Boring systematics: A genome skimmed phylogeny of ctenostome bryozoans and their endolithic family Penetrantiidae with the description of one new species.

Ctenostomes are a group of gymnolaemate bryozoans with an uncalcified chitinous body wall having few external, skeletal characters. Hence, species identification is challenging and their systematics remain poorly understood, even more so when they exhibit an endolithic (boring) lifestyle. Currently, there are four Recent families of endolithic bryozoans that live inside mineralized substrates like mollusk shells. In particular, Penetrantiidae Silén, 1946 has received considerable attention and its systematic affinity to either cheilostomes or ctenostomes has been debated. Species delimitation of penetrantiids remains difficult, owing to a high degree of colonial and zooidal plasticity. Consequently, an additional molecular approach is essential to unravel the systematics of penetrantiids, their phylogenetic placement and their species diversity. We therefore sequenced the mitochondrial (mt) genomes and two nuclear markers of 27 ctenostome species including nine penetrantiids. Our phylogeny supports the Penetrantiidae as a monophyletic group placed as sister taxon to the remaining ctenostomes alongside paludicellids, arachnidioids and terebriporids. The boring family Terebriporidae d'Orbigny, 1847 were previously considered to be among vesicularioids, but our results suggest an arachnidioid affinity instead. Ctenostome paraphyly is supported by our data, as the cheilostomes nest within them. A Multiporata clade is also well supported, including the former victorelloid genus Sundanella. Altogether, this study provides new insights into ctenostome systematics, assists with species delimitation and contributes to our understanding of the bryozoan tree of life.

Rediscovering the unusual, solitary bryozoan Monobryozoon ambulans Remane, 1936: first molecular and new morphological data clarify its phylogenetic position.

BACKGROUND: One of the most peculiar groups of the mostly colonial phylum Bryozoa is the taxon Monobryozoon, whose name already implies non-colonial members of the phylum. Its peculiarity and highly unusual lifestyle as a meiobenthic clade living on sand grains has fascinated many biologists. In particular its systematic relationship to other bryozoans remains a mystery. Despite numerous searches for M. ambulans in its type locality Helgoland, a locality with a long-lasting marine station and tradition of numerous courses and workshops, it has never been reencountered until today. Here we report the first observations of this almost mythical species, Monobryozoon ambulans. RESULTS: For the first time since 1938, we present new modern, morphological analyses of this species as well as the first ever molecular data. Our detailed morphological analysis confirms most previous descriptions, but also ascertains the presence of special ambulatory polymorphic zooids. We consider these as bud anlagen that ultimately consecutively separate from the animal rendering it pseudo-colonial. The remaining morphological data show strong ties to alcyonidioidean ctenostome bryozoans. Our morphological data is in accordance with the phylogenomic analysis, which clusters it with species of Alcyonidium as a sister group to multiporate ctenostomes. Divergence time estimation and ancestral state reconstruction recover the solitary state of M. ambulans as a derived character that probably evolved in the Late Cretaceous. In this study, we also provide the entire mitogenome of M. ambulans, which-despite the momentary lack of comparable data-provides important data of a unique and rare species for comparative aspects in the future. CONCLUSIONS: We were able to provide first sequence data and modern morphological data for the unique bryozoan, M. ambulans, which are both supporting an alcyonidioidean relationship within ctenostome bryozoans.

Phylogenomics reveals deep relationships and diversification within phylactolaemate bryozoans.

Bryozoans are mostly sessile colonial invertebrates that inhabit all kinds of aquatic ecosystems. Extant bryozoan species fall into two clades with one of them, Phylactolaemata, being the only exclusively freshwater clade. Phylogenetic relationships within the class Phylactolaemata have long been controversial owing to their limited distinguishable characteristics that reflect evolutionary relationships. Here, we present the first phylogenomic analysis of Phylactolaemata using transcriptomic data combined with dense taxon sampling of six families to better resolve the interrelationships and to estimate divergence time. Using maximum-likelihood and Bayesian inference approaches, we recovered a robust phylogeny for Phylactolaemata in which the interfamilial relationships are fully resolved. We show Stephanellidae is the sister taxon of all other phylactolaemates and confirm that Lophopodidae represents the second offshoot within the phylactolaemate tree. Plumatella fruticosa clearly falls outside Plumatellidae as previous investigations have suggested, and instead clusters with Pectinatellidae and Cristatellidae as the sister taxon of Fredericellidae. Our results demonstrate that cryptic speciation is very likely in F. sultana and in two species of Plumatella (P. repens and P. casmiana). Divergence time estimates show that Phylactolaemata appeared at the end of the Ediacaran and started to diverge in the Silurian, although confidence intervals were large for most nodes. The radiation of most extant phylactolaemate families occurred mainly in the Palaeogene and Neogene highlighting post-extinction diversification.

Ecdysis-related neuropeptide expression and metamorphosis in a non-ecdysozoan bilaterian.

Ecdysis-related neuropeptides (ERNs), including eclosion hormone, crustacean cardioactive peptide, myoinhibitory peptide, bursicon alpha, and bursicon beta regulate molting in insects and crustaceans. Recent evidence further revealed that ERNs likely play an ancestral role in invertebrate life cycle transitions, but their tempo-spatial expression patterns have not been investigated outside Arthropoda. Using RNA-seq and in situ hybridization, we show that ERNs are broadly expressed in the developing nervous system of a mollusk, the polyplacophoran Acanthochitona fascicularis. While some ERN-expressing neurons persist from larval to juvenile stages, others are only present during settlement and metamorphosis. These transient neurons belong to the "ampullary system," a polyplacophoran-specific larval sensory structure. Surprisingly, however, ERN expression is absent from the apical organ, another larval sensory structure that degenerates before settlement is completed in A. fascicularis. Our findings thus support a role of ERNs in A. fascicularis metamorphosis but contradict the common notion that the apical organ-like structures shared by various aquatic invertebrates (i.e., cnidarians, annelids, mollusks, echinoderms) are of general importance for this process.

Paracatenula, an ancient symbiosis between thiotrophic Alphaproteobacteria and catenulid flatworms.

Harnessing chemosynthetic symbionts is a recurring evolutionary strategy. Eukaryotes from six phyla as well as one archaeon have acquired chemoautotrophic sulfur-oxidizing bacteria. In contrast to this broad host diversity, known bacterial partners apparently belong to two classes of bacteria--the Gamma- and Epsilonproteobacteria. Here, we characterize the intracellular endosymbionts of the mouthless catenulid flatworm genus Paracatenula as chemoautotrophic sulfur-oxidizing Alphaproteobacteria. The symbionts of Paracatenula galateia are provisionally classified as "Candidatus Riegeria galateiae" based on 16S ribosomal RNA sequencing confirmed by fluorescence in situ hybridization together with functional gene and sulfur metabolite evidence. 16S rRNA gene phylogenetic analysis shows that all 16 Paracatenula species examined harbor host species-specific intracellular Candidatus Riegeria bacteria that form a monophyletic group within the order Rhodospirillales. Comparing host and symbiont phylogenies reveals strict cocladogenesis and points to vertical transmission of the symbionts. Between 33% and 50% of the body volume of the various worm species is composed of bacterial symbionts, by far the highest proportion among all known endosymbiotic associations between bacteria and metazoans. This symbiosis, which likely originated more than 500 Mya during the early evolution of flatworms, is the oldest known animal-chemoautotrophic bacteria association. The distant phylogenetic position of the symbionts compared with other mutualistic or parasitic Alphaproteobacteria promises to illuminate the common genetic predispositions that have allowed several members of this class to successfully colonize eukaryote cells.

Microorganisms with novel dissimilatory (bi)sulfite reductase genes are widespread and part of the core microbiota in low-sulfate peatlands.

Peatlands of the Lehstenbach catchment (Germany) house as-yet-unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic of microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a 6-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented "core" members (up to 1% to 1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparisons of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance of ∼1 to 400 km) identified that one Syntrophobacter-related and nine novel dsrAB lineages are widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-based DGGE data were not correlated with geographic distance but could be explained largely by soil pH and wetland type, implying that the distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by local environmental conditions.

Raman microspectroscopy reveals long-term extracellular activity of Chlamydiae.

The phylum Chlamydiae consists exclusively of obligate intracellular bacteria. Some of them are formidable pathogens of humans, while others occur as symbionts of amoebae. These genetically intractable bacteria possess a developmental cycle consisting of replicative reticulate bodies and infectious elementary bodies, which are believed to be physiologically inactive. Confocal Raman microspectroscopy was applied to differentiate between reticulate bodies and elementary bodies of Protochlamydia amoebophila and to demonstrate in situ the labelling of this amoeba symbiont after addition of isotope-labelled phenylalanine. Unexpectedly, uptake of this amino acid was also observed for both developmental stages for up to 3 weeks, if incubated extracellularly with labelled phenylalanine, and P. amoebophila remained infective during this period. Furthermore, P. amoebophila energizes its membrane and performs protein synthesis outside of its host. Importantly, amino acid uptake and protein synthesis after extended extracellular incubation could also be demonstrated for the human pathogen Chlamydia trachomatis, which synthesizes stress-related proteins under these conditions as shown by 2-D gel electrophoresis and MALDI-TOF/TOF mass spectrometry. These findings change our perception of chlamydial biology and reveal that host-free analyses possess a previously not recognized potential for direct experimental access to these elusive microorganisms.

A constant flux of diverse thermophilic bacteria into the cold Arctic seabed.

Microorganisms have been repeatedly discovered in environments that do not support their metabolic activity. Identifying and quantifying these misplaced organisms can reveal dispersal mechanisms that shape natural microbial diversity. Using endospore germination experiments, we estimated a stable supply of thermophilic bacteria into permanently cold Arctic marine sediment at a rate exceeding 10(8) spores per square meter per year. These metabolically and phylogenetically diverse Firmicutes show no detectable activity at cold in situ temperatures but rapidly mineralize organic matter by hydrolysis, fermentation, and sulfate reduction upon induction at 50 degrees C. The closest relatives to these bacteria come from warm subsurface petroleum reservoir and ocean crust ecosystems, suggesting that seabed fluid flow from these environments is delivering thermophiles to the cold ocean. These transport pathways may broadly influence microbial community composition in the marine environment.

Reverse dissimilatory sulfite reductase as phylogenetic marker for a subgroup of sulfur-oxidizing prokaryotes.

Sulfur-oxidizing prokaryotes (SOP) catalyse a central step in the global S-cycle and are of major functional importance for a variety of natural and engineered systems, but our knowledge on their actual diversity and environmental distribution patterns is still rather limited. In this study we developed a specific PCR assay for the detection of dsrAB that encode the reversely operating sirohaem dissimilatory sulfite reductase (rDSR) and are present in many but not all published genomes of SOP. The PCR assay was used to screen 42 strains of SOP (most without published genome sequence) representing the recognized diversity of this guild. For 13 of these strains dsrAB was detected and the respective PCR product was sequenced. Interestingly, most dsrAB-encoding SOP are capable of forming sulfur storage compounds. Phylogenetic analysis demonstrated largely congruent rDSR and 16S rRNA consensus tree topologies, indicating that lateral transfer events did not play an important role in the evolutionary history of known rDSR. Thus, this enzyme represents a suitable phylogenetic marker for diversity analyses of sulfur storage compound-exploiting SOP in the environment. The potential of this new functional gene approach was demonstrated by comparative sequence analyses of all dsrAB present in published metagenomes and by applying it for a SOP census in selected marine worms and an alkaline lake sediment.

Biophysical controls on community succession in stream biofilms.

Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as "ecosystem engineers," modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms.

Cohn's Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase.

135 years ago Ferdinand Cohn, the founder of bacteriology, microscopically observed a conspicuous filamentous bacterium with a complex life cycle and described it as Crenothrix polyspora. This uncultured bacterium is infamous for mass developments in drinking water systems, but its phylogeny and physiology remained unknown. We show that C. polyspora is a gammaproteobacterium closely related to methanotrophs and capable of oxidizing methane. We discovered that C. polyspora encodes a phylogenetically very unusual particulate methane monooxygenase whose expression is strongly increased in the presence of methane. Our findings demonstrate a previously unrecognized complexity of the evolutionary history and cell biology of methane-oxidizing bacteria.

16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales".

For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.

The impact of surface water exchange on the nutrient and particle dynamics in side-arms along the River Danube, Austria.

Results of two monitoring programs obtained in the free-flowing section of the Danube downstream of Vienna were used to evaluate the effects of river restoration designed to increase surface water inputs into side-arms. Functional descriptors like hydrochemical parameters and plankton react immediately to restored hydrological conditions and offer the opportunity to elucidate the hydrological control on organic processing as an important ecosystem function in fluvial landscapes. Two hydraulic parameters were estimated and linked to basic ecological properties. The level of hydrological connectivity was defined as the average annual duration (days per year) of upstream surface connection and can be used as a 'simple to estimate' parameter within the planning phase. Water age, an adapted measure of residence time based on more detailed information, allow description of the temporal development in side-arms. Greater hydrological connectivity leads to lower conductivity levels and increased nutrient concentrations due to the shift of the dominating source to river water. The contribution of river flow is indicated by higher suspended solid concentrations in side-arms than disconnected water bodies. The phytoplankton biomass shows the highest mean values at a duration of integration of 1 month a(-1) and decrease with increasing connectivity. The relationships point to a more 'main channel like' hydrochemical situation in the side-arms, with a medium level of phytoplankton biomass and increased autochthonous carbon export. No evidence of eutrophication was found due to the shift of the side-arm from an organic matter sink to a source. On a more detailed level, water age demonstrates the temporal patterns of riverine input, the development of plankton production and the shift between hydrological and biological control of phytoplankton vs. riverine flow in a side-arm. The hydrologic parameters were useful predictors for evaluating the effects of restoration measures in river floodplain systems.