POLYAMINES INFLUENCE MATURATION IN REPRODUCTIVE STRUCTURES OF GRACILARIA CORNEA (GRACILARIALES,RHODOPHYTA)1

Naturally occurring levels of putrescine, spermidine, and spermine were analyzed in female gametophyte (nonfertilized) and reproductive tissues (cystocarps) at two different stages of development in the marine red algae Gracilaria cornea J. Agardh. Endogenous polyamine levels changed at differential stages of cystocarp maturation. Highest polyamine values were found on tissue from the early post‐fertilization stage, decreasing as the cystocarp matured. Incubation experiments revealed that exogenous polyamines induced cystocarp maturation and promoted carpospore liberation, developing cell masses within 4 to 7 days in treatments with spermine. This is the first report on the effect of polyamines on cystocarp maturation in marine algae.     

DIURNAL FLUCTUATION OF NITRATE REDUCTASE ACTIVITY IN THE MARINE RED ALGA GRACILARIA TENUISTIPITATA (RHODOPHYTA)1

The biochemical characteristics and diurnal changes in activity of the enzyme nitrate reductase (NR; EC 1.6.6.1) from the marine red alga Gracilaria tenuistipitata var. liui Zhang et Xia are described. Different assay conditions were tested to determine the stability of NR. The crude extract of G. tenuistipitata has a NR specific activity of 10.2 U.mg⁻¹, which is higher than the NR activities found for other algae, plants, and fungi. This NR is highly active at a slightly alkaline pH and is stable over a wide range of temperature, with an optimal activity at 20° C. The apical portions of the thallus contain 64.9 ± 6.6% of the total NR specific activity. The apparent Michaelis-Menten (K_m) constant found for KNO₃ was 197 μM, and it was 95 μM for NADH. The NR from G. tenuistipitata can be included in the NADH-specific group, because no activity was found when NADPH was used as an electron donor. In extracts of algae grown under either continuously dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at the middle of the light phase, when activity is 30-fold higher than during the night phase.     

MOLECULAR IDENTIFICATION OF TWO SIBLING SPECIES UNDER THE NAME GRACILARIA CHILENSIS (RHODOPHYTA,GRACILARIALES)1,3

Molecular markers belonging to three different genomes, mitochondrial (cox2‐3 spacer), plastid (RUBISCO spacer), and nuclear (internal transcribed spacer 1), were used to compare Gracilaria chilensis samples collected along the Chilean coast with samples ascribed to G. chilensis from the West Pacific Ocean (New Zealand and Australia). Our data are in agreement with previous studies suggesting two sibling species currently going under the name G. chilensis that co‐occur in New Zealand. One of these, a New Zealand sample previously examined by Bird and others in 1990, is conspecific with G. chilensis from Chile. Finally, our results demonstrate clearly that most of the sequences in GenBank reported as G. chilensis are based on misidentified material.     

THE ULTRASTRUCTURE OF GALLS ON THE RED ALGA GRACILARIA EPIHIPPISORA1

A strain of Gracilaria epihippisora Hoyle produces gall-like cell proliferations in culture. These growths can be excised and grown separately, where they retain an undifferentiated morphology and reach 5mm in diameter. The gall tissue consists of a single morphological cell type without any differentiation between surface and internal cells as is characteristic of normal thallus tissue. Gall cells are typically 20–40 μm in diameter and contain the usual complement of organelles and a prominent vacuole, although there are several distinct features. The large multilobed plastids have an extensive proliferation of thylakoid membranes, which form an arrangement of loops and spirals. The thallus outer cell wall layer is highly reduced. The gall growths contain intracellular virus-like particles (ca. 80 nm in diameter) that occur in discrete groups.     

SPORELING COALESCENCE AND INTRACLONAL VARIATION IN GRACILARIA CHILENSIS (GRACILARIALES,RHODOPHYTA)1

This study evaluates the hypothesis that spore coalescence may cause intraclonal variation. Spore coalescence might allow the occurrence of unitary thalli that in fact correspond to genetically different, coalesced individuals. Plant portions simultaneously derived from these chimeric individuals may exhibit dissimilar growth responses even when incubated under similar abiotic conditions. Testing of the hypothesis included various approaches. Transmission electron microscopy observations of early stages of sporeling coalescence indicated that polysporic plantlets were formed by groups of spores and their derivatives. Even though adjacent cells in two different groups may fuse, these groups maintained an independent capacity to grow and form uprights. Laboratory-grown plantlets showed a significant correlation between the initial number of spores and the total number of erect axes differentiated from the sporeling. Construction and growth of bicolor individuals indicated the chimeric nature of the coalesced individuals. Coalesced, bicolor holdfasts had green and red cells, which subsequently produced green and red uprights, respectively. Individuals fronds were also chimeric, as indicated by the production of green and red branchlets from single, red uprights. The existence of mixed tissues was further substantiated by random amplified polymorphic DNA analysis. The banding pattern produced by branchlets of a unisporic thallus was consistently monomorphic, whereas the patterns produced by the polysporic thallus were polymorphic. Growth rates of polysporic thalli had larger data dispersal and variation coefficients than oligosporic or monosporic thalli. Therefore, all results support the original hypothesis and suggest that coalescence might be ecologically more important than previously thought.     

CLATHROMORPHUM NEREOSTRATUM (CORALLINALES,RHODOPHYTA): THE OLDEST ALGA?1

The longevity of organisms is intrinsically interesting and can provide useful information on their population structure and dynamics and the dynamics of associated communities. With the exception of perennial Laminariales that have rings in the stipe, the life spans of most perennial macroalgae are unknown or based on anecdotal observations. Using morphological analyses combined with the location and time of the rise in ¹⁴C from atmospheric nuclear testing within the thallus, we determined that the growth rate of a specimen of Clathromorphum nereostratum Lebednik from Adak Island was 0.30 mm·yr^?1, the 3⁰ bands within the thallus were annual, and the specimen sampled was 61–75 years old. Living crusts of this species from the same geographic region are reported to be up to 20 cm thick. Assuming our growth rate is typical, C. nereostratum can be approximately 700 years old, the oldest known living alga. This longevity and consistent banding within the thallus suggest that smaller scale sampling and additional chemical analyses of this alga could provide a detailed long‐term record of environmental variation at high latitudes in the North Pacific.     

FIRST RECORD OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA) IN THE PO DELTA LAGOONS,MEDITERRANEAN SEA (ITALY)1

Molecular outcomes led us to report the first occurrence of the invasive alien species Gracilaria vermiculophylla (Ohmi) Papenf. in the Mediterranean Sea. This species was recorded for the first time in the Po Delta lagoons in May and October 2008, probably introduced by the importation of the Manila clam Tapes philippinarum. At present, G. vermiculophylla is spread only near some clam‐farming areas, but its diffusion is expected to increase with the colonization of other lagoons where aquaculture is spread, as already observed for other alien species such as Agardhiella subulata and Solieria filiformis. The present study supplies further information on the morphology of this species, the ecological characteristics of the colonized areas, and the most probable introduction vector, confirming that the species spreading occurs in eutrophic and turbid coastal systems.     

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.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.     

GRACILARIA VERMICULOPHYLLA (RHODOPHYTA,GRACILARIALES) IN THE VIRGINIA COASTAL BAYS,USA: COX1 ANALYSIS REVEALS HIGH GENETIC RICHNESS OF AN INTRODUCED MACROALGA

Gracilaria vermiculophylla (Ohmi) Papenfuss is an invasive alga that is native to Southeast Asia and has invaded many estuaries in North America and Europe. It is difficult to differentiate G. vermiculophylla from native forms using morphology and therefore molecular techniques are needed. In this study, we used three molecular markers (rbcL, cox2‐cox3 spacer, cox1) to identify G. vermiculophylla at several locations in the western Atlantic. RbcL and cox2‐cox3 spacer markers confirmed the presence of G. vermiculophylla on the east coast of the USA from Massachusetts to South Carolina. We used a 507 base pair region of cox1 mtDNA to (i) verify the widespread distribution of G. vermiculophylla in the Virginia (VA) coastal bays and (ii) determine the intraspecific diversity of these algae. Cox1 haplotype richness in the VA coastal bays was much higher than that previously found in other invaded locations, as well as some native locations. This difference is likely attributed to the more intensive sampling design used in this study, which was able to detect richness created by multiple, diverse introductions. On the basis of our results, we recommend that future studies take differences in sampling design into account when comparing haplotype richness and diversity between native and non‐native studies in the literature.     

PHYLOGENETIC IMPLICATIONS OF THE CAD COMPLEX FROM THE PRIMITIVE RED ALGA CYANIDIOSCHYZON MEROLAE (CYANIDIALES,RHODOPHYTA)1

The de novo pyrimidine biosynthetic pathway consists of six enzymes: carbamoyl‐phosphate synthetase II (CPS II), aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine‐5′‐monophosphate decarboxylase. The origin and organization of the first three enzymes differ markedly between Opisthokonta (Metazoa and Fungi) and the Amoebozoa and green plants. However, no information has been available regarding the characteristics of such genes in other photosynthetic eukaryotes. In this study, we examined the pyrimidine biosynthetic cluster in the primitive red alga Cyanidioschyzon merolae P. DeLuca et al. isolate 10D. Unlike the situation in green plants, the CPS II, ACT, and DHO of C. merolae were fused to form a single open reading frame (the CAD complex), as in the Opisthokonta and Amoebozoa. Phylogenetic analysis of the CPS domain sequences suggested that this red algal CAD complex did not result from a recent lateral gene transfer from Metazoa or Fungi but that the fusion of the three genes occurred before the divergence of Opisthokonta, Amoebozoa, and the red algae. These results cast doubt on the recent hypothesis that the Opisthokonta and Amoebozoa form a monophyletic group, based on the presence in both of the CAD complex.     

THE ROLE OF F-ACTIN DURING FERTILIZATION IN THE RED ALGA AGLAOTHAMNION OOSUMIENSE (RHODOPHYTA)

The actin cytoskeletons in spermatia and trichogynes of Aglaothamnion oosumiense Itono were studied using fluorescein isothiocyanate (FITC) conjugated phalloidin and the cytoskeletal inhibitors, potassium iodide (KI), cytochalasin-B, and latrunculin-A. Microfilaments were localized to the distal ends of elongated spermatia and trichogynes and were more prominent in the trichogyne before spermatium binding. The actin cytoskeleton in spermatia and trichogynes was disrupted by treatment with 0.6 M KI, 100 μM cytochalasin-B, or 10 μM latrunculin-A. The actin cytoskeleton in trichogynes recovered within 24 h of removal from the inhibitor, but no recovery was observed in spermatia. Spermatial nuclei entered mitosis as soon as spermatia attached to the trichogyne. The greatest percentage (50%– 60%) of spermatia having completed mitosis was obtained at 60 min after spermatial binding to trichogynes. During mitosis, actin accumulated in the center of the spermatium, thereby separating the two daughter nuclei. Cytoskeletal inhibitors did not affect initial binding of spermatia to trichogynes but did block subsequent stages of fertilization, including spermatial mitosis and gamete fusion. The accumulation of cellulose or β-linked polysaccharide on the spermatial surface was also blocked by treatment with actin inhibitors. Exposure of the trichogyne to actin inhibitors after gamete fusion caused spermatial nuclei in trichogynes to stop moving and to condense. These results suggest that the microfilaments involved in nuclear division, cellulose deposition into the spermatial wall, gamete fusion, and migration of spermatial nuclei in trichogynes during fertilization in Aglaothamnion oosumiense.     

Proteomic analysis of inviable salmonid hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss during early embryogenesis

Early embryos of inviable hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss at 9, 12, 15 and 20 days after fertilization were examined for protein expression profiles. A total of 44 proteins, mostly down-regulated products of house-keeping genes and those involved in nucleic acid metabolism or chromatin replication, were identified in hybrid embryos by mass spectrometry analysis and protein database searching. The identified down-regulated proteins may be responsible for the inviability in the hybrids.     

IDENTIFICATION AND CHARACTERIZATION OF THE CATALASE GENE PyCAT FROM THE RED ALGA PYROPIA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by reducing hydrogen peroxide. The full‐length catalase cDNA sequence as isolated from expressed sequence tags (ESTs) of Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi (PyCAT) through rapid amplification of cDNA ends (RACE) was identified and characterized. It encoded a polypeptide of 529 amino acids, which shared 36%–44% similarity with other known catalase proteins. Phylogenetic analysis revealed that PyCAT was closer to the catalases from plants than from other organisms. The PyCAT mRNA expression was investigated using real‐time PCR to determine life‐cycle‐specific expression and the expression pattern during desiccation. The mRNA expression level in gametophytes was significantly higher than in sporophytes, and the mRNA expression level of PyCAT was significantly up‐regulated during the desiccation process. The recombinant PyCAT protein was purified and analyzed biochemically. The recombinant PyCAT protein exhibited high enzymatic activity (28,000 U·mg^?1) with high thermal stability and a broad pH range. All these results indicate that the PyCAT is a typical member of the plant and algal catalase family and may play a significant role in minimizing the effect of oxidative damage in P. yezoensis during desiccation.     

LIFE HISTORY PHASES AND THE BIOMECHANICAL PROPERTIES OF THE RED ALGA CHONDRUS CRISPUS (RHODOPHYTA)

Chondrus crispus Stackhouse alternates between two isomorphic life history phases that differ in cell-wall phycocolloid composition. It has been long hypothesized that the gametophyte, with strong-gelling kappa-type carrageenans, is mechanically superior to the tetrasporophyte, with nongelling lambda-type carrageenans, which could contribute to the observed gametophytic dominance in many wave-swept environments. Standard mechanical tests were performed on distal tissues of C. crispus sampled from a range of environments in Narragansett Bay, Rhode Island, using a tensometer equipped with a video extensometer. Life history phase was by far the most important determinant of mechanical properties, whereas environmental factors had only modest influence (vertical distribution) or no effect (exposure); gametophytic distal tissues were 43% stronger, 21% more extensible, and 21% stiffer than tetrasporophytic distal tissues. However, the superior strength of gametophytic tissues was not evident at the stipe/holdfast junction (where breakage typically occurs), and the two phases were equally susceptible to dislodgment by a given force. The primary ecophysiological role of carrageenans in C. crispus may not be the provision of a structure to resist wave action.     

THE RED ALGA GENUS RHODOSPORA (BANGIOPHYCIDAE,RHODOPHYTA): FIRST REPORT FROM NORTH AMERICA1

We recently discovered several populations of the red alga Rhodospora sordida Geitler in samples from seep walls in Tennessee. This species is thought to be an extremely rare alga. It was reported previously only from seep walls in Europe. The last published reports of the alga were made in the mid‐1970s. A culture of the alga was made from one other site in North America (a rock wall in Arkansas) in 1977, but no publication accompanied that find. Since discovering the alga in summer 2004, we found a second population in a seep wall in northeast Ohio. We suspect that this species has a broader distribution than previously thought and has been overlooked or misidentified in past studies.     

A CELLULOSE SYNTHASE (CESA) GENE FROM THE RED ALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1

The cell walls of Porphyra species, like those of land plants, contain cellulose microfibrils that are synthesized by clusters of cellulose synthase enzymes (“terminal complexes”), which move in the plasma membrane. However, the morphologies of the Porphyra terminal complexes and the cellulose microfibrils they produce differ from those of land plants. To characterize the genetic basis for these differences, we have identified, cloned, and sequenced a cellulose synthase (CESA) gene from Porphyra yezoensis Ueda strain TU‐1. A partial cDNA sequence was identified in the P. yezoensis expressed sequence tag (EST) index using a land plant CESA sequence as a query. High‐efficiency thermal asymmetric interlaced PCR was used to amplify sequences upstream of the cDNA sequence from P. yezoensis genomic DNA. Using the resulting genomic sequences as queries, we identified additional EST sequences and a full‐length cDNA clone, which we named PyCESA1. The conceptual translation of PyCESA1 includes the four catalytic domains and the N‐ and C‐terminal transmembrane domains that characterize CESA proteins. Genomic PCR demonstrated that PyCESA1 contains no introns. Southern blot analysis indicated that P. yezoensis has at least three genomic sequences with high similarity to the cloned gene; two of these are pseudogenes based on analysis of amplified genomic sequences. The P. yezoensis CESA peptide sequence is most similar to cellulose synthase sequences from the oomycete Phytophthora infestans and from cyanobacteria. Comparing the CESA genes of P. yezoensis and land plants may facilitate identification of sequences that control terminal complex and cellulose microfibril morphology.     

STUDIES ON DEVELOPING AND RELEASED SPERMATIA IN THE RED ALGA,TIFFANIELLA SNYDERAE (RHODOPHYTA)1

Developing and released spermatia of the red alga, Tiffaniella snyderae (Farl.) Abb. were studied. Spermatia were observed under hydrodynamically defined conditions and found to be released from the exposed spermatangial heads in a spermatium-plus-strand unit that remained connected to the spermatangial head. Interactions of single-spermatial strands resulted in the formation of multi-spermatial strands as long as 600 μm with as many as 47 spermatia along their length; however, most were 100–200 μm with 8–21 spermatia. Strand length and number of spermatia were correlated. Spermatial strands contracted or extended and rotated as the water velocity past the plant was changed, and in still water the strands retracted into a clump on the spermatangial head surface. Each strand type exhibited a characteristic threshold water velocity at which it reached maximum length, and above which it broke and was carried away. Fluorescence microscopy showed that the strands did not contain nucleic acid (DNA) and could thus be differentiated from filamentous blue-green algal and bacterial epiphytes. Histochemical staining indicated that the strands and spermatial vesicles contained an acidic, sulfated polysaccharide. Chelation of Ca²⁺ with EGTA resulted in strand breakdown suggesting that this divalent cation may be involved in strand integrity. Scanning electron microscopy revealed that release from the spermatangia occurred through tears in the cuticle covering the spermatangial head if it was still present, or from exposed spermatangia. Individual spermatia were attached tangentially to a well-defined strand 0.64 μm in diameter in the contracted state to 0.2 μm in the extended state. Transmission electron microscopy of spermatangial heads showed that immature spermatangia were characterized by a centrally positioned nucleus and abundant ER cisternae filled with a moderately electron dense granular material. Later in development the spermatangia acquire two spermatial vesicles containing highly convoluted fibrillar contents. The cell becomes polarized with the nucleus displaced apically and the spermatial vesicles occupying the basal half of the spermatangium. At maturity one of the vesicles is released basally. Liberated spermatia contain a membrane-bound nucleus and mitochondria and are associated with an oblong accumulation of fibrous material similar in size and position to the strand observed with the SEM. These strands are discussed in relation to red algal fertilization and other phases of the red algal life-history.