Relationships among Eucheuma denticulatum,Eucheuma isiforme and Kappaphycus alvarezii (Gigartinales,Rhodophyta) based on nuclear ssu-rRNA gene sequences

The nuclear genes encoding small-subunit ribosomal RNAs (ssu-rDNAs) of the carrageenophytes Eucheuma denticulatum, E. isiforme and Kappaphycus alvarezii were amplified by the polymerase chain reaction, cloned and sequenced. The sequences range from 1767 (K. alvarezii) to 1781 (E. isiforme) nucleotides in length, and have guanine+cytosine contents between 51.2% (E. isiforme) and 51.5% (E. denticulatum). Pairwise sequence identities among these sequences ranged from 97.6% to 98.5%, levels comparable to some intergeneric identities within Gracilariales. In phylogenetic analyses, the two Eucheuma ssu-rDNAs group stably together vis-a-vis the ssu-rDNA of K. alvarezii, and these three ssu-rDNAs form a monophyletic group within a larger grouping of other carrageenophytes. The results demonstrate quantitatively that analysis of nuclear-encoded ssu-rDNA sequences is likely to be useful in resolving taxonomic, phylogenetic and biogeographic questions among tribe Eucheumatoideae Doty.     

MOLECULAR ANALYSIS REVEALS CRYPTIC DIVERSITY IN PORPHYRA (RHODOPHYTA)1

The small subunit ribosomal RNA (SSU rRNA) gene was amplified from 15 species of the red alga Porphyra and digested with restriction enzymes to generate data for species identification. The subset of species selected for phylogenetic analysis was P. cuneiforms (Setchell et Hus) Krishnamurthy, P. nereocystis anderson, P. schizophylla Hollenberg et Abbott, P. thuretii Setchell et Dawson and Porphyra 1674. Bangia sp. was used as an out-group. Restriction sites were mapped and used as characters in parsimony and maximum likelihood analysis. The phylogenetic hypotheses generated were compared statistically to possible alternative phylogenies based on traditional morphological taxonomic characters. The results indicate that the current subgenera in Porphyra do not represent monophyletic groups and that traditional morphological and ecological taxonomic characters alone may not be adequate for definitive species identification and cannot be relied on as an indication of Porphyra have large insertions in the SSU gene that are apparently splicesd from the final SSU rRNA molecule. The possible character, distribution and potential significance of these putative introns are discussed.     

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.     

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.     

Random amplified polymorphic DNA (RAPD) identification of genetic variation in three species ofPorphyra (Bangiales,Rhodophyta)

The random amplified polymorphic DNA (RAPD) technique was used to characterize three species ofPorphyra from the western North Atlantic and adjacent Gulf of Mexico. Twenty 10-mer primers were screened for DNA amplification usingPorphyra template DNA. Nine of these oligonucleotide primers, all (G+C)-rich, were positive or band-producing, but yielded poor or variable band resolution. Subsequent use of the universal 20-mer M 13 primer resulted in both clear band resolution with a minimum of secondary bands and a high degree of reproducibility. Amplification products for DNA from six regional isolates ofPorphyra carolinensis Coll et Cox,P. leucosticta Thuret in Le Jolis andP. rosengurttii Coll et Cox were compared to each other and toBangia atropurpurea (Roth) C. Agardh. Results provide evidence of both genetically hetero- and homogeneous populations. Use of the RAPD method with the M 13 primer yields amplification products which can be used to fingerprint specific genotypes. This procedure could be used to discriminate between hetero- and homokaryotic fusion products from previously characterized donor strains.     

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.     

Rapid DNA extraction from conchocelis and ITS-1 rDNA sequences of seven strains of cultivated Porphyra yezoensis (Bangiales, Rhodophyta)

In order to extract DNA rapidly from cultivated Porphyra, we extracted total DNA from conchocelis using the ISOPLANT II kit (Nippon Gene) without liquid nitrogen treatment or CsCl-gradient ultracentrifugation. By confirming the reproducibility of RAPD patterns, it is concluded that the quality of the extracted DNA is sufficient to use as a template for molecular investigation. Using this rapid method, the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions was amplified from seven strains of cultivated Porphyra, which had been maintained as free-living conchocelis by subculturing in the laboratory. From the amplified DNAs, the ITS-1 sequences were determined in order to identify the species and genetic relationship of the strains. The sequences were identical in the seven strains, and all the strains were identified as P. yezoensis. Furthermore, the gametophytic blades of these strains showed long linear or oblanceolate shapes in the laboratory culture. It was concluded that these strains are P. yezoensis form. narawaensis. This rapid DNA extraction method from conchocelis will be a powerful tool for phylogenetic analysis and for genetic improvement of cultivated Porphyra.     

149 Distribution and Invasion of Bangia Atropurpurea (Rhodophyta) in the Laurentian Great Lakes

Müller et al. (1998) noted that freshwater collections of the genus Bangia formed a distinct group separate from marine entities in gene sequence analyses. Recently, the species epithet B. atropurpurea has been resurrected to represent this freshwater lineage. This taxon is one of many invasive species within the Laurentian Great Lakes. B. atropurpurea was first observed in Lake Erie in 1964 and by 1982 was observed in all of the Great lakes except Lake Superior. The present study was initiated to examine the further spread of B. atropurpurea and determine the origin of these populations. Hence, a survey of all the Great Lakes was conducted in 1995 (86 sites) and again in 2002 (104 sites). Bangia was observed at 43 sites in 1995 and 39 sites in 2002. For the first time, this alga has been observed to be present in the St. Lawrence River (1995), Georgian Bay on Lake Huron (2002) and Lake Simcoe (eastern shore, 2002) and hence this alga appears to be spreading into new locations. Cluster analyses of morphological data reveal three distinct groupings that do not separate according to location or lake basin. Preliminary analyses of ITS 1 and 2 sequences show differences among samples within Lake Ontario and among all Lakes; however, collections from Lake Simcoe are very similar in sequence. We are continuing to examine the relationship of Great Lakes populations with freshwater collections from Europe.     

Babesia Canis Canis, Babesia Canis Vogeli, Babesia Canis Rossi: Differentiation of the Three Subspecies By A Restriction Fragment Length Polymorphism Analysis On Amplified Small Subunit Ribosomal Rna Genes

The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis, B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu-rDNA) of each subspecies amplified in vitro with primers derived from a semi-conserved region of the ssu-rDNA genes in other Babesia species. The polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using HinfI and TaqI restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species.     

Sequence analysis of rDNA intergenic spacer (IGS) of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

The intergenic spacer region (IGS) has been used for the first time to analyze the genetic variability of Porphyra haitanensis from different areas. In order to determine that whether the IGS sequences could be used for classification and identification in intraspecies of Porphyra, the partial IGS sequences of cultivated strains of P. haitanensis (isolated from Putian-Fujian Province, Shantou-Guangdong Province and Ningbo-Zhejiang Province), were amplified, sequenced and analyzed. The sequence analysis indicated that the partial IGS sequences from the three stains were the external transcribed spacers (ETS) of 3′ end of the IGS gene. In the three stains, the length of IGS sequences ranged from 1,085 to 1,100 bp and the G + C content varied from 50.88% to 51.27%. There were 55 variable sites which occupied approximately 5% of the ETS sequences. Similarity analysis and multisequencing alignment of sequences indicated that the partial IGS sequences of the three stains of P. haitanensis had notable variabilities. Therefore, the IGS sequence could be used as the critical genetic marker in intraspecies of P. haitanensis. Furthermore, IGS sequence analysis will be a powerful tool for genetic diversity and classification in intraspecies of other Porphyra species.     

Essential Oil Composition of Centaurea atropurpurea and Centaurea orientalis Inflorescences from the Central Balkans – Ecological Significance and Taxonomic Implications

The essential oil composition of Centaurea atropurpurea and Centaurea orientalis flowering heads (capitula) from Central Balkans have been determined by GC‐FID and GC/MS analyses. In total, 121 compounds were identified, representing on average 97.7% of the oil composition. In all samples, sesquiterpenes were most abundant group, representing 53.9 – 74.0% of the total oil. Sesquiterpene hydrocarbons dominated in all studied populations of C. orientalis and C. atropurpurea, except C. atropurpurea f. flava in which essential oil was characterized with high level of oxygenated sesquiterpenes. The dominant components differed between species, and also between typical C. atropurpurea and C. atropurpurea f. flava. The most abundant compounds of essential oil of C. orientalis were germacrene D and α‐cadinol. In C. atropuruprea, germacrene D and β‐caryophyllene were the most abundant, while caryophyllene oxide and β‐caryophyllene were dominant in C. atropurpurea f. flava oil. Taxonomical and ecological implications are further discussed.     

Genetic variation in 14 Porphyra lines using restriction site amplified polymorphism (RSAP)

Restriction site amplified polymorphism (RSAP) is a molecular marker technique which just requires a simple polymerase chain reaction to amplify fragments around restriction sites. The RSAP analytic system was set up and applied to Porphyra genetic variation analysis in this study for the first time. Fourteen Porphyra lines were screened by the RSAP analytic system with 30 primer combinations, 12 of which produced stable and reproducible amplification patterns in three repeated experiments. The 12 primer combinations produced 408 amplified fragments, 402 of which (98.53%) were polymorphic, with an average of 33.5 polymorphic fragments for each primer combination, ranging in size from 50 to 500 bp. The 408 fragments were scored one by one and then used to develop a dendrogram of the 14 Porphyra lines with unweighted pair-group method arithmetic average. The genetic distance among these Porphyra lines ranged from 0.10 to 0.50. These Porphyra lines were divided into two major groups at the 0.71 similarity level: one group contained only Porphyra haitanensis lines and the other group contained Porphyra yezoensis lines. In addition, some specific RSAP markers were acquired from each Porphyra line apart from P. yezoensis Yqd-2-1, and five of them were sequenced. One of the specific markers, R1/R3-8₁₁₉ from P. yezoensis Y-9101, was successfully converted into sequence characterized amplification region marker. The result suggested that TRAP was a simple, stable, polymorphic, and reproducible molecular marker technique for the classification and resource protection of Porphyra lines.     

Carbohydrate regulation of attachment, encystment, and appressorium formation by Pythium porphyrae (Oomycota) zoospores on Porphyra yezoensis (Rhodophyta)

Pythium porphyrae Takahashi et Sasaki, a facultative parasite of Porphyra spp., is the common microbial agent responsible for red rot disease of this red alga in Japan. Host infection by this species and other plant parasitic members of the Pythiaceae is initiated by motile biflagellate zoospores. Factors regulating host specificity and the initial steps involved in the infection process, consisting of attachment, encystment and appressorium formation, are not known. Zoospore encystment and appressorium formation of P. porphyrae were monitored by staining of the fungal cell walls using calcofluor. The zoospores infected only Porphyra spp. and Bangia atropurpurea (Roth) C. Agardh thalli, although they attached to, and encysted on, many other members of the Rhodophyceae (Stylonema alsidii[Zanardini] Drew, Gelidium elegans Kützing, Pterocladiella capillacea[Gmelin] Santelices et Hommersand, Carpopeltis affinis[Harvey] Okamura, Gloiosiphonia capillaris[Hudson] Carmichael in Berkeley, Grateloupia turuturu Yamada, Callophyllis adhaerens Yamada, Gracilaria spp., Lomentaria hakodatensis Yendo, Rhodymenia intricata[Okamura] Okamura, Griffithsia subcylindrica Okamura, Wrangelia tanegana Harvey, and Polysiphonia morrowii Harvey). No attachment or encystment was observed on the red alga Kappaphycus striatum (Schmitz) Doty ex Silva in Silva et al., the brown algae Undaria pinnatifida (Harvey) Suringar, Scytosiphon sp., and Sargassum thunbergii (Mertens ex Roth) Kuntze as well as members of the Ulvaceae (green algae). Sequential extraction of carbohydrates from Porphyra yezoensis Ueda thalli and the addition of diverse monosaccharides, polysaccharides, and amino acids to zoospore suspensions indicated that encystment and appressorium formation were induced only by sulfated galactans (porphyran, commercial agar, agarose, and carrageenans). Zoospore attachment and encystment on thalli of P. yezoensis was abolished by periodate oxidation of the thallus surface and was reduced by 80–90% after enzymatic removal of sulfated galactan (porphyran). It appears that the interaction of zoospore surface receptors with sulfated galactan (porphyran) determinants on the thallus surface induced specific attachment and encystment on Porphyra spp. thalli. Zoospores encysted, germinated, and formed appressoria on sulfated galactan films and in suspensions of this carbohydrate. Attachment and encystment were induced on commercial agar and agarose films, but appressoria were not induced on agarose films. Supplementation of agarose media with both cold and hot water fractions and with porphyran from P. yezoensis–induced appressoria implicated sulfated galactans (porphyran) in appressorium formation.     

MOLECULAR EVOLUTION OF GLUTAMINE SYNTHETASE II AND III IN THE CHROMALVEOLATES1

Glutamine synthetase (GS) is encoded by three distinct gene families (GSI, GSII, and GSIII) that are broadly distributed among the three domains of life. Previous studies established that GSII and GSIII isoenzymes were expressed in diatoms; however, less is known about the distribution and evolution of the gene families in other chromalveolate lineages. Thus, GSII cDNA sequences were isolated from three cryptophytes (Guillardia theta D. R. A. Hill et Wetherbee, Cryptomonas phaseolus Skuja, and Pyrenomonas helgolandii Santore), and GSIII was sequenced from G. theta. Red algal GSII sequences were obtained from Bangia atropurpurea (Mertens ex Roth) C. Agardh; Compsopogon caeruleus (Balbis ex C. Agardh) Mont.; Flintiella sanguinaria F. D. Ott and Porphyridium aerugineum Geitler; Rhodella violacea (Kornmann) Wehrmeyer and Dixoniella grisea (Geitler) J. L. Scott, S. T. Broadwater, B. D. Saunders, J. P. Thomas et P. W. Gabrielson; and Stylonema alsidii (Zanardini) K. M. Drew. In Bayesian inference and maximum‐likelihood (ML) phylogenetic analyses, chromalveolate GSII sequences formed a weakly supported clade that nested among sequences from glaucophytes, red algae, green algae, and plants. Red algal GSII sequences formed two distinct clades. The largest clade contained representatives from the Cyanidiophytina and Rhodophytina and grouped with plants and green algae. The smaller clade (C. caeruleus, Porphyra yezoensis, and S. alsidii) nested within the chromalveolates, although its placement was unresolved. Chromalveolate GSIII sequences formed a well‐supported clade in Bayesian and ML phylogenies, and mitochondrial transit peptides were identified in many of the sequences. There was strong support for a stramenopile‐haptophyte‐cryptophyte GSIII clade in which the cryptophyte sequence diverged from the deepest node. Overall, the evolutionary history of the GS gene families within the algae is complex with evidence for the presence of orthologous and paralogous sequences, ancient and recent gene duplications, gene losses and replacements, and the potential for both endosymbiotic and lateral gene transfers.     

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.     

Molecular phylogeny of the extinct cave lion Panthera leo spelaea

To reconstruct the phylogenetic position of the extinct cave lion (Panthera leo spelaea), we sequenced 1 kb of the mitochondrial cytochrome b gene from two Pleistocene cave lion DNA samples (47 and 32 ky B.P.). Phylogenetic analysis shows that the ancient sequences form a clade that is most closely related to the extant lions from Africa and Asia; at the same time, cave lions appear to be highly distinct from their living relatives. Our data show that these cave lion sequences represent lineages that were isolated from lions in Africa and Asia since their dispersal over Europe about 600 ky B.P., as they are not found among our sample of extant populations. The cave lion lineages presented here went extinct without mitochondrial descendants on other continents. The high sequence divergence in the cytochrome b gene between cave and modern lions is notable.     

Karyological evolution and molecular phylogeny in Macaronesian dendroid spurges (Euphorbia subsect. Pachycladae)

 Euphorbia subsect. Pachycladae is a taxon of primarily Macaronesian distribution, defined by morphological and biogeographical criteria. On the basis of morphological data, it is a heterogeneous group within which at least three complexes of species can be distinguished. To ascertain whether it is a natural group and discover its phylogenetic relations, we performed a cladistic analysis of the sequences of ribosomal nuclear DNA and a karyological study. The results of the two studies are concordant and show that the sub-section is polyphyletic and includes three different groups. The first monophyletic group is made up of the Macaronesian endemics E. atropurpurea complex and E. lamarckii complex, which form a polytomy with E. dendroides as the basal species. The lauroid species E. longifolia and E. stygiana represent the second monophyletic group, which derive from Mediterranean forms of E. sect. Helioscopia Dumort. Both species are paleopolyploid (2n=44) with highly symmetrical karyotypes. Finally, E. balsamifera, with a Canarian, African and Arabian distribution, remains isolated in a basal position. Its karyotype, with 2n=20 chromosomes, differs from the Macaronesian model and displays analogies with African cactiform spurges. On the basis of the results, some hypotheses are formulated about speciation processes in the three groups. Received March 3, 2001 Accepted October 28, 2001     

Diversity of bladed Bangiales (Rhodophyta) in western Mediterranean: recognition of the genus Themis and descriptions of T. ballesterosii sp. nov., T. iberica sp. nov., and Pyropia parva sp. nov.

The diversity of the bladed species of the red algal order Bangiales from the Iberian Mediterranean shores has been reassessed after a detailed study of this region. Prior to this study, 11 bladed species of Bangiales had been reported from Mediterranean waters: Porphyra atropurpurea, P. cordata, P. coriacea, P. dioica, P. linearis, P. purpurea, P. umbilicalis, Pyropia leucosticta, Pyropia koreana (as P. olivii), Py. elongata (as P. rosengurttii) and Py. suborbiculata. A combined analysis of the nuclear nSSU and the plastid rbcL genes together with detailed morphological studies has confirmed the presence of species within the genera Porphyra and Pyropia and also revealed a third, undescribed genus, Themis gen. nov. Porphyra linearis, Pyropia elongata and the introduced Pyropia koreana had been previously listed for the Mediterranean and were recorded in this study. An additional four species, including the introduced Pyropia suborbiculata and three new species: Pyropia parva sp. nov., Themis ballesterosii sp. nov., and Themis iberica sp. nov. were also observed. Hence, most of the Porphyra species traditionally reported along these shores were not reported in this survey. This new floristic Bangiales composition confirms the importance of the Mediterranean basin as a hotspot for biodiversity, possible endemics of ancient origin and high proportion of introductions. Our data also continue to confirm the extent of Bangiales diversity at regional and worldwide levels.     

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta)

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.     

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.