Genetic differentiation and distribution of Pyropia acanthophora (Bangiales,Rhodophyta) in the Philippines

Pyropia acanthophora is a foliose Bangiales with widely known endemic populations in Indo-Pacific region. This alga has expanded its range recently as a consequence of introduction. In an attempt to explore the genetic diversity of Py. acanthophora within the Philippines and the impact of the introduction of the species from elsewhere, an examination of molecular differentiation and distribution was undertaken using the mitochondrial COI-5P and plastid rbcL gene sequences. The results revealed that the populations of Py. acanthophora in the Philippines exhibited high haplotypic and genetic diversities, and were found to be distinct from those previously reported as conspecific populations found in Taiwan, India, Japan, Hawaii, and from those introduced populations from Brazil. The network analyses as inferred from rbcL and from the combined COI-5P and rbcL genes showed evidence that the Philippine populations of Py. acanthophora exhibited a chaotic patchiness pattern characterized by a population with highly site-exclusive haplotypes, wide genetic variability and lack of local geographic patterns. The distribution of Py. acanthophora within the Philippines was also found to be greater than what was previously known, ranging from the extreme northwest to extreme northeast mainland Luzon coasts, including Camiguin Is., Cagayan. Understanding the genetic diversity and distribution of Py. acanthophora in the Philippines provides valuable information in relation to the conservation and effective resource management of native populations of Py. acanthophora in the tropical Asian region.     

Growth rate of the carrageenophyte Kappaphycus alvarezii (Rhodophyta, Gigartinales) in vitro

Unialgal cultures were established trom a 2.5 g branch ot a brown variant ot Kappaphycus alvarezii (Doty) Doty ex P. Silva, with the main objective to produce branches for monthly outplanting in the sea. Ditterent conditions were tested to optimize production ot branches in the laboratory. The best growth was obtained under culture conditions of 25 ± 2°C, 170–210 μmol photons m² s^?1, 14:10 LD photoperiod and salinity 32–35‰. Three culture media (Provasoli,‘F/2’and von Stosch) were tested. Deleterious ettects were observed in branches incubated continuously in full‐ or halt‐strength Provasoli's enriched seawater medium (PES). Exponential growth rates ot about 3% day^?1 were obtained using PES, pulse‐fed 24 h per week, or other diluted media used continuously (one‐quarter strength ‘F/2’and half‐strength von Stosch). Laboratory‐grown branches with mean weights from 2.97 to 4.25 g were successfully introduced into the sea at Ubatuba, SP, Brazil (23°26.9′S, 45°0.3′W) an area with mean monthly seawater temperature from 20.3 to 28.5°C (extremes: 17.0–31.0°C). Transplantation of branches produced in unialgal culture, as done in the present study, avoids the risk of introduction of unwanted species into new areas.     

Time-dependent attachment mechanism of bacterial pathogen during ice-ice infection in Kappaphycus alvarezii (Gigartinales, Rhodophyta)

The mechanism of infection by Vibrio sp. P11 promoting the ice-ice disease in Kappaphycus alvarezii was investigated in vitro. Its intensity of infection differs from that of another ice-ice promoter (Cytophaga sp. P25) by promoting the disease much faster. However, when secondary infection by other bacteria starts, its ability to compete with these bacteria gradually diminishes, whereas, infection by P25, although not displaying such drastic effects as P11, shows consistent competitive ability against other bacteria. Time-series infection experiments with application of polyclonal antibodies to specifically detect Vibrio sp. P11 revealed that this bacterium has a high affinity for the seaweed especially when the latter is stressed. It promotes the disease after a rapid increase in cell density of up to 10⁷ g⁻¹ (wet wt.) in the first 24 h. This bacterial cell build-up may take only 1–2 h on stressed thalli, but takes about 24 h on non-stressed thalli. Build-up is not sustainable in non-stressed thalli as high density is usually followed by a sudden decline in cell number believed to result from an algal defence against potential pathogens. Inoculation of the bacterium on thalli incubated in continuous culture system extends the time of bacterial attachment due to laminar flow and, possibly, competition by existing bacteria on the seaweed surface and in ambient seawater medium. Motility-driven cell attachment by this bacterium is suggested as an important factor for infection. This revised version was published online in June 2006 with corrections to the Cover Date.     

Introduction of non-native species of Kappaphycus (Rhodophyta, Gigartinales) in subtropical waters: Comparative analysis of growth rates of Kappaphycus alvarezii and Kappaphycus striatum in vitro and in the sea in south-eastern Brazil

We compared the growth rates of Kappaphycus alvarezii (Doty) Doty ex P. Silva and Kappaphycus striatum (Schmitz) Doty, both, in vitro under different conditions of light and temperature, and in the sea. Temperature was the most important factor controlling the growth of both species, in vitro and in the field. In the sea there was a clear seasonal pattern in growth rate, attributed to temperature and salinity variation. The lower growth rates were registered in winter and spring, and the highest in summer and autumn months for both species. Based on growth rate in the field, and the production of viable tetraspores during the summer in Kappaphycus striatum, we conclude that is more profitable, and ecologically safer, to only continue with the introduction program of Kappaphycus alvarezii.     

Hypnea species (Gigartinales,Rhodophyta) from the southeastern coast of Brazil based on molecular studies complemented with morphological analyses,including descriptions of Hypnea edeniana sp. nov. and H. flava sp. nov.

The red algal genus Hypnea (Gigartinales) has a wide geographical distribution along tropical and subtropical coasts around the world. The relatively simple and plastic morphology, often influenced by the conditions of its habitat, complicates the identification of Hypnea species. Therefore, the number and status of some species remain in doubt. Molecular studies have been performed to supplement traditional studies based on morphology, mainly for Hypnea species occurring in Asia. In the present study, sequence data from the DNA barcode COI-5P for 114 samples from the southeastern coast of Brazil, indicated the occurrence of six taxa. Additionally, sequence data from the UPA and rbcL markers for representatives of each of those taxa confirmed the existence of six different species. After morphological analysis and comparison with sequences available in GenBank, these species were named as follows: H. aspera, H. cervicornis, H. cf. musciformis, H. spinella, and two new species, H. flava Nauer, Cassano & M.C. Oliveira and H. edeniana Nauer, Cassano & M.C. Oliveira. Hypnea cervicornis, often considered as a later synonym of H. spinella, should be considered as a distinct species based on morphology and divergence of the three molecular markers used. Hypnea aspera is a new record for the Atlantic Ocean.     

Laboratory-induced development of the ice-ice disease of the farmed red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The most common perception of unfavorable environmental factors causing the ice-ice disease in the farmed seaweeds, Kappaphycus and Eucheuma, was demonstrated in this study for the first time using stressful conditions of abiotic factors in a continuous culture system. Light intensity of less than 50 mol photon m^–2 s^–1 and salinity of 20% or less induced ice-ice whitening characterized by short segments at midbranches which were similar to those observed in the Philippine seaweed farms, while temperatures of up to 33–35 °C resulted in wide-scale whitening leading to complete damage of the branches. These effects were preceded by slow growth rates from an optimum of 3.7% d^–1 to almost –2.0% d^–1. Mechanical stress by wound injury did not result to ice-ice whitening similar to the above. Environmental factors observed to trigger ice-ice in the laboratory, although may not necessarily parallel those in the field, may act synergestically to produce similar effects.     

Effect of ammonium on growth and carrageenan content in Kappaphycus alvarezii (Gigartinales,Rhodophyta)

Kappaphycus alvarezii, cultured in a region deficient in nitrogen with intermittent application of 10 mM ammonium at three-day intervals for 1 hour, attained a daily growth rate of 4.6%, which was considered to be the optimum growth rate. Under such conditions, the C/N ratio was 29 with a carrageenan content of 58% and gel strength of 45–70 g cm^–2. Gel strength of carrageenan was high when nitrogen content was high and vice versa.     

Species delimitation methods reveal cryptic diversity in the Hypnea cornuta complex (Cystocloniaceae,Rhodophyta)

The simple and convergent morphologies of many red algae make these species difficult to identify using traditional morphological characters. Many cryptic species have been described in recent years based on molecular datasets, and this has led to the application of an integrative taxonomy approach in species delimitation. Here, we performed several species delimitation methods (mBGD, ABGD, SPN, PTP, GMYCs and GMYCm) based on two different loci (COI-5P and rbcL) in species of the Hypnea cornuta complex. These methods were combined with morphological and phylogenetic data, extensive sampling, analysis of topotype material, and historically relevant herbarium samples. Our findings demonstrate that the groups morphologically assigned to H. cornuta and H. stellulifera consist of five different cryptic species. H. cornuta is a polyphyletic taxon composed of three well-separated lineages, thus requiring sequencing of type or topotype specimens to determine which one is Hypnea cornuta sensu stricto. We have revealed that the distribution of H. stellulifera is limited to Asia, while the Brazilian specimens initially assigned to this species were clarified as a new endemic species: Hypnea cryptica sp. nov. Our results indicated that only an integrative approach combining several lines of evidence, including morphology, nomenclature history, molecular data, biogeography and ecology can correctly solve the taxonomic status of widely distributed cryptic species.     

A molecular assessment of species diversity and generic boundaries in the red algal tribes Polysiphonieae and Streblocladieae (Rhodomelaceae,Rhodophyta) in Canada

Sequence data generated during a Canadian barcode survey (COI-5P) of the tribes Polysiphonieae and Streblocladieae, a large and taxonomically challenging group of red algae, revealed significant taxonomic confusion and hidden species diversity. Polysiphonia pacifica Hollenberg, P. paniculata Montagne, P. stricta (Dillwyn) Greville and Vertebrata fucoides (Hudson) Kuntze were all complexes of two or more genetically distinct yet overlooked species. One variety of P. pacifica was elevated to the rank of species as P. determinata (Hollenberg) Savoie & Saunders, stat. nov. Several new additions to the Canadian flora were recorded including P. kapraunii Stuercke & Freshwater and P. morrowii Harvey. Subsequent multi-gene (COI-5P, LSU and rbcL) phylogenetic analyses confirmed that the genus Polysiphonia Greville was polyphyletic, and currently assigned species resolved with many other genera. Polysiphonia sensu stricto was restricted to a group of species that formed a monophyletic lineage with the type, Polysiphonia stricta. Carradoriella P.C.Silva was resurrected based on the South African species Carradoriella virgata (C.Agardh) P.C.Silva. Species previously attributed to Polysiphonia were transferred to Carradoriella, Leptosiphonia and Vertebrata as well as to three new genera described here: Acanthosiphonia gen. nov., based on A. echinata (Harvey) comb. nov.; Eutrichosiphonia gen. nov. for E. confusa (Hollenberg) comb. nov. and E. sabulosia (B.Kim & M.S.Kim) comb. nov.; and Kapraunia gen. nov., which includes K. schneideri (Stuercke & Freshwater) comb. nov. and three additional species.     

Growth rate of the carrageenophyte Kappaphycus alvarezii (Rhodophyta, Gigartinales) introduced in subtropical waters of São Paulo State, Brazil

Twenty branches of Kappaphycus alvarezii (Doty) Doty ex P. Silva produced in unialgal culture and weighing about 3 g each were transferred into the sea monthly from October 1995 to October 1996 at Ubatuba Bay, São Paulo State, Brazil (23°26.9′S, 45°0.3′W), an area with mean monthly seawater temperature from 20.3 to 28.5°C (extremes: 17.0–31.0°C). All plants were harvested after the second month and a new series of growth experiments was performed using cuttings weighing from 100 to 150 g. The cultivation experiments were carried out using a floating system. Daily growth rates were calculated based on monthly weight measurements of each individual. Small branches produced in vitro showed high survival rates when introduced into the sea during all seasons. Growth rates observed for the transplanted branches during the first and second months in the sea were higher (6.5–10.7% day‐¹) than for subsequent cuttings produced in the sea (4.5–8.2% day‐¹). The latter values are in the range reported for other regions. Seasonal variation of growth rates was clearly related to seawater temperature. These results show that the commercial cultivation of K. alvarezii is technically feasible at Ubatuba Bay using a floating raft culture method.     

Seasonal Occurrences of Epiphytic Algae on the Commercially Cultivated Red Alga Kappaphycus Alvarezii (Solieriaceae, Gigartinales, Rhodophyta)

Common problems faced in farming of the red algal genus Kappaphycus/Eucheuma are “ice-ice disease” and the occurrence of epiphytes. Considerable work has been documented on “ice-ice disease” and it's mode of infection but limited information is available on the emergence of epiphytes. The present study addresses the phenomenon of epiphyte infection, its prevalence in commercially cultivated red alga, Kappaphycus alvarezii, and their variability associated with seasonality. Cultured seaweed became susceptible to epiphytes in the dry seasons (1) between March – June and (2) September – November. Findings revealed Neosiphonia savatieri (Hariot) M. S. Kim et I. K. Lee, as the dominant infecting epiphyte, representing up to 80–85% of the epiphyte present during peak seasons. Besides N. savatieri, Neosiphonia apiculata, Ceramium sp., Acanthophora sp. and Centroceras sp. were observed in smaller quantities. SEM (Scanning Electron Microscope) micrographs revealed the epiphyte's attachment to the host. Further histological study showed the extent of penetration of epiphytes into the host's cortex tissues and condition of its surrounding tissues. The outbreak of epiphytic filamentous red algae also correlated with drastic changes in seawater temperature and salinity during March– June and September – November.     

Occasional pathogenic bacteria promoting ice-ice disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The bacterial isolates from normal and diseased branches of Kappaphycus alvarezii and Eucheuma denticulatum in the Philippines were examined for possible role in the development of the ice-ice disease. The numbers of bacteria on and in ice-iced branches were 10–100 times greater than those from normal, healthy ones. Gram-positive bacteria predominated in almost all branch sources, but with an increasing proportion of agar-lysing bacteria in branches suffering from the ice-ice disease. These agar-lysing bacteria were composed of yellow and non-pigmented, spreading colonies identified to the Cytophaga-Flavobacterium complex and the Vibrio group. Among isolates which mainly appeared on ice-iced branches, two strains, designated as P11 (Vibrio sp.) and P25 (Cytophage sp.), which showed pathogenic activity, were obtained. These strains caused early ice-ice whitening of K. alvarezii especially when subjecting branches to environmental stress, such as reduced salinity and light intensity, suggesting that these bacteria were occasionally pathogenic. This paper offers new evidence of bacterial role in the development of so-called ice-ice disease among farmed species of Kappaphycus.     

Kappaphycus malesianus sp. nov.: a new species of Kappaphycus (Gigartinales,Rhodophyta) from Southeast Asia

A new species, Kappaphycus malesianus, is established as a new member of the genus Kappaphycus. Locally known as the “Aring-aring” variety by farmers in Malaysia and the Philippines, this variety has been commercially cultivated, often together with Kappaphycus alvarezii due to the similarities in morphology. Despite also producing kappa-carrageenan, the lower biomass of the K. malesianus when mixed with K. alvarezii ultimately affects the carrageenan yield. Morphological observations, on both wild and cultivated plants, coupled with molecular data have shown K. malesianus to be genetically distinct from its Kappaphycus congeners. The present study describes the morphology and anatomy of this new species as supported by DNA data, with additional morphological features for distinguishing between commercial Kappaphycus cultivars.     

Phylogeography of the red alga Gracilariopsis tenuifrons (Gracilariales) along the Brazilian coast

Changes in the sea level during the Holocene are regarded as one of the most prevalent drivers of the diversity and distribution of macroalgae in Brazil, influenced by the emergence of the Vitória-Trindade seamount chain (VTC). Gracilariopsis tenuifrons has a wide geographic distribution along the Brazilian coast, from Maranhão state (2°48′64.3" S) to Santa Catarina state (27.5°73′83" S). The knowledge of historical processes affecting diversity may allow the development of conservation strategies in environments against anthropogenic influence. Therefore, knowledge about phylogeography and populational genetic diversity in G. tenuifrons is necessary. Six populations were sampled along the northeastern tropical (Maranhão—MA, Rio Grande do Norte—RN, Alagoas—AL, and Bahia—BA States) and southeastern subtropical (São Paulo “Ubatuba”—SP1 and São Paulo “Itanhaém”—SP2 States) regions along the Brazilian coast. The genetic diversity and structure of G. tenuifrons were inferred using mitochondrial (COI-5P and cox2-3 concatenated) DNA markers. Gracilariopsis tenuifrons populations showed an evident separation between the northeast (from 2°48′64.3" S to 14°18′23" S; 17 haplotypes) and the southeast (from 23°50′14.9" S to 24°20′04.7" S; 10 haplotypes) regions by two mutational steps between them. The main biogeographical barrier to gene flow is located nearby the VTC. The southeast region (São Paulo State) is separated by two subphylogroups (SP1, three haplotypes and SP2, six haplotypes), and Santos Bay (estuary) has been considered a biogeographical barrier between them. The presence of genetic structure and putative barriers to gene flow are in concordance with previous studies reporting biogeographic breaks in the southwest Atlantic Ocean, including the genetic isolation between northeast and southeast regions for red and brown algae in the vicinity of the VTC.     

Callus ontogeny of the Kappaphycus alvarezii (Rhodophyta, Gigartinales) brown tetrasporophyte strain

This study analyzes the callus ontogeny of the Kappaphycus alvarezii brown strain tetrasporophyte using micropropagation and tissue culture. Calli were induced from axenic explants and grown for 2 months. Ontogenic analysis was made in explants collected from days?7 to 60 of culture and observed under light and transmission electron microscopy (TEM). At the end of the experimental period, 60-day samples were also analyzed by confocal microscopy. Formation of calli was initiated in the first week from medullary and cortical cells of the explant region in air contact. Alterations in the apical region of the cells were observed in TEM as cell wall thickening, proliferation of convoluted membranes, increased number of mitochondria and chloroplast alterations, indicating the dedifferentiation of these cells. Throughout the experiment, the thick cell wall of the cells which compose callus filaments showed the presence of acidic polysaccharides, suggesting a large amount of carrageenan and neutral polysaccharides. The cytoplasm showed the following main characteristics: large amount of starch grains, presence of chloroplasts with altered plastoglobules, as well as disorganized thylakoids, presence of several convoluted membranes, and vacuoles. These filaments maintained their uniseriate organization and irregular branching with many intercellular connections between them. Despite the initial stress caused by explant isolation and the beginning of callus formation, results showed that filaments were capable of reorganization and development, retaining their dedifferentiated cells, which, in turn, strongly suggested the potential of callus-based micropropagation.     

Non-enzymatic isolation of somatic cells from Kappaphycus spp. and Eucheuma denticulatum (Solieriaceae,Rhodophyta)

Cell culture technology is immensely useful in somatic hybridization, induction of mutations, cloning of specific isolates and maintenance of strains of defined genotypes. However, its application in strain improvement of some tropical red macroalgae has been limited due to the difficulty of isolating viable cells from their complex intercellular matrices. A simple, non-enzymatic technique of isolating somatic cells was developed for Kappaphycus spp. and Echeuma denticulatum. Surface-sterilized tissues (0.1 g fresh weight, 2.0 mm thick discs) from subcortical and medullary layers were treated with 3% NaOH, 3% KOH, or hydrogen peroxide in phosphate buffer solution (PBH₂O₂). Tissues of K. alvarezii treated with PBH₂O₂ softened after 5 h of treatment and completely dissociated after 12 h. Viable cell counts (VCC), determined through staining with Evan’s blue, were significantly higher (2.4 × 10⁵ cells g^?1 fresh weight tissue) in K. alvarezii (‘tambalang’ strain) treated with PBH₂O₂ compared with tissues treated with carrageenase from a marine bacterium.     

Systematics and genetic variation in commercial shape Kappaphycus and shape Eucheuma (Solieriaceae, Rhodophyta)

The systematics and taxonomy of Kappaphycus and Eucheuma (Solieriaceae) is confused and difficult due to morphological plasticity, lack of adequate characters to identify species and commercial names of convenience. These taxa are geographically widely dispersed through cultivation. Commercial, wild and herbarium sources were analysed; molecular markers provided insights into taxonomy and genetic variation, and where sources of genetic variation may be located. The mitochondrial cox2-3 and plastidal RuBisCo spacers were sequenced. There is a clear genetic distinction between K. alvarezii (“cottonii”) and K. striatum (“sacol”) samples. Kappaphycus alvarezii from Hawaii and some samples from Africa are also genetically distinct. Our data also show that all currently cultivated K. alvarezii from all over the world have a similar mitochondrial haplotype. Within Eucheuma denticulatum (“spinosum”) most African samples are again genetically distinct. Our data also suggest that currently cultivated E. denticulatum may have been “domesticated” several times, whereas this is not evident for the cultivated K. alvarezii. The present markers used do not distinguish all the morpho-types known in cultivation (e.g. var. tambalang, “giant” type) but do suggest that these markers may be useful to assess introductions and species identification in samples.     

The farmed Eucheuma species (Gigartinales,Rhodophyta) in Danajon Reef,Philippines: carrageenan properties

Six cultured strains of Eucheuma denticulatum and E. alvarezii, from which stocks can be selected for the development of a Eucheuma seedling bank, were tested for their carrageenan quality from June to November 1988. Percent yield of all the varieties taken together was apparently higher in June, becoming lower in November (regression, r –0.785, probability, p 0.001). Stepwise regression analysis was done to determine the existence of any relationship between any of the following parameters: gel strength, viscosity, sulfate content, month of sampling, and yield, whether taken individually or in combination. Results show variations of the yield with the month of sampling. ANOVA was performed to test whether there are differences in sulfate levels, gel strength, and viscosity between the Eucheuma alvarezii morphotypes. There was no significant difference between the green and the brown types.     

Strain selection in Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) using tetraspore progeny

A brown strain of Kappaphycus alvarezii from the Philippines produced tetraspores in the summer and autumn (December 1995 to May 1996) in cultivation experiments in the sea at Ubatuba, São Paulo State, Brazil. In vitro tetraspore release and germination experiments showed a mass mortality two to four days after release. Only 20 plants derived from tetraspores were grown successfully for over a year in the laboratory. Large differences in morphology, colour, size and growth rates were observed amongst these plants. The individual plants differed from one another in one or more characteristic. Differences appeared in the early developmental stages and persisted through time. After ten months, the plants that grew best in laboratory culture were transferred into the sea, but the others remained very small (3 to 5 mm), even after two years. In the sea, the plants also showed individual differences in their ability to survive and grow. These results emphasise the potential of the tetraspore progeny for strain selection in K. alvarezii. The results also suggest that the tetrasporophyte used in these studies is of hybrid origin.