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.     

Confirmation of cultivated Porphyra tenera (Bangiales, Rhodophyta) by polymerase chain reaction restriction fragment length polymorphism analyses of the plastid and nuclear DNA

Polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis of the plastid ribulose‐1,5‐bisphosphate carboxylase (RuBisCo) spacer region was developed for a more reliable and rapid species identification of cultivated Porphyra in combination with PCR‐RFLP analysis of the nuclear internal transcribed spacer (ITS) region. From the PCR‐RFLP analyses of the plastid and nuclear DNA, we examined seven strains of conchocelis that were used for cultivation as Porphyra tenera Kjellman but without strict species identification. The PCR‐RFLP analyses suggested that two strains, C‐32 and 90‐02, were cultivated P. tenera and that the other five strains, C‐24, C‐28, C‐29, C‐30 and M‐1, were Porphyra yezoensis f. narawaensis Miura. To identify species more accurately and to reveal additional genetic variation, the two strains C‐32 and 90‐02 were further studied by sequencing their RuBisCo spacer and ITS‐1 regions. Although RuBisCo spacer sequences of the two strains were identical to each other, each of their ITS‐1 sequences showed a single substitution. The sequence data again confirmed that the two strains (C‐32 and 90‐02) were cultivated P. tenera, and suggested that the two strains showed some genetic variation. We concluded that PCR‐RFLP analysis of the plastid and nuclear DNA is a powerful tool for reliable and rapid species identification of many strains of cultivated Porphyra in Japan and for the collection of genetically variable breeding material of Porphyra.     

MORPHOLOGICAL AND MOLECULAR ANALYSIS OF THE ENDANGERED SPECIES PORPHYRA TENERA (BANGIALES,RHODOPHYTA)1

Porphyra tenera Kjellman, widely cultivated in nori farms before the development of artificial seeding, is currently listed as an endangered species in Japan. To confirm whether a wild‐collected gametophytic blade was P. tenera or the closely related species P. yezoensis Ueda, morphological observations and molecular analyses were made on the pure line HGT‐1 isolated from a wild blade. This pure line was identified as P. tenera based on detailed morphological features. Sequences of the nuclear internal transcribed spacer region 1 and the plastid RUBISCO spacer revealed that P. tenera HGT‐1 was clearly different from P. yezoensis f. narawaensis Miura, the main species cultivated in Japan. PCR‐RFLP analysis of the internal transcribed spacer region was found to be a convenient method for rapid discrimination between P. tenera and cultivated P. yezoensis. The restriction patterns generated by the endonucleases Dra I and Hae III were useful for differentiating between both gametophytic and conchocelis stages of P. tenera HGT‐1 and P. yezoensis f. narawaensis strains. Thus, PCR‐RFLP analysis will serve as a valuable tool for rapid species identification of cultivated Porphyra strains, culture collections of Porphyra strains for breeding material and conservation of biodiversity, and, as codominant cleaved amplified polymorphic sequence markers for interspecific hybridization products between P. tenera and P. yezoensis f. narawaensis. Under the same culture conditions, rate of blade length increase and the blade length‐to‐width ratio were lower in P. tenera HGT‐1 than in P. yezoensis f. narawaensis HG‐4. The HGT‐1 became mature more rapidly than HG‐4 and had thinner blades.     

ALLOPOLYPLOIDY IN NATURAL AND CULTIVATED POPULATIONS OF PORPHYRA (BANGIALES,RHODOPHYTA)1

To confirm whether allopolyploidy occurs in samples of previously identified Porphyra yezoensis Ueda, P. tenera Kjellm., and P. yezoensis × P. tenera from natural and cultivated populations, we examined these samples by using PCR‐RFLP and microsatellite analyses of multiple nuclear and chloroplast regions [nuclear regions: type II DNA topoisomerase gene (TOP2), actin‐related protein 4 gene (ARP4), internal transcribed spacer (ITS) rDNA and three microsatellite loci; chloroplast region: RUBISCO spacer]. Except for the ITS region, these multiple nuclear markers indicated that the wild strain MT‐1 and the cultivated strain 90‐02 (previously identified as P. yezoensis × P. tenera and cultivated P. tenera, respectively) are heterozygous and possess both genotypes of P. tenera and P. yezoensis in the conchocelis phase. Furthermore, gametophytic blades of two pure lines, HG‐TY1 and HG‐TY2 (F₁ strains of MT‐1 and 90‐02, respectively), were also heterozygous, and six chromosomes per single cell could be observed in each blade of the two pure lines. These results demonstrate that allopolyploidy occurs in Porphyra strains derived from both natural and cultivated populations, even though ITS genotypes of these strains showed homogenization toward one parental ITS.     

Porphyra and Bangia (Bangiaceae, Rhodophyta) in warm temperate waters of eastern Australia: Morphological and molecular analyses

Morphological observations confirm the presence of only three species of the Bangiaceae (Rhodophyta) in warm temperate waters of eastern Australia: Bangia atropurpurea, Porphyra columbina and Porphyra denti-culata. Analyses of DNA sequence data from the inter-generic spacer region between the large- and small-sub-unit ribulose-l,5-bisphosphate carboxylase/oxygenase gene (rbcL and rbcS, respectively) and portions of the flanking regions, confirm these taxonomic conclusions for the two Porphyra species: there is clear sequence divergence between the two species, and strong genetic similarity between P. columbina isolates over a wide geographical region. Sequence analyses also reveal a strong similarity between Bangia isolates over a wide geographical range, but the taxonomy of B. atropurpurea may need to be re-examined in light of sequence differences between these and northern hemisphere isolates of B. atropurpurea. Molecular analyses support the view that Bangia and Porphyra species are sufficiently closely related to be placed in a single genus.     

A simple restriction fragment length polymorphism (RFLP) assay to discriminate common Porphyra (Bangiophyceae, Rhodophyta) taxa from the Northwest Atlantic

The identification of Porphyra species hashistorically been difficult because of the lack of distinguishing morphologicaland ecological characters. We developed a restriction fragment lengthpolymorphism (RFLP) assay, based on inter-specific sequence variation inthe ribulose bisphosphate carboxylase oxygenase largesubunit (rbcL) gene andrbcL-rbcS intergenic spacer, toprovide a simple and effective tool for screening and sorting large collectionsof Porphyra from the Northwest Atlantic. A singlerestriction digest (Hae III) discriminates betweenmultiplePorphyra species including one cryptic taxon; anadditionalenzyme (Hind III) was necessary to distinguish between theclosely related P. leucosticta and an introducedspecies P. yezoensis.     

Comparative study of wild and cultivated <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) based on molecular and morphological data

We compared the wild Porphyra strain OGATSU from northeastern Japan with cultivated Porphyra yezoensis f. narawaensis using the RuBisCO spacer, rbcL, and ITS-1 DNA sequences as well as early gametophyte development. Based on the molecular analyses and detailed morphological observations, OGATSU was identified as P. yezoensis, but also revealed important differences from the cultivated form. Under the same culture conditions, gametophytic blades of OGATSU produced more archeospores than P. yezoensis f. narawaensis strain HG-4. The length of blades and their length-to-width ratios were significantly lower in OGATSU than in HG-4, and the color of OGATSU blades was darker than that of HG-4. The first lateral cell division in conchospore germlings occurred significantly earlier in the OGATSU strain than in the HG-4 strain, resulting in the rounder shape of the OGATSU blade compared to that of P. yezoensis f. narawaensis. These results suggested that wild strains such as OGATSU can provide useful characters that could enhance cultivated varieties in a careful breeding program.     

PCR-RFLP and AP-PCR of rbcL and ITS of rDNA Show That ×Taxodiomeria peizhongii (Taxodium×Cryptomeria) Is not an Intergeneric Hybrid

×Taxodiomeria peizhongii Z. J. Ye, J. J. Zhang et S. H. Pan was regarded as a new intergeneric hybrid between Taxodium mucronatum Tenore (as the female donor) and Cryptomeria fortunei Hooibrenk ex Otto et Dietr (as the male donor). To confirm the authenticity of the intergeneric hybrid, we analyzed the rbcL gene and the internal transcribed spacer (ITS) of 26S‐18S ribosomal RNA gene of the three species using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and arbitrarily primed PCR (AP‐PCR), and obtained the following results: i) Taxodiomeria peizhongii had the same RFLP maps of the rbcL gene and the ITS as Taxodium mucronatum, but was different from C. fortunei; ii) a 311‐bp PCR amplification product was obtained in C. fortunei by AP‐PCR of ITS, but was not found in Taxodiomeria peizhongii. Our results have demonstrated that C. fortunei did not provide any genome for Taxodiomeria peizhongii, implying that T. peizhongii is not an intergeneric hybrid between the two species. (Managing editor: Wei Wang)     

Cryptic diversity,biogeography and genetic variation in Northeast Pacific species of <Emphasis Type="Italic">Porphyra</Emphasis> sensu lato (Bangiales,Rhodophyta)

We sequenced the chloroplast rubisco large subunit (rbcL) gene in 236 samples of Porphyra sensu lato from the northeast Pacific. Comparisons of sequences within the study area as well as comparisons with published sequences revealed up to five cryptic species among the 22 named species: a species closely related to Porphyra abbottiae, a species previously identified as P. pseudolinearis, a species closely related to P. pseudolanceolata and previously identified as that species, a previously unknown species from the eastern Aleutian Islands, and a species closely related to P. schizophylla and previously identified as that species. All of these previously unrecognized species had high bootstrap values separating them from the other species. In addition, our wide geographic sampling allowed us to extend, curtail or clarify the geographic ranges of a number of the species. We also provide published sequences for P. gardneri and P. smithii for the first time. We compared amount of sequence divergence within species grouped on the basis of sexuality (monoecious, sectored into separate male and female “halves”, or dioecious), habitat (high, mid, or low intertidal/subtidal), and seasonality (winter, spring, or summer) using Tukey’s HSD t test, but we observed no significant differences between species grouped in this manner. Different species showed different levels of genetic variation in the rbcL gene apparently unrelated to these traits. Also, we observed no differences in the patterns of genetic variation in a species based on whether the specimens were collected from outside or from within the region covered by ice during Pleistocene glaciations.     

rbcL gene sequences reveal relationships among north-east Pacific species of Porphyra (Bangiales, Rhodophyta) and a new species, P. aestivalis

Phylogenetic analyses of the rbcL (chloroplast Rubisco large subunit) gene from 23 newly sequenced species of Porphyra, primarily from the north‐east Pacific, one Bangia and previously published sequences from both genera resolve relationships among most species of Porphyra and reveal five clades of species with Porphyra‐type morphologies among a number of Bangia lineages: (1) P. papenfussii V. Krishnam; (2) P. mumfordii S. C. Lindstrom et K. M. Cole and P. rediviva Stiller et Waaland together with a group of north Atlantic species, including the type of the genus, P. purpurea (Wahl‐enb.) C. Agardh; (3) P. cuneiformis (Setch. et Hus) V. Krishnam., P. occidentalis Setch. et Hus, P. schizo‐phylla Hollenb., and P. variegata (Kjellm.) Kjellm. and their Atlantic sibling species, all distromatic; (4) P. aestivalis sp. nov. and its north Atlantic sibling, P. birdiae C. D. Neefus et A. C. Mathieson; and (5) a speciose clade containing both Pacific and Atlantic representatives. Close relationships are confirmed between sibling species previously identified by iso‐zymes, morphology and chromosomal features. The morphologically similar dioecious P. pseudolanceolata V. Krishnam., P. conwayae (S. C. Lindstrom et K. M. Cole) stat. nov., and P. lanceolata (Setch. et Hus) G. M. Smith occur in a strongly supported subclade in clade 5 together with the monoecious P. fallax S. C. Lindstrom et K. M. Cole. Results presented here highlight the need for intensive taxon sampling and for examination of different parts of the genome to understand more fully relationships among species and higher level taxa in the Bangiales.     

Development of an expression system using the heat shock protein 70 promoter in the red macroalga, Porphyra tenera

Porphyra is a commercially valuable source of food and drugs and an important model organism for algal research. However, genetic research on Porphyra tenera has been limited by a lack of a heterologous gene expression system. In this study, we isolated native promoter PtHSP70 for the efficient expression of foreign genes in this organism. This promoter lies approximately 1 kb upstream of the heat shock protein 70 coding sequence and was isolated using adapter ligation-mediated genomic polymerase chain reaction. Promoter activity was evaluated using the synthetic GUS gene (PyGUS) with optimized codons for Porphyra yezoensis. Interestingly, the PtHSP70 promoter allowed the efficient expression of PyGUS in P. tenera and P. yezoensis, whereas the PyGAPDH promoter from P. yezoensis was not fully functional in P. tenera. The PtHSP70 promoter may have a more conserved regulatory mechanism than the PyGAPDH promoter between these species, suggesting that PtHSP70 could serve as a universal promoter for Porphyra species. We also established an efficient transient transformation system for P. tenera by evaluating transformation parameters including gold particle quantity, helium and vacuum pressure, developmental stages of leafy gametophytes, and target distance. Under optimal conditions of transient transformation, the frequency of GUS expression was determined by histochemical staining as 30–50 cells per bombardment. In addition, PyGUS expression was detected during the regeneration of monospores in P. tenera, indicating successful genetic transformation. Therefore, the new transient transformation system using the PtHSP70 promoter can be used for foreign gene expression in P. tenera, which may advance the development of P. tenera as a model organism.     

Genetic and morphological differentiation of Porphyra and Pyropia species (Bangiales,Rhodophyta) coexisting in a rocky intertidal in Central Chile

A recent molecular taxonomic study along the Chilean coast (18° S–53° S) described 18 candidate species of bladed Bangiales of which only two were formally described. Few studies focused on local genetic and morphological diversity of bladed Bangiales and attempted to determine their intertidal distribution in contrasting habitats, and none were performed in Chile. To delimit intertidal distributions of genetic species, 66 samples of bladed Bangiales were collected at Maitencillo (32° S) in four zones: a rocky platform, a rocky wall, and two boulders zones surrounded by sandy and rocky bottoms, respectively. These samples were identified based on sequences of the mitochondrial COI and chloroplast rbcL markers. We also collected 87 specimens for morphological characterization of the most common species, rapidly assessing their putative species identity using newly developed species‐diagnostic (PCR‐RFLP) markers. Eight microscopic and two macroscopic morphological traits were measured. We described and named three of four species that predominate in Maitencillo (including Pyropia orbicularis): Pyropia variabilis Zapata, Meynard, Ramírez, Contreras‐Porcia, sp. nov., Porphyra luchea Meynard, Ramírez, Contreras‐Porcia sp. nov., and Porphyra longissima Meynard, Ramírez, Contreras‐Porcia, sp. nov. With the exception of Po. longissima restricted to boulders surrounded by sandy bottom, and a morphotype of Py. variabilis restricted to rocky walls, the other species/morphotypes have overlapping intertidal distributions. Except for Po. longissima, which is clearly differentiated morphologically (longest and thinnest blades), we conclude that morphology is not sufficient to differentiate bladed Bangiales. Our findings underscore the importance of refining our knowledge of intrinsic and environmental determinants on the distribution of bladed Bangiales.     

Rapid discrimination of <Emphasis Type="Italic">Porphyra tenera</Emphasis> Kjellman var. <Emphasis Type="Italic">tamatsuensis</Emphasis> Miura by PCR-RFLP

To allow to discriminate rapidly the strains of Porphyra tenera var. tamatsuensis, cultivars of which grow more vigorously than strains of P. tenera var. tenera, strains of both varieties were examined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis using mitochondrial DNA related to the ATP synthase F0 subunit 6 (ATP6) gene. The lengths of all sequences in this region of three strains of each variety were 670 bp and had just a single nucleotide substitution. Digestion with the restriction enzyme of TaaI yielded three visible bands which appeared in the three strains of P. tenera var. tamatsuensis, whereas two bands appeared in the three strains of P. tenera var. tenera. We therefore conclude that PCR-RFLP analysis is a valuable tool for discrimination of P. tenera var. tamatsuensis among the stock of P. tenera strains used for mariculture.     

Morphological and AFLP variation of Porphyra yezoensis Ueda form, narawaensis Miura (Bangiales, Rhodophyta)

Detailed morphological observations were made on two strains of cultivated Porphyra: HG‐1 (pure line isolated from Dai‐1) and Noriken‐4 (parental strain of a pure line HG‐4). The two strains were identified as P. yezoensis f. narawaensis based on their macroscopic and microscopic features, such as long linear or oblanceolate blades up to 50 cm in maximum length, division formulae of spermatangia and zygotosporangia, shape of trichogynes and carpogonia, and the second transverse divisional plane formed at the division from c/2 to c/4 in zygotosporangia. Gametophytic blades from two completely homozygous conchocelis strains isolated in this study (HG‐1 and HG‐4) were cultured under the same conditions and compared to confirm whether the differences in their shapes are genetically determined. The shape of blades from both of conchospores and monospores was always more slender in HG‐4 than in HG‐1 at the same blade age, suggesting that the difference in the blade shape between the two pure lines is due to genetic variation. To estimate the level of genetic variation the two pure lines were subjected to amplified fragment length polymorphism fingerprint analysis. A total of 230 bands were detected in HG‐1 and HG‐4 using eight selective primer pairs, and the number of polymorphic bands was only two in HG‐1. These results indicate that the two pure lines certainly show genetic variation, which is, however, at an extremely low level. The importance of pure‐line breeding and the origin of currently cultivated Porphyra are discussed. This is the first report to identify currently cultivated Porphyra strains in Japan based on combined results of detailed morphological observations and molecular analysis.     

Sensory and fatty acid analyses of two Atlantic species of Porphyra (Rhodophyta)

Sensory analyses were conducted to determine levels of consumer acceptability of Porphyra yezoensis, P. umbilicalis, and P. amplissima to select appropriate species for aquaculture development in Maine (USA). The subjects included children (n = 67) and adults (n = 84); the children participated in study design by helping to select the 9 point hedonic scale used in the affective sensory tests. Two substrates were used; Porphyra was baked in crackers and also used as a coating for popcorn. No significant differences (p > 0.5) in acceptability of one species over another were observed in either trial, which suggests that native Atlantic species of Porphyra such as P. amplissima and P. umbilicalis have developmental potential in foods for North American consumers. Fatty acids were analyzed in the taste test material and in freshly collected P. umbilicalis; eicosapentaenoic acid [EPA; 20:5 (n-3)] and palmitic acid were the most common fatty acids. Quantitative analysis of EPA determined that freshly collected (January 2005) P. umbilicalis contained 3.2 mg EPA g dry wt⁻¹ (74 mg EPA 100 g fresh wt⁻¹). This concentration is not high enough to make P. umbilicalis a primary source of daily omega-3 fatty acids, but the favorable n-3/n-6 ratio (2-3:1) in these species contributes to their nutritional value.     

Molecular features of a defined genetic marker for the determination of the Porphyra tenera lineage

Nucleotide sequences of the nuclear SSU rDNA and ITS1 are presented as a defined genetic marker for Porphyra tenera as a species. Exon nucleotide sequences were identical within all the P. tenera specimens. Intron nucleotide sequences varied between populations. The introns and ITS1 variations are presented as defined genetic markers to establish the Porphyra tenera strains. Wild-collected thalli identified by morphological systematics, from five populations of Porphyra tenera throughout Japan, were discriminated by comparing sequences of the various regions utilizing the results of this and previous studies.