MOLECULAR PHYLOGENY OF GRACILARIA SPECIES INFERRED FROM MOLECULAR MARKERS BELONGING TO THREE DIFFERENT GENOMES 1 Madhu Pareek, Avinash Mishra, and Bhavanath Jha 2 Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), G. B. Marg, Bhavnagar- 364021, Gujarat, India The nucleotide sequence data of molecular markers 18S rRNA, RUBISCO spacer, and cox2-3 intergenic spacer were integrated to infer the phy- logeny of Gracilaria species, collected from the western coast of India, reducing the possibility of misidentification and providing greater phyloge- netic resolution. A phylogenetic tree was con- structed using cox2-3 and RUBISCO spacer sequences, exhibiting the same clustering but differing slightly from that of the rRNA-based phy- logenetic tree. The phylogeny inferred from the combined data set confers an analogous pattern of clustering, compared with those of trees con- structed from individual data sets. The combined data set resulted in a phylogeny with better resolu- tion, which supported the clade with higher consis- tency index, retention index, and bootstrap values. It was observed that Gracilaria foliifera (Forssk.) Børgesen is closer to G. corticata (J. Agardh) J. Agardh varieties, while G. salicornia (C. Agardh) E. Y. Dawson and G. fergusonii J. Agardh both originated from the same clade. The position of G. textorii (Suringar) De Toni faltered and toppled between G. salicornia and G. dura (C. Agardh) J. Agardh; however, G. gracilis (Stackh.) M. Steentoft, L. M. Irvine et W. F. Farnham was evidently distant from the rest of the species. Key index words: cox; Gracilaria; phylogeny; rRNA; RUBISCO; seaweed; systematic Abbreviations: BLAST, Basic Local Alignment Search Tool; cox, cytochrome oxidase; ME, min- imum evolution; MEGA, Molecular Evolutionary Genetics Analysis; MP, maximum parsimony; NJ, neighbor joining; rbc L ⁄ S, RUBISCO large ⁄ small subunit Gracilaria is a taxonomically challenging genus due to structural simplicity, high morphological plasticity, and great species diversity. Gross morpho- logical characters provide the basis of identification, and incorrect applications have led to a number of misidentifications. Moreover, species delineation of Gracilaria is problematic due to the limitations of distinct morphological and reproductive charac- teristics (Bird 1995), and their relationship to other groups remains uncertain. Taxonomic investigations of Gracilaria species from Indian waters, especially from the Gujarat coast, where the majority of the species diversity is found (Jha et al. 2009), are limited and inadequate because of seasonal and environmental variations in morphological charac- ters, which often lead to misidentification of the species. Molecular phylogenetic studies in the family Gracilariaceae are largely based on the sequence analysis of the SSU rRNA gene, RUBISCO spacer, and cox2-3 spacer. The mitochondrial cox1 and plastid rbcL genes were sequenced recently for the identification and phylogeny of red algal species (Guillemin et al. 2008, Gurgel et al. 2008, Yang et al. 2008). However, plastid protein-coding genes such as rbcL (often slow-evolving genes) offer low resolution of population within species, whereas nucleotide spacer regions such as ITS (fast-evolv- ing regions) are sometimes inconsistent in align- ment because of insertions and deletions. Mitochondrial protein-coding cytochrome c oxi- dase subunit I gene (cox1) is verified as a suitable marker for barcoding of red algae (Saunders 2005, Geraldino et al. 2006, Robba et al. 2006). Based on the analysis of cox1 obtained from differ- ent samples of red algae, Robba et al. (2006) demonstrate that, in comparison to the plastid RUBISCO spacer, cox1 is a more sensitive marker for revealing the population structure and the hid- den diversity of red algal species. Nucleotide sequences of nuclear genes encoding SSU rRNAs (especially 18S rRNA) have been used in phyloge- netics for more than a decade (Bird et al. 1992, Bellorin et al. 2002). This use is still increasing, particularly to determine the position of red alga in wider phylogenetic schemes. Sequence data have helped in solving various dis- putes of the systematics and phylogeny of Gracilaria (Bellorin et al. 2002, Gargiulo et al. 2006); however, a true phylogeny with greater confidence may remain as an elusive goal for systematists. With the expansion of molecular techniques to numerous 1 Received 9 October 2009. Accepted 30 June 2010. 2 Author for correspondence: e-mail bjha@csmcri.org. J. Phycol. 46, 1322–1328 (2010) Ó 2010 Phycological Society of America DOI: 10.1111/j.1529-8817.2010.00903.x 1322