Using gigartinine to track the distribution of an alien species of <Emphasis Type="Italic">Gracilaria</Emphasis> in New Zealand

The presence of gigartinine has been used previously as a taxonomic marker for the positive identification in Manukau Harbour (west coast, Auckland) of Gracilaria sp., which has apparently been introduced to New Zealand waters and is easily confused morphologically with the native species, G. chilensis. Thirty two estuarine/harbour populations of Gracilaria in New Zealand were screened for the presence of gigartinine to further test the utility of gigartinine as a reliable species marker. DNA fingerprinting was used to confirm the identity of a subset of the specimens surveyed. Using genetic rather than chemical characterisation, it was discovered that Gracilaria sp. is also present in Orakei Basin (east coast, Auckland). Although a sample from the wild did not have the anticipated gigartinine content, tank cultivation of thalli from this population in an artificially elevated nitrogen environment allowed the plant to accumulate nitrogen as gigartinine. This confirmed the unusual ability of this species of Gracilaria to store nitrogen in this form, unlike the widespread, morphologically similar, G. chilensis.     

Agar quality of commercial agarophytes from different geographical origins: 1. Physical and rheological properties

Six economically important species ofGracilaria, from a number of commercial sources around the world, andGracilariopsis lemaneiformis, collected from two Japanese localities, were used as the sources of raw material for the evaluation of agar quality. Agar-agar was extracted by pretreatment with various concentratrions of NaOH (0%, 3%, 5%, 7%, 10%) incubated at 80 °C for 2 h. Agar yield, viscosity, dynamic gelling and melting temperature and gel texture were determined for 1.5% agar gels. The highest agar yield was obtained fromG. gracilis from Argentina (39.5%), while the lowest was from BrazilianG. gracilis (13.37%). Dynamic gelling temperature was highest in the agar fromG. gracilis from Turkey (59 °C) and lowest in the non-alkali treated agar isolated fromG. edulis from Indonesia (46 °C). Melting temperature ranged from 96 °C in the agars from the JapaneseGracilariopsis andG. chilensis from Chile to 69 °C in the non-alkali treated agar fromG. edulis from Indonesia. In general, all species produced an agar with high gel strength after treatment with 5% NaOH, except forG. chilensis and the twoGracilariopsis species, which produced an agar with high gel strength after treatment with 3, 7 and 10% NaOH. The highest gel strength (2056 ± 13.6 cm^–2) and hardest gel (261 ± 19.89 g mm^–2) were obtained fromG. lemaneiformis from Japan (Oita Prefecture) after treatment with 7 and 10% NaOH respectively. The lowest gel strength (351 ± 93 cm^–2) was obtained fromG. gracilis from Brazil after treatment with 3% NaOH. The softest gel (66.31 ± 9.63 g mm^–2) was isolated fromG. tenuistipitata from China, after treatment with 3% NaOH. The most flexible gel (11.62 ± 0.31 g mm^–2 × 10²) was obtained fromG. chilensis from Chile after treatment with 3% NaOH.Author for correspondence     

Comparison of growth rate in culture,dry matter content,agar content and agar quality of two New Zealand red seaweeds,Gracilaria chilensis Bird,McLachlanet Oliveira andGracilaria truncata Kraft

Plant growth rates and agar characteristics were compared for two agarophytes,Gracilaria chilensis (Gracilariales, Rhodophyta) andG. truncata, so that the suitability ofG. truncata for mariculture could be assessed.G. chilensis plants grew steadily in the laboratory culture system at rates of 5–8% day^-1 (mean Relative Growth Rate) throughout the 6 week experimental period, with no decline in health.G. truncata plants grew at rates of 2–4% day^-1 for 5 weeks, but during the sixth week their apical tips became necrotic and growth rates fell to zero. There was no significant difference in the dry matter content (as a percentage of their wet weight) between the two species ofGracilaria, with values falling in the range of 16–19%. Slightly higher agar yields were obtained from alkali-treatedG. chilensis (17–20% dry matter) than from untreatedG. truncata (16–18%) although the agar fromG. truncata formed softer gels from which it was more difficult to recover. The gel strength of untreated agar extracted fromG. chilensis was very low (ca. 100 g cm^-2 for a 1% gel) but when an alkali treatment step was included in the extraction process, it increased to 520 g cm^-2 for a 1% gel. Contrary to an earlier report, untreatedG. truncata agar also had a very low gel strength (ca. 100 g cm^-2 for a 1.5% gel), but it rose to only 167 g cm^-2 after alkaline treatment.     

Influence of enhanced CO2 on growth and photosynthesis of the red algaeGracilaria sp. andG. chilensis

The influence of elevated CO₂ concentrations on growth and photosynthesis ofGracilaria sp. andG. chilensis was investigated in order to procure information on the effective utilization of CO₂. Growth of both was enhanced by CO₂ enrichment (air + 650 ppm CO₂, air + 1250 ppm CO₂, the enhancement being greater inGracilaria sp. Both species increased uptake of NO₃ ^– with CO₂ enrichment. Photosynthetic inorganic carbon uptake was depressed inG. chilensis by pre-culture (15 days) with CO₂ enrichment, but little affected inGracilaria sp. Mass spectrometric analysis showed that O₂ uptake was higher in the light than in the dark for both species and in both cases was higher inGracilaria sp. The higher growth enhancement inGracilaria sp. was attributed to greater depression of photorespiration by the enrichment of CO₂ in culture.     

Deviations in the life-history of Gracilaria sp. (Rhodophyta,Gigartinales), from Coquimbo,Chile, under different culture conditions

Cystocarpic branches of a species of Gracilaria from Coquimbo, Chile, were cultured in vitro. A Polysiphonia-like life history was completed in about 6 months, but some abnormalities were observed: i. carpospores gave rise to plants producing either tetrasporangia and spermatangia, or tetrasporangia only; ii. tetraspores cultured without aeration developed into plants bearing spermatangia only; tetraspores cultured with aeration developed into 1:1 female and male gametophytes; iii. plant originated from tetraspore produced spermatangia and tetrasporangia; one of these tetraspores developed into a male gametophyte; iv. some tetraspores gave rise to spherical bodies instead of the ordinary cylindrical branches; one of them bore spermatangia after three months. The results show that environmental factors seem to be interfering with the mechanism of sex determination and induce the development of spermatangia on putative female gametophytes, or on putative tetrasporophytes. Noted added in proof: The Gracilaria sp. studied here was recently described as a new species, G. chilensis by Bird C. J., McLachlan, J & Oliveira, E. C. de, 1986. Can. J. Bot. 64: 2928–2934. Noted added in proof: The Gracilaria sp. studied here was recently described as a new species, G. chilensis by Bird C. J., McLachlan, J & Oliveira, E. C. de, 1986. Can. J. Bot. 64: 2928–2934.     

Acclimation to UV irradiance in Gracilaria chilensis Bird,McLachlan & Oliveira (Gigartinales,Rhodophyta)

Photoautotrophs can cope with an increase in ultraviolet (UV) irradiance in the aquatic environment, through protection and acclimation mechanisms (i.e. synthesis of UV-absorbing compounds). This capacity has been proven to vary according to the organism's sensitivity. To quantify variations of this capacity between the different parts of macroalgae, an in vitro study was performed with the tips, cystocarps and thalli segments of Gracilaria chilensis. Whole algae incubated during 3 days at high and low PAR, supplying UV B (4.6 µW cm^–2) during 2 hours showed, as predicted, an increase in absorption (OD) at 320 nm of the different parts, after the first day of exposure to UV B. The tips presented the highest increase in the standardized OD at 320 nm relative to cystocarps and thalli segments; their mean percentage of increase was 38% and 29% at low and high PAR, respectively. The lowest sensitivity was consistently found in the thalli segments, while the highest was in the tips. The tips are important for growth and therefore they play a major role in the maintenance of the Gracilaria populations. Acclimation mechanisms that occurred in a short time scale, and mainly in the tips, may allow Gracilaria to have an almost immediate protection to increases in UV B fluxes.     

Development of microsatellites DNA markers in the cultivated seaweed,Gracilaria chilensis (Gracilariales,Rhodophyta)

The red algae Gracilaria chilensis is extensively cultivated for agar production. In spite of its commercial significance as the first algal resource in Chile, no information is available on the pattern of genetic diversity. In this paper, we isolated six polymorphic microsatellite markers from a G. chilensis‐enriched DNA library. Genetic diversity was assessed in two natural populations revealing relatively low levels of heterozygosity ranging from 0.00 to 0.51. The six loci developed here are good candidates to assess the level of genetic resources within this species, which probably suffered from over‐exploitation in several localities along the Chilean coast.     

DNA base composition heterogeneity in some agarophytes (Gracilariales,Rhodophyta) from Mexico and the Philippines

Estimates of nuclear DNA base composition by determination of thermal denaturation temperatures (T_m) indicate guanine + cytosine (G + C) levels of 35.4–46.8% for ten species of the Gracilariaceae, representing the generaGracilaria andHydropuntia. T_m values were found to be reproducible with variation among most samples and replicates of less than 1 °C and 2 mol%. Interspecific variation in G + C values was less than 11.4% amongGracilaria species. Calculation of intragenomic base pair composition distribution based on mid-resolution thermal denaturation (A 1 °C/min with 4s interval H and dT logging) indicated an inverse relationship between maximum similarity values and taxonomic rank. Intraspecific (population level) maximum similarity (homology) values were estimated to range from 79–90% inGracilaria tikvahiae (4 isolates). Interspecific values of 46–69% were found in 13 species ofGracilaria. Nucleotide distribution similarity values for the Gracilariaceae are compared with previous information for genome organization and complexity, genome size and karyotype patterns.Author for correspondence     

Effect of nutrient pulse concentration and frequency on growth ofGracilaria chilensis plants and levels of epiphytic algae

The effect of nutrient pulse concentration and frequency onGracilaria chilensis Bird, McLachlanet O Oliveira growth and epiphyte abundace was investigated for plants grown in an indoor culture facility. The frequency of nutrient pulses (which ranged from 1 pulse to 4 pulses per 14 days) had a strong influence on plant growth, while pulse concentration (from 72 to 143 µM as ammonium) had a lesser influence. Growth became a function of total N flux only when plants received nutrient pulses at least twice per 14 days. Both pulse frequency and pulse concentration affected the abundance of epiphytic algae found attached toGracilaria thalli, but pulse frequency was the more significant of the two factors. Their effects could be combined into the single factor, total N flux. Both reasonableG. chilensis growth and low levels of epiphytes were achieved under these conditions (20 °C, 25 µ mol photon m^–2 s^–1 PAR) if ammonium was pulsed at relatively high concentrations (up to 150 µM) once every 7 days into otherwise nitrogen-depleted seawater.     

Ecological differences among Chilean populations of commercialGracilaria

Field farming ofGracilaria is gradually replacing use of the wild crop in Chile. The most popular planting method consists of establishing underground thallus systems for patches ofGracilaria on wave-sheltered, soft-bottom habitats. Commercial cultivation often involves transplanting vegetative material to distant places along the coast. This study shows that native populations possess some ecological differences that might affect cultivation success. Seven environmental factors were tested for their effects on growth of four geographically separated populations belonging to two species ofGracilaria. No specific or population differences were found under various temperature or salinity regimes. Specific differences occurred in responses to irradiance, daylength, sand burial and sand abrasion. Responses to epiphytes differed among both population and species. Epiphyte recruitment was inhibited by some populations and stimulated by others. Given the farming methods presently used in Chile, these results have ecological and economical importance, as they suggest that transplanting randomly from one to other type of habitat does not assure cultivation success.     

Organellar DNA restriction fragment length polymorphism (RFLP) and nuclear random amplified polymorphic DNA (RAPD) analyses of morphotypes of Gracilaria (Gracilariales,Rhodophyta) from Chile

The extreme phenotypic variability recognized among the species of Gracilaria has highlighted the need for the application of refined methods to help solve taxa identifications. In Chile, there still exists uncertainty about the exact number of Gracilaria species. Our investigations are centered on DNA analyses of morphotypes collected from different geographical locations, namely Lenga and Isla Santa María, Region VIII (36°00 S to 38°00 S), and Maullín, Region X (39°30 S to 43°40 S). These two regions of Chile are considered as areas of confluence of G. chilensis, G. verrucosa, and a species of Gracilariopsis. In this study four morphotypes, from a natural bed located in Maullín, were analyzed for RFLP of plastid DNA and the results compared with data of four morphotypes from a bed in Lenga. The DNA banding patterns from each enzyme digest were identical irrespective of morphotypes and/or locations. In an attempt to unravel the nature of the morphological differences found among Lenga and Maullín morphotypes, RAPD analyses of nuclear DNA were also performed; however, no polymorphism has been found yet. Therefore, the data of this study, as well as concurrent data from preliminary interfertility tests, suggest that all morphotypes belong to a single taxon, Gracilaria chilensis.Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas     

MOLECULAR IDENTIFICATION OF TWO SIBLING SPECIES UNDER THE NAME GRACILARIA CHILENSIS (RHODOPHYTA,GRACILARIALES)1,3

Molecular markers belonging to three different genomes, mitochondrial (cox2‐3 spacer), plastid (RUBISCO spacer), and nuclear (internal transcribed spacer 1), were used to compare Gracilaria chilensis samples collected along the Chilean coast with samples ascribed to G. chilensis from the West Pacific Ocean (New Zealand and Australia). Our data are in agreement with previous studies suggesting two sibling species currently going under the name G. chilensis that co‐occur in New Zealand. One of these, a New Zealand sample previously examined by Bird and others in 1990, is conspecific with G. chilensis from Chile. Finally, our results demonstrate clearly that most of the sequences in GenBank reported as G. chilensis are based on misidentified material.     

Effects of stock loading and planting distance on the growth and production ofGracilaria chilensis in rope culture

Through experimental manipulation of stock loading and planting distances in rope culture, this study evaluates the hypothesis that optimum density for growth and production ofGracilaria chilensis vary within a bed. Small packages ofGracilaria disposed at short distances exhibit higher growth and production rates in the landward ends of ropes than larger packages of seaweed disposed at longer distances. In these experiments, the landward end of ropes faces estuarine conditions, with reduced salinities and increased sediment loads during low tides. The interacting effects of stocks loading and position in the rope can affect growth and production rates so much as to nullify the 3.5-fold summer increments in production that occur in these estuarine beds of southern Chile. It is concluded that through manipulation of planting distances and stock loading, the production of this species could be increased up to 35% in some areas.     

Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content

Select species of the agarophyte Gracilaria were studied from southeastern Australia. The morphology and anatomy of species is described and molecular relations are inferred based on plastid and mitochon‐drial DNA sequence data. Agar yields and qualities are determined for each species. Gracilaria chilensis, found in Tasmania and Victoria, is morphologically and molecularly similar to G. chilensis from New Zealand and Chile and has low agar yields of 11–16%. Gracilaria cliftonii from Victoria, has high crude agar yield (52%) and is molecularly uniform. Gracilaria perplexa sp. nov., known only from Botany Bay, New South Wales, has an agar yield of 39%. The agar of G. perplexa is unusual in requiring the addition of 0.1 mol L^?1 NaCl for alcohol precipitation and is cold‐water (25°C) soluble because of the very high sulfate ester content. Molecular phylogeny shows that G. perplexa is closely related to Gracilaria preissiana from western Australia, but differs from the latter in its reduced branching and narrower more terete axes.     

Interaction mechanisms between Gracilaria chilensis (Rhodophyta) and epiphytes

Epiphytes can have a considerable effect on Gracilaria production, and Ulva is one of the commonest algal species identified as an epiphyte, reaching loads of 60% (g of epiphytes per g of Gracilaria) in the intertidal cultures of southern Chile. This study evaluates the relative importance of light reduction, addition of weight to the host thalli and nutrient depletion, as mechanisms determining the interaction effects of Ulva epiphytes on Gracilaria cultivation. Using field experiments undertaken during the main Gracilaria growth season (spring), we evaluate the mechanisms of epiphyte-host algae interaction by manipulating artificial epiphytes. The results indicate that Ulva can significantly depress Gracilaria biomass production and that the addition of weight to the host algae and the consequent dislodgement increase, appear to be the main mechanisms involved in the Ulva-Gracilaria interaction. However, the light reduction caused by the epiphytes can also partially explain the reduction in Gracilaria production. Nutrients depletion would not appear to fully account for the detrimental effects of Ulva over Gracilaria in intertidal farming areas of southern Chile.     

Opportunities and challenges for the development of an integrated seaweed-based aquaculture activity in Chile: determining the physiological capabilities of <Emphasis Type="Italic">Macrocystis</Emphasis> and <Emphasis Type="Italic">Gracilaria</Emphasis> as biofilters

Seaweed production is a reality in Chile. More than ten species are commercially used to produce phycocolloids, fertilizers, plant growth control products, human food or animal fodder and feed additives. These multiple uses of algae offer a number of possibilities for coupling this activity to salmon, abalone and filter-feeder farming. In this context, different experiments carried out in Chile have demonstrated that Gracilaria chilensis and Macrocystis pyrifera have great potential in the development of an integrated aquaculture strategy. The present Integrated Multi-Trophic Aquaculture (IMTA) approach study showed that Gracilaria can be cultured best at 1 m depth whereas Macrocystis has an especially good growth response at 3 m depth. Both species use available nitrogen efficiently. On the other hand, high intensities of solar radiation (UV and PAR) can be critical at low depths of cultivation, and our results indicate that both species show photosynthetic susceptibility mainly at noon during the summer. The demand of Macrocystis for abalone feeding is increasing, thus improving the opportunity for developing an integrated nutrient waste recycling activity in Chile. Although Gracilaria shows a higher nitrogen uptake capacity than Macrocystis, its market value does not yet allow a massive commercial scaling.     

Quantification and characterization of nuclear genomes in commercial red seaweeds (Gracilariales) from the Philippines

Eight species of Gracilariaceae from the Philippines, representing the generaGracilaria, Gracilariopsis andHydropuntia, were investigated to quantify and characterize their nuclear genomes. DNA reassociation kinetics were used to determine nuclear genome organization and complexity in six of these species. Results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–74% and unique DNA ranged from 26–84%. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA contents. Comparisons of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in an estimate of 0.38–0.43 pg/2 C genomes for seven of the species investigated. Preliminary analyses of agar content and quality confirm the economic potential ofGracilaria firma, Gracilaria sp. 2 from Sorsogon andGracilariopsis bailinae. Nuclear genome profiles developed from data for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity.Author for correspondence     

A critical comparison ofGracilaria chilensis andG. sordida (Rhodophyta,Gracilariales)

Gracilaria chilensis Bird, McLachlanet Oliveira from Chile andG. sordida Nelson from New Zealand have been compared with respect to reproductive anatomy, chromosome number, interfertility, and organellar DNA restriction profiles. No differences were found in reproductive anatomy, which in these species is distinguished by deeptextorii-type spermatangial conceptacles and prominent tubular nutritive cells directed only to the floor of the cystocarp. The species share a chromosome number ofn = 24 and are readily interfertile. Electrophoretic profiles of organellar DNA digested with four different restriction endonucleases were virtually identical between the species except for bands that represented accompanying plasmids. However, previous research has indicated that the four plasmid bands inG. chilensis and the single one inG. sordida have a common origin. On these groundsG. chilensis andG. sordida are     

Attenuation of water velocity and incident light as a function of shape parameters inGracilaria

Characteristic shape parameters, light intensities and relative water velocities were estimated in thalli of free-moving seaweeds, using threeGracilaria-like species:G. cornea, G. conferta andGracilariopsis lemaneiformis. Relative velocities over the branches were determined mostly by rotation of the algae in the water as opposed to linear translation. The tree major shape parameters of the thallus, weight, volumetric specific weight and areal specific weight, explain the differences in relative velocity. Relative velocities near the center of the thallus were about 50% to 80% of the external velocity. Light intensities at the vicinity of the center ofG. cornea ranged between 60% to 90% of the external light intensity. Light attenuation in the center was proportional to the weight of the thallus. The significance of light and velocity attenuation as a function of shape parameters is discussed.Author for correspondence     

Assessment of allozyme variation among New Zealand populations of Gracilaria chilensis (Gracialiares,Rhodophyta) using starch-gel electrophoresis

New Zealand populations of Gracilaria chilensis are uniform in anatomical reproductive characteristics but vary morphologically and have been found to separate into two distinct groups with respect to agar methylation level, namely low (24–30%) and high (43–47%). To investigate the genetic variation within New Zealand populations of this species, 14 isozyme loci detected by starch-gel electrophoresis were examined in 17 wild populations from a wide range of localities, and in cultures derived from these populations. Five of these loci were polymorphic, but the genetic variation within populations was low: of the 17 populations examined, 15 were fixed at all loci (heterozygosity 0.000) and in the remaining two populations the observed heterozygosity was still low (0.004 and 0.011). The genetic distances between the populations ranged from 0.00 to 0.43. UPGMA cluster analysis separated the populations into two groups, a northern group and a group found throughout the country. Although these two groups do not correlate with the two groups based on agar methylation level at every locality, the correlation is sufficiently striking to merit further investigation.