Comparison of three techniques for identifying isomorphic phases of <Emphasis Type="BoldItalic">Chondrus crispus</Emphasis> (Gigartinaceae)

Chondrus crispus, a member of the economically important family Gigartinaceae, alternates between two free-living isomorphic life history phases. The carrageenan composition of the gametophyte and tetrasporophyte phases has been used to identify non-reproductive material, and thus provide a better understanding of the ecology of the species. The aim of this study was to compare three methods for identifying the isomorphic phases. The results obtained by the widely used resorcinol test and an iridescence method are compared with those obtained by a technique that involves the acquisition of mid-infrared absorbance spectra from dried samples by Fourier transform infrared spectroscopy (FT-IR). Both the resorcinol test and FT-IR produced similar results with a calculated gametophyte to tetrasporophyte ratio of 2.1 and 2.3:1, respectively. In contrast the iridescence method was less reliable, with a calculated gametophyte to tetrasporophyte ratio of 1.3:1 and a low predictive value (0.70) for selecting tetrasporophytes. The FT-IR technique provides a rapid assessment, does not involve the use of hazardous chemicals, has the potential to be applied to members of the Gigartinaceae for which the resorcinol and iridescence tests are less applicable and it has greater flexibility in that it can identify additional carrageenan fractions.     

Open water Chondrus crispus Stackhouse cultivation

Selected strains of Chondrus crispus were acclimated to relativelywarm Pacific waters off the Baja California peninsula, Mexico, and werecultured in semi-exposed oceanic conditions. Specific techniques weredeveloped for culturing this species in semi-exposed oceanic environment,in an effort to establish the basis for its commercial farming. This is the firsttime that Chondrus crispus has been cultured in the ocean and outsideits natural Atlantic environment. The information generated in this studyis being used to feed an economic model to test the feasibility of thecommercial culturing of Chondrus crispus.     

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.     

Annual net primary production calculated from eastern Canadian Irish moss fishery data

Western Prince Edward Island Irish moss (Chondrus crispus) has been intensively dragraked since 1966. As well, most unattached fronds removed by wave surge, ice, etc. are brought to shore by waves and currents, where they are harvested eagerly. Accurate annual fishing yields were recorded between 1966 and 1981 inclusive. Given that herbivore densities are reduced, likely due to the intensive raking, and that mean annual bycatch (non-Irish moss seaweeds) (23.4 %) and commercial bed sizes (873 ha) were known, the fishing yields thus were considered a unique database from which to calculate net primary production (NPP). Factors used to convert from wet to dry wt (DW), and from dry wt to carbon were 0.22 an 0.31, respectively. Calculated mean annual NPP values were as follows: 2.101 ± 0.654 t (DW) ha^–1 y^–1; 210.1 g DW m^–2 yr^–1 and 63.0 g C m^–2 yr^–1. These values are much lower than those calculated for northwest Atlantic kelp and rockweed but similar to that determined for northeast Atlantic Gracilaria verrucosa. The interannual variability pattern for NPP was similar for both the wild Irish moss harvest and that of experimental Chondrus crispus outplants placed in one of the 14 commercial beds.     

CUTICLES FROM CHONDRUS CRISPUS (RHODOPHYTA)1

A simple, nondestructive physical process was developed for routinely isolating the outermost layers from female, male, and sporophyte fronds of Chondrus crispus Stack-house. Yields of pure cuticles from apical segments ranged from 0.74 to 2.35% on a dry weight basis after 5–7 d of culture. These undegraded cuticles were examined by electron microscopy (scanning and transmission electron microscopy), spectroscopy (infrared and X-ray), and chemical means. Cuticles isolated from female or male fronds were characterized by parallel arrays of electron-dense lamellae (typically 6–14) alternating with more electron-transparent regions. The thickness and uniformity of these lamellae provide the physical basis for the iridescence characteristic of C. crispus fronds. Sporophyte fronds are not iridescent. This phenomenon may be explained by the fewer electron-dense cuticular lamellae (usually three to seven) and the fact that these lamellae anastomose freely to form a thin cuticle with a highly irregular substructure. Elements detected by X-ray analysis, in addition to carbon and oxygen, included Mg, Br, S, and Ca in both gametophyte and sporophyte cuticles. Major features of FTIR spectra of all cuticles were absorbances due to proteins. A strong band, indicative of sulfate ester, occurred near 1250 cm^?1 in all cuticle preparations. Gametophyte, but not sporophyte, cuticles absorbed at 935, 846, and 800 cm^?1 consistent with the presence of kappa and/or iota carrageenan. Amino acid analyses showed that sporophyte and gametophyte cuticles were generally similar in gross composition. All contained proline as the principal residue together with significant amounts of cysteine, methionine, and lysine. Protein contents calculated from these analyses ranged from 37.6 to 44.4% on a dry weight basis as compared to 51.5–56.7% calculated from total nitrogen values. Up to 75% of the cuticle mass was solubilized by sodium dodecyl sulfate-β-mercaptoethanol. Three similar migrating bands were seen in female and male cuticle extracts on sodium dodecyl sulfate–polyacrylamide gel electrophoresis; however, none of the three weaker bands from sporophyte cuticles comigrated with those from gametophytes. Chloroform-methanol extraction removed < 3.3% of the cuticle mass, suggesting that lipids were minor components.     

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.     

ENDOPHYTIC ALGAE OF CHONDRUS CRISPUS (RHODOPHYTA). III. HOST SPECIFICITY1

Susceptibility of rhodophycean macroalgae to infection by the green endophytes, Acrochaete operculata Correa & Nielsen and A. heteroclada Correct & Nielsen was studied. Cross-infection experiments showed that A. operculata is host specific and developed only in sporophytic fronds of Chondrus crispus Stackh. and Iridaea cordata (Turn.) Bory. Although A. operculata penetrated equally the multilamellar outer cell wall of sporophytic and gametophytic fronds of C. crispus, subsequent development was arrested in the gametophytic fronds. Susceptibility of the sporophytic phase of C. crispus was detected early in the development of the host, at a discoid stage that is structurally distinct from the adult fronds. The evidence strongly suggests that host specificity in A. operculata is determined by cell-wall composition of the hosts, likely the carrageenan fraction. In contrast, A. heteroclada was not host specific, infecting all offered hosts, including carrageenophytes and agarophytes. Germination occurred on the surface of the hosts and led to the development of an epiphytic stage. Subsequent penetration in many cases involved total displacement of cortical tissue in the infected frond.     

SPORELING COALESCENCE IN CHONDRUS CRISPUS (RHODOPHYCEAE)1

Coalescence of developing sporelings of Chondrus crispus Stackhouse was observed. Juvenile tetra-sporophytes showed a higher proportion of coalescence than developing gametophytes. Stages of complete coalescence between different sporelings are illustrated. Coalesced sporelings exhibit vertical and horizontal alignment of cells, as well as “cuticular” continuity and secondary pit connections between adjacent, coalesced sporelings. Ultimately the cells in the center of the coalesced sporelings produce upright, multiaxial fronds that grow more rapidly than fronds of non-coalesced sporelings. Other red algae, such as Gracilaria verrucosa (Hudson) Papenfuss and Gigartina stellata (Stackhouse) Batters also show a similar sequence of sporeling coalescence and enhanced growth. The ecological significance of sporeling coalescence is discussed.     

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.     

Phylogeography of the Northern Atlantic species Chondrus crispus (Gigartinales, Rhodophyta) inferred from nuclear rDNA internal transcribed spacer sequences

Eighteen isolates of the red algae Chondrus crispus were collected from Northern Atlantic sites, together with C. ocellatus, C. yendoi and C. pinnulatus from the North Pacific. The nuclear rDNA internal transcribed spacer (ITS) was sequenced and compared, spanning both the ITS regions and the 5.8S rRNA gene. Percentage of nucleotide variation for C. crispus ranged from 0.3% to 4.0%. Phylogenetic analyses were performed using maximum parsimony (MP), neighbor-joining (NJ) and minimum evolution methods. They showed that two main clades existed within the C. crispus samples examined and that suggested C. crispus had a single Atlantic origin. The clustering however did not follow the geographic origin. We hypothesized that the current distribution of C. crispus populations might be a result of three main factors: temperature boundaries, paleoclimate and paleoceanography. ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies. Handling editor: C. Sturmbauer     

WATER MOTION AND MORPHOLOGY IN CHONDRUS CRISPUS (RHODOPHYTA)1

Morphological variability of intertidal Chondrus crispus Stackh. fronds along a small open rocky coast was related to wave exposure and emersion. Cluster analysis revealed two well-defined morphologies: filiform and planiform, named the N morphotype and B morphotype, respectively. We propose a rapid method of classifying fronds based on the morphology of the cross section at half the height on the thallus. The N morphotype is characterized by fewer dichotomies per unit length, a circular cross section with a large inner cortex, and narrow fronds. It is abundant at low intertidal and exposed sites. The B morphotype is characterized by more dichotomies, smaller sizes, a subelliptical or flattened cross section, and broad fronds. It is abundant at high intertidal sites in sheltered areas. Regression analysis revealed a major effect of water movements on frond morphology with respect to tidal level, which was more evident at high intertidal levels. No relationships were observed between morphology and life history phases.     

Seasonal acclimatization of thallus proline contents of Mastocarpus stellatus and Chondrus crispus: intertidal rhodophytes that differ in freezing tolerance

Mastocarpus stellatus and Chondrus crispus often co‐occur in the lower intertidal of Northern Atlantic rocky shorelines. At our field site along the Maine coast (USA), Mastocarpus stellatus thalli possessed greater contents of proline when compared with thalli of Chondrus crispus. In addition, M. stellatus thalli acclimated to colder growth conditions in winter/early spring by increasing proline content several fold; no seasonal acclimation in proline content was observed in C. crispus. Proline accumulates in the tissues of a broad diversity of freezing‐tolerant organisms and is among the most common cryoprotectant molecules. Thus, our observations provide a basis for the previously well‐documented greater freezing tolerance of Mastocarpus stellatus when compared with Chondrus crispus.     

GENETIC POPULATION STRUCTURE AND MATING SYSTEM IN CHONDRUS CRISPUS (RHODOPHYTA)1

Chondrus crispus Stackh. has been intensely studied, yet no study to date has elucidated its population structure or mating system despite many populations in which there was a haploid bias and lack of male gametophytes. Therefore, 12 nuclear microsatellite loci were identified in this red alga. Microsatellite markers were developed and tested against a panel of specimens collected from two shore levels at two sites in Brittany, France: Pointe de Primel and Pointe de la Jument, Concarneau. Single locus genetic determinism was verified at eight polymorphic loci, as only one band was observed for haploid genotypes, whereas one or two bands were observed for diploids. These markers enabled the detection of unique genotypes within sampled populations, indicating that very few fronds shared the same multilocus genotype. This finding suggests that asexual reproduction was not the prevailing mode of reproduction. In addition, we explored the hierarchical population structure showing that gene flow is restricted at small spatial scales (<50 m) between upper and lower Chondrus‐range populations within a shore. Sexual reproduction predominated in the populations of C. crispus studied, but probably due to fine‐scale spatial substructuring, inbreeding was also significant. In conclusion, this study reveals that fine‐scale genetic variation is of major importance in C. crispus, suggesting that differences between microhabitats should be essential in understanding evolutionary processes in this species.     

Isolation of humic acid from the brown algaeAscophyllum nodosum,Fucus vesiculosus,Laminaria saccharina and the marine angiospermZostera marina

Recent discovery of humic acid (HA) in the free-living, brown algaPilayella littoralis has prompted a search for HA in other live plants. Marine algaeCodium fragile andMonostoma oxyspermum (greens),Chondrus crispus,Palmaria palmata andPolysiphonia lanosa (reds),Ascophyllum nodosum, Fucus vesiculosus andLaminaria saccharina (browns) andZostera marina (marine angiosperm) were investigated for their HA content. Only the brown algae and the marine angiosperm contained HA, which was extracted by a standard procedure augmented with necessary removal of alginic acid (where applicable). The isolated products were identified as HA by comparison of their analytical data, uv-visible, FTIR,¹H NMR spectra and morphologies with those of authentic HA isolated from municipal compost.Authors for correspondence     

The demography of fronds of Chondrus crispus Stackhouse

A demographic analysis was done of an intertidal stand of Chondrus crispus Stackhouse at Pubnico Point, Nova Scotia. The distribution of gametophytic and tetrasporophytic fronds was assessed. Fronds were placed in one of four size-classes. Size-specific measurements for fronds are presented. Gametophytic fronds dominated the stand. Fronds in Size-Class 2 exhibited high survivorship. Survivorship fell sharply when fronds entered Size-Class 3 and Size-Class 4. Fronds became fertile and highly branched when in the third and fourth size-classes. It is suggested that these two factors contribute to the higher mortality rates of such individuals.Monthly spore release rates·m^?2 for gametophytes and tetrasporophytes were measured. The annual release rate of carpospores exceeded the annual release rate of tetraspores by a ratio of 4.7:1. Rates did not fall below 10⁶ spores released·m^?2·month^?1 for either phase.     

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.     

Recent changes in the seaweed community of Western Prince Edward Island: implications for the seaweed industry

Prior to 1980, seaweed beds of western Prince Edward Island, Canada, were dominated by Chondrus crispus Stackhouse, associated with 37 other species of common algae. Furcellaria lumbricalis (Huds.) Lamour. occurred in 4.6% of the samples from the Pleasant View bed but never reached measurable levels of cover. By 1991 F. lumbricalis was reported in 67% of the samples from this bed. Furcellaria lumbricalis biomass was negatively correlated with C. crispus biomass and positively correlated with Phyllophora Grev. spp. biomass. F. lumbricalis biomass and cover peaked in subtidal areas below 4 m at 359 ± 299 SD fresh g 0.25 m^–2. Harvesters avoid concentrations of F. lumbricalis, resulting in a 5% content of this species in the commercial harvest. Systematic harvesting located concentrations of F. lumbricalis in seven of 10 commercial beds. Proposed causes for change in species dominance from C. crispus to F. lumbricalis range from general environmental change, natural succession to excessive human perturbation.     

An assessment of the AFLP method for investigating population structure in the red alga Chondrus crispus Stackhouse (Gigartinales, Florideophyceae)

The appropriateness of the Amplified Fragment Length Polymorphism (AFLP) technique for investigating Chondrus crispus Stackhouse populations in the Maritime Provinces of Canada was assessed. The AFLP procedure was first subjected to reproducibility testing and three shortcomings were noted: 1) failure to reproduce band intensity between replicate runs for the same individual and primer pair; 2) failure of some bands to replicate; 3) lack of reproducibility for complete replicate runs for some individuals and primer pairs. In the last-mentioned case, the lack of reproducibility resulted in characteristic electropherograms indicative of weak reactions. These weak runs can be attributed to poor restriction digest/ligation reactions and/or substandard PCR, these failures ultimately resulting from low and inconsistent DNA quality. We recommend that reproducibility testing should be completed routinely in studies using the AFLP technique. In the current work, only fragments and individuals that gave reproducible results were used in subsequent analyses. The AFLP method resulted in highly variable markers within and between the populations of C. crispus included in this investigation, which prevented successful resolution of population structure. This situation could result from a lack of suitability for AFLP markers in population genetic studies, and/or too extensive genetic variation for C. crispus populations to be discerned by the AFLP technique. These two possible explanations are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.