MOLECULAR SYSTEMATICS OF THE GRACILARIACEAE (GRACILARIALES,RHODOPHYTA) WITH EMPHASIS ON SOUTHERN AFRICA1

Southern Africa has economically exploited populations of terete gracilarioids on the cool temperate west coast and numerous species of endemic and Indo‐Pacific tropical Gracilariaceae on the south and east coasts. Gross morphological characters have been the main means of identification, and incorrect applications have led to a number of misidentifications. In this study, small subunit rDNA and RUBISCO spacer sequences were used to determine phylogenetic relationships. Whereas rDNA sequences successfully differentiate major groups within the family as well as species belonging to the Gracilariopsis and the Curdiea/Melanthalia clade, RUBISCO spacer sequencing was required to distinguish between species of Gracilaria. The southern African gracilarioid complex (stringy, terete, elongate members of the Gracilariaceae) was resolved into three species: Gracilaria gracilis, Gracilariopsis longissima, and Gracilariopsis funicularis. South African Gracilaria protea was shown to be conspecific with tropical Indian Ocean G. corticata. Apart from G. gracilis and G. corticata, South African Gracilaria species were differentiated into a temperate‐tropical terete grouping and a temperate‐tropical flattened grouping.     

PHYLOGENY AND SYSTEMATICS OF THE MARINE ALGAL FAMILY GRACILARIACEAE (GRACILARIALES,RHODOPHYTA) BASED ON SMALL SUBUNIT rDNA AND ITS SEQUENCES OF ATLANTIC AND PACIFIC SPECIES1

We sequenced the small subunit rDNA and internal transcribed spacer region of Gracilariaceae from the tropical Atlantic and Pacific, with emphasis on flattened or compressed species. Sequence comparisons confirmed three main lineages of Gracilariaceae: Curdiea/Melanthalia, Gracilariopsis/Gracilariophila, and Gracilaria. The Curdiea/Melanthalia diverged early in the family. Gracilariopsis was paraphyletic, because at least one Gracilariophila species evolved from it. The Atlantic Gracilariopsis were monophyletic and separated from the Pacific lineages. The Gracilaria included all species referable to its own species and to Hydropuntia, which was paraphyletic, formed by distantly related lineages. The new combination Gracilaria pauciramosa (N. Rodríguez Ríos) Bellorin, M. C. Oliveira et E. C. Oliveira is proposed for Polycavernosa pauciramosa N. Rodríguez Ríos. Recognition of subgenera within Gracilaria, based on spermatangial arrangement, was not supported. Instead, infrageneric groups were delineated by geographic origins and combinations of reproductive characters. Most Pacific species with either “textorii” or “verrucosa” type spermatangia were deeply separated from Atlantic species. Within the Atlantic Gracilaria, a lineage encompassing mostly tropical cylindrical species with “henriquesiana” type spermatangia and distinctive cystocarp anatomy was recognized. A lineage was also retrieved for cold water stringy species with verrucosa type spermatangia. Several species from the western Atlantic are closely related to Gracilaria tikvahiae McLachlan with nearly identical morphology. On the other hand, most flattened species from the tropical Atlantic were closely related despite their diverse morphologies. The interpretation of our data in addition to the literature indicates that more populations from the Indo‐Pacific must be studied before a general picture of Gracilariaceae evolution can be framed.     

COMPARATIVE MORPHOLOGY AND TAXONOMIC STATUS OF GRACILARIOPSIS (GRACILARIALES,RHODOPHYTA)1

The vegetative organization and reproductive development of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto et Foldvik [including Gracilaria sjoestedtii Kylin] were investigated. Our observations on spermatangial development and post-fertilization features establish that Gracilariopsis Dawson is distinct at the generic level from Gracilaria Greville, and ice propose the resurrection of Gracilariopsis Dawson as a result. Spermatangial parent cells of Gracilariopsis are superficial, initiated in pairs or groups of three by concavo-convex longitudinal and transverse divisions. Each spermatangial parent cell cuts off a single, colorless spermatangium distally by a transverse division. The female reproductive apparatus consists of a supporting cell that bears a two-celled carpogonial branch flanked by two sterile branches, as in Gracilaria. Likewise, up to six sterile cells fuse with the carpogonium after fertilization to produce a primary fusion cell that generates the gonimoblasts; however, a secondary fusion cell is absent. Inner gonimoblast cells unite with cytologically modified cells of the inner pericarp by means of secondary pit-connections. Tubular nutritive cells are absent. The gonimoblast consists of a central sterile tissue interconnected throughout by secondary pit-connections surmounted by a fertile layer composed of carposporangia aligned in straight chains. The distribution of Gracilariopsis is extended to Western Europe.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.     

Gracilaria vermiculophylla: A western Pacific species of Gracilariaceae (Rhodophyta) first recorded from the eastern Pacific

We report Gracilaria vermiculophylla (Ohmi) Papenfuss from the Pacific coast of North America based on a morphoanatomical revision and comparison of sequences of the nuclear gene coding for the small subunit of ribosomal RNA and the internal transcribed spacers of populations from Baja California, Mexico and Hiroshima, Japan. It is the first convincing report of this species out of its center of distribution in western north Pacific, where it has been considered as a synonym of former ‘G. verrucosa’ records. G. vermiculophylla also occurs in central California as indicated by internal transcribed spacer sequences of a previously unknown Gracilaria Greville material. In the northeastern Pacific G. vermiculophylla is characterized by a robust somewhat vermiform, well‐branched cylindrical thallus, with gradual cell size transition from cortex to medulla, deep spermatangial conceptacles, regular chains of carposporangia and carposporangial initials, downwardly oriented tubular nutritive cells, but rarely with upwardly oriented tubular nutritive cells. In some features of its cystocarp anatomy G. vermiculophylla is related to Gracilariopsis E. Y. Dawson or Hydropuntia Montagne and it is a relevant species for discussions about Gracilariaceae genera.     

Inter- and intraspecific variation of nuclear DNA reassociation kinetics in the Gracilariales (Rhodophyta)

DNA reassociation kinetics were used to determine inter- and intraspecific variation in genome organization and complexities in species ofGracilaria andGracilariopsis. Results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repeated sequences varied from 13–95%. Three geographic populations ofGracilaria tikvahiae were similar with 13–27% repeated sequences.Gracilaria sp. cultivars G-1 and G-6 with 35% and 95% repeated sequences, respectively, were distinct from each other andG. tikvahiae. No pattern of genome organization and complexity was found which permitted a distinction betweenGracilaria andGracilariopsis. Comparison of the percent of unique and repetitive sequences (U/R) indicated a wide range of ratios, withGracilaria tikvahiae populations having the highest values (2.7–7.3) andGracilaria sp. cultivar G-6,G. blodgettii andGracilariopsis lemanieformis the lowest (0.05–1.80). Unique component complexities varied one order of magnitude, from 10⁸ forGracilaria takvahiae to 10⁷ forGracilaria sp. cultivar G-6,G. blodgettii andGracilariopsis lemanieformis. Information for genome size, organization and complexity is used to develop a nuclear genome profile forGracilaria blodgettii andGracilariopsis lemanieformis which are characterized by commercial grade agars having high gel strengths (> 700 g cm^?2) and elevated melting temperatures (99 °C).     

Karyology and cytophotometric estimation of nuclear DNA content variation in Gracilaria,Gracilariopsis and Hydropuntia (Gracilariales,Rhodophyta)

Chromosome numbers of 1 N=24 were determined for three species of Gracilaria (G. flabelliforme P. Crouan et H. Grouan ex Schramm et Maze, G. mammillaris Montagne and G. tikvahiae McLachlan) and 1 N=32 for two species of Gracilariopsis (G. lemaneiformis (Bory) Dawson, Acleto et Folvik and G. tenuifrons (Bird et Oliveira) Fredericq et Hommersand). Karyotypes for these species exhibit a characteristic size difference between largest and smallest chromosomes. Polyvalents were a common feature of meiotic nuclei. Microspectrophotometry with the DNA-localising fluorochrome DAPI was used to quantify nuclear genome sizes. A 2 C genome size of 0·37–0·40 pg was determined for five species of Gracilaria (G. chilensis Bird, McLachlan et Oliveira, G. flabelliforme, G. mammillaris, G. pacifica Abbott, G. tikvahiae) and 0·33 pg for an isolate of G. verrucosa (Hudson) Papenfuss from Pas de Calais, France. Species of Hydropuntia (H. cornea (J. Agardh) Wynne and H. dentata (J. Agardh) Wynne) and Gracilariopsis (G. lemaneiformis and G. tenuifrons) were found to have slightly larger 2 C genome contents of 0·42–0·47 pg. No intraspecific variation in 2 C genome sizes was found in regional populations of Gracilaria tikvahiae and Gracilariopsis tenuifrons.     

Molecular characterisation and development of rapid molecular methods to identify species of Gracilariaceae from the Atlantic coast of Morocco

In Gracilariaceae, species identification is traditionally based on gross morphology; therefore the taxonomic status of terete individuals remains frequently problematic due to the lack of diagnostic characters to identify specimens. Different morphospecies have been recorded along the Atlantic coast of Morocco; however, no clear diagnostic characters were available to discriminate between terete species. Rapid molecular techniques have been developed recently to resolve many taxonomic problems and to re-assess the global diversity and biogeography in algae. In this study, molecular markers were used as DNA barcoding to characterise species. The sequence of the Rubisco spacer allowed identification of six species of Gracilariaceae: Gracilaria gracilis, Gracilaria dura, Gracilaria conferta, Gracilaria vermiculophylla, Gracilaria multipartita and Gracilariopsis longissima. In order to identify species with certainty, two simple and rapid methods based on the amplification of rDNA ITS and PCR-RFLP of the large subunit of the Rubisco were developed.     

FATTY ACID DISTRIBUTION AMONG SOME RED ALGAL MACROPHYTES1

Macrophytic members of the Rhodophyta, which were grown under controlled conditions, were analyzed for their fatty acid distribution. Significant differences were found between some species of the Gelidiales and Gracilariales. Two dominant polyunsaturated fatty acids of the 20 carbon chain group, 20:4ω6 and 20:5ω3, were found in all the Gelidiales species, whereas only 20:4ω6 was found in the Gracilariales species. An inverse relationship between the content of these two polyunsaturated fatty acids in Gelidiales species and among the two groups of species are reported and discussed. All other fatty acid characteristics were more or less similar in these two orders. We cannot draw any solid conclusion about the relationship between Gracilariopsis cf. lemaneiformis (formerly Gracilaria lemaneiformis) and the genus Gracilaria, since our results (the existence of 20:4ω6 and 20:5ω3 in Gracilariopsis) contradict results found by other researchers. The diverse growth conditions of photon flux density and temperature caused some differences in the distribution of the fatty acids in each species. These differences could not explain the different results in similar species reported in the literature.     

DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE‐REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR‐ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.     

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.     

Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content

Select species of the agarophyte Gracilaria were studied from southeastern Australia. The morphology and anatomy of species is described and molecular relations are inferred based on plastid and mitochon‐drial DNA sequence data. Agar yields and qualities are determined for each species. Gracilaria chilensis, found in Tasmania and Victoria, is morphologically and molecularly similar to G. chilensis from New Zealand and Chile and has low agar yields of 11–16%. Gracilaria cliftonii from Victoria, has high crude agar yield (52%) and is molecularly uniform. Gracilaria perplexa sp. nov., known only from Botany Bay, New South Wales, has an agar yield of 39%. The agar of G. perplexa is unusual in requiring the addition of 0.1 mol L^?1 NaCl for alcohol precipitation and is cold‐water (25°C) soluble because of the very high sulfate ester content. Molecular phylogeny shows that G. perplexa is closely related to Gracilaria preissiana from western Australia, but differs from the latter in its reduced branching and narrower more terete axes.     

Seasonal growth activity of local and foreign gracilarioid strains in Israel

Six gracilarioid strains originating from different climatical environments were cultured in two cultivation systems: a short-term indoor one with a cross gradiant table, and a long-term outdoor one. Seasonal growth performances of the different strains were determined. The growth results in the two culture systems showed similar trends. The tropical species.Gracilaria cornea andG. cornea mutant, showed highest growth rates during summer and no growth at all during winter. The temperate species,Gracilaria verrucosa andGracilariopsis lemaneiformis, showed best growth performances during winter with small fluctuations between seasons. The subtropical speciesGracilaria conferta (local) showed seasonal growth fluctuations all over the year. The foreign species definitely did not acclimate under local conditions, but successfully preserved their original response to temperature. Regression equations confirmed that temperature was the dominant environmental variable in most of the gracilarioid strains. The growth rate results obtained showed encouraging prospects for high algal productivity as compared to other cultivation systems. Seasonal cultivation strategy ofGracilaria spp. in Israel is discussed.     

GRACILARIOPSIS MCLACHLANII SP. NOV. AND GRACILARIOPSIS PERSICA SP. NOV. OF THE GRACILARIACEAE (GRACILARIALES,RHODOPHYCEAE) FROM THE INDIAN OCEAN1

Two new species of Gracilariopsis from the Indian Ocean are proposed—Gracilariopsis (Gp.) mclachlanii Buriyo, Bellorin et M. C. Oliveira sp. nov. from Tanzania and Gracilariopsis persica Bellorin, Sohrabipour et E. C. Oliveira sp. nov. from Iran—based on morphology and DNA sequence data (rbcL gene and SSU rDNA). Both species fit the typical features of Gracilariopsis: axes cylindrical throughout, freely and loosely ramified up to four orders, with an abrupt transition in cell size from medulla to cortex, cystocarps lacking tubular nutritive cells and superficial spermatangia. Nucleotide sequence comparisons of rbcL and SSU rDNA placed both species into the Gracilariopsis clade as distinct species from all the accepted species for this genus, forming a deeply divergent lineage together with some species from the Pacific. The new species are very difficult to distinguish on morphological grounds from other species of Gracilariopsis, stressing the importance of homologous molecular marker comparisons for the species recognition in this character‐poor genus.     

LOW GENETIC VARIABILITY OF SARGASSUM MUTICUM (PHAEOPHYCEAE) REVEALED BY A GLOBAL ANALYSIS OF NATIVE AND INTRODUCED POPULATIONS1

Sargassum muticum (Yendo) Fensholt is one of the most well‐known invasive species in the world. There have, however, been few genetic investigations on both its introduced and native populations. There are also some questions about the taxonomic status of this species. This study is the first to assess the genetic diversity of S. muticum on a global scale, by utilizing one marker each from the extranuclear genomes, namely, plastidial RUBISCO and mitochondrial TrnW_I spacers, as well as the nuclear internal transcribed spacer 2 (ITS2). Based on the markers investigated, both the invasive as well as the native populations of this species appeared very homogenous, when compared with other invasive and brown macroalgae. No variation in ITS2 and RUBISCO spacer was revealed in S. muticum populations, including those from its native ranges in Asia and the introduced ranges in Europe and North America. Two TrnW_I spacer haplotypes with a fixed two‐nucleotide difference were found between the populations of eastern Japan and the other 15 populations examined. This study confirms that there is no cryptic diversity in the introduced range of this species. All the materials collected globally are indeed S. muticum. Results depicting the distribution range of the two TrnW_I spacer haplotypes also support the earlier suggestion that the source of the introduced S. muticum populations is most likely western and central Japan (Seto Inland Sea), where the germlings of S. muticum were likely to have been transported with the Pacific oysters previously introduced for farming in Canada, UK, and France in earlier years.     

SINGLE LOCUS MICROSATELLITES IN GRACILARIALES (RHODOPHYTA): HIGH LEVEL OF GENETIC VARIABILITY WITHIN GRACILARIA GRACILIS AND CONSERVATION IN RELATED SPECIES1

Four single locus microsatellites identified in the red alga Gracilaria gracilis (Stackhouse) Steentoft, Irvine, et Farnham (Rhodophyta) were examined for allelic diversity at different spatial and taxonomic levels. First, because simple morphological diagnostic characters are often missing within the Gracilariaceae, we developed a simple and rapid method based on rDNA ITS size variation in order to verify the taxonomic status of the samples used in this study. All European (including Mediterranean samples), Argentinian, and Namibian samples used in our study were confirmed to be a homogenous G. gracilis group. By contrast, our results on rDNA ITS sizes showed that Gracilaria from Japan, initially identified as G. gracilis, was different from the rest of the G. gracilis group. Secondly, microsatellite polymorphism and conservation at the species level was tested on the worldwide collection of G. gracilis and within a single population. The loci Gv1AAG and Gv1AAC showed no allelic variation, whereas two others, Gv1CT and Gv2CT, were highly polymorphic. All microsatellite loci were conserved within G. gracilis, except in the sample from Japan. The taxonomic status of G. gracilis from Japan is thus questionable. This study revealed a high level of within-population polymorphism (52 alleles for Gv1CT and 12 for Gv2CT). Moreover, the combination of these two loci was shown to be very powerful for identifying individuals within a population, that is, 93% of the individuals were characterized by a unique genotype. Finally, conservation of the four loci was tested in taxonomically related species of Gracilaria (G. chilensis, G. pacifica, and G. tikvahiae) and two Gracilariopsis species (Gs. sp. and Gs. longissima). The results suggest that the polymorphic locus Gv2CT may provide a valuable genetic marker within the different species of the Gracilariaceae.     

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.     

Quantification and characterization of nuclear genomes in commercial red seaweeds (Gracilariales) from the Philippines

Eight species of Gracilariaceae from the Philippines, representing the generaGracilaria, Gracilariopsis andHydropuntia, were investigated to quantify and characterize their nuclear genomes. DNA reassociation kinetics were used to determine nuclear genome organization and complexity in six of these species. Results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–74% and unique DNA ranged from 26–84%. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA contents. Comparisons of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in an estimate of 0.38–0.43 pg/2 C genomes for seven of the species investigated. Preliminary analyses of agar content and quality confirm the economic potential ofGracilaria firma, Gracilaria sp. 2 from Sorsogon andGracilariopsis bailinae. Nuclear genome profiles developed from data 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.Author for correspondence