Ammonium uptake by Chondrus crispus Stackhouse (Gigartinales,Rhodophyta) in culture

Cultivated Chondrus crispus was used in N-NH₄ uptake experiments in the laboratory. An elevation of temperature increased the apparent rate of uptake, especially up to 11 °C. Uptake in the dark was found to be 83 % of that in the light. The apparent uptake decreased with increasing internal N pool; rates were 26.5, 22.2 and 20.2 µg N g dry wt^–1 min^–1 for internal N pools of 2.7, 3.5 and 4.6%, respectively. Apparent uptake increased with the substrate N concentration. The resulting curve has two components: an active uptake and a diffusion component at high (> 5000 µg N L^–1) external N levels. Ks and V _max were calculated by deducting the diffusion component from the uptake curve: these were of 497 µg N L ^–1 and 14.4 µg N g dry wt^–1 min^–1. respectively, and reflect a low substrate affinity. This could be the result of 10 years of continuous culture of C. crispus. Uptake was similarly followed in the culture tanks and showed comparable results; nighttime would be the most appropriate time to supply nutrients.     

DNA sequence,structure, and phylogenetic relationship of the mitochondrial small-subunit rRNA from the red alga Chondrus crispus (Gigartinales,Rhodophytes)

The entire nucleotide sequence containing the small-subunit ribosomal RNA gene (SSU rRNA) from the mitochondrial genome of Chondrus crispus was determined. To our knowledge, this is the first sequence of a mitochondrial 16S-like rRNA from a red alga. The length of this gene is 1,376 nucleotides. Its secondary structure was constructed and compared with other known secondary structures from eubacteria and from mitochondria of land plants, green and brown algae, and fungi. Phylogenetic trees were built upon SSU rRNA sequence alignment from mitochondria and eubacteria. The results show that rhodophytes and chromophytes provide additional links in the evolution of mitochondria between the green plant lineage and the nonplant lineages.Correspondence to: C. Boyen     

Relationship between cover of Chondrus crispus (Gigartinales,Rhodophyta) and Phymatolithon (Corallinales,Rhodophyta) on friable rock substrata

Field observations in sublittoral Prince Edward Island, Canada, indicated that on a friable sandstone substratum Chondrus crispus was more commonly associated with Phymatolithon sp. than with bare rock. Thus, a substantial proportion of the population of Irish moss along the coast of Prince Edward Island occurs on this encrusting coralline. These observation may be explained on the basis of the relative stability of the substratum in contrast to other studies in which sloughing of epithallial cells by species of both Phymatolithon and Lithothamnium has been reported to limit epiphytism by fleshy macrophytes.Issued as NRCC 31426.     

Etiology of infectious diseases in cultivated Chondrus crispus (Gigartinales,Rhodophyta)

The appearance of cavities and holes in fronds of commercially cultivated Chondrus crispus is described. These ultimately arise from the ravages of a green spot or green rot disease system in which several biotic agents can participate. Nematodes capable of bacterial grazing were recovered from necrotic lesions and we suggest that the nematodes can facilitate wound healing in diseased tissues. Bacteria isolated from disease lesions and from the surfaces of healthy fronds were screened for pathogenic strains. A particularly virulent one, the DOR isolate, was purified from small dark orange colored colonies grown on dilution plates. It was present in necrotic tissue and also recovered from surface scrapings of old healthy fronds, but not from their apical regions. Growth of the DOR isolate appeared to be inhibited by other bacteria colonizing the algal surface. It was shown to be a facultative pathogen, the virulence of which depended on the availability of ammonium or constituents easily metabolized to ammonium. It induced green rot disease in healthy C. crispus and was recoverable in pathogenic form from experimentally infected frond apices. A wound, disease and recovery cycle is discussed to illustrate potential interrelationships involving animal grazers, algal endophytes, Petersenia pollagaster, bacteria and nematodes.     

Influence of tissue source and growth rates on dry weight and carrageenan composition of Chondrus crispus (Gigartinales,Rhodophyta)

Carrageenan analyses were conducted on vegetative female clones of Chondrus crispus that were cultured to provide tissues with differing growth rates. Tissue dry weights increased from apex to base of fronds. Total carrageenan contents were lower in apical 1 to 2 cm segments than elsewhere in the frond, except when the alga was grown at high photon irradiances. Clone 373A contained more carrageenan than clone G8. The proportion of 0.3 M KCl-soluble polymers in the total native carrageenans varied from 44 to 92%, being highest in older tissues of fronds cultured at high photon irradiances. The apical 1 cm segments contained less KCl-soluble carrageenans than other tissues from the corresponding fronds. The KCl-soluble carrageenans, when alkali-modified and refractionated, afforded the expected kappa-iota carrageenan in > 79% yields. The remainder consisted of a polymer containing 23.1% SO₃Na and 8.4% 3,6-anhydrogalactose. Lambda carrageenan was not detected. Variations in carrageenan distribution between the apical region and other parts of the frond may reflect the increasing influence of medullary tissue developed as the immature cells differentiate.     

Amplified Fragment Length Polymorphism (AFLP) as a source of genetic markers for red algae

A recent PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP), was successfully applied to the red alga Chondrus crispus Stackh. This is apparently the first account to describe the application of AFLP methodology to an alga. Six isolates of C. crispus were analyzed by AFLP. A total of twenty-five primer pairs were screened and six primer pairs were selected for further investigation. Both conservative and variable markers were identified within and between populations; some markers were unique to individuals. As such, AFLP should prove useful as a source of genetic markers in algae for applications as diverse as genome mapping to population genetic investigations. This revised version was published online in June 2006 with corrections to the Cover Date.     

The use of acetic acid as a source of carbon by cultured Chondrus crispus (Gigartinales,Rhodophyta) stackhouse

When growing seaweeds in tanks, pH and carbon source supply have to be controlled in order to maximize photosynthesis. pH can be controlled either by adding various inorganic acids which requires the extra addition of carbon, or by combining pH control and carbon source with for instance CO₂ or an organic acid such as acetic acid (CH₃COOH). We have found comparable productivity of Chondrus using CO₂ or CH₃COOH in tank culture with an increase in production of 25.0 and 27.5%, respectively, over the control. Laboratory experiments showed that acetic acid enabled us to maintain a steady state total inorganic carbon in the medium, the algae displaying an active photosynthesis. Experiments using labelled acetic acid CH₃-¹⁴COOH showed that the acid molecule or at least the -¹⁴COOH group is taken up by Chondrus although the mechanism was not elucidated. Preliminary extractions with hot ethanol showed that 67.9% of the label was solubilized from labelled tissue. Few counts were found in the carrageenans (< 1 %) and between 25.6 and 45.1% were found in the cellulosic residues. Acetic acid is suggested as an interesting means of regulating the pH and adding carbon in macrophyte culture.     

Reproduction ofChondrus crispus Stackhouse (Rhodophyta,Gigartinales) in sublittoral Prince Edward Island,Canada

The influence of size (as biomass), morphology and depth on reproductive patterns was examined in populations ofChondrus crispus in the sublittoral zone of Prince Edward Island, Canada. Fronds of ≥ 10 mg wet wt were considered, and a minimum biomass could not be related to reproduction. However, as frond biomass increased, the frequency of reproductive structures also increased. Reproductive maturity was related to the number of dichotomies. Fronds with less than two dichotomies were only occasionally reproductive, while those with two or more dichotomies could bear sori. Depth in the Prince Edward Island environment had no apparent influence on reproductive maturity, and there was no apparent relationship between depth and the vertical distribution of cystocarpic or tetrasporic fronds.     

A molecular investigation of polymorphism in the North Atlantic red alga Chondrus crispus (Gigartinales)*

Seven samples of Chondrus crispus Stackhouse, representing widely contrasting forms from both sides of the North Atlantic, were compared by restriction digestion of their plastid DNA. The similar banding patterns confirmed that the seven forms were conspecific and distinct from Chondrus ocellatus Holmes f. ocellatus from Japan, used as an outgroup. Nucleotide sequences of the internal transcribed spacers (ITS1 and ITS21 and the intervening 5.8S rRNA gene of the nuclear rRNA operon were investigated as a potential indicator of genetic divergence among morphological variants of C. crispus. The combined ITS regions were relatively short in Chondrus (between 719 and 731 base pairs [bp] in C. crispus and 724 bp in C. ocellatus f. ocellatus), and the sequence of the 5.8s rDNA fragment (152 bp) was identical in both species. In the aligned ITS regions, there were 0–18 base pair differences (0–2.18% divergence) in pairwise comparisons of the seven forms of C. crispus but no consistent pattern of variation according to gross morphology or geographic origin. However, the ITS sequence differed at 41–54 sites (6.22-7.56%) between C. crispus and C. ocellatus f. ocellatus, again illustrating the genetic distinctiveness of the latter species.     

Delineation of Chondrus (Gigartinales, Florideophyceae) in China and the origin of C. crispus inferred from molecular data

Complete nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences were obtained for 18 Chondrus populations collected at 15 sites from eight countries worldwide. Pairwise comparisons with the multiple alignment revealed that intraspecific divergences of ITS sequences ranged from 0.3 to 1.8% in C. crispus Stackhouse (except for the entity SVLH from France) and from 0.0 to 0.6% in C. ocellatus Holmes, whereas interspecific divergences in Chondrus varied from 1.4 to 5.0%. Three phylogenetic methods (neighbour joining, maximum parsimony and maximum likelihood) confirmed three main lineages: the North Atlantic lineage containing entities of C. crispus from Canada, France, Germany, England, Portugal, Ireland and Wales; a second lineage comprising three species: C. sp. 1, C. armatus (Harvey) Yamada et Mikami, and C. pinnulatus (Harvey) Okamura from the Northern Pacific; and a third lineage containing just one species: C. ocellatus from the Northern Pacific. Chondrus yendoi Yamada et Mikami separated from other Chondrus species singly. nrDNA ITS data indicate that a previous assignment of C. sp. 2 to Mazzaella japonica (Mikami) Hommersand may be incorrect, and additional evidence is needed to resolve the generic placement of this entity. It is inferred from the nrDNA ITS data that three Chondrus species are presently known in China with two, C. ocellatus and C. nipponicus, in Qingdao and two, C. armatus and C. nipponicus, in Dalian. We hypothesize that the ancestor of North Atlantic C. crispus had a Pacific origin, and that the present distribution of C. crispus in the Atlantic Ocean correlates with a trans-Arctic dispersal and vicariance events associated with Pleistocene glaciation maxima.     

Taxonomic features of the red alga Meristotheca coacta (Solieriaceae, Gigartinales)

Vegetative and reproductive structures of the little‐known red alga, Meristotheca coacta Okamura (Solieriaceae, Gigartinales) were described on the basis of specimens collected from three localities in southern Japan, including an island close to the lectotype locality. Meristotheca coacta was shown to be a strongly supported monophyletic clade in our rbcL analyses. The taxonomic features of this species were reassessed. This species is characterized as follows: (i) the thalli consist of relatively thick (300–1400 μm from the uppermost to basal regions), elastic and rose‐red blades; (ii) the blades are irregularly lobed and branched into variously shaped segments with undulate and crispate margins; (iii) tetrasporangial initials are laterally attached to their parental cells and the lateral pit‐connections remain in mature tetrasporangia; (iv) gametophytes are dioecious; (v) carpogonial branches are three‐celled (occasionally two‐ or four‐celled); (vi) a darkly staining auxiliary cell complex is present and recognizable prior to diploidization; and (vii) cystocarps are produced along (or near) the margins of the blades and on the marginal proliferations and lack spinous outgrowths.     

AFLP reveals cryptic population structure in migratory European red admirals (Vanessa atalanta)

1. The genetic differentiation in a migratory butterfly, the red admiral (Vanessa atalanta), was investigated to discern patterns of migratory routes used across Europe. AFLP profiles showed significant differences between almost all sampled locations, but there was no clear pattern of isolation‐by‐distance. 2. Using the software STRUCTURE 2.2, we found two distinct genotype clusters present in different frequencies at all study sites. The frequencies of these genotypic clusters varied significantly between years within the same site. Remarkably few individuals were of mixed ancestry, indicating that some isolating mechanisms are present. Twenty‐seven mtDNA haplotypes were identified but they showed no geographic structure, nor were they related to either of the two genotype clusters identified in the AFLP data. 3. Most field observations of migrating red admirals suggest a regular north–south migration pattern in Europe. Our data indicate both long‐distance migration and a more variable pattern in orientation, since the composition of the two genotypic clusters shows dramatic variation between sites and years in the northern part of the distribution range.     

Characterization of the edible red alga Meristotheca papulosa (Solieriaceae, Gigartinales) from Japan

The vegetative and reproductive morphology of the edible red alga Meristotheca papulosa (Montagne) J. Agardh (Solieriaceae) was reexamined based on material collected from various localities in Japan. Although the habit of the blades is variable according to the length and width of the axes, the frequency of branching and the abundance of proliferations, rbcL sequence analyses indicate their conspecificity. M. papulosa displays four distinctive reproductive features (presence of an auxiliary cell complex, occurrence of cystocarps on marginal proliferations and the blade surface (although very rare) in addition to the margins of axes, frequent production of spinose outgrowths on the pericarp and tetrasporangial initials typically basally attached to their parental cells) that have not been reported for M. papulosa from other areas. Although these features might warrant recognition of the Japanese entity as a separate species, a better understanding of their possible taxonomic value requires comparisons with M. papulosa from other geographic regions, including the type locality.     

On the life history ofTurnerella mertensiana (Rhodophyta,Gigartinales)

Turnerella (Gigartinales) withT. mertensiana known from northern Japan was studied to determine its life history. Carpospores cultured from foliose female plants gave rise on germination to crustose plants containing tetrasporangia, as noted previously inT. pennyi from the Atlantic. The crusts were slow to develop and required 3–5 years to achieve reproductive maturity. Tetraspores liberated in culturedT. mertensiana gave rise on germination eventually to thalli similar to gametophyticT. mertensiana. Thus this species may be said to adhere to the pattern of life history in which a large, foliose gametophyte alternates with a small crustose tetrasporophyte. The erect filaments of the small sporophytes branch laterally and tetrasporangial mother cells are found there, exemplifying an unusual method for crustose species to produce tetrasporangia.     

A nearly idealized 6'-O-methylated iota-carrageenan from the Australian red alga Claviclonium ovatum (Acrotylaceae, Gigartinales)

The polysaccharides extracted from Claviclonium ovatum were studied by a combination of compositional assays, reductive partial hydrolysis, linkage analysis, Fourier Transform infrared (FTIR) spectroscopy, and 13C, 1H, and 13C/1H heteronuclear multiple quantum correlation (HMQC) two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The chemical and spectroscopic data showed that the alkali-modified C. ovatum polysaccharides are composed of a nearly idealized repeating unit of 6'-O-methylcarrabiose 2,4'-disulfate (the repeating unit of 6'-O-methylated iota-carrageenan), although some minor components were also present. The C. ovatum galactans are the most highly methylated carrageenans reported.     

Protoplast isolation and regeneration in the marine red alga Porphyra nereocystis

We have developed a method for isolating viable protoplasts from the blade phase of the epiphytic marine red alga Porphyra nereocystis Anderson, using a two-step enzymatic digestion with commercially available enzymes. The first step uses papain, the second step uses abalone acetone powder. The method is rapid and gives a high yield of viable protoplasts. In liquid culture in enriched seawater medium, the protoplasts can undergo regeneration along three pathways: they directly form filaments resembling the conchocelis phase of Porphyra; they form calli with relatively thick-walled, pigmented cells; and they indirectly form blades from the edges of these calli. Porphyra nereocystis protoplasts also may serve as an alternative propagation method in aquaculture and be useful for studies of cell-wall formation, cell division, and thallus differentiation. They may also be used in somatic selection, somatic hybridization and gene-transfection experiments.Abbreviations AAP abalone acetone powder - PAP papain - FDA fluorescein diacetate This paper is dedicated to the memory of the late Dr. Munenao Kurogi (1921–1988), Professor Emeritus of Hokkaido UniversityThis research was supported by the Washington Sea Grant Program (National Oceanic and Atmospheric Administration). We thank Professor Y. Fujita (Nagasaki University, Japan), Professor S.-J. Wang (Shanghai University of Fisheries, P.R. China) and Dr. H. Kito (Seikai Regional Fisheries Research Laboratory, Nagasaki, Japan) for sharing their experience with Porphyra protoplast production with us. We thank J.S. Charleston for expert technical assistance in preparation of the electron-microscopy specimens. We also thank Dr. S.K. Herbert and John Carrier (Friday Harbor Laboratories) and Dr. John Merrill and D. Gillingham (American Sea Vegetable Co. and Applied Algal Research, Seattle) for collections of P. nereocystis.     

Genetic diversity and population structure of spottedtail goby (Synechogobius ommaturus) based on AFLP analysis

Larval dispersal may have an important impact on genetic structure of benthic fishes. To examine population genetic structure of spottedtail goby Synechogobius ommaturus, samples from five different locations of China and Kunsan population in Korea were analyzed by using amplified fragment length polymorphism (AFLP) technology. A total of 253 bands were identified from 91 individuals by 5 primer combinations and the percentage of polymorphic bands was 43.87%. The average gene diversity was 0.0794 ± 0.1470 and Shannon’s information index was 0.1279 ± 0.2138. The pairwise F_st values ranged from 0.022 to 0.201. The results of AMOVA analysis indicated that 90.54% of the genetic variation contained within populations and 9.46% occurred among populations. The gene flow estimates (Nm) demonstrated that different gene flow existed among populations from 0.994 to 11.114. No significant genealogical branches or clusters were recognized on the UPGMA tree. The results support the hypothesis that larval dispersal ability can be responsible for the genetic diversity and population structuring.