IN SITU UPTAKE KINETICS OF AMMONIUM AND PHOSPHATE AND CHEMICAL COMPOSITION OF THE RED SEAWEED GRACILARIA TIKVAHIAE1

The uptake kinetics of ammonium and phosphate by Gracilaria tikvahiae McLachlan were studied under field conditions. Seaweeds, pulse fed once a week for 6 h over a 4-week period, had maximum uptake rates of 19 μmol·g fwt^?1·h^?1 for ammonium and 0.28 μmol·g fwt^?1·h^?1 for phosphate. For both nutrients there was a positive linear correlation between uptake rate (v) and concentration (S) over the entire range of concentration tested. In a nutrient depletion experiment, the phosphate uptake curve determined over a wide range of concentrations consisted of two stages of saturation at low concentrations, and a linear phase at high concentrations. Ash free dry weight, chlorophyll a, phycoerythrin, and protein content were higher in pulse fed plants than in control plants receiving no nutrient additions, while the reverse held true for carbohydrate contents and the C/N ratios. The C/N ratio inversely correlated with ammonium and phosphate uptake rate as well as protein and phycoerythrin content, and positively with carbohydrate content.     

Increased potential for wound activated production of Prostaglandin E2 and related toxic compounds in non-native populations of Gracilaria vermiculophylla

The capacity of the East Asian seaweed Gracilaria vermiculophylla (“Ogonori”) for production of prostaglandin E₂ from arachidonic acid occasionally causes food poisoning after ingestion. During the last two decades the alga has been introduced to Europe and North America. Non-native populations have been shown to be generally less palatable to marine herbivores than native populations. We hypothesized that the difference in palatability among populations could be due to differences in the algal content of prostaglandins. We therefore compared the capacity for wound-activated production of prostaglandins and other eicosatetraenoid oxylipins among five native populations in East Asia and seven non-native populations in Europe and NW Mexico, using a targeted metabolomics approach. In two independent experiments non-native populations exhibited a significant tendency to produce more eicosatetraenoids than native populations after acclimation to identical conditions and subsequent artificial wounding. Fourteen out of 15 eicosatetraenoids that were detected in experiment I and all 19 eicosatetraenoids that were detected in experiment II reached higher mean concentrations in non-native than in native specimens. Wounding of non-native specimens resulted on average in 390% more 15-keto-PGE₂, in 90% more PGE₂, in 37% more PGA₂ and in 96% more 7,8-di-hydroxy-eicosatetraenoic acid than wounding of native specimens. Not only PGE₂, but also PGA₂ and dihydroxylated eicosatetraenoic acid are known to deter various biological enemies of G. vermiculophylla that cause tissue or cell wounding, and in the present study the latter two compounds also repelled the mesograzer Littorina brevicula. Non-native populations of G. vermiculophylla are thus more defended against herbivory than native populations. This increased capacity for activated chemical defense may have contributed to their invasion success and at the same time it poses an elevated risk for human food safety.     

EFFECTS OF PHOTON FLUENCE RATE AND LIGHT SPECTRUM COMPOSITION ON GROWTH,PHOTOSYNTHESIS AND PIGMENT RELATIONS IN GRACILARIA SP.1

The optimal photon fluence rate for growth of tha llus tips of Gracilaria sp. was low (about 100 μE·^–2·¹); higher photon fluence rates inhibited growth. Both phycoerythrin (PE) and chlorophyll (chl) contents decreased with increasing photon fluence rates (up to 100 μE·–m^–2s^–1) in a fashion inverse to the growth response. Chl/PE ratios varied directly as the growth response over a larger photon fluence rate range. The peak chl/PE ratios were obtained at a photon fluence rate optimal for growth, suggesting that this parameter may be used to estimate in situ growth rates. A low compensation point (about 7 μE·^–2s^–1) was observed for low light (15 μE·^–2s^–1) grown plants. This compensation point was also obtained for growth in the long–term (5–6 weeks) experiments. Plants grown at 60 and 140 μE·^–2s^–1 showed higher light compensation and saturation points, suggesting that the variations in pigment composition found between the different treatments determine the photosynthetic responses at sub–optimal photon fluence rates. Photosynthetic rates at light saturation were the same, on a biomass basis, for plants grown at the various photon fluence rates. Thus, the photosynthetic dark reactions were not influenced by previous light regimes. It is suggested that maximal photosynthetic rates expressed on a biomass basis better reflect the potential productivity at tight saturation than if expressed on a pigment basis. Gracilaria sp. grew better under non–filtered fluorescent and greenish than under reddish and blue–enriched light of equal and sub–optimal photon, fluence rate. However, the pigment relations of the algae did not change in a direction complementary to the light composition at which they grew. This, together with the relatively higher photosynthetic rates under reddish and blueish light for plants previously grown under reddish and blueish light, suggests that adaptations to variouslight spectra are based on mechanisms different from complementary chromatic adaptation of the pigments.     

SPORELING COALESCENCE AND INTRACLONAL VARIATION IN GRACILARIA CHILENSIS (GRACILARIALES,RHODOPHYTA)1

This study evaluates the hypothesis that spore coalescence may cause intraclonal variation. Spore coalescence might allow the occurrence of unitary thalli that in fact correspond to genetically different, coalesced individuals. Plant portions simultaneously derived from these chimeric individuals may exhibit dissimilar growth responses even when incubated under similar abiotic conditions. Testing of the hypothesis included various approaches. Transmission electron microscopy observations of early stages of sporeling coalescence indicated that polysporic plantlets were formed by groups of spores and their derivatives. Even though adjacent cells in two different groups may fuse, these groups maintained an independent capacity to grow and form uprights. Laboratory-grown plantlets showed a significant correlation between the initial number of spores and the total number of erect axes differentiated from the sporeling. Construction and growth of bicolor individuals indicated the chimeric nature of the coalesced individuals. Coalesced, bicolor holdfasts had green and red cells, which subsequently produced green and red uprights, respectively. Individuals fronds were also chimeric, as indicated by the production of green and red branchlets from single, red uprights. The existence of mixed tissues was further substantiated by random amplified polymorphic DNA analysis. The banding pattern produced by branchlets of a unisporic thallus was consistently monomorphic, whereas the patterns produced by the polysporic thallus were polymorphic. Growth rates of polysporic thalli had larger data dispersal and variation coefficients than oligosporic or monosporic thalli. Therefore, all results support the original hypothesis and suggest that coalescence might be ecologically more important than previously thought.     

QUANTIFICATION OF THE EFFECTS OF SPORELING COALESCENCE ON THE EARLY DEVELOPMENT OF GRACILARIA CHILENSIS (RHODOPHYTA)1

Sporeling coalescence in species of Gracilariales and Gigartinales is predicted to result in larger basal areas of growing disks as well as earlier initiation, increased abundance, and faster growth rates of erect shoots as compared to noncoalescent sporelings. These responses have been interpreted as providing mutual benefits for organisms living in aggregation, counterbalancing disadvantages associated with crowding. Quantitative evaluations of sporelings of Gracilaria chilensis failed to support several of these predictions. Sporelings were grown in the laboratory from a range of single sporelings to coalescent masses of 20 sporelings. Coalescent sporeling masses of G. chilensis exhibited larger basal areas than noncoalescent ones, but because the specific growth rates were inversely related to the original number of carpospores, no significant differences in actual area increments, during most of the experiment, were found among sporelings derived from one, two, or three to five coalescing sporelings. Initiation of erect shoots occurred at a similar time, regardless of their origin, i.e. coalescent or noncoalescent. Abundance of erect shoots was only loosely related to the number of coalescing sporelings. Even though by the end of the experiment (week 18), the total length of the longer erect shoots arising from coalescent sporeling masses was significantly greater than that of shoots arising from noncoalescent sporelings, total length was independent of the original number of coalescing sporelings. Furthermore, specific elongation rates between week 12 and week 18 were significantly greater for noncoalescent sporelings than for coalescent sporeling masses. Quantitative screening of other species seems necessary before generalizations on the ecological advantages of sporeling coalescence in seaweeds can be made.     

Photosynthetic response of P-deficient Gracilaria tenuistipitata under two different phosphate treatments

Phosphorus-deficient Gracilaria tenuistipitata Zhang et Xia was cultured for 15 days at two different inorganic phosphate (P_i) concentrations: 3 μM (low P_i treatmenl) or 30 μM phosphate (high P_i treatment). The amount of ribulose-l,5-bisphosphate carboxy-lase/oxygenase (Rubisco), phycobiliproteins, Chl a and total soluble proteins were higher in the high P_i than in the low P_i treatment. The total N content of the low P_i plants was lower than in plants grown at high P_i concentrations whereas the amount of total C was highest in low P_i plants. The increase of Rubisco content in the high P_i treatment (3-fold) was parallel to the enhancement of the maximum photosynthetic rate which increased 5-fold. This correspondence was also found in the low P_i treatment in which Rubisco content and maximum photosynthesis did not change significantly from the initial values. The photosynthetic efficiency was also higher at high than at low P_i. The high P_i plants also showed higher dark respiration and growth rates. The data presented here suggest that marine macroalgae submitted to P_i deficiency exhibit a decrease in growth caused not only by P_i implication on energy transfer in photosynthesis and respiration, but also by the diminution of the amount of photosynthetic pigments and Rubisco.     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

Observations on Flattened Species of Gracilaria (Gracilariaceae, Rhodophyta) from Taiwan

Four flattened Gracilaria species have been reported from Taiwan: G. spinulosa, G. vieillardii, G. textorii and G. punctata, identified based on branching pattern, the presence or absence of spines, and characters that often vary seasonally. Gracilaria spinulosa was originally described from the type locality, Tainan. Species with toothed margins are usually referred to G. “vieillardii”; those with smooth margins to G. “textorii”, and those with smooth margins and dark spots scattered over the blade to G. “punctata”. Molecular analyses show that specimens with marginal teeth cluster in three different groups: a G. “vieillardii” clade, a G. spinulosa clade, and a clade sister to G. spinulosa. An undescribed species comprises the third clade, which is distinguished by its relatively large gonimoblast cells and weakly developed tubular nutritive cells. The three clades can be separated by the character of the tubular nutritive cells, the size of gonimoblast cells and certain vegetative features. Plants with entire margins form a single clade characterized by cystocarps with basal tubular nutritive cells and their absence in the cystocarp cavity. They are nested in the Hydropuntia complex and are referred to as Gracilaria “punctata” here. The records of G. textorii and G. punctata from Taiwan require reinvestigation in comparison with the Japanese species.     

Comparison of three gracilarioids: growth rate, agar content and quality

Three gracilarioid species, Gracilariopsis bailiniae and Gracilaria tenuistipitata from Vietnam and Gracilaria gracilis from Russia, were studied in order to determine whether Gracilaria gracilis might be a superior species for cultivation in brackish-water ponds for agar production compared with the Vietnamese species. The effects of different salinity levels on the growth rate and agar production as well as agar properties of three gracilarioid species were compared in controlled laboratory experiments. Gracilaria tenuistipitata and G. gracilis were tolerant to low salinity (∼10‰), whereas Gp. bailiniae died under these conditions. G. tenuistipitata showed superior growth among the three species examined. Gracilaria gracilis had the highest agar content [36.8–46.6% dry weight (dw)]. Agar yield from Vietnamese gracilarioids did not exceed 30% dw. Gel strength of native agar from Gracilaria gracilis was two-fold higher that from Vietnamese species (278 g cm⁻² vs 130 g cm⁻²). Alkali pretreatment increased gel strength significantly for Gracilaria gracilis (1.4-fold), and G. tenuistipitata and Gp. bailiniae (2.3-fold) compared with native agar. The results suggest that Gracilaria gracilis may be a suitable species for production of reasonably good quality agar.