Phykos 44 (1): 52-58 (2014) Morphological and molecular assessment of Hypnea valentiae ©Phycological Society, India 52 Morphological and molecular assessment of native carrageenophyte Hypnea valentiae (Cystocloniaceae, Gigartinales) in Indian Subcontinent Felix Bast*, Satej Bhushan and Aijaz Ahmad John Centre for Biosciences, Central University of Punjab, Bathinda, Punjab, 151001, India * Corresponding author; Telephone: +91 98721 52694; Fax: +91 164 2430586. Email: felix.bast@cup.ac.in Abstract Hypnea valentiae is an important red alga commercially cultivated in various parts of the world for the production of carrageenan. Presented in this report is findings of morphological and molecular observations of naturally-occurring populations of this alga collected from west and east coasts of India. Both the isolates had similar external as well as microscopic morphology. Nuclear ribosomal DNA Internal Transcribed Spacer-1 (ITS1) sequences from these geographical isolates had 4.35 x 10 -1 Tamura-3-Parameter (T3P) pairwise distance between them, which indicate significant evolutionary differences accumulated over time. In comparison, T3P distance between related genera Kappaphycus and Eucheuma was 1.85 x 10 -1 . In our phylogeny reconstruction using Bayesian Inference, both the isolates formed a well- supported clade along with the only available accession of this genus at ITS1 locus, indicating affiliation of both the isolates in this genus. Interestingly, isolate from the west coast was more basal in the phylogram, which suggests phylogenetically primitive position of this population. Newly generated DNA barcodes of the geographic isolates of this native carragenophyte in this study is expected to be a key in tracing its further dispersal routes, either natural or deliberate. This is the first report on the comparative morphological and molecular assessment of Hypnea from India. Keywords: Red algae; Seaweed; Carrageenan; Genetic Diversity; Indian Ocean, ITS Introduction Hypnea J.V. Lamouroux (Cystocloniaceae, Gigartinales) is an important red algal genus cultivated worldwide for the commercial production of carrageenan; cultivation of which is ranked second after Eucheuma (Mshigeni and Chapman 1994). This genus encompasses some of the most abundant intertidal seaweeds of tropical coasts of Indo-Pacific region (Geraldino et al. 2010). Due to high morphological variability exhibited by this genus, morphometric species delineation is very challenging and unreliable (Yamagishi and Masuda 2000). There are about 50 species in this genus currently recognized in the world (Masuda et al. 1997), of which two are reportedly present in India, although no definitive taxonomical identification attempt have ever been made. These include H. musciformes that are being commercially cultivated in South-East coast for Carrageenan (Ganesan et al. 2006) and H. valentiae, extracts of which were studied for a number of biochemical activities that involved cholinesterase inhibitory effect (Suganthy et al. 2010) and the snake venom detoxification (Vasanthi et al. 2003). None of these studies reported how species-level identification has been made and, therefore, possibility of mistaken identification cannot be ruled out. Given the high morphological plasticity of Hypnea and lack of clear species-delineating synapomorphies, we sought out for the taxonomic assessment of this genus in India based on molecular evidences in addition to the existing morphometry- based keys. DNA barcoding-which include sequencing of a barcode locus and comparing the sequence with public repositories- is now a standard technique adapted worldwide for the reliable taxonomic identification of plants (Hollingsworth et al. 2009). While no consensus exists among phycologists on which barcode should be universally adapted for algae, a number of studies suggested effectiveness of nuclear ribosomal DNA Internal Transcribed Spacer-1 (nrDNA ITS1) to bring out molecular evolution at intraspecific levels, especially for red algal order Gigartinales (Hughey et al. 2002). Recently DNA barcoding have been successfully employed for the taxonomic assessment of this genus based on plastid RUBISCO Large subunit (rbcL) locus (Yamagishi and Masuda 2000; Geraldino et al. 2006) and mitochondrial cytochrome c oxidase subunit-1 (cox1) locus (Geraldino et al. 2009; Wolf et al. 2011). There had been no previous attempts made to barcode this important red algal genus in India. In addition, this is for the first time that barcode based on nuclear rDNA ITS region have been employed for phylogeny reconstruction in this genus. We, therefore, sought out for barcoding H. valentiae collected from west and east coasts of India and reconstruction of evolutionary heritage of these isolates using robust statistical framework of Bayesian Inference. Materials and methods Algal thalli growing attached to intertidal rocks were collected from Bekal, Kerala, India (12.2329N; 75.1512E) and Mandapam, Tamil Nadu, India (9.1658N; 79.1127E). Thalli were brought to the laboratory in the ice box (-4°C to -8°C) and processed immediately. Morphological features were recorded using an upright microscope (BX53, Olympus, Japan) with an attached digital camera (E450, Olympus, Japan). Pressed vouchers were prepared and deposited in the Central National Herbarium,