Development and application of LSU rRNA probes for Karenia brevis in the Gulf of Mexico, USA

The brevetoxin producing dinoflagellate, Karenia brevis, is the target of several monitoring and research programs in the Gulf of Mexico, where it forms extensive and frequently long-lived annual blooms that can cause human intoxication and fish kills, as well as severe economic losses to coastal communities. Rapid, reliable methods for the detection and enumeration of K. brevis cells, as well as their discrimination from morphologically similar species, are valuable tools for managers and scientists alike. Our aim was to produce a species-specific molecular probe that would serve as a tool to facilitate the efficient and reliable detection of K. brevis in the Gulf of Mexico. We sequenced a fragment of the large-subunit ribosomal RNA gene (LSU rDNA) from five K. brevis cultures isolated from the Texas Gulf coast, the Florida Gulf coast, and the Atlantic coast of Florida, and detected no differences among these isolates. A consensus sequence was thus compiled and compared to a previously published sequence from Karenia mikimotoi, the closest known phylogenetic relative to K. brevis, for the purpose of identifying unique K. brevis signature sequences. Fluorescently-labeled (FITC) oligonucleotide probes targeting these regions of the K. brevis LSU rRNA were designed to include at least two base pair differences, as compared to K. mikimotoi. Among seven probes designed, one uniquely identified all K. brevis isolates to the exclusion of all other species tested (Kbprobe-7), including a Gulf of Mexico K. mikimotoi isolate (Sarasota, FL) and several additional Gymnodinium species, as well as other dinoflagellate, diatom, and raphidophyte taxa. Importantly, K. brevis cells in samples taken during a 2001 bloom, fixed with a mixture of modified saline ethanol and 10% formalin, and stored at 4 °C for 7 months were successfully labeled with Kbprobe-7. In addition, preliminary analysis of labeled cells by flow cytometry revealed that K. brevis could be distinguished from K. mikimotoi in solution, suggesting other potential applications of this probe.     

A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia brevis in the Rookery Bay National Estuarine Research Reserve

A DNA hybridization assay was developed in microtiter plate format to detect the presence of toxic dinoflagellates in coastal waters. Simultaneous detection of multiple species was demonstrated using Karenia brevis, Karenia mikimotoi, and Amphidinium carterae. Molecular probes were designed to detect both K. brevis and K. mikimotoi and to distinguish between these two closely related species. The assay was used to detect K. brevis in coastal waters collected from the Rookery Bay National Estuarine Research Reserve. Assay results were verified by species-specific PCR and sequence analysis. The presence/absence of K. brevis was consistent with microscopic observation. Assay sensitivity was sufficient to detect K. brevis in amounts defined by a regional monitoring program as “present” (≤1000 cells/L). The assay yielded quick colorimetric results, used a single hybridization temperature, and conserved the amount of genomic DNA utilized by employing one set of PCR primers. The microplate assay provides a useful tool to quickly screen large sample sets for multiple target organisms.     

Development of microsatellite markers in the marine phytoplankton Karenia mikimotoi (Dinophyceae)

The marine phytoplankton, Karenia mikimotoi, causes severe red tides which are associated with mass mortality of marine fish, and have expanded their distributions in the coastal waters of western Japan. To assess the dispersal mechanism, a population genetic study using highly polymorphic genetic markers is one of the crucial approaches. Here we developed 12 polymorphic microsatellite markers from K. mikimotoi. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from 5 to 23, and the estimate of gene diversity was from 0.551 to 0.933 across the 12 microsatellites. We consider these loci potentially useful for detailing the genetic structure and gene flow among K. mikimotoi populations.     

A satellite and field portrait of a Karenia mikimotoi bloom off the south coast of Ireland,August 1998

An extensive surface bloom of the dinoflagellate Karenia mikimotoi occurred off southwestern Ireland during August, 1998. The bloom was evident both from remotely sensed satellite ocean colour data and as visibly discoloured water, from the mouth of Bantry Bay around towards Cork, extending some 60 km offshore. The timing of the bloom co-incided with a field survey in the area. This paper compares the surface distributions of chlorophyll and K. mikimotoi concentrations with satellite ocean colour and thermal infra-red sea surface temperature images, from which may be derived the origins of the bloom. It would appear that weak coastal upwelling transported a thermocline population of K. mikimotoi up to the surface in the region of the Fastnet Rock, where it was wind-dispersed eastwards across the northern Celtic Sea.     

Molecular variation in Galdieria sulphuraria (Galdieri) Merola and its bearing on taxonomy

Cyanidium caldarium, Cyanidioschyzon merolae and Galdieria sulphuraria are three unicellular algae characteristic, of acid thermal environments. Recently, on the basis of morphological characters, three new species of Galdieria (G. partita, G. daedala, G. maxima ) isolated from acid-thermal springs in Russia have been instituted. A selected region of rbcL and the sequence of the intergenic spacer between the rbcL and rbcS have been amplified and sequenced from different Galdieria species and strains, in order to define molecular relationship among these interesting algae. The obtained cladogram shows that Cyanidium caldarium and Cyanidioschyzon merolae form a sister group which, in turn, is in a sister group relationship with Galdieria. This last genus is divided in two clades, one of which includes G. sulphuraria accessions from Naples (Italy), California, and Yellowstone and the other one includes G. sulphuraria accessions from Java (Indonesia) and from the Russian species. These results support the status of the genus Galdieria and suggest that G. daedala, G. maxima and G. partita are three very similar strains of G. sulphuraria; the rbcL variation within Galdieria accessions has a pattern which is broadly connected to the geographial distribution. The data obtained from the intergenic rbcL-rbcS spacer partly confirm those from the rbcL analysis.     

Chlorella chloroplast DNA sequence containing a gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a part of a possible gene for the β′ subunit of RNA polymerase

The sequence of a 2782 bp fragment of the chloroplast genome of Chlorella ellipsoidea has been determined. The region includes the entire gene (rbcL) for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase and a sequence (rpoC-like) similar to part of the gene for the subunit of E. coli RNA polymerase which is oriented in same direction as rbcL. The arrangement is rpoC-like — 446 bp — rbcL. The rbcL gene codes for a polypeptide of 475 amino acids whose sequence shows 88% homology with those of tobacco and spinach, 94% homology with that of Chlamydomonas, and 85% homology with that of Anacystis. The putative rbcL promoter sequence has homology with E. coli promoter sequences and its putative terminator sequence is capable of forming a stem-and-loop structure.     

Phylogenetic analysis of Fosterella L.B. Sm. (Pitcairnioideae, Bromeliaceae) based on four chloroplast DNA regions

The about 31 species of Fosterella L.B. Sm. (Bromeliaceae) are terrestrial herbs with a centre of diversity in the central South American Andes. To resolve infra- and intergeneric relationships among Fosterella and their putative allies, we conducted a phylogenetic analysis based on sequence data from four chloroplast DNA regions (matK gene, rps16 intron, atpB-rbcL and psbB-psbH intergenic spacers). Sequences were generated for 96 accessions corresponding to 60 species from 18 genera. Among these, 57 accessions represented 22 of the 31 recognized Fosterella species and one undescribed morphospecies. Maximum parsimony and Bayesian inference methods yielded well-resolved phylogenies. The monophyly of Fosterella was strongly supported, as was its sister relationship with a clade comprising Deuterocohnia, Dyckia and Encholirium. Six distinct evolutionary lineages were distinguished within Fosterella. Character mapping indicated that parallel evolution of identical character states is common in the genus. Relationships between species and lineages are discussed in the context of morphological, ecological and biogeographical data as well as the results of a previous amplified fragment length polymorphism (AFLP) study.     

A molecular and morphological investigation of Batrachospermum arcuatum (Batrachospermales, Rhodophyta) in China

Batrachospermum arcuatum specimens were analysed from seven stream segments in North China. Morphological characteristics were observed and cluster analysis was used to evaluate the divergence among thalli from. Sequence data of the rbcL gene (chloroplast gene) and cox2-3 spacer region (mitochondrial gene) were also utilized to evaluate genetic variation in specimens among stream segments. The specimens from four of the streams were monoecious, while the individuals at the other three locations were dioecious. Cluster analysis showed that the monoecious specimens were not separated from the dioecious specimens, based on morphology, but rather the specimens were grouped by geographical closeness and habitat similarity. Likewise, the combined analyses of rbcL and the cox2-3 spacer data from provided more evidence that breeding system (monoecy vs. dioecy) is not a good morphological character to distinguish species.     

The potential threat of algal blooms to the abalone (Haliotis midae) mariculture industry situated around the South African coast

Toxic algal blooms are common world-wide and pose a serious problem to the aquaculture and fishing industries. Dinoflagellate species such as Karenia brevis, Karenia mikimotoi, Heterosigma akashiwo and Chatonella cf. antiqua are recognised toxic species implicated in various faunal mortalities. Toxic blooms of Karenia cristata were observed on the south coast of South Africa for the first time in 1988 and were responsible for mortalities of wild and farmed abalone. K. cristata and various other dinoflagellate species common along the South African coast, as well as K. mikimotoi (Isolation site: Norway, Univ. of Copenhagen) and K. brevis (Isolation site: Florida, BIGELOW), were tested for toxicity by means of a bioassay involving Artemia larvae as well as abalone larvae and spat. K. cristata, like K. brevis, contains an aerosol toxin; however, the toxin present in K. cristata has not yet been isolated and remains unknown. K. brevis was, therefore, used to determine which developmental phase of the bloom would affect abalone farms most, and whether ozone could be used as an effective mitigating agent. Of the 17 dinoflagellate species tested, K. cristata, Akashiwo sanguinea, K. mikimotoi and K. brevis pose the greatest threat to the abalone mariculture industry. K. brevis was most toxic during its exponential and stationary phases. Results suggest that ozone is an effective mitigation agent but its economic viability for use on abalone farms must still be investigated.     

A modified assay to determine hemolytic toxin variability among Karenia clones isolated from the Gulf of Mexico

Karenia brevis (formerly Gymnodinium breve) is a toxic marine dinoflagellate generally restricted to the Gulf of Mexico and is the main causative organism in fish kills, shellfish intoxications and respiratory distress in humans following bloom events. K. mikimotoi is a morphologically similar co-occurring species which is toxic in other parts of the world oceans, but has not been recognized as a major contributor in toxicity of blooms within the Gulf of Mexico. Recently there has been increasing evidence of the simultaneous production of a variety of bioactive compounds in addition to potent neurotoxins (brevetoxin) in Karenia brevis isolates. These compounds are potentially ichthyotoxic and have been shown to cause hemolysis in several bioassays [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519; Kirkpatrick, B., Fleming, L.E., Squicciarini, D., Backer, L.C., Clark, R., Abraham, W., Benson, J., Cheng, Y.S., Johnson, D., Pierce, R., Zaias, J., Bossart, G.D., Baden, D.G., 2004. Literature review of Florida red tide: implications for human health effects. Harmful Algae 3, 99–115]. Presence of hemolytic compounds may therefore add to the overall toxicity levels of bloom events. Current monitoring methods include assays which are highly sensitive in brevetoxin detection and yet may not target other harmful compounds.By adapting protocols developed by Eshbach et al. [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519], Red drum (Sciaenops ocellatus) erythrocytes were used to create a modified bioassay to detect hemolytic activity of crude algal extracts. Red drum was selected because it is endemic to coastal areas throughout the Gulf of Mexico and is sensitive to Karenia blooms, and thus makes this species a valid ecological target. Preliminary data has shown this method is sensitive for use in assessing hemolysis induced by laboratory cultures down to levels of 1 × 10³ cells mL⁻¹. Results showed an unexpectedly high level of hemolytic activity among K. mikimotoi clones, with one Texas strain inducing significantly higher hemolysis compared to Florida K. brevis isolates. Using this approach, future research efforts will examine the difference in production of hemolytic compounds among various Karenia clones.     

Implications ofrbcL sequence data for higher order relationships of theLoasaceae and the anomalous aquatic plantHydrostachys (Hydrostachyaceae)

Subclass and ordinal relationships ofLoasaceae, a small predominately New World family, are examined usingrbcL sequence data. Sequences were examined for eight of the fifteen genera of theLoasaceae and the morphologically anomalous aquatic genusHydrostachys (Hydrostachyaceae). Parsimony analyses of these sequences, combined with previously publishedrcbL data, indicate thatLoasaceae belong in theCornales, and are the sister group ofHydrangeaceae. This agrees with phylogenies based on chloroplast DNA inverted repeat restriction site, morphological and chemical data. TherbcL trees support the monophyly of theLoasaceae and most generic relationships correspond to current subfamily divisions. TherbcL phylogeny also provides the first suggestion thatHydrostachys is allied with theHydrangeaceae in theCornales.     

rbcL sequences support exclusion ofRetzia,Desfontainia, andNicodemia from theGentianales

The taxonomic positions ofRetzia, Desfontainia, andNicodemia have been much discussed, and all three genera have been included inLoganiaceae (Gentianales). We have made a cladistic analysis ofrbcL gene sequences to determine the relationships of these taxa toGentianales. Four newrbcL sequences are presented; i.e., ofRetzia, Desfontainia, Diervilla (Caprifoliaceae), andEuthystachys (Stilbaceae). Our results show thatRetzia, Desfontainia, andNicodemia are not closely related toLoganiaceae or theGentianales. Retzia is most closely related toEuthystachys and is better included inStilbaceae. The positions ofDesfontainia andNicodemia are not settled, butDesfontainia shows affinity for theDipsacales s.l. andNicodemia for theLamiales s.l.     

Relative rates of nucleotide substitution at the rbcl locus of monocotyledonous plants

Summary We subjected 35 rbcL nucleotide sequences from monocotyledonous taxa to maximum likelihood relative rate tests and estimated relative differences in rates of nucleotide substitution between groups of sequences without relying on knowledge of divergence times between taxa. Rate tests revealed that there is a hierarchy of substitution rate at the rbcL locus within the monocots. Among the taxa analyzed the grasses have the most rapid substitution rate; they are followed in rate by the Orchidales, the Liliales, the Bromeliales, and the Arecales. The overall substitution rate for the rbcL locus of grasses is over 5 times the substitution rate in the rbcL of the palms. The substitution rate at the third codon positions in the rbcL of the grasses is over 8 times the third position rate in the palms. The pattern of rate variation is consistent with the generation-time-effect hypothesis. Heterogenous rates of substitution have important implications for phylogenetic reconstruction.Offprint requests to: M.T. Clegg     

哀牢山兜兰,中国兰科一新天然杂种

报道了中国云南省兰科（Orchidaceae）兜兰属（Paphiopedilum）一新天然杂交种：哀牢山兜兰（Paphiopedilum×ailaoshanense B. Liu & S. P. Chen）。哀牢山兜兰与白旗兜兰（P. spicerianum）和沧源兜兰（P. gratrixianum var. cangyuanense）近缘,与前者的区别在于哀牢山兜兰花梗和子房有毛,中萼片带紫色晕以及紫色斑点,花瓣上侧暗绿色带紫色晕,下侧黄绿色;与后者的区别在于中萼片具1条宽阔的紫褐色中带,退化雌蕊紫色。哀牢山兜兰与天然杂种泸水兜兰（Paphiopedilum×lushuiense）相似,而哀牢山兜兰的中萼片下部边缘不后卷,近基部具紫红色晕,合萼片具2条明显紫色粗脉,花瓣匙形,上边缘波状,长6.0~6.2 cm,唇瓣长5.0~5.5 cm。为厘清哀牢山兜兰,沧源兜兰及白旗兜兰之间的关系,基于3个叶绿体基因片段（matK、rbcL、trnL）,构建了兜兰属部分植物系统发育树。鉴于形态和分子数据,认为哀牢山兜兰是天然杂交种。凭证标本存放于福建农林大学标本馆。     

Phylogenetic utility of the nuclear rDNA ITS region in subfamilyIxoroideae (Rubiaceae): Comparisons with cpDNArbcL sequence data

ITS of the nrDNA were sequenced for 21 taxa inIxoroideae and outgroups (Rubiaceae) and compared with sequences of the cp-generbcL. Separate and combined analyses were performed. ITS-variation was extensive and, because of alignment ambiguities, some sites were excluded from the analyses. Several topologies from therbcL analysis that conflicted with earlier classifications are corroborated by the ITS data: 1)Posoqueria should be excluded fromGardenieae. 2) The disputed genusBertiera, previously inGardenieae, is basal in an extendedCoffeeae, includingTricalysia. 3)Ixora should be excluded fromPavetteae. 4)Vangueria, (Antirheoideae), belongs toIxoroideae. This affiliation ofAntirheoideae tribes withIxoroideae is also shown by new ITS andrbcL data forAlberta. Incongruities found between the two data sets may be caused by density of taxon sampling, different evolutionary rates, phylogenetic sorting, homoplasy caused by functional constraints, or sampling of non-orthologous ITS types.     

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.     

Intralineage variation in the pattern ofrbcL nucleotide substitution

Variation in chloroplastrbcL sequences was studied in representative species of four different lineages: the tribeRubieae (Rubiaceae), and the generaDrosera (Droseraceae),Nothofagus (Nothofagaceae) andIlex (Aquifoliaceae). Each lineage has its particular non-overlapping set ofrbcL polymorphic sites, indicating that common unconstrainedrbcL sites are not shared. Large differences in the rate and pattern of nucleotide substitution are observed among the four lineages. The genusIlex has the lowest rate of substitution, the lowest transition/transversion ratio, the lowest synonymous/replacement ratio and the lowest number of substitutions at the third codon position. An apparent relationship of these measures to the age of the lineages is observed. The A + T content and codon use among the four lineages are very similar and, apparently, cannot account for the observed differences in patterns of nucleotide substitution. However, the A + T content of the two bases immediately flanking the polymorphic sites is higher inIlex than in the other lineages. This could be correlated with the transversion/transition bias observed inIlex. The particularly low synonymous/replacement ratio found inIlex could also be explained by the small population sizes of species in this genus.     

Systematic studies of the antarctic species of the phyllophoraceae (Gigartinales,Rhodophyta) based on rbcL sequence analysis

The taxonomic placement of four antarctic species of the marine red algal family Phyllophoraceae (Gigartinales) is assessed within a preliminary molecular phylogeny of the family based on direct sequence analysis of the chloroplast gene rbcL. Parsimony analysis of rbcL sequences indicates that Gymnogongrus antarcticus and Gymnogongrus turquetii cluster in a clade consisting predominantly of southern hemisphere species currently placed in Gymnogongrus and Ahnfeltiopsis, whereas Phyllophora ahnfeltioides and Phyllophora antarctica cluster in a separate clade that is widely divergent from the northern hemisphere Phyllophora clade. Results from molecular and morphological data challenge the current taxonomic concept that type of life history is a phylogenetically valid criterion for recognition of genera in the Phyllophoraceae.     

Invasion of protein coding genes by green algal ribosomal group I introns

The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns.     

Phylogeny and biosystematics ofPseudomonotes (Dipterocarpaceae) based on molecular and morphological data

The placement of a recently discovered South American monotypic genus,Pseudomonotes tropenbosii, in subfam.Monotoideae (Dipterocarpaceae) extends the geographical range of the subfamily from Africa to the Neotropics. Although morphological and anatomical evidence suggest similarities betweenPseudomonotes andMonotes, the close alliance of these two genera was questionable due to their disjunct distribution and a lack of phylogenetic analysis. In the present study, we reconstructed the phylogeny ofPseudomonotes and other putatively related taxa usingrbcL sequence data. The analysis ofrbcL sequences of 20 taxa belonging to 15 genera and eight families recovered a single most parsimonious tree. The genusSarcolaena (Sarcolaenaceae) formed a clade sister to the monophyleticDipterocarpaceae clade.Monotes andPseudomonotes formed a strongly supported group, sister to the monophyletic clade withPakaraimaea and the remaining Asiatic dipterocarp species studied. The study strongly supports the placement ofPseudomonotes within subfam.Monotoideae of theDipterocarpaceae.