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.     

Distribution of polyunsaturated fatty acids in red algae of the genus Gracilaria, a promising source of prostaglandins

The fatty acid (FA) composition and the content of the prostaglandin PGE₂ were determined in the red algae Gracilaria vermiculophylla and G. austramaritima from Peter the Great Bay (Sea of Japan), as well as in G. tenuistupitata, G. ?hangii, and G. bailiniae from lagoons in southern Vietnam (in the South China Sea). Polyunsaturated FA (PUFA) comprised 49?C56% of the total FAs in algae from the Sea of Japan, while in algae from the South China Sea their share was 20% at most. The content of arachidonic acid (20:4n-6) in the total FAs of G. vermiculophylla was as high as 45.4%, while the level of 20:4n-6 in Gracilaria from coastal waters of Vietnam did not exceed 12.5%. G. austramaritima stood out for its high content of eicosapentaenoic acid 20:5n-3 (33.5%). The ratios of 20:4n-6/20:5n-3 in G. vermiculophylla, G. austramaritima, G. tenuistupitata, G. changii, and G. bailiniae were 10.6, 0.3, 3.9, 4.0, and 1.5, respectively. The content of PGE₂ was the highest (286 ??g/g dry weight) in G. vermiculophylla from the Sea of Japan and did not exceed 20 ??g/g dry weights in other Gracilaria species. This study showed that it is possible to introduce G. vermiculophylla from the Sea of Japan into the mariculture of northern Vietnam. In the experiment, during 3 weeks of cultivation, the biomass of introduced Gracilaria increased 1.2?C1.3 times in a sea lagoon in Vietnam and 1.5?C2 times in an aquarium; the algal growth rates were 1.57 ± 0.21% per day. In cultivated Gracilaria, the level of 20:4n-6 decreased to 5.9% (20:4n-6/20:5n-3 = 2.3) and the level of PGE₂ decreased to 12 ??g/g in dry weight. The PUFA compositions of G. vermiculophylla from various natural populations differed insignificantly; however, the stress caused by introduction led to a sharp reduction in the content of 20:4n-6, which was probably connected with a decreased biosynthesis rate of PUFAs of the n-6 series. At the same time, approximately equal amounts of PGE₂ methyl ester were extracted from natural and cultivated G. vermiculophylla after treatment by a method proposed for obtaining prostaglandins. Thus, the cultivation conditions evidently did not influence the prostaglandin biosynthesis enzyme complex in G. vermiculophylla; this species, when grown in mariculture, can be used as a source of prostaglandins.     

Delimitating cryptic species in the Gracilaria domingensis complex (Gracilariaceae,Rhodophyta) using molecular and morphological data

Species in the genus Gracilaria that display conspicuously flattened vegetative morphologies are a taxonomically challenging group of marine benthic red algae. This is a result of their species richness, morphological similarity, and broad phenotypic plasticity. Within this group, the Gracilaria domingensis complex is one of the most common, conspicuous, and morphologically variable species along the tropical western Atlantic Ocean. Previous research has identified that members of this complex belong to two distantly related clades. However, despite this increased phylogentic resolution, species delimitations within each of these clades remain unclear. Our study assessed the species diversity within this difficult complex using morphological and molecular data from three genetic markers (cox1, UPA, and rbcL). We additionally applied six single‐marker species delimitation methods (SDM: ABGD, GMYCs, GMYCm, SPN, bPTP, and PTP) to rbcL, which were largely in agreement regarding species delimitation. These results, combined with our analysis of morphology, indicate that the G. domingensis complex includes seven distinct species, each of which are not all most closely related: G. cervicornis; a ressurected G. ferox; G. apiculata subsp. apiculata; a new species, Gracilaria baiana sp. nov.; G. intermedia subsp. intermedia; G. venezuelensis; and G. domingensis sensu stricto, which includes the later heterotypic synonym, G. yoneshigueana. Our study demonstrates the value of multipronged strategies, including the use of both molecular and morphological approaches, to decipher cryptic species of red algae.     

Cultivation of seaweed Gracilaria lemaneiformis enhanced biodiversity in a eukaryotic plankton community as revealed via metagenomic analyses

Plankton diversity reflects the quality and health of waters and should be monitored as a critical feature of marine ecosystems. This study applied a pair of 28S rRNA gene‐specific primers and pyrosequencing to assess the effects of large‐scale cultivation of the seaweed Gracilaria lemaneiformis on the biodiversity of eukaryotic plankton community in the coastal water of Guangdong, China. With 1 million sequences (2,221 operational taxonomic units [OTUs]) obtained from 51 samples, we found that the biodiversity of eukaryotic plankton community was significantly higher in the seaweed cultivation area than that in the nearby control area as reflected in OTU richness, evenness (Shannon–Wiener index) and dominance (Simpson index) for total plankton community and its four subcategories when Gracilaria biomass reached the maximum, while no such a significant difference was observed before seaweed inoculation. Our laboratory experiment using an artificial phytoplankton community of nine species observed the same effects of Gracilaria exposure. Principal component analysis and principal coordinates analysis showed the plankton community structure in cultivation area markedly differed from the control area when Gracilaria biomass reached its maximum. Redundancy analysis showed that G. lemaneiformis was the critical factor in controlling the dynamics of eukaryotic plankton communities in the studied coastal ecosystem. Our results explicitly demonstrated G. lemaneiformis cultivation could enhance biodiversity of plankton community via allelopathy, which prevents one or several plankton species from blooming and consequently maintains a relatively higher biodiversity. Our study provided further support for using large‐scale G. lemaneiformis cultivation as an effective approach for improving costal ecosystem health.     

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.     

Gracilaria and its epiphytes: 4. The response of two Gracilaria species to Ulva lactuca in a bacteria-limited environment

The responses of Gracilaria lemaneiformis, an easily epiphytized host,and the relatively resistant G. cornea mutant, to the green alga Ulva lactuca were studied using biculture experiments with and withoutantibiotics. Both Gracilaria species grown with and without U.lactuca showed different levels of growth rate, release of hydrogenperoxide and of halogenated hydrocarbons. These quantitative differencesled to a successful response against Ulva lactuca in the case of G.cornea mutant and to a failure in response in the case of G.lemaneiformis. The response of each Gracilaria species to U.lactuca was qualitatively similar to its response to bacteria. This suggeststhe involvement of oligosaccharide elicitors produced in the presence ofepiphytes and bacteria. A clear Gracilaria inhibition was demonstratedwith extracts of the culture medium. It appears that hydrogen peroxide,halogenated hydrocarbons and oligosaccharides may be components of theinhibitory activity of the extracts. The responses of Gracilaria speciesto the presence of U. lactuca suggest the characterization of adefence response.     

Comparison of three gracilarioids: growth rate, agar content and quality

Three gracilarioid species, Gracilariopsis bailiniae and Gracilaria tenuistipitata from Vietnam and Gracilaria gracilis from Russia, were studied in order to determine whether Gracilaria gracilis might be a superior species for cultivation in brackish-water ponds for agar production compared with the Vietnamese species. The effects of different salinity levels on the growth rate and agar production as well as agar properties of three gracilarioid species were compared in controlled laboratory experiments. Gracilaria tenuistipitata and G. gracilis were tolerant to low salinity (∼10‰), whereas Gp. bailiniae died under these conditions. G. tenuistipitata showed superior growth among the three species examined. Gracilaria gracilis had the highest agar content [36.8–46.6% dry weight (dw)]. Agar yield from Vietnamese gracilarioids did not exceed 30% dw. Gel strength of native agar from Gracilaria gracilis was two-fold higher that from Vietnamese species (278 g cm⁻² vs 130 g cm⁻²). Alkali pretreatment increased gel strength significantly for Gracilaria gracilis (1.4-fold), and G. tenuistipitata and Gp. bailiniae (2.3-fold) compared with native agar. The results suggest that Gracilaria gracilis may be a suitable species for production of reasonably good quality agar.     

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.     

Gracilaria corallicola and G. multipartita (Gracilariales,Rhodophyta), two related flattened European species

Two European species of Gracilaria possess flattened blades borne on cylindrical axes, namely, G. multipartita, known primarily from the Atlantic coast, and G. corallicola from the Mediterranean Sea. They are sister species that cluster with G. armata, G. bursa-pastoris and G. longa in rbcL analyses with strong bootstrap support. Blades of G. multipartita taper towards the tips, whereas those of G. corallicolla have broadly rounded tips. Spermatangia of G. corallicola are borne in shallow conceptacles (textorii-type) and data from the literature indicate that the same is true of G. multipartita. Cystocarp morphology is similar, with the gonimoblast filaments initially elongated, narrow and densely filled with cytoplasm, and with tubular nutritive cells issuing initially from lower gonimoblast cells and fusing with cells in the lowermost regions of the outer pericarp. Tetrasporangia are initiated terminally and displaced laterally with the production of side branches from the subterminal cell. The diagnostic characters of the Gracilariaceae are reviewed from a developmental perspective.     

Using gigartinine to track the distribution of an alien species of <Emphasis Type="Italic">Gracilaria</Emphasis> in New Zealand

The presence of gigartinine has been used previously as a taxonomic marker for the positive identification in Manukau Harbour (west coast, Auckland) of Gracilaria sp., which has apparently been introduced to New Zealand waters and is easily confused morphologically with the native species, G. chilensis. Thirty two estuarine/harbour populations of Gracilaria in New Zealand were screened for the presence of gigartinine to further test the utility of gigartinine as a reliable species marker. DNA fingerprinting was used to confirm the identity of a subset of the specimens surveyed. Using genetic rather than chemical characterisation, it was discovered that Gracilaria sp. is also present in Orakei Basin (east coast, Auckland). Although a sample from the wild did not have the anticipated gigartinine content, tank cultivation of thalli from this population in an artificially elevated nitrogen environment allowed the plant to accumulate nitrogen as gigartinine. This confirmed the unusual ability of this species of Gracilaria to store nitrogen in this form, unlike the widespread, morphologically similar, G. chilensis.     

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.     

Observations on Flattened Species of Gracilaria (Gracilariaceae, Rhodophyta) from Taiwan

Four flattened Gracilaria species have been reported from Taiwan: G. spinulosa, G. vieillardii, G. textorii and G. punctata, identified based on branching pattern, the presence or absence of spines, and characters that often vary seasonally. Gracilaria spinulosa was originally described from the type locality, Tainan. Species with toothed margins are usually referred to G. “vieillardii”; those with smooth margins to G. “textorii”, and those with smooth margins and dark spots scattered over the blade to G. “punctata”. Molecular analyses show that specimens with marginal teeth cluster in three different groups: a G. “vieillardii” clade, a G. spinulosa clade, and a clade sister to G. spinulosa. An undescribed species comprises the third clade, which is distinguished by its relatively large gonimoblast cells and weakly developed tubular nutritive cells. The three clades can be separated by the character of the tubular nutritive cells, the size of gonimoblast cells and certain vegetative features. Plants with entire margins form a single clade characterized by cystocarps with basal tubular nutritive cells and their absence in the cystocarp cavity. They are nested in the Hydropuntia complex and are referred to as Gracilaria “punctata” here. The records of G. textorii and G. punctata from Taiwan require reinvestigation in comparison with the Japanese species.     

GRACILARIA HUMMII SP. NOV. (GRACILARIALES,RHODOPHYTA), A NEW NAME FOR THE AGAROPHYTE “GRACILARIA CONFERVOIDES” HARVESTED IN NORTH CAROLINA DURING WORLD WAR II1

Gracilaria hummii Hommers. et Freshwater is proposed as a new name for the inshore cylindrical species found in North Carolina that was treated as Gracilaria confervoides (L.) Grev. during World War II, and more recently as G. verrucosa (Huds.) Papenf. Molecular evidence places G. hummii in the Gracilis‐group in Gracilaria together with G. gracilis (Stackh.) Steentoft, L. M. Irvine et Farnham, the name currently applied to specimens formerly identified as G. confervoides and G. verrucosa. G. hummii differs from G. gracilis in possessing shallower male conceptacles in which the spermatangial filament originates from a surface cortical cell rather than from a subcortical cell. The cystocarps are similar, except that the gonimoblasts of G. hummii are attached to the base of the pericarp by numerous, prominent thickened terminal tubular cells and because terminal tubular cells are absent above the midregion of the cystocarp or in the vicinity of the ostiole. The gonimoblasts are subtended by a bundle of longitudinally oriented, thick‐walled secondary filaments of a type that has not been described before in Gracilaria but that may be characteristic of some other species. G. hummii occupies a basal position in the Gracilis‐group and is distinct from all other cylindrical North Carolina Gracilaria species, according to the molecular and morphological evidence.     

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.     

Distribution and Identification of an Invasive Gracilaria Species that is Hampering Commercial Fishing Operations in Southeastern North Carolina, USA

A newly abundant Gracilaria species in the sounds of southeastern North Carolina has become a problem for commercial fishing and industries drawing water from the lower Cape Fear River. DNA sequence analyses have shown that this species is Gracilaria vermiculophylla, a taxon originally described from East Asia. Surveys for G. vermiculophylla have shown that it has a discontinuous distribution in the sounds of southeastern North Carolina, and suggest that it is spreading. Gracilaria vermiculophylla meets 6 of the 10 criteria used to help identify invasive species in that it has only recently appeared in southeastern North Carolina; is associated with human mechanisms of dispersal; has a restricted distribution; has disjunct populations in isolated oceans; no means of active dispersal, and an exotic evolutionary origin. The species may also meet two additional criteria as its local range is believed to be expanding, and it is filling a previously unoccupied seasonal niche. These factors taken together strongly suggest that G. vermiculophylla is an invasive species in southeastern North Carolina.     

The yield,physical and chemical properties of agar gel from Gracilaria species (Gracilariales,Rhodophyta) of the Kenya coast

Six species of Gracilaria, G. corticata J. Agardh, G. crassa Harvey, G. millardetii J. Agardh, G. salicornia (J. Ag.) Dawson, G. verrucosa (Huds.) Papenfuss and Gracilaria sp, collected from different stations along the Kenya coast were studied. The yield of hot water native agar extract ranges from 8.1–30% of dry weight, with G. verrucosa and G. salicornia having the greatest and the least yield, respectively. The gel-strength of 1.5% agar solution was highest in G. verrucosa (220 g cm^–2) and lowest in G. corticata (< 60 g cm – 2) whereas the highest gelling temperature was recorded for Gracilaria sp. (40.4 °C) and the lowest in G. verrucosa (28.9 °C). 3,6 anhydrogalactose content was the highest in G. verrucosa and the lowest in G. corticata while sulphate content was higher in the latter.     

The presence of gigartinine in a New Zealand Gracilaria species

Gigartinine, 5-(3-amidinoureido)-2-aminovaleric acid, serves as achemotaxonomic marker to distinguish two species of Gracilaria withvery similar morphologies. Gigartinine was identified by ¹³C-NMRspectroscopy and amino acid analysis of a cold-water extract from Gracilaria sp. nov., collected from a sheltered harbour localityat Blockhouse Bay, Auckland, New Zealand. Levels of this amino acid,naturally ca. 5% by dry weight of seaweed, were able to be depleted andthen restored during a nitrogen pulsing experiment. In contrast, native andpulsed samples of Gracilaria chilensis from Point Arthur, Wellingtonshowed no extractable gigartinine. Although these two species are unableto be distinguished in the field by morphological characteristics, they canbe separated by the presence or absence of gigartinine.