Microsatellite markers from expressed sequence tags (ESTs) of seaweeds in differentiating various Gracilaria species

Gracilaria is a red seaweed that has been cultivated worldwide and is commercially used for food, fertilizers, animal fodder, and phycocolloids. However, the high morphological plasticity of seaweeds often leads to the misidentification in the traditional identification of Gracilaria species. Molecular markers are important especially in the correct identification of Gracilaria species with high economic value. Microsatellite markers were developed from the expressed sequence tags of seaweeds deposited at the National Center for Biotechnology Information database and used for differentiating Gracilaria changii collected at various localities and two other Gracilaria species. Out of 33 primer pairs, only one primer pair gave significant results that can distinguish between three different Gracilaria species as well as G. changii from various localities based on the variation in repeated nucleotides. The unweighted pair group method using arithmetic mean dendrogram analysis grouped Gracilaria species into five main clades: (a) G. changii from Batu Besar (Malacca), Sandakan (Sabah), Bintulu (Sarawak), Batu Tengah (Malacca), Gua Tanah (Malacca), Middle Banks (Penang), Sungai (Sg.) Merbok (Kedah), Teluk Pelandok (Negeri Sembilan), Pantai Dickson (Negeri Sembilan), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore); (b) Gracilaria manilaensis from Cebu, Philippines; (c) G. changii from Morib (Selangor); (d) Gracilaria fisheri from Pattani, Thailand; and (e) G. changii from Pantai Dickson (Negeri Sembilan), Gua Tanah (Malacca), Sg. Merbok (Kedah), Sg. Kong-Kong (Johore), and Sg. Pulai (Johore). This result shows that this primer pair was able to distinguish between three different species, which are G. changii from Morib (Malaysia), G. fisheri from Pattani (Thailand), and G. manilaensis from Cebu (Philippines), and also between different genotypes of G. changii. This suggested that the simple sequence repeat primer we developed was suitable for differentiating between different Gracilaria species due to the polymorphisms caused by the variability in the number of tandem repeats.     

Morphological and molecular characterisation and differentiation of <Emphasis Type="Italic">Sargassum baccularia</Emphasis> and <Emphasis Type="Italic">S. polycystum</Emphasis> (Phaeophyta)

Two Sargassum species (S. baccularia and S. polycystum) collected from Teluk Kemang and Cape Rachado, Port Dickson, Negeri Sembilan, Malaysia, which are alike in morphology except for the rhizoidal system and vesicles, were characterised using random amplified polymorphism DNA (RAPD). The genomic DNA of both species was isolated from the leaves using a modified CTAB method. Four random primers, that is, OPA2, OPA3, OPA4 and OPA13, successfully amplified the DNA. The polymorphisms generated by these four primers were analysed using the Dice Coefficient of Similarity and cluster analysis was carried out using GelCompar II Version 2.0 (Applied Maths, Kortrijk, Belgium) based on UPGMA. DNA analysis showed that three primers were able to differentiate the two species. Morphological analysis using Principal Components Analysis (PCA) and discriminant function analysis supported the molecular data. Both species are characterised by heavily muricate main branches, oblong-lanceolate leaves with dentate margins and discoid holdfasts and spherical vesicles; both are dioecious. The only difference is that S. polycystum has secondary holdfasts transformed into stolons. This last characteristic is therefore a very important criterion and may contribute to the difference shown by DNA analysis.     

Interactive Effects of Temperature and UV Radiation on Photosynthesis of Chlorella Strains from Polar,Temperate and Tropical Environments: Differential Impacts on Damage and Repair

Global warming and ozone depletion, and the resulting increase of ultraviolet radiation (UVR), have far-reaching impacts on biota, especially affecting the algae that form the basis of the food webs in aquatic ecosystems. The aim of the present study was to investigate the interactive effects of temperature and UVR by comparing the photosynthetic responses of similar taxa of Chlorella from Antarctic (Chlorella UMACC 237), temperate (Chlorella vulgaris UMACC 248) and tropical (Chlorella vulgaris UMACC 001) environments. The cultures were exposed to three different treatments: photosynthetically active radiation (PAR; 400–700 nm), PAR plus ultraviolet-A (320–400 nm) radiation (PAR + UV-A) and PAR plus UV-A and ultraviolet-B (280–320 nm) radiation (PAR + UV-A + UV-B) for one hour in incubators set at different temperatures. The Antarctic Chlorella was exposed to 4, 14 and 20°C. The temperate Chlorella was exposed to 11, 18 and 25°C while the tropical Chlorella was exposed to 24, 28 and 30°C. A pulse-amplitude modulated (PAM) fluorometer was used to assess the photosynthetic response of microalgae. Parameters such as the photoadaptive index (E_k) and light harvesting efficiency (α) were determined from rapid light curves. The damage (k) and repair (r) rates were calculated from the decrease in ΦPSII_eff over time during exposure response curves where cells were exposed to the various combinations of PAR and UVR, and fitting the data to the Kok model. The results showed that UV-A caused much lower inhibition than UV-B in photosynthesis in all Chlorella isolates. The three isolates of Chlorella from different regions showed different trends in their photosynthesis responses under the combined effects of UVR (PAR + UV-A + UV-B) and temperature. In accordance with the noted strain-specific characteristics, we can conclude that the repair (r) mechanisms at higher temperatures were not sufficient to overcome damage caused by UVR in the Antarctic Chlorella strain, suggesting negative effects of global climate change on microalgae inhabiting (circum-) polar regions. For temperate and tropical strains of Chlorella, damage from UVR was independent of temperature but the repair constant increased with increasing temperature, implying an improved ability of these strains to recover from UVR stress under global warming.     

Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

Environmental Control of Vanadium Haloperoxidases and Halocarbon Emissions in Macroalgae

Vanadium-dependent haloperoxidases (V-HPO), able to catalyze the reaction of halide ions (Cl^?, Br^?, I^-) with hydrogen peroxide, have a great influence on the production of halocarbons, which in turn are involved in atmospheric ozone destruction and global warming. The production of these haloperoxidases in macroalgae is influenced by changes in the surrounding environment. The first reported vanadium bromoperoxidase was discovered 40 years ago in the brown alga Ascophyllum nodosum. Since that discovery, more studies have been conducted on the structure and mechanism of the enzyme, mainly focused on three types of V-HPO, the chloro- and bromoperoxidases and, more recently, the iodoperoxidase. Since aspects of environmental regulation of haloperoxidases are less well known, the present paper will focus on reviewing the factors which influence the production of these enzymes in macroalgae, particularly their interactions with reactive oxygen species (ROS).     

<Emphasis Type="Italic">Halymenia johorensis</Emphasis> sp. nov. (Halymeniaceae,Rhodophyta), a new foliose red algal species from Malaysia

A new Halymenia species, Halymenia johorensis sp. nov., from southern Peninsular Malaysia is proposed based on plastid-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) gene analyses and detailed morphological observations. The new species is characterized by having (1) elliptical, oblong, or irregularly shaped blades, incised with some perforations, arising from a narrow-cuneate stipe attached to a discoid holdfast; (2) blades with a cartilaginous and gelatinous texture, a smooth to rugose surface, and irregularly dentate and cleft margins; and (3) isodiametric outer cortical cells and rounded to stellate inner cortical cells. RbcL sequence analyses have shown H. johorensis to be genetically distinct from other Halymenia species. Although H. johorensis is sister to Halymenia plana, these two species can be distinguished both molecularly and morphologically. Further studies are necessary to investigate the phylogenetic relationships and species diversity in this genus.     

Influence of carbon source on growth,biochemical composition and pigmentation of Ankistrodesmus convolutus

The unicellular chlorophyte Ankistrodesmus convolutus Corda was grown in the light using inorganic medium (Bold's Basal Medium, BBM) and BBM enriched with 0.1% w/v of glucose, sodium acetate, sodium citrate or sodium bicarbonate. Glucose supported the highest specific growth rate (μ = 0.93 d^-1) and gave the highest biomass (453 mg dry weight L^-1) at the time of harvest. Of four glucose concentrations (0.05, 0.1, 0.25, 0.5% w/v), best growth was attained at 0.1% w/v. At 0.5% w/v glucose, the cells had high carbohydrates but low lipids and proteins. The relative amounts of 16:0, 18:0, 18:1 and 18:2 increased at the expense of 18:3(n-3) in the carbon-supplemented cultures and at glucose concentrations higher than 0.1% w/v. Cultures grown on glucose had less chlorophyll and carotenoid contents than cultures grown on other carbon sources. Chlorophyll and carotenoid contents decreased with increasing glucose concentrations in the medium.     

New records of Malaysian marine algae

Checklists of the marine algae of Malaysia have been published. The last checklist included the marine algae of Singapore. The checklist recorded 212 taxa for the region. This paper lists an additional 7 taxa of Chlorophyta, 18 taxa of Rhodophyta and 5 taxa of Phaeophyta, which had been left out of the last checklist. These include marine algae from Pulau Pinang, Pulau Redang and Pulau Sibu. In addition, new records of Cyanophyta (2 species), Chlorophyta (4 species), Rhodophyta (10 species) and Phaeophyta (3 species) are reported.