PHYSIOLOGICAL INVESTIGATIONS ON ALARIA ESCULENTA (LAMINARIALES,PHAEOPHYCEAE). III. EXUDATION BY THE BLADE1,2,3

Comparison of exudation rate of medullary conducting cells in the midrib of Alaria esculenta (L.) Grev. showed lowest rates for the blade meristem and highest rates in the non-growing region, 300–500 mm from the meristem. Holding plants under continuous darkness or severing the wings from the midrib reduced exudation rate by 26 and 37%, respectively. Osmotic pressure of exudate in sink (meristem) and source (non-growing region, up to 500 mm from the meristem) were similar (34.6–36.1 · 10²kpa). Pressure flow mechanism of translocation is evaluated in Alaria.     

SOURCE-SINK RELATIONSHIPS IN THE BLADE OF ALARIA ESCULENTA (LAMINARIALES,PHAEOPHYCEAE)1

The translocation of ¹⁴C-labelled photoassimilate was studied in blades of the kelp, Alaria esculenta (L.) Grev., which had been surgically modified to produce source and sink regions of various sizes. Thirty cm above the blade base a 2.5 cm dia. section of the blade received a 1 h pulse of H¹⁴CO₃^?. Efflux of ¹⁴C-labelled photoassimilate from this “source disc” was monitored over the next 8 days using a Geiger-Müller detector probe. Accumulation of ¹⁴C-labelled solutes by a “sink disc” of similar diameter, 20 cm below the source disc, was also monitored. Rate of ^l4C efflux from the source disc was governed by two factors: (1) total sink size and (2) feed-back from competing sources. In the latter case, source export was depressed if the portion of the blade, just distal to the source disc, was present. While the initial ¹⁴C influx rate into the sink disc was greater when competing sinks were present, the total accumulation of ¹⁴C-photoassimilates was 2–3 times higher in the sink disc when competing sources were not present. Basipetal translocation velocity (1.3–1.7 cm h^?1) was unaffected by competing sources and sinks.     

PHYSIOLOGICAL INVESTIGATIONS ON ALARM ESCULENTA (LAMINARIALES. PHAEOPHYCEAE). IV. INORGANIC AND ORGANIC NITROGEN IN THE BLADE1,2

Seasonal variations in nitrate and organic nitrogen content along the wing and midrib of Alaria esculenta (L.) Grev. lamina have been compared with the NO₃^- cycle in the sea and yearly growth pattern of the blade. Throughout the year, organic N is highest in blade meristem, while NO₃^- distribution is less consistent. NO₃^-in blades reaches a peak in March (ca. 25–28 μM), whereas maximum relative accumulation, 3,300X ambient seawater level, occurs in October. Content of NO₃^- and organic N in the blade decreases in concert with the decline of seawater NO₃^- in April. The three periods of rapid blade growth are not correlated with a specific organic N content in the blade meristem. Laboratory experiments suggest that low NO₃^- and elevated seawater temperature are not the major factors retarding Alaria blade growth during summer and early fall in nature.     

THE RATE OF TRANSLOCATION IN ALARIA ESCULENTA (LAMINARIALES,PHAEOPHYCEAE)12

The rate of translocation of organic carbon in blades of Alaria esculenta (L.) Grev. (Laminariales) was calculated from blade growth data. The cross sectional area of sieve filaments in the midrib medulla was estimated from light microscopic examination of fresh material. Files of these filaments form a perimedullar ring occupying ca. 2/3 of the medulla. Values computed for specific mass transfer of carbon into the blade meristem ranged from 36.2 to 60.8 mg C.wk^?1.0.1 mm^?2 sieve filaments.     

PHYSIOLOGICAL INVESTIGATIONS ON ALARIA ESCULENTA (L.) GREV. (LAMINARIALES). I. ELONGATION OF THE BLADE1,2,3

A seasonal rhythm in blade elongation rate was observed in Alaria esculenta. Maximum (3.0–3.6 cm/week) and minimum (0.5–1.0 cm/week) rates were recorded in June and September, respectively. Translocation of growth-promoting metabolites occurs within the main blade and between the sporophylls and blade. Bidirectional flow of growth-promoting metabolities within the stipe was demonstrated. Seasonal effects of nutrients (NO₃- and PO₄^3-) and photoperiod on blade elongation were shown in long-term laboratory experiments. Control of seasonal blade growth by exogenous and endogenous factors is discussed.     

ULTRASTRUCTURE OF SWARMERS IN THE LAMINARIALES (PHAEOPHYCEAE). II. SPERM1

The ultrastructure of sperm from 13 species in 11 genera of Laminariales collected in the northeast Pacific Ocean is unique in the brown algae. The sperm are elongate, and possess a nucleus, several mitochondria and two or three chloroplasts, but no eyespot. The anterior flagellum bears mastigonemes on the proximal half of its length; a distal “whiplash” portion lacks mastigonemes and is an extension of only the two central singlet microtubules of the axoneme. A peculiar feature of these sperm is the posterior flagellum, which is longer than the anterior flagellum and tapers distally as the doublet microtubules become singlets and decrease in number. This feature contrasts with the laminarialean zoospore, which possesses a short posterior flagellum with the usual “9 + 2” axoneme. The structure of these sperm differs from that reported for Chorda, the sperm of which resembles a primitive brown algal zoospore. The facts support the concept that Chorda is the most primitive member of the Laminariales.     

TRANSLOCATION OF 11C-PHOTOASSIMILATE IN THE BLADE OF MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Radioactive bicarbonate was pulse fed to blades of Macrocystis pyrifera (L.) C. A. Ag. and the movement of the ¹¹C-labelled photoassimilates was monitored in vivo using an externally mounted array of Geiger-Müller detectors. Results of experiments conducted in August 1982 and February 1983 showed kinetic transport profiles composed of short pulses of ¹¹C (periods of two to three minutes and six to eight minutes) and a mass flow component travelling with a speed of 6–22 cm · h^?1. The pulse-like movement of ¹¹C-photoassimilates, revealed for the first time in a kelp, may be driven by an energy-assisted transport mechanism. Light microscopy revealed a putative symplastic transport pathway from the photo synthetic meristoderm to the medullary sieve cells in the M. pyrifera blade. Of particular importance were the connections between the inner cortical cells and thin-walled medullary sieve cells. Electron microscopy showed sieve plate pore diameters ranging between 35–60 nm in the cortex and ca. 40 nm in the end walls of the thin-walled sieve cells.     

ULTRASTRUCTURE OF SWARMERS IN THE LAMINARIALES (PHAEOPHYCEAE). I. ZOOSPORES1

Zoospores of 17 species in 14 genera of Laminariales, collected in the northeast Pacific Ocean, were studied by electron microscopy. These zoospores are unique in the brown algae in lacking both an eyespot in the single chloroplast and any associated swelling at the base of the shorter, posterior flagellum. Spores of all species examined possess a distal whiplash portion on the longer, mastigoneme-bearing anterior flagellum. This appendage may sometimes be as long as the mastigoneme-bearing portion of the flagellum, but it is only seldom preserved in the preparations for electron microscopy. A microtubular cytoskeleton is probably responsible for maintaining the shape of the spore. It consists of a short band of about 10 microtubules between the two basal bodies, scattered tubules converging at the anterior of the spore, a band of 7–9 tubules directed anteriorly from the anterior basal body, and a band directed posteriorly from the posterior basal body. These anterior and posterior bands may form one continuous band looping around the periphery of the spore. Variation with possible taxonomic significance was found in the ultrastructure of vesicles which apparently contain adhesive material, and which are extruded through the plasmalemma when the zoospores settle.     

TRANSLOCATION OF 14C IN MACROCYSTIS INTEGRIFOLIA (PHAEOPHYCEAE)1,2

Translocation patterns in the giant kelp, Macrocystis integrifolia Bory, were investigated in situ using ¹⁴C tracer; sources and sinks were identified. Export was first detected after 4 h of labeling; experiments were routinely 24 h continuous ¹⁴C application. Mature blades exported ¹⁴C to young blades on the same frond and on younger fronds, as well as to sporophylls and frond initials at the bases of the fronds. Blades <0.3 m from the apex imported and did not export; this distance did not change seasonally. In spring export from blades 0.3–1.25 m from the apex was exclusively upwards; older blades also exported downwards. In fall downward export began 0.5 m from the apex, and blades >2 m from the apex exported exclusively downwards. Carbon imported by frond initials, young fronds, and sporophylls in fall may partly be stored for growth in early spring. No translocation was seen in very young plants until one blade (secondary frond initial) bad been freed from the apical blade; this blade exported to the apical blade for a time, but imported when it began to develop into a frond. The second and third formed blades on the primary fronds (sporophylls also exported when <0.3 m from the apex, and later stopped. Frond initials and sporophylls on later-formed fronds did not export at all. The translocation pattern in M. integrifolia differs from that previously reported in M. pyrifera in seasonal change and in distances from the apex at which the changes take place.     

REPRODUCTIVE PLASTICITY IN THE KELP ALARIA NANA (PHAEOPHYCEAE)1

Unlike terrestrial plants, reproductive control in algae has not been attributed to biotic factors or internal chemical signals. However, experimental removal of reproductive structures (sporophylls) from the kelp Alaria nana Shrader resulted in the development of functional reproducing structures on the vegetative frond in 45% (10/22) of the experimental plants but not on the control plants where sporophylls were left intact (n = 35). In naturally occurring Alaria nana, this compensatory reproductive response occurred at a low incidence and was restricted primarily to larger individuals in the population. Thus, compensation and developmental plasticity, mediated In plant size, may play an important role in how algae respond to reproductive tissue loss.     

THE RELEASE,SETTLEMENT AND GERMINATION OF ZOOSPORES IN CHORDA TOMENTOSA (PHAEOPHYCEAE,LAMINARIALES)1,2

Details of zoospore germination in Chorda tomentosa Lyngb. are outlined. Uninucleate zoospores, when released are embedded in a mucilaginous mass of carbohydrate which dissolves and the biflagellate zoospores become motile. The long anterior flagellum is composed of a highly coiled terminal region and a rigid lower section bearing mastigonemes. The rigid, short posterior flagellum lacks mastigonemes. After initial contact by the tightly coiled region of the anterior flagellum, the zoospore draws itself to the substrate by flagellar resorbtion. After deposition of 3 wall layers the germling produces a germ tube. During this time the disc-shaped chloroplast enlarges undergoing changes in shape. As the germ tubes reach ca. 15 μm they cease forward growth and swell at their tips. The majority of cytoplasm of the original zoospore moves into the tube. Just before the nucleus enters the tube, centriole replication occurs. Mitosis is presumed to take place somewhere in the germ tube so that at 24 h, 2-celled gametophytes are produced.     

LIFE HISTORY AND SYSTEMATIC POSITION OF PSEUDOCHORDA GRACILIS SP. NOV. (LAMINARIALES,PHAEOPHYCEAE)1

Pseudochorda gracilis sp. Nov. (Pseudochordacease, Laminariales) is described from the Japan Sea coast of Hokkaido/ the species is subtidal, epilithic and annual, appearing in spring and maturing in winter. Erect thalli grow solitary or in tufts on a small discoid holdfast. They are simple, cord-shaped and hollow, with inner hyphal filaments, cylindrical medullary cells and paraphyses consisting of 3–6 cells. Hair tufts are observed only in young thalli. Unilocular sporangia are sessile and narrowly ovate. In culture, P. gracilis shows a heteromorphic life history with oogamy, characteristic of the order Laminariales. Gametophytes are dioecious and dimorphic. Gametophytes mature under lower temperature conditions (usually below 10°C), and sporophytes mature under low temperature and short-day conditions (5°C, SD). The seasonal growth pattern of the species results from the photoperiod-temperature conditions controlling saprophyte maturation.     

BLUE LIGHT PHOTOREACTIVATION IN ULTRAVIOLET-IRRADIATED YOUNG SPOROPHYTES OF ALARIA ESCULENTA AND LAMINARIA SACCHARINA (PHAEOPHYTA)1

Exposure of ultraviolet (UV)-irradiated young sporophytes of Alaria esculenta (L.) Grev. and Laminaria saccharina (L.) Lamour. to visible light resulted in recovery from UV damage that would otherwise cause much higher mortality. For this photoreactivation, blue light was highly effective, whereas negligible reactivation was produced in green or red light. The blue quantum requirements for a 50% response were 1.9 mol·m^?2 in A. esculenta and 1.2 mol·m^?2 in L. saccharina, which were comparable to those reported for other blue light responses requiring high energy found in brown algae.     

CRYOPRESERVATION OF THE GAMETOPHYTIC CELLS OF LAMINARIALES (PHAEOPHYTA) IN LIQUID NITROGEN1

The gametophytic cells of six species of Laminariales, Laminaria japonica Areschoug, L. longissima Miyabe, Kjellmaniella crassifolia Miyabe, Ecklonia stolonifera Okamura, E. kurome Okamura, and Undaria pinnatifida (Harvey) Suringar, were subjected to cryopreservation in liquid nitrogen. The cells were suspended in various cryoprotective solutions and slowly cooled to –40°C over a period of 4 h. After this slow cooling step, the suspensions were immediately immersed in liquid nitrogen. All the species of Laminariaceae used in the present study survived maximally in a mixture of ethylene glycol and proline. On the other hand, the gametophytic cells of Undaria pinnatifida, a member of the Alariaceae, survived maximally in the mixture of glycerol and proline. The viability of the thawed gametophytic cells decreased during postthawing incubation. The decrease in viability continued for 4–6 days, and the minimum levels ranged from 36.2% to 67.2%. After 4–6 days of incubation, the percentage viability of all strains began to increase due to the renewal of cell division.     

THE RECLASSIFICATION OF LESSONIA LAMINARIOIDES (LAMINARIALES,PHAEOPHYCEAE): PSEUDOLESSONIA GEN. NOV.1

A new genus, Pseudolessonia, is proposed for the kelp Lessonia laminarioides Postels et Ruprecht (Laminariales, Phaeophyceae), which occurs on the northwest side of the Sea of Okhotsk, in the northwest Pacific Ocean. Pseudolessonia is monotypic and differs from Lessonia in its short primary stipes and its corrugated, unilaterally arranged blades with entire margins. This species is transferred on the basis of morphology and plastid gene sequence comparisons. We determined psaA and rbcL gene sequences from 17 taxa of Pseudolessonia, Lessonia, and putative relatives. Analyses of individual and combined data sets resulted in congruent trees showing a clear separation of Pseudolessonia laminarioides from Lessonia, but suggesting its sister relationships with the clade of Nereocystis, Macrocystis, Pelagophycus, and Postelsia in the North Pacific Ocean. On the other hand, Lessonia species from the South Pacific Ocean formed a strongly supported clade. The results indicate that the basal splitting of the blade, which has been considered a diagnostic character for the family Lessoniaceae, is a result of convergent evolution.     

AN EVALUATION OF METHODS USED TO ASSESS INTERGENERIC HYBRIDIZATION IN KELP USING PACIFIC LAMINARIALES (PHAEOPHYCEAE)1

Kelp intergeneric laminarialean hybridizations and hybridization protocol were assessed using seven northeast Pacific kelp species: Alaria marginata Postels and Ruprecht, Costaria costata (C. A. Agardh) Saunders, Eisenia arborea Areschoug, Laminaria saccharina (L) Lamouroux, Lessoniopsis littoralis (Tilden) Reinke, Macrocystis integrifolia Bory, and Nereocystis leutkeana (Mertens) Postels and Ruprecht. Survival and development of sporophyte morphologies derived from selfings, separate males and females, and reciprocal crosses were evaluated over 30 weeks of cultivation. All cultures were initiated from cloned gametophytes. Two closely related species, Laminaria angustata Kjellman and L. japonica Areschoug, demonstrated the efficacy of long‐term (up to 30 years) cloned gametophytes in hybridization studies. Sporophyte morphologies appeared in 34%–69% of control and hybridization trials, and 6%–16% of all trials produced sporophytes in control and hybridization conditions that persisted through 30 weeks of cultivation. Sporophytes in control and hybridization conditions could appear normal or abnormal. Usually, the morphology of sporophytes in hybridizations and female controls resembled the female parent, whereas the sporophytes in male controls often had an abbreviated morphology, lacking definitive generic features. Species‐specific rDNA internal transcribed spacer molecular primers were used to determine the parentage of five putative hybrids. Only the L. japonica♀/L. angustata♂ hybrid bore both parental genomes. That negative controls could produce persistent and normal‐appearing sporophytes negates their value and emphasizes the importance of molecular confirmation in hybridization studies. These findings were applied to critique the only known wild intergeneric hybrid, Pelagophycus/Macrocystis.     

COORDINATNE NUCLEAR AND CHLOROPLAST DIVISION IN UNILOCULAR SPORANGIA OF LAMINARIA ANGUSTATA (LAMINARIALES,PHAEOPHYCEAE)1

Changes in the number of nuclei and chloroplasts were examined during the process of unispore formation in unilocular sporangia of Laminaria angustata. Just before meiosis, eight chloroplasts were always present in unilocular sporangial mother cells. The number of chloroplasts remained constant through meiosis. After the resulting four nuclei divided again (third nuclear division), a close association between a nucleus and a chloroplast developed among each of the eight nuclei and eight chloroplasts. The eight chloroplasts divided ahost synchronously before the synchronous division of the eight nuclei. Following the 16 nucleate stage with 16 chloroplasts and the final 32 nucleate stage with 32 chloroplasts, 32 unispores, each with a nucleus and a chloroplast, were fomd in unilocular sporangza of L. angustata. Immunofluorescence microscopy using an anti-centrin antibody showed that two anticentrin-stained structures (as future mitotic poles) occurred adjacent to each of the premitotic four nuclei, and each spot was located near a chloroplast. Therefore, after the third division, each of the eight nuclei established close contact with a chloroplast presumably mediated by the centrosomes.     

ROLE OF SETTLEMENT DENSITY ON GAMETOPHYTE GROWTH AND REPRODUCTION IN THE KELPS PTERYGOPHORA CALIFORNICA AND MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Laboratory studies were used to examine how variation in the density of spore settlement influences gametophyte growth, reproduction, and subsequent sporophyte production in the kelps Pterygophora californica Ruprecht and Macrocystis pyrifera (L.) C. Ag. In still (non-aerated) cultures, egg maturation in both species was delayed when spores were seeded at densities 300 · mm^?2. Although the density at which this inhibition was first observed was similar for both species, the age at which their eggs matured was not. P. californica females reached sexual maturity an average of 4 days (or ～ 30%) sooner than did M, pyrifera. As observed previously in field experiments, per capita sporophyte production was negatively density dependent for both species when seeded at spore densities of 10 · mm^?2. Total sporophyte production (i.e. number · cm^?2) for both species, however, was greatest at intermediate densities of spore settlement (～ 50 spores · mm^?2). In contrast, total sporophyte production by P. californica steadily increased with increasing spore density in aerated cultures; highest sporophyte density was observed on slides seeded at a density of 1000 spores · mm^?2. Preliminary experiments with P. californica involving manipulation of aeration and nutrients indicate that inhibition of gametophyte growth and reproduction at higher densities of spore settlement in non-aerated cultures was probably caused by nutrient limitation.     

A MULTI‐GENE MOLECULAR INVESTIGATION OF THE KELP (LAMINARIALES,PHAEOPHYCEAE) SUPPORTS SUBSTANTIAL TAXONOMIC RE‐ORGANIZATION1

Every year numerous ecological, biochemical, and physiological studies are performed using members of the order Laminariales. Despite the fact that kelp are some of the most intensely studied macroalgae in the world, there is significant debate over the classification within and among the three “derived” families, the Alariaceae, Laminariaceae, and Lessoniaceae (ALL). Molecular phylogenies published for the ALL families have generated hypotheses strongly at odds with the current morphological taxonomy; however, conflicting phylogenetic hypotheses and consistently low levels of support realized in all of these studies have resulted in conservative approaches to taxonomic revisions. In order to resolve relationships within this group we have sequenced over 6000 bp from regions in the nuclear, chloroplast, and mitochondrial genomes and included 42 taxa in Bayesian, neighbor‐joining, and parsimony analyses. The result is the first comprehensive and well‐supported molecular phylogeny for the ALL complex of the Laminariales. We maintain the three recognized families (Alariaceae, Laminariaceae, and Lessoniaceae), but with vastly different compositions, as well as propose the Costariaceae fam. nov. for Agarum, Costaria, Dictyoneurum, and Thalassiophyllum, the only genera in the Laminariales with flattened, occasionally terete, stipes and either a perforate or reticulate blade. In addition, our data strongly support a split of the genus Laminaria. We resurrect the genus Saccharina Stackhouse for the Laminaria clade that does not contain L. digitata (Hudson) J.V. Lamouroux, the type of the genus.     

EFFECTS OF NITRATE CONCENTRATION ON THE GROWTH AND PHYSIOLOGY OF LAMINARIA SACCHARINA (PHAEOPHYTA) IN CULTURE1,2

Laminaria saccharina Lamour. sporophytes were grown in enriched and synthetic media through a range of nitrate concentrations, There was an approximately linear relationship between growth and nutrient concentration up to 10 μ substrate concentration. The half-saturation constant (K_{2) was ca. 1.4 μ NO3-. The internal levels of NO3- increased at substrate concentrations above 10 μM b>3- and reached levels several thousand times higher than the surrounding medium. Thus there is evidence for luxury consumption of NOsb>3}^-. The chlorophyll content and photosynthetic capacities of plants also increased with increasing external NO₃^- The ecological implications of this work are considered.