Molecular Phylogenetic Survey Research Articles

Taxonomy of the Genus Halophila Thouars (Hydocharitaceae): A Review.

The seagrass genus Halophila Thouars has more than twenty described species and is predominately distributed over a wide geographic range along the tropical and the warm temperate coastlines in the Indo-West Pacific Oceans. A brief history of the Halophila taxonomic development is presented. Based on reproductive and vegetative morphology, the genus is divided into eight sections including three new sections: section Australes, section Stipulaceae and section Decipientes. A rewritten taxonomic description of the type species for the genus Halophila, H. madagascariensis Steudel ex Doty et B.C. Stone, is provided. The lectotype of H. engelmannii Asch. as well as neotypes of H. hawaiiana Doty et B.C. Stone and H. spinulosa (Br.) Asch. are designated. Furthermore, H. ovalis ssp. bullosa, ssp. ramamurthiana and ssp. linearis together with H. balforurii have been recognised as distinct species. Nomenclature, typification, morphological description and botanical illustrations are presented for each taxon. Recent molecular phylogenetic surveys on certain Halophila taxa are also discussed. Field surveys for the deep water Halophila in West Pacific regions are suggested. Morphological studies combined with molecular investigations for the Halophila on the east coast of Africa and the West Indian Ocean are urgently needed and highly recommended.

Six new species and reports of Hydnum (Cantharellales) from eastern North America.

Five species of Hydnum have been generally recognized from eastern North America based on morphological recognition: H.albidum, H.albomagnum, H.repandum and varieties, H.rufescens, and H.umbilicatum. Other unique North American species, such as H.caespitosum and H.washingtonianum, are either illegitimately named or considered synonymous with European taxa. Here, seventeen phylogenetic species of Hydnum are detected from eastern North America based on a molecular phylogenetic survey of ITS sequences from herbarium collections and GenBank data, including environmental sequences. Based on current distribution results, sixteen of these species appear endemic to North America. Of these, six species are described as new: H.alboaurantiacum, H.cuspidatum, H.ferruginescens, H.subconnatum, H.subtilior, and H.vagabundum. Geographic range extensions and taxonomic notes are provided for five additional species recently described as new from eastern North America. A new name, H.geminum, is proposed for H.caespitosum Banning ex Peck, non Valenti. Overall, species of Hydnum are best recognized by a combination of morphological and molecular phylogenetic analyses. Taxonomic descriptions are provided for seventeen species, including epitype designations for H.albidum, H.albomagnum, and H.umbilicatum, taxa described more than 100 years ago, and molecular annotation of the isotype of H.washingtonianum. Photographs and a key to eastern North American Hydnum species are presented.

Equilibrium Bird Species Diversity in Atlantic Islands

Half a century ago, MacArthur and Wilson proposed that the number of species on islands tends toward a dynamic equilibrium diversity around which species richness fluctuates [1]. The current prevailing view in island biogeography accepts the fundamentals of MacArthur and Wilson's theory [2] but questions whether their prediction of equilibrium can be fulfilled over evolutionary timescales, given the unpredictable and ever-changing nature of island geological and biotic features [3-7]. Here we conduct a complete molecular phylogenetic survey of the terrestrial bird species from four oceanic archipelagos that make up the diverse Macaronesian bioregion-the Azores, the Canary Islands, Cape Verde, and Madeira [8, 9]. We estimate the times at which birds colonized and speciated in the four archipelagos, including many previously unsampled endemic and non-endemic taxa and their closest continental relatives. We develop and fit a new multi-archipelago dynamic stochastic model to these data, explicitly incorporating information from 91 taxa, both extantand extinct. Remarkably, we find that all four archipelagos have independently achieved and maintained a dynamic equilibrium over millions of years. Biogeographical rates are homogeneous across archipelagos, except for the Canary Islands, which exhibit higher speciation and colonization. Our finding that the avian communities of the four Macaronesian archipelagos display an equilibrium diversity pattern indicates that a diversity plateau may be rapidly achieved on islands where rates of insitu radiation are low and extinction is high. This study reveals that equilibrium processes may be more prevalent than recently proposed, supporting MacArthur and Wilson's 50-year-old theory.

New distributional record of Chelonibia testudinaria (Linnaeus, 1758) from the island of Gökçeada, northern Aegean Sea, Turkey

* Correspondence: ogonulal@istanbul.edu.tr Chelonibia testudinaria (Linnaeus, 1758) is a cosmopolitan species of barnacle common in warm seas and all over the Mediterranean as an epibiont of marine turtles and other hosts (Newman and Abbott, 1980; Koukouras and Matsa, 1998; Zardus et al., 2014). C. testudinaria is the most often reported sea turtle barnacle and it has been commonly found on Caretta caretta (Linnaeus, 1758) (loggerhead) and Chelonia mydas (Linnaeus, 1758) (green turtle) dating as far back as the Miocene epoch (Blick et al., 2011). The barnacle is considered an obligate commensal with narrow host specialization and is reported from all species of sea turtles (Zardus et al., 2013). Relini (1980) has noted that this species is also rarely found on inanimate objects. The effects of epibionts on marine turtles are poorly known. Zardus et al. (2013) conducted a worldwide molecular phylogenetic survey of Chelonibia. They found four species (C. testudinaria, C. patula, C. manati, and C. caretta); apart from C. caretta these are genetically indistinguishable. The three taxa are morphotypes of the same species and synonymized under C. testudinaria. The only record known to date from Turkish seas is from the eastern Mediterranean coast (Geldiay et al., 1982). C. testudinaria has been reported from C. caretta from the Strymonikos Gulf (Aegean Sea, Greece) (Koukouras and Matsa, 1998). The geographical distributions recorded for C. testudinaria in the Mediterranean Sea are given in Table 1. In the present study, C. testudinaria is reported for the first time from the Turkish coast of the Aegean Sea. Description: C. caretta, five pairs of lateral scutes, anterior pair touching the precentral scute, two claws on each flipper (Fischer et al., 1987). C. testudinaria, the aperture is oval, exceeding in length one third of the longer basal diameter. The mandibles have five teeth. The shell is strong, conical, and depressed, with more or less deep notches on it (Gruvel, 1965). In September 2014, two dead loggerhead turtles (C. caretta) washed ashore on the island of Gokceada in the northern Aegean Sea, Turkey (Figure 1). I carefully analyzed the body of the turtles completely. It was not clear how long the turtles’ carcasses had stayed in the sea before becoming stranded. Firstly the weights were measured with handheld luggage scales. The turtles were measured according to Bolten (1999). Measurements were taken either with calipers (straight-line measurements) or with a flexible tape measure (curved measurements). Straight carapace length (SCL) is measured from the anterior edge of the carapace to the posterior tip of the supracaudals. Also anterior and posterior locations are on the same side of the carapace. Straight carapace width (SCW) is measured at the widest point; there are no anatomical reference points. Chelonibia barnacles were photographed and counted and their diameters were measured with calipers to the nearest 0.1 mm. Chelonibia barnacles are usually oval rather than round and so measurements are taken of the longest axis, called the rostro-carinal length. The number of Chelonibia individuals and morphological analysis of both turtles are given in Table 2. Abstract: A total of 420 specimens of Chelonibia testudinaria (Linnaeus, 1758) were found on two dead loggerhead turtles (Caretta caretta) washed ashore on the island of Gokceada (northern Aegean Sea). The species is reported for the first time from the Turkish coast of the Aegean Sea and is the second record since 1982 for the Turkish Seas.

Deconstructing an assemblage of “turtle” barnacles: species assignments and fickle fidelity in Chelonibia

Barnacles in the genus Chelonibia are commensal with a variety of motile marine animals including sea turtles, crustaceans, and sirenians. We conducted a worldwide molecular phylogenetic survey of Chelonibia collected from nearly all known hosts to assess species relationships, host-fidelity, and phylogeographic structure. Using DNA sequences from a protein-coding mitochondrial gene (COI), a mitochondrial rRNA gene (12S), and one nuclear rRNA gene (28S), we found that of four species, three (C. testudinaria, C. patula, and C. manati) are genetically indistinguishable. In addition, we show each utilizes a rare androdioecious mode of reproduction involving complemental males. In contrast, the fourth species (C. caretta), which is hermaphroditic and specializes on turtles, is genetically distinct—leading to the conclusion that the three former taxa are morphotypes of the same species and should be synonymized under C. testudinaria. Phylogenetic analysis resulted in three geographic clades (Atlantic, Indian Ocean/western Pacific, and eastern Pacific) with haplotype parsimony networks revealing no shared haplotypes among geographic regions. Analysis of molecular variance detected significant differences among sequences by region (p < 0.005); conversely, there were no significant differences among sequences when grouped by host or taxonomic designation. Average pairwise genetic distances were lower between the eastern Pacific and Atlantic clades (0.053 ± 0.006) than between the eastern Pacific and Indian Ocean/western Pacific clades (0.073 ± 0.008), suggesting Atlantic and eastern Pacific populations were connected more recently, perhaps until the rise of the Isthmus of Panama. Host use by Chelonibia morphotypes is discussed along with speculation on possible ancestral hosts and support for a “turtle-first” hypothesis.

Phototrophic Phylotypes Dominate Mesothermal Microbial Mats Associated with Hot Springs in Yellowstone National Park

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.

Conundrums in species concepts: the discovery of a new cryptic species segregated from Parmelina tiliacea(Ascomycota: Parmeliaceae)

AbstractParmelina tiliacea is a common, widely distributed species in south-western Europe, easily identifiable by morphology and much used as an air pollution bioindicator in many regions. A molecular phylogenetic survey of samples from many geographical areas, using Maximum Parsimony and Bayesian inference of nuITS and mtLSU rDNA regions, revealed a group of samples geographically restricted to a small region of the Iberian Peninsula and genetically separated from the other P. tiliacea specimens studied. These samples are morphologically indistinguishable from P. tiliacea, apart from subtle anatomical characters in the ascomata (hyphae of the exciple and ascospore width), which are frequently absent. Although geographically different, the two taxa occupy similar habitats and are even sympatric in some areas, indicating that they do not exchange genetic material. This previously overlooked, and apparently endemic lineage, is described as P. cryptotiliacea sp. nov., and the name Lichen tiliaceus is epitypified by a sequenced specimen to fix the application of Parmelina tiliacea to the widespread genotype. A second unexpected result was the discovery that the morphologically distinct P. pastillifera was nested within P. tiliacea. These two cases stress the need to use molecular tools to elucidate species concepts even within widespread morphologically well-characterized macrolichens. Such investigations are necessary to improve our understanding and estimation of biodiversity, and to facilitate the development of sound biodiversity conservation strategies for lichens.

Archaeal Diversity and Distribution along Thermal and Geochemical Gradients in Hydrothermal Sediments at the Yonaguni Knoll IV Hydrothermal Field in the Southern Okinawa Trough

A variety of archaeal lineages have been identified using culture-independent molecular phylogenetic surveys of microbial habitats occurring in deep-sea hydrothermal environments such as chimney structures, sediments, vent emissions, and chemosynthetic macrofauna. With the exception of a few taxa, most of these archaea have not yet been cultivated, and their physiological and metabolic traits remain unclear. In this study, phylogenetic diversity and distribution profiles of the archaeal genes encoding small subunit (SSU) rRNA, methyl coenzyme A (CoA) reductase subunit A, and the ammonia monooxygenase large subunit were characterized in hydrothermally influenced sediments at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa Trough. Sediment cores were collected at distances of 0.5, 2, or 5 m from a vent emission (90 degrees C). A moderate temperature gradient extends both horizontally and vertically (5 to 69 degrees C), indicating the existence of moderate mixing between the hydrothermal fluid and the ambient sediment pore water. The mixing of reductive hot hydrothermal fluid and cold ambient sediment pore water establishes a wide spectrum of physical and chemical conditions in the microbial habitats that were investigated. Under these different physico-chemical conditions, variability in archaeal phylotype composition was observed. The relationship between the physical and chemical parameters and the archaeal phylotype composition provides important insight into the ecophysiological requirements of uncultivated archaeal lineages in deep-sea hydrothermal vent environments, giving clues for approximating culture conditions to be used in future culturing efforts.

A molecular phylogenetic survey of caprimulgiform nightbirds illustrates the utility of non-coding sequences

The order Caprimulgiformes comprises five bird families adapted to nocturnal activity. The order has been regarded as monophyletic, but recent evidence suggests that swifts and hummingbirds (Apodiformes) belong within it. To explore the group's phylogeny, we obtained more than 2000 bp of DNA sequence from the cytochrome b and c-myc genes for 35 taxa, representing all major lineages and outgroups. Non-coding sequences of the c-myc gene were unsaturated, readily alignable and contained numerous informative insertions and deletions (indels), signalling broad utility for higher level phylogenetics. A 12 bp insertion in c-myc links Apodiformes with owlet-nightjars, confirming paraphyly of the traditional Caprimulgiformes. However, even this rare genomic change is homoplasious when all birds are considered. Monophyly of each of the five traditional families was strongly confirmed, but relationships among families were poorly resolved. The tree structure argues against family status for Eurostopodus and Batrachostomus, which should be retained in Caprimulgidae and Podargidae, respectively. The genus Caprimulgus and both subfamilies of Caprimulgidae appear to be polyphyletic. The phylogeny elucidates the evolution of adaptive traits such as nocturnality and hypothermia, but whether nocturnality evolved once or multiple times is an open question.

Cryptic species in Gemmuloborsonia (Gastropoda: Conoidea)

During a broad molecular taxonomic and phylogenetic survey of the gastropod superfamily Conoidea, 80 specimens of several species of the genus Gemmuloborsonia were sequenced for the cytochrome c oxidase subunit I gene. The genus, originally established for fossil species from the Plio-Pleistocene of the Philippines, now includes living species from bathyal depths of the Indo-Pacific Oceans. The molecular data demonstrated the presence of five separate entities, while only four ‘morphospecies’ could be isolated by visual examination. The two largest groups, representing separate species from the molecular data, were impossible to distinguish with certainty using shell or anatomical characters. To examine shell morphology in more detail the shape of the last whorl was analysed by Fourier analysis, and the Fourier coordinates were used in canonical variate analysis. The majority of the specimens were separated into two groups, but 21.6% of the specimens were impossible to distinguish by morphological characters. One of these two forms was attributed to the known species Gemmuloborsonia moosaiSysoev & Bouchet, 1996, while the other is described as a new species Gemmuloborsonia clandestina. Bathytoma colorataSysoev & Bouchet, 2001 is transferred to Gemmuloborsonia on the basis of molecular analysis and radular morphology. Another species, represented in our material by a single specimen, remains undescribed.

Green lacewing phylogeny, based on three nuclear genes (Chrysopidae, Neuroptera)

Abstract Systematic relationships among higher taxa within Chrysopidae, a large and agriculturally significant neuropteran family, are poorly understood. A molecular phylogenetic survey of Chrysopidae was performed with three nuclear genes, namely wingless (546 bp), phosphoenolpyruvate carboxykinase (483 bp), and sodium/potassium ATPase alpha subunit (410 bp). We examined 83 species in 24 genera, mainly from Japan, Eurasia and Africa. Parsimony and Bayesian analyses of combined datasets of a total of 1439 bp demonstrated that (1) monophyly of the subfamily Chrysopinae was supported but the relationship between Nothochrysinae and Apochrysinae was unclear, although the two subfamilies together may constitute the sister taxon of Chrysopinae; (2) of the three tribes examined within Chrysopinae (Ankylopterygini, Belonopterygini and Chrysopini), monophyly of Ankylopterygini and Belonopterygini was supported, but the relationships among the three remain unclear; (3) seven sub‐clades in Chrysopini were indicated, namely (i) Brinckochrysa, (ii) Chrysemosa + Suarius, (iii) Chrysotropia + Nineta, (iv) Mallada + Chrysoperla + Peyerimhoffina, (v) Cunctochrysa + Meleoma + Nipponochrysa + Apertochrysa albolineatoides, (vi) Chrysopa + Plesiochrysa, and (vii) Dichochrysa + Apertochrysa eurydera; and (4) most genera were monophyletic, except for Apertochrysa and Cunctochrysa, each of which was shown to have two distinct origins. Our molecular analysis allowed the assignment of several species of uncertain affinities to known genera. There was some disagreement between the molecular and previously published morphological phylogenies, but in general our results confirmed existing morphological hypotheses of evolution within the family.

Taxonomic Relationships within the Pan-Oriental Narrow-mouth Toad Microhyla ornata as Revealed by mtDNA Analysis (Amphibia, Anura, Microhylidae)

A molecular phylogenetic survey was conducted using mtDNA sequences of 12S and 16S rRNA, and cyt-b genes to examine taxonomic relationships among populations of the Pan-Oriental microhylid, Microhyla ornata, from India, Bangladesh, Thailand, Laos, China, Taiwan, and the Ryukyu Archipelago of Japan. Two discrete clades are recognized within this species, one consisting of populations from India and Bangladesh, and the other encompassing the remaining populations. In the latter clade, populations from the Ryukyu Archipelago are clearly split from the rest (populations from Taiwan and the continent) with considerable degrees of genetic differentiations. Each of the three lineages is judged to represent a good species, and the name Microhyla ornata is restricted to the South Asian populations. For the populations from Taiwan and a wide region from China to Southeast Asia, the name Microhyla fissipes should be applied, whereas the Ryukyu populations are most appropriately referred to as Microhyla okinavensis, although further substantial genetic differentiations are recognized among some island group populations within this last species.

Microbial diversity of a sulphide spire located in the Edmond deep‐sea hydrothermal vent field on the Central Indian Ridge

ABSTRACTA culture‐independent molecular phylogenetic survey was carried out for a bacterial and archaeal community of a mineralized crust coating a sulphide spire, which was collected from the Edmond vent field (23° S, 69° E, 3300 m depth) on the Central Indian Ridge. Small‐subunit rRNA genes (16S rDNA) were amplified from environmental DNA by PCR utilizing Bacteria‐specific, and Archaea‐specific 16S rDNA primers. PCR products were cloned and 26 bacterial and nine archaeal unique sequence types (phylotypes) were identified from 150 clones analysed by restriction fragment length polymorphism, representing eight and four distinct lineages, respectively. The majority (&gt;90%) of the bacterial phylotypes group with the ɛ‐Proteobacteria and confirms the global prevalence of ɛ‐Proteobacteria in deep‐sea hydrothermal environments. Among the ɛ‐Proteobacteria, &gt;40% of the phylotypes were closely related to the recently isolated deep‐sea vent thermophilic chemolithoautotrophic sulphur‐reducer, Nautilia lithotrophica. A single bacterial sequence was nearly identical (99% similarity) to the thermophilic hydrogen‐oxidizing Hydrogenobacter thermolithotrophum, and is the first report of Hydrogenobacter at deep‐sea hydrothermal vents. A majority (97%) of the archaeal phylotypes grouped with the ‘Deep‐sea Hydrothermal Vent Euryarchaeotal Group’, a phylogenetic lineage of uncultured Archaea that have only been reported from other deep‐sea hydrothermal vents on the Mid‐Atlantic Ridge, East Pacific Rise, Juan de Fuca Ridge, Isu–Ogasawara Arc, Okinawa Trough and the Manus Basin. A single sequence was closely related to the hyperthermophilic sulphur‐reducing Thermococcales frequently found in diverse deep‐sea vent environments. Scanning electron micrographs of the mineralized crust reveal abundant filamentous, rod and coccoidal forms encased in sulphur and sulphide mineral precipitate, suggesting that the thermophilic chemolithoautorophs and sulphide‐producing heterotrophs may influence the architecture and sulphur cycling of the sulphide spire.

Molecular phylogeny and surface morphology of Colpodella edax (Alveolata): insights into the phagotrophic ancestry of apicomplexans.

The molecular phylogeny of colpodellids provides a framework for inferences about the earliest stages in apicomplexan evolution and the characteristics of the last common ancestor of apicomplexans and dinoflagellates. We extended this research by presenting phylogenetic analyses of small subunit rRNA gene sequences from Colpodella edax and three unidentified eukaryotes published from molecular phylogenetic surveys of anoxic environments. Phylogenetic analyses consistently showed C. edax and the environmental sequences nested within a colpodellid clade, which formed the sister group to (eu)apicomplexans. We also presented surface details of C. edax using scanning electron microscopy in order to supplement previous ultrastructural investigations of this species using transmission electron microscopy and to provide morphological context for interpreting environmental sequences. The microscopical data confirmed a sparse distribution of micropores, an amphiesma consisting of small polygonal alveoli, flagellar hairs on the anterior flagellum, and a rostrum molded by the underlying (open-sided) conoid. Three flagella were present in some individuals, a peculiar feature also found in the microgametes of some apicomplexans.

Extremely acidophilic protists from acid mine drainage host Rickettsiales-lineage endosymbionts that have intervening sequences in their 16S rRNA genes.

During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name "Candidatus Captivus acidiprotistae." To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.

Geomicrobiology of the ocean crust: a role for chemoautotrophic Fe-bacteria.

The delicate balance of the major global biogeochemical cycles greatly depends on the transformation of Earth materials at or near its surface. The formation and degradation of rocks, minerals, and organic matter are pivotal for the balance, maintenance, and future of many of these cycles. Microorganisms also play a crucial role, determining the transformation rates, pathways, and end products of these processes. While most of Earth's crust is oceanic rather than terrestrial, few studies have been conducted on ocean crust transformations, particularly those mediated by endolithic (rock-hosted) microbial communities. The biology and geochemistry of deep-sea and sub-seafloor environments are generally more complicated to study than in terrestrial or near-coastal regimes. As a result, fewer, and more targeted, studies usually homing in on specific sites, are most common. We are studying the role of endolithic microorganisms in weathering seafloor crustal materials, including basaltic glass and sulfide minerals, both in the vicinity of seafloor hydrothermal vents and off-axis at unsedimented (young) ridge flanks. We are using molecular phylogenetic surveys and laboratory culture studies to define the size, diversity, physiology, and distribution of microorganisms in the shallow ocean crust. Our data show that an unexpected diversity of microorganisms directly participate in rock weathering at the seafloor, and imply that endolithic microbial communities contribute to rock, mineral, and carbon transformations.

Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases.

A significant number of chimeric 16S rDNA sequences of diverse origin were identified in the public databases by partial treeing analysis. This suggests that chimeric sequences, representing phylogenetically novel non-existent organisms, are routinely being overlooked in molecular phylogenetic surveys despite a general awareness of PCR-generated artefacts amongst researchers.

First insight into the genome of an uncultivated crenarchaeote from soil.

Molecular phylogenetic surveys based on the characterization of 16S rRNA genes have revealed that soil is an environment particularly rich in microbial diversity. A clade of crenarchaeota (archaea) has frequently been detected among many other novel lineages of uncultivated bacteria. In this study we have initiated a genomic approach for the characterization of uncultivated microorganisms from soil. We have developed a procedure based on a two-phase electrophoresis technique that allows the fast and reliable purification of concentrated and clonable, high molecular weight DNA. From this DNA we have constructed complex large-insert genomic libraries. Using archaea-specific 16S rRNA probes we have isolated a 34 kbp fragment from a 900 Mbp fosmid library of soil DNA. The clone contained a complete 16S/23S rRNA operon and 17 genes encoding putative proteins. Phylogenetic analyses of the rRNA genes and of several protein encoding genes (e.g. DNA polymerase, FixAB, glycosyl transferase) confirmed the specific affiliation of the genomic fragment with the non-thermophilic clade of the crenarchaeota. Content and structure of the genomic fragment indicated that the archaea from soil differ significantly from their previously studied uncultivated marine relatives. The protein encoding genes gave the first insights into the physiological potential of these organisms and can serve as a basis for future genomic and functional genomic studies.

Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal.

Most filamentous bacteria in biological nutrient removal (BNR) processes have not been identified beyond their morphotype and simple staining reactions. Furthermore, the majority of sludge filaments observed under the microscope do not hybridize to commonly used phylogenetic probes for well characterized bacterial phyla such as the Proteobacteria, Actinobacteria, Firmicutes and BACTEROIDETES: Specific 16S rRNA-targeted oligonucleotide probes were designed for the phylum Chloroflexi (green non-sulfur bacteria) and optimized for use in fluorescence in situ hybridization. Chloroflexi have been implicated in BNR systems by phylogenetic identification of filamentous bacteria isolated by micromanipulation from sludge and culture-independent molecular phylogenetic surveys. The predominant morphotype responding to the probes was filamentous and these filaments were generally abundant in 10 Australian full-scale and two laboratory-scale BNR samples examined. Filamentous bacteria responding to a subdivision 1 Chloroflexi probe were rare in the samples, whereas subdivision 3 Chloroflexi filaments were very common in some sludges. This is in direct contrast to results obtained from molecular phylogenetic surveys of BNR systems where most sludge 16S rDNA clones belong to subdivision 1 and only a few to subdivision 3. It is suggested that filamentous bacteria belonging to the Chloroflexi phylum account for a large fraction of phylogenetically uncharacterized filaments in BNR systems and are likely to be abundant in such systems on a global scale.

Environmental Diversity of Bacteria and Archaea

The microbial way of life spans at least 3.8 billion years of evolution. Microbial organisms are pervasive, ubiquitous, and essential components of all ecosystems. The geochemical composition of Earth's biosphere has been molded largely by microbial activities. Yet, despite the predominance of microbes during the course of life's history, general principles and theory of microbial evolution and ecology are not well developed. Until recently, investigators had no idea how accurately cultivated microorganisms represented overall microbial diversity. The development of molecular phylogenetics has recently enabled characterization of naturally occurring microbial biota without cultivation. Free from the biases of culture-based studies, molecular phylogenetic surveys have revealed a vast array of new microbial groups. Many of these new microbes are widespread and abundant among contemporary microbiota and fall within novel divisions that branch deep within the tree of life. The breadth and extent of extant microbial diversity has become much clearer. A remaining challenge for microbial biologists is to better characterize the biological properties of these newly described microbial taxa. This more comprehensive picture will provide much better perspective on the natural history, ecology, and evolution of extant microbial life.