stages of the life cycle of same species. For the strains of Phaeocystis sp. from coastal regions of southeast China, no motile cells have yet been observed and without a clear picture of the entire life cycle, we are unable to classify the stages of the life cycle in the fashion suggested by Kornmann (1955). The features of these strains are quite different from those of P. globosa and P. poutchii. The diameter of this strain’s colonies can attain 30 mm, much greater than either P. globosa or P. poutchii, and they grow at temperatures up to 301C (Table 3). However, 18S rDNA sequence analysis clearly showed that isolates of Phaeocystis sp. strain Santou 97 and P. globosa were remarkably similar and can be differentiated from P. cf pouchetii or other species. In addition, the rDNA ITS sequences recently obtained from several Phaeocystis species also demonstrated that Phaeocystis sp. and Phaeocystis globosa are also identical (unpublished results).
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Species
Colony morphology
Physiology
Maximum size
Shape
Cell distribution
Growth optimum
Temperature tolerance
P. globasaa P. pouchetiia
8–9 mm 1.5–2 mm
Spherical and numerous derived forms Spherical up to a colony diameter of 0.1 mm, lobed above 0.3 mm
161C 81C
0.6–221C o2–141C
P. sp (Santou)b
2–30 mm
Same as P. globosa
Evenly along the periphery Only in the curves of the lobes of larger colonies, and mostly regular; four cells form a square, cell free mucilage in between Same as P. globosa
201C
? to 301C
a b
Jahnke and Baumann (1987). He et al. (1999).
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Table 3 Colony features in morphology and physiology of Phaeocystis
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There is circumstantial evidence that colony formation is dependent on environmental conditions and may vary with growth stage although a clear relationship has not yet been established. Several explanations have been put forward to explain the mechanism of Phaeocystis related blooms on the coast of China, including the increases in the level of pollution of Chinese coastal waters, global warming etc. (Liang et al., 1999), but the origin of causative Phaeocystis species remained unknown. Is the ‘‘red-tide’’ forming species a previously unnoticed endemic species or an introduced flora? To distinguish between endemic and introduced flora, historical records of species abundance in the region are required. The absence of such data is difficult to exclude the possibility that Phaeocystis species were already present in a given area. To our knowledge, P. globosa has never been reported in Chinese coastal waters, thus dispersal of an endemic organism must be inferred from other data. On the basis of an earlier phylogenetic analysis (Medlin et al., 1994), it was concluded that Phaeocystis most probably originated as a warm-water genus with a global distribution. Medlin et al. (1994) estimated that the separation of the cold-water species, such as P. pouchetii and the warm-water Phaeocystis species, such as P. globosa most likely occurred about 50 million years ago (mya). At this time when the world’s oceans were thermodynamically more homogeneous than today and the climate was generally very warm (Frakes et al., 1992), consistent with the hypothesis that Phaeocystis originated as a warm-water cosmopolitan genus, which means P. globosa is a cosmopolitan species too. According to this hypothesis, the Phaeocystis species (P. globosa) from southeast coast of China would be endemic rather than a newly introduced species. This implies that the outbreaks of Phaeocystis blooms in the southeast Chinese coast might have a direct connection with the increase in pollution of Chinese coastal waters and the prevalence of a previously unnoticed endemic species which can propagate rapidly and reach high cell densities in these polluted waters. The recent alarming increase in algal blooms in Chinese waters could be due to the growing pollution that can make these endemic but normally low level populations evenmore dangerous in the future. In conclusion, our results clearly showed that phenotypes of the genus Phaeocystis may vary in response to changing environmental conditions as well as growth stage. Because of this phenotypic plasticity, it is critical to develop DNA probes to distinguish between the different Phaeocystis species and to facilitate their identification in field samples. It was further demonstrated that molecular phylogeny data could be used not only to assess ecological/taxonomic problems, but also to test theories of geographical distribution of taxa. Such an approach would be extremely useful to explain the dispersal mechanism of ‘‘red-tide’’ species.
Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 39770058 and 39970063) and Funds for Young Scholar from the Education
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Ministry of China. We are grateful to Dr. L.K. Medlin at Alfred Wegener Institute of Polar and Marine Research, Germany for providing isolates of Phaeocystis globosa (strain CCMP627) and Phaeocystis pouchetii (strain P361), and Dr. Lu SongHui at The Institute of Hydrobiology, Jinan University, Guangzhou for providing isolates of Phaeocystis sp. (strain Santou 97) from the coastal region of southeast China. Special thanks go to Dr. H.M. Krisch of the LMGM CNRS, Toulouse, France for revising the text of the manuscript.
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