Genetic diversity of Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta) in Southeast Asia

The commercial importance of carrageenophytes Kappaphycus and Eucheuma is well known, with much interest in terms of cultivation, marketing, and research. Considering the many lucrative prospects, these red seaweeds were introduced into various parts of the world for farming, where merely a few were comprehensively documented. Despite being extensively cultivated throughout Southeast Asia, the genetic diversity of Kappaphycus and Eucheuma is poorly studied, where heavy reliance is placed on the use of local or commercial names for identifications. This study used the mitochondrial-encoded cox1 and cox2–3 spacer genetic markers to investigate the Kappaphycus and Eucheuma haplotypes, cultivated and wild, available throughout Southeast Asia. Concatenated cox1–cox2–3 spacer datasets were also analyzed. The near full-length cox1 gene is preferred at revealing the genetic diversity of Kappaphycus and Eucheuma, provided a larger reference database is available. Both molecular markers were capable of delineating common members of the genus Kappaphycus (i.e., Kappaphycus alvarezii, Kappaphycus striatus, and Kappaphycus cottonii) and Eucheuma denticulatum, and revealed interesting genotypes and new species which may be potential alternatives to the common cultivars as well as materials for research. The relative scarcity of Eucheuma species is discussed and future sites for sampling are recommended.     

Assessment of Four Molecular Markers as Potential DNA Barcodes for Red Algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta)

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.     

Genetic differentiation in wild Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae,Rhodophyta) from East Malaysia reveals high inter- and intraspecific diversity with strong biogeographic signal

Journal of Applied Phycology - The region of Eastern Sabah, Malaysia, harbours a rich diversity of eucheumatoid (i.e. Kappaphycus and Eucheuma spp.) algae. The global cultivation of this group of...     

Systematics and genetic variation in commercial shape Kappaphycus and shape Eucheuma (Solieriaceae, Rhodophyta)

The systematics and taxonomy of Kappaphycus and Eucheuma (Solieriaceae) is confused and difficult due to morphological plasticity, lack of adequate characters to identify species and commercial names of convenience. These taxa are geographically widely dispersed through cultivation. Commercial, wild and herbarium sources were analysed; molecular markers provided insights into taxonomy and genetic variation, and where sources of genetic variation may be located. The mitochondrial cox2-3 and plastidal RuBisCo spacers were sequenced. There is a clear genetic distinction between K. alvarezii (“cottonii”) and K. striatum (“sacol”) samples. Kappaphycus alvarezii from Hawaii and some samples from Africa are also genetically distinct. Our data also show that all currently cultivated K. alvarezii from all over the world have a similar mitochondrial haplotype. Within Eucheuma denticulatum (“spinosum”) most African samples are again genetically distinct. Our data also suggest that currently cultivated E. denticulatum may have been “domesticated” several times, whereas this is not evident for the cultivated K. alvarezii. The present markers used do not distinguish all the morpho-types known in cultivation (e.g. var. tambalang, “giant” type) but do suggest that these markers may be useful to assess introductions and species identification in samples.     

Non-enzymatic isolation of somatic cells from Kappaphycus spp. and Eucheuma denticulatum (Solieriaceae,Rhodophyta)

Cell culture technology is immensely useful in somatic hybridization, induction of mutations, cloning of specific isolates and maintenance of strains of defined genotypes. However, its application in strain improvement of some tropical red macroalgae has been limited due to the difficulty of isolating viable cells from their complex intercellular matrices. A simple, non-enzymatic technique of isolating somatic cells was developed for Kappaphycus spp. and Echeuma denticulatum. Surface-sterilized tissues (0.1 g fresh weight, 2.0 mm thick discs) from subcortical and medullary layers were treated with 3% NaOH, 3% KOH, or hydrogen peroxide in phosphate buffer solution (PBH₂O₂). Tissues of K. alvarezii treated with PBH₂O₂ softened after 5 h of treatment and completely dissociated after 12 h. Viable cell counts (VCC), determined through staining with Evan’s blue, were significantly higher (2.4 × 10⁵ cells g^?1 fresh weight tissue) in K. alvarezii (‘tambalang’ strain) treated with PBH₂O₂ compared with tissues treated with carrageenase from a marine bacterium.     

Occasional pathogenic bacteria promoting ice-ice disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The bacterial isolates from normal and diseased branches of Kappaphycus alvarezii and Eucheuma denticulatum in the Philippines were examined for possible role in the development of the ice-ice disease. The numbers of bacteria on and in ice-iced branches were 10–100 times greater than those from normal, healthy ones. Gram-positive bacteria predominated in almost all branch sources, but with an increasing proportion of agar-lysing bacteria in branches suffering from the ice-ice disease. These agar-lysing bacteria were composed of yellow and non-pigmented, spreading colonies identified to the Cytophaga-Flavobacterium complex and the Vibrio group. Among isolates which mainly appeared on ice-iced branches, two strains, designated as P11 (Vibrio sp.) and P25 (Cytophage sp.), which showed pathogenic activity, were obtained. These strains caused early ice-ice whitening of K. alvarezii especially when subjecting branches to environmental stress, such as reduced salinity and light intensity, suggesting that these bacteria were occasionally pathogenic. This paper offers new evidence of bacterial role in the development of so-called ice-ice disease among farmed species of Kappaphycus.     

Laboratory-induced development of the ice-ice disease of the farmed red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The most common perception of unfavorable environmental factors causing the ice-ice disease in the farmed seaweeds, Kappaphycus and Eucheuma, was demonstrated in this study for the first time using stressful conditions of abiotic factors in a continuous culture system. Light intensity of less than 50 mol photon m^–2 s^–1 and salinity of 20% or less induced ice-ice whitening characterized by short segments at midbranches which were similar to those observed in the Philippine seaweed farms, while temperatures of up to 33–35 °C resulted in wide-scale whitening leading to complete damage of the branches. These effects were preceded by slow growth rates from an optimum of 3.7% d^–1 to almost –2.0% d^–1. Mechanical stress by wound injury did not result to ice-ice whitening similar to the above. Environmental factors observed to trigger ice-ice in the laboratory, although may not necessarily parallel those in the field, may act synergestically to produce similar effects.     

Strain selection in Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) using tetraspore progeny

A brown strain of Kappaphycus alvarezii from the Philippines produced tetraspores in the summer and autumn (December 1995 to May 1996) in cultivation experiments in the sea at Ubatuba, São Paulo State, Brazil. In vitro tetraspore release and germination experiments showed a mass mortality two to four days after release. Only 20 plants derived from tetraspores were grown successfully for over a year in the laboratory. Large differences in morphology, colour, size and growth rates were observed amongst these plants. The individual plants differed from one another in one or more characteristic. Differences appeared in the early developmental stages and persisted through time. After ten months, the plants that grew best in laboratory culture were transferred into the sea, but the others remained very small (3 to 5 mm), even after two years. In the sea, the plants also showed individual differences in their ability to survive and grow. These results emphasise the potential of the tetraspore progeny for strain selection in K. alvarezii. The results also suggest that the tetrasporophyte used in these studies is of hybrid origin.     

Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae)

Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and experimental system for in vitro culture. Five strains were tested: brown (BR), green (GR) and red (RD) tetrasporophytes, brown female gametophyte (BFG), and a strain originating from tetraspore germination (“Edison de Paula”, EP). The effects of three culture media were tested on callus formation, regeneration from explants and from callus in the three tetrasporophytic and EP strains: seawater enriched with half-strength of von Stosch’s (VS 50) and Guillard & Ryther’s (F/2 50) solutions, plus synthetic ASP 12-NTA medium, with or without gelling agent. Explants of the EP strain were treated with glycerol and the phytoregulators indole-3-acetic acid (IAA); 2,4-diclorophenoxyacetic acid (2,4-D); and benzylaminopurine (BA), alone or in combination. The effects of colchicine (0.01%) during 24, 48, 72 hours and 14 days were analyzed in the BFG and EP strains. The EP strain showed the highest percentage of explants forming callus and regeneration from explants in VS 50, indicating its high potential for micropropagation in comparison to the other strains. Regeneration from callus was very rare. Treatments with glycerol and IAA:BA (5:1 mg L⁻¹) stimulated the regeneration from explants. Significant differences were observed in the percentages of regeneration of EP strain explants treated with colchicine for 14 days. Our results indicate that IAA and BA stimulated the regeneration process, and that colchicine produced explants with high potential for regeneration, being useful for improving the micropropagation of K. alvarezii.     

Spatial variations in nutrient supply to the red algae Eucheuma serra (J. Agardh) J. Agardh

The nutrient supply rates within the canopy of the economically important red algal species, Eucheuma serra J. Agardh were determined experimentally in a recirculating flow-chamber. A single individual was placed in the working section of the 2000 × 200 × 250 mm³ acrylic flow-chamber and subjected to unidirectional water velocities from 1.0 to 9.3 cm s⁻¹. Rates of nutrient supply were determined using 9.7 mm diameter CaSO₄ (gypsum) spheres that were attached to the thallus inside and outside of the canopy. The supply rates within the canopy were 56% less than outside of the canopy. Increases in internal and external water velocity asymptotically increased the nutrient supply rates regardless of location. A model was developed to examine how changes in ammonium and nitrate supply compared with the physiologically maximum uptake rates of these nutrients. The results suggest that when the ammonium concentration in the water was 20 µmol L⁻¹ uptake rates were limited by the supply rate especially at velocities below 5 cm s⁻¹, whereas in the case of 20 µmol L⁻¹ of nitrate, the supply of nitrate was more than adequate to maximize nutrient uptake.     

Optimization of culture conditions for the direct regeneration of Kappaphycus alvarezii (Rhodophyta,Solieriaceae)

Cultivation of seaweeds on a commercial scale requires a large number of propagules with desirable phenotypic traits which include high growth rates and resistance to diseases. Seaweed micropropagation can be considered as one of the best methods to provide a large amount of seedlings for commercial cultivation. This study was carried out to optimize the parameters known to affect the growth of Kappaphycus alvarezii in vitro and subsequently improve the production of seedlings through micropropagation. Suitability of media, concentration of phytoregulators, types and concentration of fertilizers, culture density, light intensity, interval of aeration activity, salinity, and pH were found to be critical factors for the growth of K. alvarezii. The optimum condition for direct regeneration of K. alvarezii in a culture vessel was found to be cultivation of explants in Provasoli's enriched seawater (PES) media supplemented with 2.5 mg L^?1 6-benzylaminopurine (BAP), 1.0 mg L^?1 indole-3-acetic acid (IAA), and 3.0 mg L^?1 natural seaweed extract (NSE) with culture density of 0.4 %?w/v, under light intensity of 75 μmol photons m^?2 s^?1, continuous aeration of 30.0 L h^?1, salinity of 30.0 ppt, and pH 7.5. An airlift photobioreactor was constructed for the mass propagation of K. alvarezii explants with optimum culture conditions obtained from the study. The optimum growth rates of the K. alvarezii explants in culture vessels (5.5 % day^?1) and photobioreactor (6.5 % day^?1) were found to be higher than the growth rate observed in field trials in the open sea (3.5 % day^?1). The information compiled during the course of this study will be of utility to commercial seaweed cultivators.     

Relationships among Eucheuma denticulatum,Eucheuma isiforme and Kappaphycus alvarezii (Gigartinales,Rhodophyta) based on nuclear ssu-rRNA gene sequences

The nuclear genes encoding small-subunit ribosomal RNAs (ssu-rDNAs) of the carrageenophytes Eucheuma denticulatum, E. isiforme and Kappaphycus alvarezii were amplified by the polymerase chain reaction, cloned and sequenced. The sequences range from 1767 (K. alvarezii) to 1781 (E. isiforme) nucleotides in length, and have guanine+cytosine contents between 51.2% (E. isiforme) and 51.5% (E. denticulatum). Pairwise sequence identities among these sequences ranged from 97.6% to 98.5%, levels comparable to some intergeneric identities within Gracilariales. In phylogenetic analyses, the two Eucheuma ssu-rDNAs group stably together vis-a-vis the ssu-rDNA of K. alvarezii, and these three ssu-rDNAs form a monophyletic group within a larger grouping of other carrageenophytes. The results demonstrate quantitatively that analysis of nuclear-encoded ssu-rDNA sequences is likely to be useful in resolving taxonomic, phylogenetic and biogeographic questions among tribe Eucheumatoideae Doty.     

Germination and survival of tetraspores of Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) introduced in subtropical waters of Brazil

The carrageenophyte Kappaphycus alvarezii was introduced in 1995 and vegetatively propagated in Ubatuba, São Paulo State, Brazil, for the purpose of commercial cultivation. This species produces tetraspores mainly in the austral summer and fall. Tetraspore germination and survival were studied under different conditions of temperature, photon flux density, and photoperiod in the laboratory. Field experiments were also carried out. Although tetraspores of K. alvarezii germinated, they had low survival rates, most dying after 20 days. Recruitment of K. alvarezii tetraspores did not occur in experiments conducted in the field. The results indicated that the establishment of K. alvarezii via spore production in the natural environment of the south‐east coast of Brazil is rather remote.     

Evaluation of steam explosion as pretreatment in agar extraction fromGracilaria dura (C. Agardh) J. Agardh (Gracilariaceae,Rhodophyta)

Steam explosion was investigated as a pretreatment step in the isolation of agar from the macroalgaGracilaria dura. As compared to conventional procedures, the yield of agar obtained using this method on alkali (Na₂CO₃) conditioned algal material was higher. Extractions performed first at 95 °C and then at 121 °C showed that the major fraction of the agar was extracted at 95 °C, independently of the pretreatment. The efficiency of sulphate hydrolysis during steam explosion ofG. dura previously conditioned in Na₂CO₃, was similar to that of a NaOH based alkali pretreatment. Except for a lower nitrogen content of the sample obtained after NaOH based alkali pretreatment and a higher 6-O-methyl--d-galactose content in the agar after steam explosion, the chemical composition of the agars showed no significant difference. Agars extracted after steam explosion had melting temperature, gel strength and apparent modulus of elasticity lower than those of corresponding native and alkali (NaOH) pretreated samples, but comparable to those of a commercial sample.Author for correspondence     

Effect of drying temperature on carrageenan yield and quality of Kappaphycus alvarezii (Rhodophyta,Solieriaceae) cultivated in Brazil

This study evaluates the effect of different drying temperatures on the properties of semi-refined (SR) and refined (R) carrageenan extracted from Kappaphycus alvarezii cultivated in Brazil. Drying kinetics was studied in seaweeds under the following treatments: sun drying (control) and drying at 40, 60, and 90 °C in convective air dryer. Drying was carried out until the moisture content of seaweeds reached values below of 30 % on wet basis. Significant reductions in drying time were observed with the increase of temperature. At 90 °C, 30 % moisture content was reached in 100 min, as compared with the 1,440 min required by the sun-drying treatment. SR yields showed no significant differences when compared to the control, varying from 40 to 44 %, while R had a significantly higher yield (30 %) at 90 °C in relation to control (26 %). Gel strength of SR was significantly higher in the sun-dried samples (1,685.1 g cm^?2) and 60 °C samples (1,727.2 g cm^?2), but no significant differences were observed in R gel strengths. Lowest syneresis was observed in both SR (9.8 %) and R (10.3 %) after the treatment at 90 °C. Significantly lower viscosity values were observed for SR at 60 °C (233 mPa s) and at 90 °C (175 mPa s), as for R, the lowest value was observed at 90 °C (205 mPa s). Based on these results, it was concluded that best results for both types of carrageenan are obtained drying at 60 °C.     

Effects of low temperature stress on the antioxidant system and photosynthetic apparatus of Kappaphycus alvarezii (Rhodophyta,Solieriaceae)

To understand the effects of low temperature stress on Kappaphycus alvarezii and the responses of antioxidant systems and photosystem II (PSII), behaviour in K. alvarezii thalli exposed to low temperatures (20°C, 17°C and 14°C) for 2 hours was evaluated. Compared with the control at 26°C, activities of some antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX) and the level of antioxidant substance (reduced glutathione) increased in K. alvarezii thalli when exposed to lowered temperatures (20°C, 17°C). Hydroxyl free radical (·OH) scavenging activity of K. alvarezii thalli also increased at 20°C and 17°C compared with the control. This indicated that the resistance to low temperature stress in the antioxidant system of K. alvarezii increased at lowered temperatures of 20°C and 17°C. However, at the lowest temperature (14°C), no significant increases of this algal antioxidant were observed. Under low temperature stress, the maximum quantum yield of PSII photochemistry (F_V/F_M) and PSII actual photochemical efficiency (Φ_PSII) decreased in K. alvarezii thalli, suggesting that the photosynthetic capacity declined. Components of the photosynthetic apparatus (such as the oxygen-evolving complex, light absorption antennas, reaction centres, electron acceptor sides and electron donor sides of PSII) were damaged by low temperature stress to varying degrees. In addition, it was found that low temperature stress led to decreases of both D1 protein and Rubisco LSU (Rubisco large subunit) protein levels. This work is a significant contribution towards understanding the basic mechanism involved in the resistance and the adaptation of K. alvarezii to low temperature stress conditions.     

Seasonal Occurrences of Epiphytic Algae on the Commercially Cultivated Red Alga Kappaphycus Alvarezii (Solieriaceae, Gigartinales, Rhodophyta)

Common problems faced in farming of the red algal genus Kappaphycus/Eucheuma are “ice-ice disease” and the occurrence of epiphytes. Considerable work has been documented on “ice-ice disease” and it's mode of infection but limited information is available on the emergence of epiphytes. The present study addresses the phenomenon of epiphyte infection, its prevalence in commercially cultivated red alga, Kappaphycus alvarezii, and their variability associated with seasonality. Cultured seaweed became susceptible to epiphytes in the dry seasons (1) between March – June and (2) September – November. Findings revealed Neosiphonia savatieri (Hariot) M. S. Kim et I. K. Lee, as the dominant infecting epiphyte, representing up to 80–85% of the epiphyte present during peak seasons. Besides N. savatieri, Neosiphonia apiculata, Ceramium sp., Acanthophora sp. and Centroceras sp. were observed in smaller quantities. SEM (Scanning Electron Microscope) micrographs revealed the epiphyte's attachment to the host. Further histological study showed the extent of penetration of epiphytes into the host's cortex tissues and condition of its surrounding tissues. The outbreak of epiphytic filamentous red algae also correlated with drastic changes in seawater temperature and salinity during March– June and September – November.     

Investigation of the application of Acadian Marine Plant Extract Powder (AMPEP) to enhance the growth, phenolic content, free radical scavenging, and iron chelating activities of Kappaphycus Doty (Solieriaceae, Gigartinales, Rhodophyta)

An optimization study on concentration (viz. 0.01, 0.1, and 1.0 g L^?1) and dipping time (i.e., 30 and 60 min) was conducted on three different color morphotypes (i.e., reddish brown, yellowish brown and purple) of the commercial carrageenophyte Kappaphycus alvarezii (Doty) Doty. The study tested the efficacy of Acadian Marine Plant Extract Powder (AMPEP) on the growth rate and occurrence of macro-epiphytes from August to November, representing the wet season of the Philippines. The optimum concentration and dipping time were obtained at 0.1 g L^?1 and 30 min, respectively. These optimum parameters were then further verified in a commercial nursery using the yellowish brown morphotype. In another experiment, K. alvarezii (tambalang purple morphotype) and Kappaphycus striatum (Schmitz) Doty (sacol green morphotype) with, and without, AMPEP dippings were tested for their total phenolic content, free radical scavenging and iron chelating activities. Seaweed dipped in AMPEP demonstrated higher growth rates than the control. Lower concentrations (i.e., 0.01-0.1 g L^?1) and shorter dipping time (e.g., 30 min) produced higher growth rates than the highest concentration (1.0 g L^?1) and longer (60 min) dipping time. The presence of macro-epiphytes such as filamentous Ulva did not adversely affect the robust growth of the three color morphotypes of K. alvarezii. The lowest and highest growth rates obtained in a commercial seaweed nursery using the optimum concentration and dipping time of AMPEP were observed in July and January with 0.8% and 6.7% day^?1, respectively. The antioxidant content of K. alvarezii (tambalang purple) and K. striatum (sacol green) responded differently to AMPEP dipping. The changes in total antioxidant activity followed almost the same trend as in phenolic content, in both K. alvarezii (tambalang purple) and K. striatum (sacol green), whereas, the iron chelating ability of both seaweeds with and without AMPEP dipping varied monthly. The results obtained for the use of AMPEP dips for commercial Kappaphycus cultivation demonstrated an effective management tool for improved farming protocols.     

A morphological and molecular assessment of the genus Prionitis J. Agardh (Halymeniaceae, Rhodophyta)

A critical reassessment of the morphological features of two closely related red algal genera, Grateloupia C. Agardh and Prionitis J. Agardh (Halymeniaceae), shows that members of the two genera share very similar reproductive (including the Grateloupia‐type auxiliary‐cell ampullae) and vegetative characters. Diagnostic features hitherto used for distinguishing these two genera, the texture of blades (lubricous to leathery in Grateloupia vs cartilaginous in Prionitis) and the position of reproductive structures (scattered over the entire blade in Grateloupia vs confined to particular portions of the blade in Prionitis), are continuous across some 75 species of both genera, thus making it difficult to draw a clear‐cut distinction between the two genera. In ribulose‐1,5‐bisphosphate carboxylase/oxygenase gene (rbcL) sequence analyses, the species of Grateloupia and Prionitis, including the two generitypes, constitute a large monophyletic clade in the Halymeniaceae. It is therefore proposed that Prionitis be included in the synonymy under Grateloupia and the appropriate combinations are proposed.