Total V9 rDNA information organized at the metabarcode level (Database W4)

The present data set provides a tab separated text file compressed in a zip archive. The file includes metadata for each TaraOceans V9 rDNA metabarcode including the following fields: md5sum = unique identifier; lineage = taxonomic path associated to the metabarcode; pid = % identity to the closest reference barcode from V9_PR2; sequence = nucleotide sequence of the metabarcode; refs = identity of the best hit reference sequence(s); TARA_xxx = number of occurrences of this barcode in each of the 334 samples; totab = total abundance of the barcode ; cid = identifier of the OTU to which the barcode belongs; and taxogroup = high-taxonomic level assignation of this barcode. The file also includes three categories of functional annotations: (1) Chloroplast: yes, presence of permanent chloroplast; no, absence of permanent chloroplast ; NA, undetermined. (2) Symbiont (small partner): parasite, the species is a parasite; commensal, the species is a commensal; mutualist, the species is a mutualist symbiont, most often a microalgal taxon involved in photosymbiosis; no the species is not involved in a symbiosis as small partner; NA, undetermined. (3) Symbiont (host): photo, the host species relies on a mutualistic microalgal photosymbiont to survive (obligatory photosymbiosis); photo_falc, same as photo, but facultative relationship; photo_klep, the host species maintains chloroplasts from microalgal prey(s) to survive; photo_klep_falc, same as photo_klep, but facultative; Nfix, the host species must interact with a mutualistic symbiont providing N2 fixation to survive; Nfix_falc, same as Nfix, but facultative; no, the species is not involved in any mutualistic symbioses; NA, undetermined. For example, the collodarian/Brandtodinium symbiosis is annotated: Chloroplast, "no"; Symbiont (small), "no"; Symbiont (host), "photo", for the collodarian host; and: Chloroplast, "yes"; Symbiont (small), "mutualist"; Symbiont (host), "no", for the dinoflagellate microalgal endosymbiont.chloroplast = "yes", "no" or "NA"; symbiont.small = "parasite", "commensal", "mutualist", "no" or "NA"; symbiont.host = "photo", "photo_falc", "photo_klep", "Nfix", no or NA; benef = "Nfix", "no" or "NA"; trophism = Metazoa , heterotroph , NA , photosymbiosis , phototroph according to the previous fields.

Molecular characterization of the microbial species that colonize human ileal and colonic mucosa by using 16S rDNA sequence analysis

Aim: The aim of this study was to characterize the bacterial community adhering to the mucosa of the terminal ileum, and proximal and distal colon of the human digestive tract. Methods and Results: Pinch samples of the terminal ileum, proximal and distal colon were taken from a healthy 35-year-old, and a 68-year-old subject with mild diverticulosis. The 16S rDNA genes were amplified using a low number of PCR cycles, cloned, and sequenced. In total, 361 sequences were obtained comprising 70 operational taxonomic units (OTU), with a calculated coverage of 82.6%. Twenty-three per cent of OTU were common to the terminal ileum, proximal colon and distal colon, but 14% OTU were only found in the terminal ileum, and 43% were only associated with the proximal or distal colon. The most frequently represented clones were from the Clostridium group XIVa (24.7%), and the Bacteroidetes (Cytophaga-Flavobacteria-Bacteroides ) cluster (27.7%). Conclusion: Comparison of 16S rDNA clone libraries of the hindgut across mammalian species confirms that the distribution of phylogenetic groups is similar irrespective of the host species. Lesser site-related differences within groups or clusters of organisms, are probable. Significance and Impact: This study provides further evidence of the distribution of the bacteria on the mucosal surfaces of the human hindgut. Data contribute to the benchmarking of the microbial composition of the human digestive tract.

A preliminary phylogenetic analysis of the Capsalidae (Platyhelminthes : Monogenea : Monopisthocotylea) inferred from large subunit rDNA sequences

Phylogenetic relationships within the Capsalidae (Monogenea) were examined Using large subunit ribosomal DNA sequences from 17 capsalid species (representing 7 genera, 5 subfamilies), 2 outgroup taxa (Monocotylidae) plus Udonella caligorum (Udonellidae). Trees were constructed using maximum likelihood, minimum evolution and maximum parsimony algorithms. An initial tree, generated from sequences 315 bases long, Suggests that Capsalinae, Encotyllabinae, Entobdellinae and Trochopodinae are monophyletic, but that Benedeniinae is paraphyletic. Analyses indicate that Neobenedenia, currently in the Benedeniinae, should perhaps be placed in 2 separate subfamily. An additional analysis was made which omitted 3 capsalid taxa (for which only short sequences were available) and all outgroup taxa because of alignment difficulties. Sequence length increased to 693 bases and good branch support was achieved. The Benedeniinae was again paraphyletic. Higher-level classification of the Capsalidae, evolution of the Entobdellinae and issues of species identity in Neobenedenia are discussed.

Multiple origins of parasitism in lice: phylogenetic analysis of SSU rDNA indicates that the Phthiraptera and Psocoptera are not monophyletic

The Paraneoptera (Hemipteroid Assemblage) comprises the orders Thysanoptera (thrips), Hemiptera (bugs), Phthiraptera (lice) and Psocoptera (booklice and barklice). The phylogenetic relationships among the Psocodea (Phthiraptera and Psocoptera), Thysanoptera and Hemiptera are unresolved, as are some relationships within the Psocodea. Here, we present phylogenetic hypotheses inferred from SSU rDNA sequences; the most controversial of which is the apparent paraphyly of the Phthiraptera, which are parasites of birds and mammals, with respect to one family of Psocoptera, the Liposcelididae. The order Psocoptera and the suborder that contains the Liposcelididae, the Troctomorpha, are also paraphyletic. The two remaining psocopteran suborders, the Psocomorpha and the Trogiomorpha, are apparently monophyletic. The Liposcelididae is most closely related to lice from the suborder Amblycera. These results suggest that the taxonomy of the Psocodea needs revision. In addition, there are implications for the evolution of parasitism in insects; parasitism may have evolved twice in lice or have evolved once and been subsequently lost in the Liposcelididae.

Unexpected prevalence of parasite 18S rDNA sequences in winter among Antarctic marine protists

Parasites are not typically considered to be important components of polar marine ecosystems. It was therefore surprising when 18S rDNA surveys of protists in the West Antarctic Peninsula in winter revealed high abundances of parasite sequences. Parasite sequences made up, on average, over half (52%) of sequence reads in samples from deep water in winter. Winter surface water and sediment samples contained relatively fewer, but still strikingly high, parasite sequence reads (13 and 9%, respectively), while surface water samples in summer contained fewer parasite sequences (1.8%). A total of 1028 distinct parasite Operational Taxonomic Units were observed in winter, with the largest abundances and diversities within Syndiniales groups I and II, including Amoebophrya. Less abundant parasite sequence groups included Apicomplexa, Blastodinium, Chytriodinium, Cryptocaryon, Paradinium, Perkinsidae, Pirsonia and Ichthyophonae. Parasite sequence distributions suggested interactions with known hosts, such as diatom parasites which were mainly in the sediments, where resting spores of Chaetoceros spp. diatoms were abundant. Syndiniales sequences were correlated with radiolarian sequences, suggesting parasite–host interactions. The abundant proportions of parasite sequences indicate a potentially important role for parasites in the Antarctic marine ecosystem, with implications for plankton population dynamics, the role of the microbial loop, carbon flows and ecosystem responses to ongoing anthropogenic climate change.

Unexpected prevalence of parasite 18S rDNA sequences in winter among Antarctic marine protists

Parasites are not typically considered to be important components of polar marine ecosystems. It was therefore surprising when 18S rDNA surveys of protists in the West Antarctic Peninsula in winter revealed high abundances of parasite sequences. Parasite sequences made up, on average, over half (52%) of sequence reads in samples from deep water in winter. Winter surface water and sediment samples contained relatively fewer, but still strikingly high, parasite sequence reads (13 and 9%, respectively), while surface water samples in summer contained fewer parasite sequences (1.8%). A total of 1028 distinct parasite Operational Taxonomic Units were observed in winter, with the largest abundances and diversities within Syndiniales groups I and II, including Amoebophrya. Less abundant parasite sequence groups included Apicomplexa, Blastodinium, Chytriodinium, Cryptocaryon, Paradinium, Perkinsidae, Pirsonia and Ichthyophonae. Parasite sequence distributions suggested interactions with known hosts, such as diatom parasites which were mainly in the sediments, where resting spores of Chaetoceros spp. diatoms were abundant. Syndiniales sequences were correlated with radiolarian sequences, suggesting parasite–host interactions. The abundant proportions of parasite sequences indicate a potentially important role for parasites in the Antarctic marine ecosystem, with implications for plankton population dynamics, the role of the microbial loop, carbon flows and ecosystem responses to ongoing anthropogenic climate change.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis : C. parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis . In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Phylogenetic placement of the sugarcane orange rust pathogen Puccinia kuehnii in a historical and regional context

Sugarcane orange rust, caused by Puccinia kuehnii, was once considered a minor disease in the Australian sugar industry. However, in 2000 a new race of the pathogen devastated the high-performing sugarcane cultivar Q124, and caused the industry Aus$150–210 million in yield losses. At the time of the epidemic, very little was known about the genetic and pathogenic diversity of the fungus in Australia and neighbouring sugar industries. DNA sequence data from three rDNA regions were used to determine the genetic relationships between isolates within two P. kuehnii collections. The first collection comprised only recent Australian field isolates and limited sequence variation was detected within this population. In the second study, Australian isolates were compared with isolates from Papua New Guinea, Indonesia, China and historical herbarium collections. Greater sequence variation was detected in this collection and phylogenetic analyses grouped the isolates into three clades. All isolates from commercial cane fields clustered together including the recent Australianfield isolates and the Australian historical isolate from 1898.The other two clades included rust isolates from wild and garden canes in Indonesia and PNG. These rusts appeared morphologically similar to P. kuehnii and could potentially pose a quarantine threat to the Australian sugar industry. The results have revealed greater diversity in sugarcane rusts than previously thought.

Culture independent analysis of greyback canegrub-associated microflora and microbial community comparison using subtractive hybridisation

Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putativelysaprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens. Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubsassociated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community.

Molecular phylogeny supports the paraphyletic nature of the genus Trogoderma (Coleoptera: Dermestidae) collected in the Australasian ecozone

To date, a molecular phylogenetic approach has not been used to investigate the evolutionary structure of Trogoderma and closely related genera. Using two mitochondrial genes, Cytochrome Oxidase I and Cytochrome B, and the nuclear gene, 18S, the reported polyphyletic positioning of Trogoderma was examined. Paraphyly in Trogoderma was observed, with one Australian Trogoderma species reconciled as sister to all Dermestidae and the Anthrenocerus genus deeply nested within the Australian Trogoderma clade. In addition, time to most recent common ancestor for a number of Dermestidae was calculated. Based on these estimations, the Dermestidae origin exceeded 175 million years, placing the origins of this family in Pangaea.

Metabolic and ecological study of environmental pentose utilizing bacteria (E-PUB)

Lignocellulosic materials, such as sugar cane bagasse, a waste product of the sugarcane processing industry, agricultural residues and herbaceous crops, may serve as an abundant and comparatively cheap feedstock for largescale industrial fermentation, resulting in the production of marketable end-products. However, the complex structure of lignocellulosic materials, the presence of various hexose and pentose sugars in the hemicellulose component, and the presence of various compounds that inhibit the organisms selected for the fermentation process, all constitute barriers that add to the production costs and make full scale industrial production economically less feasible. The work presented in this thesis was conducted in order to screen microorganisms for ability to utilize pentose sugars derived from the sugar mill industrial waste. A large number of individual bacterial strains were investigated from hemi-cellulose rich material collected at the Proserpine and Maryborough sugar mills, notably soil samples from the mill sites. The research conducted to isolation of six pentose-capable Gram-positive organisms from the actinomycetes group by using pentose as a sole carbon source in the cultivation process. The isolates were identified as Corynebacterium glutamicum, Actinomyces odontolyticus, Nocardia elegans, and Propionibacterium freudenreichii all of which were isolated from the hemicellulose-enriched soil. Pentose degrading microbes are very rare in the environment, so this was a significant discovery. Previous research indicated that microbes could degrade pentose after genetic modification but the microbes discovered in this research were able to naturally utilize pentose. Six isolates, identified as four different genera, were investigated for their ability to utilize single sugars as substrates (glucose, xylose, arabinose or ribose), and also dual sugars as substrates (a hexose plus a pentose). The results demonstrated that C. glutamicum, A. odontolyticus, N. elegans, and P. freudenreichii were pentose-capable (able to grow using xylose or other pentose sugar), and also showed diauxie growth characteristics during the dual-sugar (glucose, in combination with xylose, arabinose or ribose) carbon source tests. In addition, it was shown that the isolates displayed very small differences in growth rates when grown on dual sugars as compared to single sugars, whether pentose or hexose in nature. The anabolic characteristics of C. glutamicum, A. odontolyticus, N. elegans and P. freudenreichii were subsequently investigated by qualitative analysis of their end-products, using high performance liquid chromatography (HPLC). All of the organisms produced arginine and cysteine after utilization of the pentose substrates alone. In addition, P. freudenreichii produced alanine and glycine. The end-product profile arising from culture with dual carbon sources was also tested. Interestingly, this time the product was different. All of them produced the amino acid glycine, when grown on a combination substrate-mix of glucose with xylose, and also glucose with arabinose. Only N. elegans was able to break down ribose, either singly or in combination with glucose, and the end-product of metabolism of the glucose plus ribose substrate combination was glutamic acid. The ecological analysis of microbial abundance in sugar mill waste was performed using denaturing gradient gel electrophoresis (DGGE) and also the metagenomic microarray PhyloChip method. Eleven solid samples and seven liquid samples were investigated. A very complex bacterial ecosystem was demonstrated in the seven liquid samples after testing with the PhyloChip method. It was also shown that bagasse leachate was the most different, compared to all of the other samples, by virtue of its richness in variety of taxa and the complexity of its bacterial community. The bacterial community in solid samples from Proserpine, Mackay and Maryborough sugar mills showed huge diversity. The information found from 16S rDNA sequencing results was that the bacterial genera Brevibacillus, Rhodospirillaceae, Bacillus, Vibrio and Pseudomonas were present in greatest abundance. In addition, Corynebacterium was also found in the soil samples. The metagenomic studies of the sugar mill samples demonstrate two important outcomes: firstly that the bagasse leachate, as potentially the most pentose-rich sample tested, had the most complex and diverse bacterial community; and secondly that the pentose-capable isolates that were initially discovered at the beginning of this study, were not amongst the most abundant taxonomic groups discovered in the sugar mill samples, and in fact were, as suspected, very rare. As a bioprospecting exercise, therefore, the study has discovered organisms that are naturally present, but in very small numbers, in the appropriate natural environment. This has implications for the industrial application of E-PUB, in that a seeding process using a starter culture will be necessary for industrial purposes, rather than simply assuming that natural fermentation might occur.

A molecular phylogeny of the checkered beetles and a description of Epiclininae a new subfamily (Coleoptera: Cleroidea: Cleridae)

We provide the first molecular phylogeny of the clerid lineage (Coleoptera: Cleridae, Thanerocleridae) within the superfamily Cleroidea to examine the two most recently-proposed hypotheses of higher-level classification. Phylogenetic relationships of checkered beetles were inferred from approximately ~5,000nt of both nuclear and mitochondrial rDNA (28S, 16S, and 12S) and the mitochondrial protein-coding gene COI. A worldwide sample of ~70 genera representing almost a quarter of generic diversity of the clerid lineage was included and phylogenies were reconstructed using Bayesian and Maximum Likelihood approaches. Results support the monophyly of many proposed subfamilies but were not entirely congruent with either current classification system. The subfamilial relationships within the Cleridae are resolved with support for three main lineages. Tillinae are supported as the sister group to all other subfamilies within the Cleridae, whereas Thaneroclerinae, Korynetinae and a new subfamily formally described here, Epiclininae subf. n, form a sister group to Clerinae + Hydnocerinae.