GENETIC DATA HINT AT A COMMON DONOR REGION FOR INVASIVE ATLANTIC AND PACIFIC POPULATIONS OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA)1

Gracilaria vermiculophylla (Ohmi) Papenf., an agar‐producing red alga introduced from northeast Asia to Europe and North America, is often highly abundant in invaded areas. To assay its genetic diversity and identify the putative source of invasive populations, we analyzed the mitochondrial cytochrome c oxidase subunit I (cox1) gene from 312 individuals of G. vermiculophylla collected in 37 native and 32 introduced locations. A total of 19 haplotypes were detected: 17 in northeast Asia and three in Europe and eastern and western North America, with only one shared among all regions. The shared haplotype was present in all introduced populations and in ～99% of individuals in the introduced areas. This haplotype was also found at three native locations in east Korea, west Japan, and eastern Russia. Both haplotype and nucleotide diversities were extremely low in Europe and North America compared to northeast Asia. Our study indicates that the East Sea/Sea of Japan is a likely donor region of the invasive populations of G. vermiculophylla in the east and west Atlantic and the east Pacific.     

A MORPHOLOGICAL STUDY AND TAXONOMIC REASSESSMENT OF THE GENERITYPE SPECIES IN THE GRACILARIACEAE1

We investigated the reproductive morphology of representative material corresponding to the type species of each of the described genera presently placed in synonymy under Gracilaria. From these observations and published studies of recognized genera, 10 species groups are identified in the Gracilariaceae based on spermatangial type and cystocarp development. Actual or potential generic names are given in brackets after each group: 1) abscissa group (Melanthalia), 2) flabellata group (Curdiea), 3) lemaneiformis group (Gracilariopsis), 4) chilensis group, 5) edulis group (Plocaria/Polycavernosa), 6) urvillei group (Hydropuntia), 7) crassissima group, 8) salicornia group (Corallopsis), 9) gracilis group, and 10) bursa‐pastoris group (Gracilaria). Tyleiophora was shown to belong to the bursa‐pastoris group. The type species of the parasitic genera Gracilariophila and Congracilaria are closely related to their host species. Species assemblages recognized here based on morphological evidence received moderate to strong bootstrap support in recently published molecular phylogenies. Further studies may show that some groups correspond to genera, whereas others do not merit generic status.     

Agar Analysis,nuclear genome quantification and characterization of four agarophytes (Gracilaria) from the Mexican Gulf Coast

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in four species of Gracilaria (Gracilariales, Rhodophyta). In Gracilaria tikvahiae, G. caudata, G. cervicornis and G. divaricata, results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–46% and unique DNA ranged from 45–78%, Thermal denaturation (T _m) indicated guanine + cytosine (G + C) levels of 41.9–46.0 mol % G + C. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA content. Comparisons of mean nuclear DNA (I _f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.37–0.40 pg/2C genomes for the four Gracilaria species. Total agar content following alkaline pretreatment ranged from 7–15% dry weight. Gel strengths were generally below commercial levels, ranging from 40–260 g cm⁻² Nuclear genome profiles developed from information for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity as previously speculated.     

Apical callus formation and plant regeneration controlled by plant growth regulators on axenic culture of the red alga Gracilariopsis tenuifrons (Gracilariales, Rhodophyta)

Axenic cultures of Gracilariopsis tenuifrons (Bird et Oliveira) Fredericq et Hommersand (Gracilariales, Rhodophyta) were established in ASP12‐NTA solid medium (0.4% agar and 1.0% sucrose) supplemented with plant growth regulators to evaluate the effects on apical callus formation and plant regeneration. Indole‐3‐acetic acid (IAA), 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 6‐benzylaminopurine (BA) were added individually or in combinations (IAA : BA) over a range of concentrations from 0.5 to 5 mg L^?1. Growth of apical and intercalary segments was stimulated by high concentrations of 2,4‐D (5 mg L^?1) and a high IAA to BA ratio (IAA : BA = 5:1 mg L^?1) respectively. Apical calluses were originated from divisions of apical and cortical cells located at apical regions of thallus segments and lateral branches. Low concentration of IAA (0.5 mg L^?1) or a high IAA to BA ratio (IAA : BA = 5:1 mg L^?1) were the optimal treatments for inducing apical callus formation in apical segments, while high concentration of IAA (5 mg L^?1) stimulated the highest callus induction rate in intercalary segments. Conversely, equal parts IAA and BA (IAA : BA = 1:1 mg L^?1) and low concentration of 2,4‐D (0.5 mg L^?1) stimulated growth of apical calluses from apical and intercalary segments, respectively. Two processes of regeneration were observed: direct regeneration (upright axis originated from cells of proximal region of intercalary segments) and indirect regeneration (adventitious plantlet originated from cells of apical calluses). Direct regeneration was promoted significantly by treatment with a low IAA to BA ratio (IAA : BA= 1:5 mg L^?1), and treatments with IAA (0.5 mgL^?1) or 2,4‐D (0.5 or 5 mg L^?1) significantly stimulated the elongation of upright axis. Plant growth regulators are essential to inducing indirect regeneration, and a high concentration of IAA (5 mg L^?1) and BA (5 mg L^?1) were the optimal treatments for inducing the regeneration of plantlets from apical calluses in apical and intercalary segments, respectively. Regenerating plantlets grew into plants morphologically similar to those formed from germinating spores, and became fertile after 6 weeks. The results suggest that auxins and cytokinins are involved in developmental regulatory processes in G. tenuifrons. The regeneration process from calluses in species of Gracilariales was observed for the first time in the present study. The culture system described for G. tenuifrons could be useful for micropropagation and for biotechnological applications in agarophytic algae.     

Effect of dark treatment on the starch degradation and the agar quality of cultivated Gracilariopsis lemaneiformis (Rhodophyta,Gracilariales) from Venezuela

The effect of darkness and ammonia on Gracilariopsis lemaneiformis agar yield and quality was studied. Plants were cultivated under controlled conditions in the laboratory and fertilized with ammonia prior to a month storage in the dark. Starch inclusions are known to interfere with the mechanical properties of the isolated agar. The starch content of algal tissue and the activities of the floridean starch phosphorylase and -glucosidase were measured during this storage period as a way to follow the enhancement of starch degradation in the absence of light, and to measure the effect of darkness on the content and quality of the synthesized agar. The agar yield, gel strength, 3,6-anhydrogalactose, sulphate and starch content were considerably affected by this dark treatment. The values obtained reflect a pattern of optimization of agar quality when Gracilariopsis lemaneiformis was stored in the dark. The isolated agar following dark treatment is less contaminated by starch inclusions. This is shown by the improvement in the agar yield and quality, the nutrient status of the plant and the reduction of the starch content.Abbreviations DW: Dry weight - FW: Fresh weight - S: Sulphate - 3,6-AG: 3,6-anhydrogalactose - GS: Gel strenght at 1.5% concentration - CX: Cold extract at 25 °C - A: Autoclaved extract at 121 °C - MW: Molecular weight - PFD: Photon flux density     

PROPOSAL OF THE GRACILARIALES ORD. NOV. (RHODOPHYTA) BASED ON AN ANALYSIS OF THE REPRODUCTIVE DEVELOPMENT OF GRACILARIA VERRUCOSA1

The mode of division of vegetative cells, formation of spermatangial parent cells, initiation of the carpogonial branch apparatus, and formation of tetrasporangial initials are homologous developmental processes that are documented for the first time in the type species of the economically important family Gracilariaceae, Gracilaria verrucosa (Hudson) Papenfuss from the British Isles. G. verrucosa is characterized by a supporting cell of intercalary origin that bears a 2-celled carpogonial branch flanked by two sterile branches, direct fusion of cells of sterile branches onto the carpogonium, formation of an extensive carpogonial fusion cell through the incorporation of additional gametophytic cells prior to gonimoblast initiation, gonimoblast initials produced from fusion cell lobes, schizogenous development of the cytocarp cavity, inner gonimoblast cells producing tubular nutritive cells that fuse with cells of the pericarp or floor of the cystocarp, absence of cytologically modified tissue in the floor of the cystocarp, and carposporangial initials produced in clusters or irregular chains. Spermatangial parent cells are generated in flaments from intercalary cortical cells that line an intercellular space forming a ‘pit’ or ‘conceptacle’. Tetrasporangial initials are transformed from terminal cells derived through division of an outer cortical cell. Tetrasporangia are cruciately divided. The Gracilariaceae is removed from Gigartinales and transferred to the new order Gracilariales. Their closest living relatives appear to be agarophytes belonging to the Gelidiales and Ahnfeltiales.     

Quantification and characterization of nuclear genomes in commercial red seaweeds: Gracilariales and Gelidiales

Nuclear genome profiles were developed for representative species of the Gelidiales and Gracilariales using information from present and previous studies of cytogenetics, cytophotometry and DNA reassociation kinetics. Results indicate that species of Gracilaria and Gracilariopsis are characterized by distinct chromosome complements of n = 24 and n = 32, respectively, a narrow range of small genome sizes (2C = 0.35–0.45 pg) and a wide range of values for repeated and unique genome sequences. In contrast, the Gelidiales investigated are characterized by a wide range of chromosome complements, n = 6–29, a wider range of genome sizes (2C = 0.42–0.68 pg) and a large proportion of unique genome sequences. Nuclear genome sizes for species of the Gelidiales and Gracilariales are compared with estimates of other red algal orders including the Bangiales, Ceramiales and Gigartinales.     

DNA base composition heterogeneity in some agarophytes (Gracilariales,Rhodophyta) from Mexico and the Philippines

Estimates of nuclear DNA base composition by determination of thermal denaturation temperatures (T_m) indicate guanine + cytosine (G + C) levels of 35.4–46.8% for ten species of the Gracilariaceae, representing the generaGracilaria andHydropuntia. T_m values were found to be reproducible with variation among most samples and replicates of less than 1 °C and 2 mol%. Interspecific variation in G + C values was less than 11.4% amongGracilaria species. Calculation of intragenomic base pair composition distribution based on mid-resolution thermal denaturation (A 1 °C/min with 4s interval H and dT logging) indicated an inverse relationship between maximum similarity values and taxonomic rank. Intraspecific (population level) maximum similarity (homology) values were estimated to range from 79–90% inGracilaria tikvahiae (4 isolates). Interspecific values of 46–69% were found in 13 species ofGracilaria. Nucleotide distribution similarity values for the Gracilariaceae are compared with previous information for genome organization and complexity, genome size and karyotype patterns.Author for correspondence