Internal Transcribed Spacer Ribosomal DNA Research Articles

16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal DNA as differential markers of Trichuris discolor populations

Comparative morphological, biometrical and molecular studies of Trichuris discolor isolated from Bos taurus from Spain and Iran was carried out. Furthermore, Trichuris ovis isolated from B. taurus and Capra hircus from Spain has been, molecularly, analyzed. Morphological studies revealed clear differences between T. ovis and T. discolor isolated from B. taurus but differences were not observed between populations of T. discolor isolated from different geographical regions. Nevertheless, the molecular studies based on the amplification and sequencing of the internal transcribed spacers 1 and 2 ribosomal DNA and 16S partial gene mitochondrial DNA showed clear differences between both populations of T. discolor from Spain and Iran suggesting two cryptic species. Phylogenetic studies corroborated these data. Thus, phylogenetic trees based on ITS1, ITS2 and 16S partial gene sequences showed that individuals of T. discolor from B. taurus from Iran clustered together and separated, with high bootstrap values, of T. discolor isolated from B. taurus from Spain, while populations of T. ovis from B. taurus and C. hircus from Spain clustered together but separated with high bootstrap values of both populations of T. discolor. Furthermore, a comparative phylogenetic study has been carried out with the ITS1and ITS2 sequences of Trichuris species from different hosts. Three clades were observed: the first clustered all the species of Trichuris parasitizing herbivores (T. discolor, T. ovis, Trichuris leporis and Trichuris skrjabini), the second clustered all the species of Trichuris parasitizing omnivores (Trichuris trichiura and Trichuris suis) and finally, the third clustered species of Trichuris parasitizing carnivores (Trichuris muris, Trichuris arvicolae and Trichuris vulpis).

Genetic diversity and phylogenetic analysis of genus Paeonia based on nuclear ribosomal DNA ITS sequence

The genus Paeonia belongs to the family Paeoniaceae having significant medicinal and ornamental importance. The present investigation was undertaken with an aim to understand phylogenetic relationships of three Paeonia species (P. lactiflora, P. obovata, and P. suffruticosa) that are widely distributed in China, Korea, and Japan, using nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequence and to compare the phylogeny results with investigations reported earlier using existed sequences of the same species. The size variation obtained among sequenced nrDNA ITS region was narrow and ranged from 722 to 726 bp. The highest interspecific genetic distance (GD) was found between P. lactiflora and P. suffruticosa or P. obovata. The phylogram obtained using our nrDNA ITS sequences showed non-congruence with previous hypothesis of the phylogeny between section Paeonia and section Moutan of genus Paeonia. This result was supported by the phylogenetic relations showed in the phylogram constructed with existed sequences in NCBI. The present study suggested that P. obovata belonging to section Paeonia was phylogenetically closer to P. suffruticosa representing section Moutan of genus Paeonia than P. lactiflora belonging to section Paeonia. The main reason of the paraphyly of section Paeonia is thought to be nucleotide additivity directly caused by origin hybridization. This study provides more sequence sources of genus Paeonia, and will help for further studies in intraspecies population, and their phylogentic analysis and molecular evolution.

Purification and characterization of extracellular dextranase from a novel producer, Hypocrea lixii F1002, and its use in oligodextran production

Fungi were isolated from natural soil samples and screened for extracellular dextranase synthesis. The strain F1002 was identified as Hypocrea lixii using a standard internal transcribed spacer ribosomal DNA analysis and was selected for extracellular dextranase synthesis. The enzyme was purified via ammonium sulfate precipitation and Sepharose 6B chromatography, which resulted in an 8.3-fold increase in the specific activity and a 10.73% recovery. This enzyme is a monomeric protein with a molecular mass of 62kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme, which was identified as an endodextranase, had an optimum pH of 5.0 and an optimum temperature of 25°C. The dextranase activity was enhanced by Mg2+, Al3+, and especially Zn2+ at a low concentration, which improved its activity to 124.22%. The enzyme has a very high hydrolytic affinity toward high-molecular weight dextrans. Setting the concentrations of the H. lixii F1002 dextranase (2.31U/mL) and dextrans (6%), as well as the reaction time (45min), allowed the dextranase to hydrolyze dextrans of controlled molecular weights (20–70kDa). Three types of oligodextrans with different molecular weights (namely, 69,376, 38,251, and 21,364Da) were obtained, with a total yield of 80.32%.

Host Associations Between Fungal Root Endophytes and Boreal Trees

Fungal root endophytes colonize root tissue concomitantly with mycorrhizal fungi, but their identities and host preferences are largely unknown. We cultured fungal endophytes from surface-sterilized Cenococcum geophilum ectomycorrhizae of Betula papyrifera, Abies balsamea, and Picea glauca from two boreal sites in eastern Canada. Isolates were initially grouped on the basis of cultural morphology and then identified by internal transcribed spacer ribosomal DNA sequencing or by PCR restriction fragment length polymorphism. Phylogenetic analysis of the sequence data revealed 31 distinct phylotypes among the isolates, comprising mainly members of the ascomycete families Helotiaceae, Dermateaceae, Myxotrichaceae, and Hyaloscyphaceae, although other fungi were also isolated. Multivariate analyses indicate a clear separation among the endophyte communities colonizing each host tree species. Some phylotypes were evenly distributed across the roots of all three host species, some were found preferentially on particular hosts, and others were isolated from single hosts only. The results indicate that fungal root endophytes of boreal trees are not randomly distributed, but instead form relatively distinct assemblages on different host tree species.

Characterisation of Phytophthora colocasiae isolates associated with leaf blight of taro in India

Leaf blight and corm rot caused by Phytophthora colocasiae are the most devastating diseases of taro. Fourteen P. colocasiae isolates collected from different states of India were characterised for mating type, metalaxyl fungicide sensitivity and aggressiveness on taro. These organisms were identified as P. colocasiae based on morphological characteristics, internal transcribed spacer (ITS) sequence homology and pathogenicity to taro plants. Ribosomal DNA (rDNA) ITS regions of P. colocasiae isolates were examined, and compared with additional Phytophthora species. All isolates of P. colocasiae fell within a single cluster in phylogenetic trees, regardless of their geographic origins. These isolates were more closely related to Phytophthora capsici. All isolates except the isolate collected from Sikkim state of India (98–111) were sensitive to metalaxyl at 100 ppm and it was also scored for causing higher average foliar disease and tuber rot ratings. Isolates tested exhibited four growth patterns in cultures: cottony, rosaceous, petaloid and stellate. P.colocasiae isolates, including an IISR isolate (PC-73), with cottony growth pattern did not grow at 36°C. The mean oospore diameter of A1 mating type isolates was greater than that of A2 mating type isolates. Six of 14 isolates tested produced chlamydospores in V8-CaCO3 liquid medium.

Molecular Detection ofLeishmaniaInfection Due toLeishmania majorandLeishmania turanicain the Vectors and Reservoir Host in Iran

An epidemiological study was carried out on the vectors and reservoirs of cutaneous leishmaniasis in rural areas of Damghan district, Semnan province, central Iran, during 2008-2009. Totally, 6110 sand flies were collected using sticky papers and were subjected to molecular methods for detection of Leishmania parasite. Phlebotomus papatasi Scopoli was the common species in outdoor and indoor resting places. Polymerase chain reaction technique showed that 24 out of 218 P. papatasi (11%) and 4 out of 62 Phlebotomus caucasicus Marzinovskyi (6.5%) were positive for parasites Leishmania major Yakimoff and Schokhor. Twenty-one rodent reservoir hosts captured using Sherman traps were identified as Rhombomys opimus Lichtenstein (95%) and Meriones libycus Lichtenstein (5%). Microscopic investigation on blood smear of the animals for amastigote parasites revealed 8 (40%) rodents infected with R. opimus. L. major infection in these animals was then confirmed by polymerase chain reaction against internal transcribed spacer ribosomal DNA (rDNA) loci of the parasite followed by restriction fragment length polymorphism. Further, sequence analysis of 297 bp of ITS1-rDNA loci revealed the presence of L. major and Leishmania turanica in P. papatasi, and L. major in R. opimus. This is the first molecular report of L. major infection in both vectors (P. papatasi and P. caucasicus) and reservoir host (R. opimus) in this region. The results indicated that P. papatas was the primary vector of the disease and circulating the parasite between human and reservoirs, and P. caucasicus could be considered as a secondary vector. Further, our study showed that R. opimus is the most important host reservoir for maintenance of the parasite source in the area.

Development of a real-time PCR assay for the identification of Gyrodactylus parasites infecting salmonids in northern Europe

Gyrodactylus salaris is a monogenean freshwater parasite that causes high mortality in wild Atlantic salmon, and a number of countries employ monitoring programmes for its presence. A TaqMan-MGB (minor groove binding) probe real-time multiplex assay targeting the internal transcribed spacer ribosomal DNA (ITS rDNA) was developed to simultaneously identify G. salaris/G. thymalli and 2 other commonly occurring Gyrodactylus species infecting salmonids in northern Europe: G. derjavinoides and G. truttae. In addition, a Gyrodactylus genus-level assay was developed to assess parasite DNA quality. The species-specific real-time PCR method correctly identified target species from a wide geographical range and from a number of salmonid hosts. It did not amplify G. lucii or G. teuchis. These species were successfully amplified using the Gyrodactylus genus real-time assay. The species-specific real-time assay proved to be significantly faster than the currently employed molecular screening method of ITS rDNA PCR amplification followed by restriction fragment length polymorphism analyses (RFLP). However, as with ITS RFLP, the real-time method did not distinguish between G. salaris and the non-pathogenic G. thymalli, its principle advantage being a significant reduction in time to achieve an initial diagnostic screen before the employment of more in-depth analyses for those specimens giving a positive G. salaris/G. thymalli real-time result.

Molecular phylogenetic hypotheses of Zoanthus species (Anthozoa:Hexacorallia) using RNA secondary structure of the internal transcribed spacer 2 (ITS2)

Previously, it has been found that species of the zoanthid genus Zoanthus (Cnidaria: Hexacorallia) possess high levels of intrageneric ITS-rDNA sequence variation both between congeners and also within one species, Zoanthus sansibaricus, resulting in an uncertain internal transcribed spacer of ribosomal DNA (ITS-rDNA) phylogeny for this group. For the first time, the secondary structures of the internal transcribed spacer 2 (ITS 2) sequences were analyzed in an attempt to further clarify and solve relationships within Zoanthus, and also with the closely related genera Acrozoanthus and Isaurus (all in family Zoanthidae). Results show that most species’ ITS2 secondary structures follow the basic four-helix-ring model proposed for most eukaryotes including anthozoans. However, not all structures had the conserved motifs present in scleractinian corals, and Z. sansibaricus had two different structural conformations from the two different ITS2 types present. The ITS2 secondary structures of Zoanthus in this study, present a well resolved and supported phylogeny that makes it an appropriate tool for solving phylogenies in this taxonomic group. Based on ITS2 secondary structure results here, we theorize that: (1) Acrozoanthus is a valid monophyly separate from Zoanthus; (2) Z. gigantus, Z. sansibaricus, and Z. praelongus do not have any compensatory base changes (CBCs) between them and form a related clade distinct from the more divergent Z. kuroshio/Z. vietnamensis clade. The results furthermore suggest that the presence of CBCs may exist only at much lower ratios between different species in zoanthids than in other investigated organism lineages.

Amplification of the Second Internal Transcribed Spacer Ribosomal DNA of Individual Trichostrongylid Nematode Larvae by Nested Polymerase Chain Reaction

The second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) is used as a genetic marker to identify trichostrongylid nematodes. However, it is often difficult to amplify by polymerase chain reaction (PCR) the ITS2 rDNA of a single trichostrongylid nematode larva or egg. A nested PCR (nPCR) assay was, therefore, developed to amplify the ITS2 from individual trichostrongylid nematode larvae. The results show that the ITS2 rDNA of a significantly greater proportion of individual larvae was amplified using nPCR compared with a standard PCR. There was also no need to column-purify the genomic DNA before nPCR, which is more time and cost effective for studies involving large sample sizes. The amplicons produced from the secondary phase of the nPCR were subjected to single-strand conformation polymorphism analyses and DNA sequencing to confirm the species identity of the larvae used in the current study as Ostertagia gruehneri. The nPCR assay was also used to amplify the ITS2 from individual trichostrongylid eggs. The ability to amplify the ITS2 rDNA from large numbers of individual nematode eggs and larvae has important implications for diagnostic testing and for conducting epidemiological studies on these parasites of veterinary importance.

Development and validation of a PCR–RFLP test to identify African Rhipicephalus ( Boophilus) ticks

The cattle tick Rhipicephalus ( Boophilus) microplus has recently invaded West Africa and caused anxiety amongst farmers in Ivory Coast, as livestock production was severely affected. The introduction of this tick species has remained unnoticed for several years, as all the members of this genus are very similar in appearance. To overcome the cumbersome morphological identification of the four closely related R. ( Boophilus) spp. in the region, a PCR–RFLP test, based on a part of the second internal transcribed spacer ribosomal DNA (ITS2), was developed. The molecular tool was successfully validated with a large number of ticks recently collected from West Africa and that were identified both morphologically and genetically. The tool developed is simple, fast, reliable and reproducible; hence it can be routinely applied for species identification.

Utility of PCR Amplification and DNA Microarray Hybridization in 16S rDNA for Rapid Diagnosis of Bacteremia Associated with Hematological Malignancies.

Abstract 1635Poster Board I-661 IntroductionThe rapid diagnosis of bacteremia is important for patient management and the choice of antimicrobial and antifungal therapy, especially in hematological malignancies. We describe a rapid detection and identification system that uses universal PCR primers and DNA microarray hybridization compared to the conventional culture method. This new DNA-microarray method could be performed within only 24 hours. Eighty-four microorganism probes are spotted onto a microarray plate containing most causal clinical pathogens (Patent No.WO2003/106676). We can detect 93% of pathogen of blood culture by the frequency of upper 28 microorganisms. Patients and MethodsWe used universal PCR primers to amplify a variable region of bacterial 16S ribosomal DNA or fungal ITS (internal transcribed spacer region) of ribosomal DNA, followed by the reverse hybridization of products to a panel of oligonucleotides. Genomic DNA was extracted from blood with a DNA isolation kit (DNeasy; QIAGEN, CA, USA) according to the manufacturer's instructions. The 16S ribosomal ribonucleic acid gene (16S rDNA) was amplified from genomic DNA using broad-spectrum PCR primers; 5'-AGAGTTTGATCCTGGCT-CAG-3', corresponding to Escherichia coli 16S rDNA positions 8 through 27, and 5'-GTATTACCGCGGCTGCTGG-3', corresponding to Escherichia coli 16S rRNA gene positions 517 through 535. On the other hand, the PCR primers; 5'-TCCGTAGGTGAACCTGCGG-3' and 5'-GCTGCGTTCTTCATCGATGC-3' are used to amplify the ITS 1 region. Thirty-five cycles of amplification were performed in a thermal cycler (Gene Amp PCR system 9600, Perkin-Elmer Cetus, CT, USA). The PCR products from samples were labeled by Cy5- primers and applied to the microarray. The microarray was scanned using a ScanArray 4000 (Axon Instruments, CA, USA) to determine the amount of Cy5-labeled PCR products. Fluorescence intensities were calculated with DNASIS Array software (HITACHI Soft Engineering, Japan). This new identification system using DNA microarray hybridization and the conventional blood culture method (BacT/ALERT, bioMerieux and Bactec, Becton-Dickinson, USA) were applied for 336 infectious episodes from 100 patients with hematological malignancy except 2 patients with aplastic anemia retrospectively from May 2004 to February 2007. The hematological malignancies included 152 episodes of acute myeloid leukemia (45.2%), 106 of non-Hodgkin lymphoma (31.5%), 38 of acute lymphoid leukemia (11.3%), 17 of multiple myeloma (5%), 12 of myelodysplastic syndrome (3.6%), 4 of aplastic anemia (1.2%) etc. ResultsThirty-nine episodes (11.6%) were positive after a few days of culturing, and 41 episodes (12.2%) were identified by the new microarray method within 24 hours. Both methods yielded positive results 10.7% (36 episodes). Pathogens in most cases were identical at the species level (21/36 episodes 58.3%), some cases were identical at the genus level (6/36 16.7%), in the other cases, several bacteria were detected by each method. A positive DNA microarray and negative blood culture occurred at a rate of 1.5% (5 episodes), with pathogens in these cases being Bacillus cereus, Staphylococcus epidermidis, Chryseobacterium indologenes, and Enterococcus faecium. A negative DNA microarray and positive blood culture occurred at a rate of 0.9% (3 episodes), and with pathogens comprising Staphylococcus aureus, á-Streptococcus, and Pseudomonas aeruginosa. Both methods yielded negative results at a rate of 86.9% (292 episodes). The sensitivity and specificity of this new microarray method were 92.3 and 98.3%, respectively, compared with the conventional blood culture method. Regarding fungal infection, only one episode out of one patient was found to be positive microarray and negative blood culture of Yeast like organism. ConclusionThis new DNA microarray method has the potential to diagnose bacteremia quickly and detect pathogens at least at the same rate as the conventional blood culture method. Regarding the possibility of improving the sensitivity and specificity, or application for fungal infection further study should be needed. In conclusion, DNA microarray is a useful method for the management of febrile patients with hematological malignancy. DisclosuresIwasaki:The ministry of Health, Labour and Welfare of Japan: Research Funding.

Characterization and Pathogenicity of Fusicladium eriobotryae, the Fungal Pathogen Responsible for Loquat Scab.

To characterize the fungal pathogen responsible for loquat scab and establish differences in pathogenicity in loquat, eight strains identified as Fusicladium eriobotryae were isolated from either loquat leaves or fruit showing scab symptoms in Spain. Loquat plants belonging to the cv. Peluche were infected via a newly developed infection system that was based on spraying susceptible loquat plants with fungal spore suspensions, keeping the plants for 1 week in 100% humidity, and then transferring the plants to ambient relative humidity in greenhouses. Scab symptoms were analyzed and pathogenic characterization of all F. eriobotryae strains revealed different degrees of aggressiveness. Based on infection progression and severity of scab symptoms, strain ST1 was confirmed as the most aggressive in cultivars in the Mediterranean region. Strain ST1, which even caused chlorotic spots in loquat stems, is so aggressive it can be used to identify highly resistant cultivars using this in vivo system. Molecular characterization of internal transcribed spacer ribosomal DNA and, particularly, the glyceraldehyde 3-phosphate dehydrogenase gene, clearly distinguished loquat strains from Venturia inaequalis. Moreover, random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction techniques were used to qualitatively discriminate between species and report the variations within fungal populations. Molecular variability was checked by comparing all the different strains and enabled the specific identification of F. eriobotryae. Although no association was observed between any pattern and phenotypic traits, such as aggressiveness, RAPD provided a specific profile that allowed fungal identification.

Vector Incrimination of Sand Flies in the Most Important Visceral Leishmaniasis Focus in Iran

The prevalence, host preference, and rate of Leishmania spp. infection of sand fly species are important parameters for incrimination of parasite vectors. We applied polymerase chain reaction (PCR)-based and enzyme-linked immunosorbent assay (ELISA) methods to detect Leishmania spp. parasites and blood meals within individual sand flies in the most important visceral leishmaniasis (VL) focus in northwestern Iran. Leishmania spp. minicircles (kinetoplast DNA) were found in 14 (0.9%) of 1,569 female specimens. Sequence analysis of 650 basepairs of an internal transcribed spacer ribosomal DNA gene identified L. infantum/L. donovani in 12 specimens and L. adleri-like parasites in 2 specimens. Nine (64.3%) of 14 of the Leishmania spp.-positive sand flies were Phlebotomus perfeliewi transcaucasicus. Blood meal identification of host DNA within sand flies by PCR-based and ELISA methods showed that 30% and 28%, respectively, were positive for human blood. Results of this study showed that P. perfeliewi transcaucasicus is the most prevalent, infected, and anthropophagic sand fly and plays a major role in VL transmission in the region studied.

Phylogenetic and Biogeographic Patterns in Gaylussacia (Ericaceae) based on Morphological, Nuclear DNA, and Chloroplast DNA Variation

Gaylussacia includes 49 species of understory shrubs in the heath family (Ericaceae) with a disjunct distri- bution in North and South America. Little is known about evolutionary relationships among the species. To investigate phylogenetic and biogeographic relationships in Gaylussacia, three data sets were constructed and analyzed cladistically using parsimony: morphology, nuclear ribosomal internal transcribed spacer DNA sequences, and chloroplast trnL intron-trnF spacer DNA sequences. Molecular data sets were analyzed in two ways to determine the phylogenetic informativeness of insertion/deletion events. Comparison of results from separate analyses of each data set showed some incongruencies be- tween the nuclear and chloroplast data sets, indicating possible past hybridization or lineage sorting. Combined analyses were conducted for ITS and morphology, and for trnL-trnF and morphology. The monophyly of the genus including G. brachycera was not strongly supported. In addition, the traditional sectional divisions of Gaylussacia were not supported in the morphological and nuclear DNA analyses. Finally, a broader perspective on the disjunct distribution of Gaylussacia includes the possibility of a North American origin for the genus, rather than a South American origin as previously hy- pothesized.

The origin and evolution of Indomalesian, Australasian and Pacific island biotas: insights from Aglaieae (Meliaceae, Sapindales)

AbstractAim The role of long‐distance dispersal in the Indomalesian, Australasian and Pacific flora is currently hotly debated. The lack of well‐resolved phylogenetic trees for Pacific plants has been a major limitation for biogeographical analysis. Here, we present a well‐resolved phylogenetic tree for the tribe Aglaieae in the mahogany family, Meliaceae, and use it to investigate the origin, evolution and dispersal history of biotas in this area. The subfamily Melioideae, including the tribe Aglaieae (Meliaceae, Sapindales), is a plant group with good representation in the region in terms of biomass and species numbers, wide ecological attributes and known animal vectors. The family has a good fossil record (especially from North America and Europe). Genera and species in the tribe Aglaieae therefore provide an excellent model group for addressing this debate.Location Indomalesia, Australasia, Pacific islands.Methods Results from nuclear internal transcribed spacer ribosomal DNA analyses of 82 taxa, based on sequence alignment guided by secondary structure models, were combined with evidence from fossils and distribution data. We used strict and relaxed molecular clock approaches to estimate divergence times within Aglaieae. Putative ancestral areas were investigated through area‐based and event‐based biogeographical approaches. Information on dispersal routes and their direction was inferred from the investigation of dispersal asymmetries between areas.Results Our study indicates that the crown group of Aglaieae dates back at least to the Late Eocene, with major divergence events occurring during the Oligocene and Miocene. It also suggests that dispersal routes existed during Miocene–Pliocene times from the area including Peninsular Malaysia, Sumatra and Borneo to Wallacea, India and Indochina, and from the area including New Guinea, New Ireland and New Britain further east to the Pacific islands at the peripheries of the distribution range. The origin of the Fijian species dates back to the Pliocene.Main conclusions Dispersal over oceanic water barriers has occurred during geological time and seems to have been a major driving force for divergence events in Aglaieae, with some old Gondwanan land masses (e.g. Australia) colonized only during recent times. Movement from the ancestral area was predominantly towards the east. Extant Fijian species of Aglaia are monophyletic and share morphological features rarely found in species of other areas, suggesting speciation within an endemic clade. Divergence of living taxa from their closest living relatives took place during both the Miocene and the Pliocene, and peaked in the Pliocene. The present‐day distribution of many species in the tribe must therefore have arisen as a result of dispersal rather than vicariance events. Furthermore, colonization from Indomalesia to Australasia and the Pacific has frequently been followed by speciation.

Phylogenetic analyses of potentially free-living Symbiodinium spp. isolated from coral reef sand in Okinawa, Japan

The existence of “free-living” Symbiodinium that can form symbioses with hosts is implied by the presence of hosts that produce Symbiodinium-free gametes and expulsion and/or expelled symbiotic algae from host. However, it is still unclear if potentially symbiotic Symbiodinium are found “free-living” in the coral reef environment. Sixteen Symbiodinium strains were established from samples taken from three sampling locations of coral reef sand in Okinawa, Japan. Phylogenetic analyses of the partial large subunit ribosomal DNA (28S-rDNA) and the internal transcribed spacer of ribosomal DNA (ITS-rDNA) conclusively showed that all 16 isolates belonged to Symbiodinium clade A sensu Rowan and Powers (1991). The lack of other Symbiodinium clades besides clade A in this study may be due to other clades not being readily culturable under culture conditions used here. The new isolates could be phylogenetically divided into four groups, though no sequences were identical to previously reported Symbiodinium. Two of the four groups were closely related to symbiotic Symbiodinium clade A isolated from a variety of host species. One isolate group formed a highly supported monophyly with Symbiodinium types that have previously been characterized as “free-living”. The remaining isolate group, although within clade A, was quite divergent from other clade A Symbiodinium. These results indicate that novel diversity of free-living Symbiodinium exists in coral sand.

Zoanthid diversity (Anthozoa: Hexacorallia) in the Galapagos Islands: a molecular examination

Although the Galapagos are famous for their unique biodiversity, many groups of marine invertebrates from this isolated archipelago remain understudied or not investigated. One such group is the zoanthids (Order Zoan- tharia, =Zoanthidea, =Zoanthiniaria), anthozoans (Cnidaria) found in marine ecosystems worldwide. Zoanthid taxonomy has been in a state of disorganization and neglect due in large part to the morphological plasticity within species and questions about the accuracy of traditionally used morpho- logical and ecological characteristics. However, recent studies utilizing molecular methodology combined with morphology have proven to be very useful in understanding zoanthid diversity. The results of a survey of zoanthids from the Galapagos and the east Pacific are reported in this study. Shallow water (\35 m) zoanthid specimens were identified using the molecular markers mitochondrial 16S ribosomal DNA (mt 16S rDNA), cytochrome oxidase subunit I (COI) gene, and the internal transcribed spacer of ribosomal DNA (ITS-rDNA). From the collected specimens seven putative zoanthid species-level clades from three known genera (Zoanthus, Palythoa, Parazoanthus) were identified at the molecular level. These identifications were further supported by morphological and ecological data. While almost all specimens belonged to known zoanthid genera, based on unique molecular and ecological data one group of speci- mens (designated unknown zoanthid sp. ''03-103'') is potentially a novel undescribed genus. Additionally, the remaining three azooxanthellate Parazoanthus clades may also be undescribed new species, but due to the overall lack of zoanthid research and descriptions from neighboring areas (East Pacific, west coast of South America) further research is needed to clearly ascertain this. Additionally, notes on the four observed nominal azooxanthellate zoanthid species and a key to all eight nominal (seven from known genera, one from a potentially new genus) shallow water zoanthid spe- cies found thus far in the Galapagos Islands are provided.

Phylogenetic relationships in the tribe Alysseae (Brassicaceae) based on nuclear ribosomal ITS DNA sequences

Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) region of 85 species (131 accessions) were used to determine the tribal limits, monophyly status, and phylogenetic intra-tribal relationships of genera within the tribe Alysseae (Brassicaceae). Both maximum parsimony and maximum likelihood analyses support the recognition of the tribe Alysseae s. str. (12 genera: Alyssoides , Alyssum , Aurinia , Berteroa , Bornmuellera , Clastopus , Clypeola , Degenia , Fibigia , Galitzkya , Hormathophylla , and Physoptychis ). Six well-supported clades were recognized within the Alysseae clade, including two Alyssum clades (one of which includes Clypeola ), an Alyssoides and allies clade (includes Alyssoides , Bornmuellera , Clastopus , Degenia , Fibigia , Hormathophylla , and Physoptychis ), a Berteroa and allies clade (includes Aurinia , Berteroa , and Galitzkya ), a Bornmuellera clade, and a Hormathophylla clade. Morphological and cytological support for these clades is reviewed. The ITS data support the exclusion of the following taxa from the Alysseae, with appropriate tribal assignment given in parentheses: Alyssum klimesii Al-Shehbaz (Camelineae), Asperuginoides (unresolved), Athysanus (Arabideae), Botschantzevia (Arabideae), Didymophysa (unresolved), Farsetia (Malcolmieae), Lobularia (Malcolmieae), and Ptilotrichum (Arabideae). Farsetia and Lobularia are inferred to be monophyletic, and based on molecular and morphological characters they are assigned to Malcolmieae, a recently described tribe.

Novel root fungal consortium associated with a dominant desert grass.

The broad distribution and high colonization rates of plant roots by a variety of endophytic fungi suggest that these symbionts have an important role in the function of ecosystems. Semiarid and arid lands cover more than one-third of the terrestrial ecosystems on Earth. However, a limited number of studies have been conducted to characterize root-associated fungal communities in semiarid grasslands. We conducted a study of the fungal community associated with the roots of a dominant grass, Bouteloua gracilis, at the Sevilleta National Wildlife Refuge in New Mexico. Internal transcribed spacer ribosomal DNA sequences from roots collected in May 2005, October 2005, and January 2006 were amplified using fungal-specific primers, and a total of 630 sequences were obtained, 69% of which were novel (less than 97% similarity with respect to sequences in the NCBI database). B. gracilis roots were colonized by at least 10 different orders, including endophytic, coprophilous, mycorrhizal, saprophytic, and plant pathogenic fungi. A total of 51 operational taxonomic units (OTUs) were found, and diversity estimators did not show saturation. Despite the high diversity found within B. gracilis roots, the root-associated fungal community is dominated by a novel group of dark septate fungi (DSF) within the order Pleosporales. Microscopic analysis confirmed that B. gracilis roots are highly colonized by DSF. Other common orders colonizing the roots included Sordariales, Xylariales, and Agaricales. By contributing to drought tolerance and nutrient acquisition, DSF may be integral to the function of arid ecosystems.

Phylogenetic patterns and conservation among North American members of the genus Agalinis (Orobanchaceae).

BackgroundNorth American Agalinis Raf. species represent a taxonomically challenging group and there have been extensive historical revisions at the species, section, and subsection levels of classification. The genus contains many rare species, including the federally listed endangered species Agalinis acuta. In addition to evaluating the degree to which historical classifications at the section and subsection levels are supported by molecular data sampled from 79 individuals representing 29 Agalinis species, we assessed the monophyly of 27 species by sampling multiple individuals representing different populations of those species. Twenty-one of these species are of conservation concern in at least some part of their range.ResultsPhylogenetic relationships estimated using maximum likelihood analyses of seven chloroplast DNA loci (aligned length = 11 076 base pairs (bp) and the nuclear ribosomal DNA ITS (internal transcribed spacer) locus (733 bp); indicated no support for the historically recognized sections except for Section Erectae. Our results suggest that North American members of the genus comprise six major lineages, however we were not able to resolve branching order among many of these lineages. Monophyly of 24 of the 29 sampled species was supported based on significant branch lengths of and high bootstrap support for subtending branches. However, there was no statistical support for the monophyly of A. acuta with respect to Agalinis tenella and Agalinis decemloba. Although most species were supported, deeper relationships among many species remain ambiguous.ConclusionThe North American Agalinis species sampled form a well supported, monophyletic group within the family Orobanchaceae relative to the outgroups sampled. Most hypotheses regarding section- and subsection-level relationships based on morphology were not supported and taxonomic revisions are warranted. Lack of support for monophyly of Agalinis acuta leaves the important question regarding its taxonomic status unanswered. Lack of resolution is potentially due to incomplete lineage sorting of ancestral polymorphisms among recently diverged species; however the gene regions examined did distinguish among almost all other species in the genus. Due to the important policy implications of this finding we are further evaluating the evolutionary distinctiveness of A. acuta using morphological data and loci with higher mutation rates.