31/08/2011
PHYLOGENETIC ANALYSIS BASED ON ITS SEQUENCES SUGGESTS RE-STRUCTURING OF D. VIRIDIS (CHLOROPHYCEAE) TAXON Patrícia Assunçãoa*, Ruth Jaén-Molinab, Juli Caujapé-Castellsb, Adelina de la Jaraa, Laura Carmonaa, Karen Freijanesa & Héctor Mendozaa*. aDepartamento bDepartamento
de Biotecnología. División de Investigación y Desarrollo Tecnológico. Instituto Tecnológico de Canarias (ITC). Pozo Izquierdo, 35119 Sta. Lucía, Gran Canaria. de Biodiversidad Molecular y Banco de ADN, Jardín Botánico Canario “Viera y Clavijo”, Apartado de correos 14 de Tafira Alta, 35017 Las Palmas de Gran Canaria *Contacts: pclemente@itccanarias.org, hmendoza@itccanarias.org
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INTRODUCTION
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Dunaliella viridis (Chlorophyceae, Dunaliellales) belongs to the Section Viridis of the subgenus Dunaliella. This halophilic species is able to tolerate hypersaline environments such as saltworks and it is widely distributed throughout the world. It has been stated that its taxonomic position needs to be investigated since several varieties and forms based largely on morphologic characteristics (cell size) have been described. In order to try to highlight the taxonomic status of this taxon we analyzed the Internal Transcribed Spacers (ITS1+ITS2) sequences [including the ITS2 secondary structure and Compensatory Base Changes (CBC) analysis] of sixteen D. viridis strains.
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A) Saltworks, habitat of D. viridis; B) Cultures of different strains of D. viridis at lab conditions; C) Microscopic morphology of D. viridis
CBC TABLE. Number of Compensatory Base Changes (CBC) in the ITS2 secondary structure (helix II and III) found between every two strains. A CBC in a pair of sequences is positively correlated with species distinctness at a confidence level of 93%; however a lack of a CBC in ITS2 secondary structure is not an indicator at all of two organisms belonging to the same species:
Chlamydomonas reinhardtii CCAP11/32D Dunaliella lateralis Nepal Dunaliella viridis var. palmelloides CCAP19/34 Dunaliella viridis var. euchlora CCAP19/21 Dunaliella acidophila CCAP19/35 Dunaliella viridis ITC5109 (Cádiz, Spain) Dunaliella viridis CONC002 89 96 Dunaliella parva CCAP19/26 Viridis Clade Dunaliella parva [viridis] UTEX1983 83 99 Dunaliella viridis DCCBC4 100 Dunaliella viridis ITC5121 (Île de Ré, France) Dunaliella viridis ITC5126 (Guérande, France) 98 61
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100
Dunaliella pseudosalina CONC010 Dunaliella viridis ITC5120 (Île de Ré, France) Dunaliella parva [maritima] SAG19-1 Dunaliella viridis ITC5115 (Isla Cristina, Huelva, Spain) Dunaliella parva fo eugametos [maritima] CCAP19/1 99 Dunaliella viridis ITC5116 (Fuerteventura, Spain) Dunaliella salina [viridis] UTEX200 Dunaliella viridis ITC5123 (Guérande, Spain) 99 Dunaliella viridis ITC5110 (Cádiz, Spain) 99 Dunaliella viridis ITC5108 (Álava, Spain)
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Viridis Clade 2
Dunaliella percei (CCAP19/2, UTEX2192) 100 Dunaliella bioculata (CCAP19/4, UTEX199)
Dunaliella minuta CCAP19/5 Dunaliella tertiolecta (all strains, see CBC Table)
70
Dunaliella salina [viridis] CCAP19/3 91 87 Dunaliella salina (all strains, see CBC Table)
ITS1+ITS2 Phylogenetic Tree. Bayesian 50% majority rule consensus tree of the Dunaliella species analyzed in this study plus Chlamydomonas reinhardtii (outgroup). The re-identification of some strains is within brackets. The values in the branches indicate posterior probabilities. It was observed great heterogeneity within the sixteen D. viridis strains analyzed. D. viridis strains were positioned in two major clades. The ITS1+ITS2 region does not contain a geographical signal overall. Genebank accession numbers of the new D. viridis strains from Spain and France will be available soon.
DQuart_CCAP19/8 DT_CCMP362 DPoly_CCAP19/7C DPerc_UTEX2192 DPrim_UTEX1000 DBioc_UTEX199 DMin_CCAP19/5 DT_CCAP19/6B DBioc_CCAP19/4 DT_CCAP19/9 DT_UTEX999 DT_ITC5111 DT_ITC5112 DT_DCCBC5 DT_CCAP19/23 DT_CCMP364 DT_CCMP1302 DT_CCAP19/27 DT_ATCC30929 DT_SAG13.86 DT_CCMP1320 DPerc_CCAP19/2 DS_AC144 DS_CONC001 DS_CCAP19/25 DS_UTEX1644 DS_CONC007 DS_9802 DS_hd6 DS_Yucatan DS_Mongolia DS_OUC21 “hd1” DS_OUC38 “hd3” DS_OUC66 “hd4” DS_Taggu DS_UTEX2538 DS_OUC36 “hd2” DS_CCAP19/12 DS_DCCBC1 DS_ATCC30861 DS_CONC006 DS_CCAP19/18 DS_CCAP19/30 DV_CCAP19/3 DP_SAG19-1 DV_ITC5115 DP_CCAP19/1 DV_ITC5116 DV_ITC5123 DV_ITC5110 DV_ITC5108 DV_UTEX200 DV_ITC5120 DPseu_CONC010 DV_ITC5121 DV_ITC5126 DV_DCCBC4 DV_ITC5109 DV_UTEX1983 DP_CCAP19/26 DV_CONC002 DV_CCAP19/21 DAcid CCAP19/35 DLat_Nepal DV_CCAP19/34 Chlamydomonas
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DV: Dunaliella viridis; DPseu: D. pseudosalina; DS: D. salina; DPoly: D. polymorpha; DQuart: D. quartolecta; DT: D. tertiolecta; DBioc: D. bioculata; DT: D. tertiolecta; DLat: D. lateralis; DPerc: D. percei; DPrim: D. primolecta; DMin: D. minuta; DP: D. parva; DAcid: D. acidophila
RESULTS & DISCUSION SOME REFERENCES USED IN THIS STUDIE: Borowitzka, M. A. & Siva, C. J. 2007. The taxonomy of the genus Dunaliella (Chlorophyta, Dunaliellales) with emphasis on the marine and halophilic species. J. Appl. Phycol. 19:56790. González, M. A., Gómez, P. I. & Polle, J. E. W. 2009. Taxonomy and Phylogeny of the genus Dunaliella. In Ben-Amotz, A., Polle, J. E.W., Subba Rao, D.V. [Eds.] The Alga Dunaliella. Biodiversity, Physiology, Genomics and Biotechnology. Science Publishers, Enfield, NH, USA, pp.15-44. Schultz, J., Wolf, M. (2009). ITS2 Sequence-Structure Analysis in Phylogenetics: A How-to Manual for Molecular Systematics . Molecular Phylogenetics and Evolution 52(2): 520-3.
1- Both the primary sequence analysis (ITS1+ITS2) and the ITS2 secondary structure analysis positioned the majority of D. viridis strains in two different major clades, revealing great heterogeneity. 2- Unexpectedly, D. viridis var. palmelloides (CCAP11/34) and D. viridis var. euchlora (CCAP19/21) only shared a similarity of 54.4%, suggesting that these strains can not belong to the same species. 3- D. viridis var. euchlora (CCAP19/21) was positioned in a different lineage of the other D. viridis specimens, but was positioned within the subgenus Dunaliella. On the contrary, D. viridis var. palmelloides (CCAP11/34) was positioned outside the subgenera Dunaliella. 4- The detection of at least three compensatory base changes (CBC) between D. viridis var. palmelloides (CCAP11/34) and the other Dunaliella strains (CBC table) suggest that this strain is a new species. In this sense we purpose the re-name of this strain as D. palmelloides. 5- The CBC results did not confirm that D. viridis var. euchlora (CCAP19/21) is a potential new species.
Supported by BANGEN (INTERREG-IIIB, MAC/1/C070) Açores, Madeira, Canarias
6- In conclusion, the primary sequence (ITS1+ITS2) and the ITS2 secondary structure results suggests the restructure of the of D. viridis taxon.
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