CULTURE AND HYBRIDIZATION STUDIES ON GIGARTINA PAPILLATA (RHODOPHYTA)1

The foliose red alga Gigartina papillata (C. Ag.) J. Ag. was studied in culture to determine its life history and possible relationship to the life history of Petrocelis middendorffii (Ruprecht) Kjellman. Carpospores cultured from individual female plants gave rise to either crustose Petrocelis-like plants that reproduced by tetraspores, or to another generation of foliose female (cystocarpic) plants that reproduced by carpospores. Apices cultured from blades of individual field-collected female plants produced either papillae with many procarps that developed cystocarps only when crossed with male plants, or papillae with few procarps that produced cystocarps in the absence of male plants. The results are interpreted to demonstrate that two types of life history occur in G. papillata: one, a sexual life history involving a crustose tetrasporophyte; the other, a possibly apomictic life history involving only cystocarpic plants. Hybridization experiments demonstrated, that G. papillata is interfertile with Gigartina-phase gametophytes cultured from tetraspores of P. middendorffii. Sexual plants of G. papillata are postulated to represent the naturally-occurring gametophyte of P. middendorffii in California. The possible relationships of the sexual and apomictic plants of G. papillata are discussed.     

CULTURE AND HYBRIDIZATION STUDIES ON PETROCELIS (RHODOPHYTA) FROM ALASKA AND CALIFORNIA1,2

Cultured tetraspores of Petrocelis middendorffii (Ruprecht) Kjellman from Amchitka Island, Alaska, gave rise to foliose, dioecious gametophytes similar to cultured gametophytes of P. franciscana Setchell & Gardner. A 1:1 ratio male:female gametophytes was obtained. Fertilized female plants produced cystocarps and carpospores that gave rise to crustose plants anatomically similar to field-collected Petro-celis sporophytes. Cultured male gametophytes of P. middendorffii were interfertile with cultured female blades of field-collected Gigartina pacifica Kjellman. Cultured P. middendorffii gametophytes from Amchitka were interfertile with cultured gametophytes of P. franciscana from 2 localities in California. Hybrid carpospores gave rise to crustose sporophytes that have not reproduced. Anatomical comparisons of P. middendorffii from Amchitka with P. franciscana from California showed no important differences in the characters originally used to separate these species. The interfertility of cultured Petrocelis gametophytes from california and Amchitka as well as the similarities of the history and anatomy suggests that a single species is involved. P. franciscana is reduced to a synonym of P. middendorfii.     

LIFE HISTORY AND HYBRIDIZATION STUDIES ON GIGARTINA STELLATA AND PETROCELIS CRUENTA (RHODOPHYTA) IN THE NORTH ATLANTIC1

Fourteen isolates of the crustose marine red alga Petrocelis cruenta J. Agardh from various localities in the British Isles, France (including the type locality), Spain and Portugal gave rise in culture to dioecious foliose plants identifiable as Gigartina stellata (Stackhouse) Batters although two isolates formed only sterile foliose blades. A total of 145 isolates of Gigartina stellata were also grown in culture from various localities in the U.S.A. (Maine), the British Isles, Iceland, Denmark, France, Spain and Portugal using both carpospores and vegetative blade apices. Two basic types of life history were found among these isolates: a direct-type life history involving the formation of further foliose plants from carpospores, some isolates of which also form spermatangia on the same papillae as the cystocarps; and a heteromorphic-type in which only crustose plants resembling Petrocelis cruenta are formed from carpospores. Only heteromorphic-type life histories were found from Spain and Portugal. Both life history types were found in plants from the U.S.A., the British Isles and northern France. Only direct-type life histories were found in plants from Iceland and Denmark. Some Petrocelis-like crusts derived from field collected G. stellata carpospores and Petrocelis crusts of hybrid progeny formed tetrasporangia in 8:16 h LD, 10° C but not in 8:16 h LD, 15° C; 16:8 h LD 10° C or 15° C; and 10:6.5:1: 6.5 h LDLD, 10° C. The spores thus formed were viable and produced normal dioecious male and female gametophytes. Short day and low temperature conditions appear necessary for tetrasporogenesis. The results from crossing experiments with 32 male and 27 female isolates of the heteromorphic-type derived from both G. stellata and P. cruenta showed that two virtually non-interbreeding populations with a high degree of geographical separation exist in the north-eastern Atlantic. Morphological differences between plants from each population are described. On the basis of culture and crossing results, Petrocelis cruenta J. Agardh is placed in synonymy with Gigartina stellata (Stackhouse in Withering) Batters.     

LIFE HISTORY OF BONNEMAISONIA CENICULATA (RHODOPHYTA): A LABORATORY AND FLELD STUDY1, 2

Cystocarpic and spermatangial plants of rarely reported red alga Bonnemaisonia geniculata Gardner, epiphytic on Odonthalia Aoccosa (Esp.) Falk, were collected from june to September 1975 at shell Beach, california. Carpospores inoculated into unialgal culture divided, upon germination, in to two daughter cells, both of which formed erect and rhizoidal axes, Erect axes were uniseriale and alternately branched with a distictive zigzag pattern of axial cells. No tetrasporangia developed in culture. The presumptive tetrasporangia developed in culture to a described genus. Plants morphologically similar to those cultured from carpospores were found at the collection site; they bore tetrasporangia from February to june. Cullured letrasporews gave rise to male and female plants similar to those of field-collected B. geniculate in ca. a I:I ratio. Fertile female plants in the presence of male plants formed cystocarps. Carpospores gave rise to the alternately branched tetrasporophyte phase. Bonnemaisonia geniculate has a heteromorphic life history involving a previously undescribed tetrasporophyte.     

ITS1 sequences of type specimens of Gigartina and Sarcothalia and their significance for the classification of South African Gigartinaceae (Gigartinales,Rhodophyta)

Uncertainties about the identity of type specimens of red algae have frequently led to taxonomic and nomenclatural confusion. A procedure for extracting PCR-amplifiable DNA from formalin-fixed material and herbarium specimens was used to investigate the taxonomic status of several South African Gigartinaceae. We compared nucleotide sequences in the internal transcribed spacer 1 (ITS1) region in type specimens and other historically important collections presently referred to Gigartina bracteata, G. radula and G. stiriata. The following opinions are supported: (1) Fucus bracteatus S.G. Gmelin, Chondrodictyon capense Kützing and Iridaea clathrata Decaisne represent a single species of South African Gigartina (G. bracteata (S.G. Gmelin) Setchell & Gardner) in which the disintegration of tetrasporangial sori results in a reticulate thallus. (2) Mastocarpus polycarpus Kützing, M. incrassatus Kützing and Iridaea lapathifolia Kützing represent a single species of South African Gigartina (G. polycarpa (Kützing) Setchell & Gardner) that has often, but erroneously, been called G. radula (Esper) J. Agardh. (3) Mastocarpus verrucosus Kützing is a later heterotypic synonym of Iridaea papillosa Bory (Sarcothalia papillosa (Bory) Leister) and was based on material that probably came from southern South America rather than from South Africa, the provenance given by Kützing. (4) Fucus stiriatus Turner and Sphaerococcus burmannii C. Agardh represent a single species of South African Sarcothalia (S. stiriata (Turner) Leister).     

Biosystematics,genetics and upper temperature tolerance ofGigartina teedii (Rhodophyta) from the Atlantic and Mediterranean

Plants ofGigartina teedii from the mediterranean isolated into laboratory culture showedPolysiphonia-type life histories with consistent formation of dioecious gametangial plants, as previously reported for Atlantic isolates. Male and female plants from the Atlantic and Mediterranean were almost completely compatible in terms of cystocarp formation on female plants, and carpospores from positive crosses always formed plants that released viable tetraspores. Sex-linked inheritance of branching pattern was found in all strains, but showed varying degrees of expression. Female plants were more branched than male plants and it is suggested that this may be an adaptation for spermatial capture.G. teedii plants showed differences in morphology in culture that are considered to be genetically-based. Preliminary studies of tip elongation showed that Mediterranean strains may have up to three times the elongation rates of Atlantic strains at 15°C, . Such genetic variation in fully-interbreeding strains suggests that populations of this species in the Atlantic and Mediterranean are genecodemic. All strains showed an upper temperature tolerance of 31°C when tested at 1°C intervals from 29—34°C. An upper temperature tolerance of 31–32°C was found for the related speciesG. intermedia from Korea and Japan, butG. johnstonii from the Gulf of California showed an upper tolerance of 32–33°C.     

CARRAGEENANS BIOSYNTHESIZED BY CARPOSPOROPHYTES OF RED SEAWEEDS GIGARTINA SKOTTSBERGII (GIGARTINACEAE) AND GYMNOGONGRUS TORULOSUS (PHYLLOPHORACEAE)1

Carrageenans biosynthesized by gametophytic and tetrasporic plants of seaweeds belonging to the Gigartinaceae and Phyllophoraceae are different: gametophytes produce carrageenans of the kappa family, whereas lambda‐carrageenans are extracted from tetrasporophytes. For Gigartina skottsbergii Setchell and Gardner and Gymnogongrus torulosus Hooker et Harvey, mature cystocarps were isolated and carrageenans were extracted. Structural determination by methylation analysis, Fourier transform infrared spectroscopy, and ¹³C‐NMR spectroscopy showed that they were kappa/iota‐carrageenans. For the extract obtained from cystocarps of Gigartina skottsbergii with water at room temperature, the ratio kappa:iota was 1:0.30 and at 90° C was 1:0.43; significant amounts of precursors were also present. The extract obtained from cystocarps of Gymnogongrus torulosus at 90° C showed prevalence of iota‐carrageenans (ratio kappa:iota 1:1.21). These extracts are similar to the polysaccharides produced by gametophytes of these seaweeds. For Gigartina skottsbergii, it was possible to separate the pericarpic tissue from the carposporophyte. Thus, they were extracted separately, and the carrageenans isolated were studied as described before, obtaining similar conclusions. These results clearly show that whereas the carposporophytes are located inside the cystocarp, they produce carrageenans of the kappa family despite of being diploid cells.     

Seasonal variations in thalli and carrageenan composition of Gigartina pistillata (Gmelin) Stackhouse (Rhodophyta, Gigartinales) harvested along the Atlantic coast of Morocco

The biology and biochemistry of Gigartina pistillata (Gmelin) Stackhouse collected monthly at Nation Beach (Morocco), was studied during one year. The biological study showed one period of active growth from April to July. The thallus composition was quite stable during the major part of the year. The dry matter was maximum in May and August and minimum in January. The maximum carrageenan content occurred in June and September (about 37%) and the minimum carrageenan content occurred in February (19.0%). The total nitrogen content varied significantly, with a maximum in January (1.98%) and a minimum in August (0.7%). The ash content was significant (23–32%) with a maximum in August and a minimum in May. The carrageenan extracted from natural populations of Gigartina pistillata was a mixture of lambda‐type and kappa‐type carrageenans. The 3,6‐anhydrogalactose varied between 4.5 mol% in June to 25 mol% in February. For industrial applications the extract could be considered as a lambda‐type. The best period for harvest of G. pistillata in Morocco is between July and August when biomass and viscosity are at their maximum. A relationship between the physical characteristics of G. pistillata carrageenans and its seasonal cycle was deduced.     

LIFE HISTORIES IN THE PHYLLOPHORACEAE (RHODOPHYTA: GIGARTINALES) FROM THE PACIFIC COAST OF NORTH AMERICA. I. GYMNOGONGRUS LINEARIS AND G. LEPTOPHYLLUS1

Carpospores of Gymnogongrus linearis (C. Ag.) J. Ag. collected from Sonoma Co., California were cultured and gave rise to crustose plants. Tetrasporogenesis could not be induced. However, tetraspores from field-collected crustose tetrasporophytes found near G. linearis from San Mateo Co., California were cultured. These field crusts superficially resemble Petrocelis middendorffii (Ruprecht) Kjellman, but differ in size, color, number of tetrasporangia per filament, and distal dichotomous branching of the perithallial filaments. Tetraspores gave rise to upright plants identical to G. linearis. Gymnogongrus leptophyllus J. Ag. collected from California and Baja California, Mexico were found as narrow and wide forms. Narrow form isolates recycled directly without producing a crustose tetrasporophyte. These are interpreted as apogamous. Carpospores of the wide form grew into crusts resembling Petrocelis (=Erythrodermis) haematis Hollenberg. Tetrasporogenesis was induced in culture by abrasion or dehydration. Tetraspores from field-collected crusts and laboratory cultured tetrasporophytes grew into plants identical to G. leptophyllus, completing a sexual life history with an alternation of heteromorphic generations.     

INDETERMINATE GROWTH OF PERICARPS IN THREE MEMBERS OF THE RHODYMENIALES (RHODOPHYTA)12

Pericarps generally are thought of as structures having determinate growth, serving as a protective covering for the developing gonimoblast. In laboratory culture, the pericarps of Champia parvula (C. Agardh) Harvey, Lomentaria baileyana (Harvey) Farlow and Lomentaria sp. exhibited indeterminate growth. These tissues could be excised and grown as separate female plants. The new plants were indistinguishable from the parent tissue, were fertile, and produced viable carpospores.     

Cultivation of Gigartina skottsbergii (Gigartinales, Rhodophyta): Recent advances and challenges for the future

This study integrates landings statistics and biological studies of the red algaGigartina skottsbergii Setchell & Gardner. The analysis of the landings and carrageenan production in Chile suggeststhat this resource will suffer a strong harvesting pressure during the nextyears. Biological results on sporulation, germination, sporeling growth and survivorship in laboratory,indoor tanks and field conditions, indicated that cultivation of this species istechnically feasible, as spores can be seeded on ropes and other substrata. Vegetative propagation of this species through tissue fragmentationis also possible. Vegetative fragments of this carrageenophyte have 20 to30% higher growth rates than whole fronds in suspended culture systems. Protoplast production can be also explored for bypassing restrictions inspore availability. Major advantages that encourage the cultivation of G. skottsbergii include its gel quantity and quality, its pathogen-freecondition, a high reproduction potential and its regeneration capacity. Onthe other hand, the major constraints are related to its relatively slowgrowth as compared to other carrageenophytes, limited availability ofspores and high mortality during juvenile stages.     

Biogeography of sexual and asexual populations in Bostrychia moritziana (Rhodomelaceae, Rhodophyta)

Bostrychia moritziana (Sonder ex Kützing) J. Agardh is recorded from many regions around the world. Our laboratory culture investigations have verified a sexual life cycle in isolates from Australia, Venezuela, Colombia, South Africa, Fiji, New Zealand and Indonesia. By contrast, asexual isolates producing successive generations of tetrasporophytes in laboratory culture and, presumably, in the field, are known from Australia. New Caledonia and Japan. In Australia, asexual reproduction is absent only in Victoria. In Western Australia, Northern Territory and Queensland, 99% of the isolates have asexual reproduction. In New South Wales (NSW), asexual and sexual populations are often intermixed. Of the 176 worldwide field collections, 58% were vegetative, 39% were tetrasporic, 2% were female and 1% were male. After several years of observations on the asexual isolates in culture, at least 30 successive asexual tetrasporophytic generations have developed. Only two asexual isolates (3558 and 3575) from NSW have formed a single male and female gametophyte in culture. In a self-cross of 3568, the carpospores developed into tetrasporophytes that recycled asexually. All outcrosses done with normal sexual isolates produced normal carposporophytes and the carpospores developed into tetrasporophytes that also recycled asexually. Asexual populations may arise repeatedly by loss of meiosis in tetrasporangia of sexual populations. Asexual reproduction apparently does not diminish the overall dispersal and abundance in the field. Our present bio-geographic data show that sexually reproducing popuiations of B. moritziana occur worldwide, while asexuaily reproducing populations are confined to the western Pacific. Bostrychia bispora West et Zuccarello, initially described on the basis of its asexual reproduction to distinguish it from B, moritziana, is now reduced to synonymy with B. moritziana.     

GENETICS OF GRACILARIA TIKVAHIAE (RHODOPHYCEAE). X. STUDIES ON A BISEXUAL CLONE12

A male done of the red alga Gracilaria tikvahiae McLachlan spontaneously produced a bisexual frond which remained bisexual in subsequent subcultures. Both male and female components of bisexual fronds were functional; however, some unusual results were obtained in crosses. When bisexual fronds were crossed with a normal haploid male, the resulting carpospores all developed into diploid male gametophytes. When bisexual plants were self fertilized, all the carpospores yielded diploid bisexual gametophytes. Only when bisexual plants were crossed to normal haploid females, did carpospores develop into diploid tetrasporophytes as they normally do. The F₁ gametophyte generation obtained from these tetrasporophytes, however, included not only females and males but also bisexual plants, in a 2:1:1 ratio. These results are consistent with the interpretation that bisexual plants have a recessive mutation of a gene other than the primary sex determining locus, and that this mutation is expressed only in male plants. It is suggested that the altered gene may ordinarily have a regulatory function in the maintenance of the dioecious condition.     

Field trials to optimise timing and frequency of pruning for cultivation of a New Zealand carrageenophyte, Gigartina atropurpurea

The endemic New Zealand red alga Gigartina atropurpurea has been identified as a candidate for aquaculture because of its high quality and value carrageenan and its broad-bladed morphology. As G. atropurpurea has not so far been exploited commercially, trials were conducted to investigate the effect of timing and frequency of pruning on the growth of plants in the wild and explants on ropes, in spring, and autumn. Plants were monitored monthly by measuring the length of the two longest blades (a proxy for biomass) and wet weights of all plant material retrieved. The results suggested five broad conclusions: (1) spring growth was much greater than autumn growth; (2) regardless of season, maximal growth rates were achieved 4–6 weeks post pruning; (3) pruning accelerated growth; (4) pruning plants twice during spring generated up to four times more biomass than when plants were pruned only once, and (5) there was no difference in the response of farmed and wild plants to pruning treatments. Based upon these preliminary trials, plant material from wild tetrasporic and female G. atropurpurea could be harvested up to three times during the growth period (August–December). However, further research on the feasibility of G. atropurpurea aquaculture is justified.     

Hybridization studies in Bostrychia. 1: B. radicans (Rhodomelaceae, Rhodophyta) from Pacific and Atlantic North America

Bostrychia radicans(Montagne) Montagne is a pantropical/temperate red alga associated with mangroves and saltmarsh plants. Collections were made from a similar north-south geographic distribution along both the Pacific and Atlantic coasts of North America. Hybridization studies were performed with cultured isolates to assess the extent of interfertility and reproductive isolation along these two coastlines. All male and female gametophytes derived from single tetrasporophytes were intercompatible. Almost all isolates extending over 1500 km of coast line from northern Pacific Mexico are compatible, forming cystocarps that released viable carpospores. Even isolates which morphologically would be placed in two species [B. radicans and B. moritziana(Sender ex Kützing) J. Agardh], based on the presence or absence of monosiphonous branches, were capable of hybridizing. Crosses of isolates from the Atlantic USA showed a greater amount of incompatibility. Certain isolates were not compatible with any other isolates including isolates collected in close proximity (North Carolina isolates), while other isolates from the same locality were compatible (South Carolina). An isolate from South Carolina formed tetrasporophytes with isolates from Pacific Mexico but tetraspores were not viable. Certain incompatible crosses formed ‘pseudocystocarps’ but viable carposporophytes did not develop. Generalizations about reproductive isolation within a species must also consider differences between populations from different biogeographic regions that may reflect different paleoclimatological histories, founder effects and unique dispersal events.     

Plant regeneration of wildGlycine species from suspension culture-derived protoplasts

Protoplasts isolated from four-week old cell suspension cultures ofGlycine canescens F. J. Herm andG. clandestina Wendl. were cultured in 8P or modified 8P to a multicellular stage. Colonies of 0.5 to 1.0 mm diameter were transferred to solid media for callus growth and regeneration. Callus consisted of friable masses with compact green nodular areas. Organogenesis of both species occurred primarily from the green nodular areas. Shoot buds ofG. clandestina did not mature, but shoots ofG. canescens proliferated on MS medium, with B5 vitamins, 0.33 mgL^–1 each BA, KN, ZN, and 0.15 mgL^–1 NAA. Shoots failed to root after multiple subcultures on four different rooting media.In vitro grafting ofG. canescens scions ontoG. max root stocks allowed plants to be transferred to soil. An overall protoplast division efficiency of 48% was achieved with moderately efficient shoot regeneration inG. canescens. Division efficiencies forG. clandestina were lower (11%). Refinements of this protocol should result in high efficiencies of regeneration which would allowin vitro manipulations of these wild soybean relatives at the single cell level and would make the derivation of somatic hybrid plants possible within the genusGlycine.Abbreviations BA 6-Benzyladenine - KN kinetin - ZN Zeatin - NAA Napthaleneacetic acid - 2,4-D 2–4-dichlorophenoxyacetic acid - PIC Picloram - CH casein hydrolysate - Gln glutamine - Met methionine - MES 2[N-morpholine] ethanesulfonic acid     

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.     

Physicochemical characterization of carrageenan fromGigartina teedii (Rooth) Lamouroux (Gigartinales,Rhodophyta)

The phycocolloids of female gametophytes ofGigartina teedii (Roth) Lamouroux harvested in Roscoff (Brittany, France) are a hybrid carrageenan resulting from juxtaposition of fragments of kappa-, iota-and nu-carrageenan. They represent 70% of the dry matter of the alga in summer. After alkaline transformation the proportion of iota-carrageenan increased to 76%, demonstrating the presence of nu-carrageenan. Absence of mu-carrageenan, the precursor of kappa-carrageenan, suggests that iota-carrageenan is desulfated enzymically to kappa-carrageenan.     

Restriction enzyme analysis of variation and taxonomy in the kelp genus Laminaria (Laminariales,Phaeophyta)

High levels of phenotypic variation in kelp species necessitate the use of taxonomic markers that are independent of morphology. Restriction fragment length polymorphism (RFLP) analysis of nuclear DNA can provide such markers. In this paper we present the results of an RFLP analysis of cytoplasmic ribosomal DNA (rDNA) in three Laminaria species (L. agardhii, L. digitata, L. groenlandica). Comparison of the restriction maps of the nontranscribed spacer (NTS) in the rDNAs suggests that this method should be useful for the differentiation of these taxa. These results are discussed, as are the applications of RFLP mapping to the identification of field-collected, morphologically variable plants.     

The life history of Petrocelis franciscana

Tetraspores from Petrocelis franciscana Setch. et Gardn. collected at Rockaway Beach, San Mateo County, California, 11 January 1971 were isolated into unialgal culture with Provasoli's enriched seawater medium at 10° and 15° C, 16:8 daily photoregime, 2000–2500 lx cool white fluorescent lighting. The germlings developed as crustose discs with marginal meristems until about 300–500 µm in diameter when erect multiaxial blades were established. The erect plants grew to reproductive maturity in 15° C but not in 10° C. By the fifth month of growth the erect axes flattened and branched dichotomously. At the end of 7 months the plants were reproductively mature. These cultured plants were morphologically similar to Gigartina agardhii Setch. et Gardn. (subgenus Mastocarpus) which occurs abundantly at Rockaway Beach. Male plants were non-papillate with spermatangia forming on the entire blade surface except at the growing tip and near the stipe. The female plants developed papillae which bore many procarps. Many of these procarps were penetrated and seemingly destroyed by intrusive filaments from adjacent vegetative branches but apparently a number were functional since a number of cystocarps developed on plants in both stationary and shake culture after 5 months. Some viable spores were released and germinated.