Do 'cheese factory-specific' mites (Acari: Astigmata) exist in the cheese-ripening cabinet?

To determine whether the mites used in the ripening process of traditional cheeses are genetically unique to cheese factories, we investigated mites from three types of traditional cheeses, that use mites in the ripening process: 'Würchwitzer Milbenkäse' from Germany and 'Mimolette' and 'Artisou' from France. In addition, traditional ripened cheeses were purchased from cheese specialty stores in France (Mimolette) and Japan ('Laguiole' from France) as well as stores in temporary markets in France ('Salers' and 'Cantal vieux') and the mites obtained from those cheeses were analyzed in this study. Partial sequences of the 28S rRNA gene (28S) were determined and used to reconstruct a phylogenetic tree. Tyrolichus casei, the dominant cheese mite species from the ripening cabinets of three traditional cheese producers and two cheese specialty stores in France and Japan, had identical partial 28S sequences. All specimens from Cantal vieux from a store in the temporary market in France had an identical sequence with Acarus siro and Acarus immobilis in the determined region of the 28S sequences. Mite individuals from Salers from a store in the temporary markets in France shared the same haplotype as Acotyledon paradoxa. For the T. casei individuals from five different localities (19 individuals in total), the nuclear loci were obtained using MIG-seq. More than several thousand genomic regions are amplified simultaneously by multiplex PCR, and targeting regions surrounded by inter-simple sequence repeats (ISSRs) in the genome were sequenced using the MiSeq system (Illumina). SNPs extracted from this genome-wide analysis showed that no genetic structure existed in the populations from any region. Among the five samples from the three regions, which were more than 500 km apart and from completely different environments, the mites had no geographic bias, but all mite individuals were genetically nearly identical. Thus, we found no evidence to support the existence of 'cheese factory-specific' T. casei mites, at least in terms of genetic analysis.

Phylogeny of Lithobiidae Newport, 1844, with emphasis on the megadiverse genus Lithobius Leach, 1814 (Myriapoda, Chilopoda).

Phylogenetic analyses based on molecular and morphological data were conducted to shed light on relationships within the mostly Palaearctic/Oriental centipede family Lithobiidae, with a particular focus on the Palaearctic genus Lithobius Leach, 1814 (Lithobiidae, Lithobiomorpha), which contains >500 species and subspecies. Previous studies based on morphological data resolved Lithobius as nonmonophyletic, but molecular-based phylogenetic analyses have until now sampled few species. To elucidate species inter-relationships of the genus, test the validity of its classification into subgenera, and infer its relationships with other Lithobiidae, we obtained molecular data (nuclear markers: 18S rRNA, 28S rRNA; mitochondrial markers: 16S rRNA, COI) and 61 morphological characters for 44 species of Lithobius representing four of its eight subgenera and nine other representatives of Lithobiidae. The data were analyzed phylogenetically using maximum-likelihood, parsimony and Bayesian inference. This study suggests that (i) a close relationship between L. giganteus and the pterygotergine Disphaerobius loricatus highlighted in recent morphological analyses is also strongly supported by molecular data, and Pterygoterginae is formally synonymized with Lithobiinae; (ii) the Oriental/Australian genus Australobius is consistently resolved as sister group to all other sampled Lithobiidae by the molecular and combined data; (iii) the subfamily Ethopolyinae may be paraphyletic; (iv) the genus Lithobius is nonmonophyletic; (v) the subgenera Lithobius, Sigibius and Monotarsobius are nonmonophyletic and should not be used in future taxonomic studies; and (vi) there are instances of cryptic species and cases in which subspecies should be elevated to full species status, as identified for some European taxa within Lithobius.

The DExD box ATPase DDX55 is recruited to domain IV of the 28S ribosomal RNA by its C-terminal region.

RNA helicases contribute to diverse aspects of RNA metabolism through their functions in re-arranging RNA structures. Identification of the remodelling targets of RNA helicases is a critical step in elucidating their cellular functions. Here, we show that, in contrast to many other ribosome biogenesis factors, the DExD box ATPase DDX55 predominantly localizes to the nucleoplasm and we identify a nuclear localization signal within the C-terminal region of the protein. DDX55 associates with pre-ribosomal subunits in human cells and is required for maturation of large subunit pre-rRNAs. Interestingly, in vitro analyses show that DDX55 selectively associates with double-stranded RNA substrates, which also stimulate its ATPase activity, and our data suggest that the C-terminal region of DDX55 contributes to this substrate specificity. The C-terminal region of DDX55 is also necessary for recruitment of the helicase to pre-ribosomes and, using in vivo crosslinking, we reveal a binding site for DDX55 in helix H62 of the 28S ribosomal RNA. Taken together, these data highlight the importance of the C-terminal region of DDX55 in substrate specificity and recruitment, and identify domain IV as a potential remodelling target of DDX55 during LSU biogenesis.

Morphological and Molecular Characterisation of Myxidium kudoi Meglitsch, 1937 from the Blue Catfish Ictalurus furcatus, Valenciennes in Oklahoma, USA.

BACKGROUND: Myxidium kudoi Meglitsch, 1937 has been described from the type host, blue catfish Ictalurus furcatus, with no additional host records or molecular data available for this species. PURPOSE: To provide molecular data and a novel host locality for this species and carry out phylogenetic analyses to infer the evolutionary relationship of the species to other members of the family Myxidiidae for which DNA sequence data is available. METHODS: These data were collected using myxospores from the gallbladder of a blue catfish, Ictalurus furcatus collected from Lake Texoma, Oklahoma, USA. Myxospores were morphologically consistent with the only other account of this species and not any other Myxidium species described from siluriform fishes. RESULTS: Myxospores were oblong with rounded ends and were 10.8-12.6 (11.6 ± 0.5) µm in length and 4.7-6.6 (5.7 ± 0.5) µm in width. Polar capsules were subspherical and 2.7-3.9 (3.4 ± 0.3) µm in length and 2.4-3.5 (3.1 ± 0.3) µm in diameter, with each capsule containing a polar filament with 3-4 coils. Molecular data consisted of a 2918-bp sequence of the partial 18S, complete ITS1, 5.8S, ITS2, and partial 28S ribosomal rRNA regions as well as a 2455-bp sequence of partial 28S ribosomal RNA. The partial 18S and 28S data was used in a concatenated Bayesian phylogenetic analysis to further infer the evolutionary relationships of the Myxidiidae. Additionally, the partial 18S data was used in a separate phylogenetic analysis. CONCLUSIONS: The present work reports novel morphological and molecular data for Myxidium kudoi as well as a novel locality of occurrence for this species. In concatenated phylogenetic analysis using 18S and 28S data and other molecular data from Myxozoa, M. kudoi grouped with other freshwater Myxidiidae. In the single-locus, 18S analysis, M. kudoi grouped with Myxidium rhodei from Rutilus rutilus and Myxidium amazonense from Corydoras melini, the only other Myxidium species of catfish for which molecular data are available.

Phylogeny and biogeography of the Cavernicola (Platyhelminthes: Tricladida): Relicts of an epigean group sheltering in caves?

The planarian suborder Cavernicola Sluys, 1990 was originally created to house five species of triclad flatworms with special morphological features and a surprisingly discontinuous and broad geographic distribution. These five species could not be accommodated with any degree of certainty in any of the three taxonomic groups existing at that moment, viz., Paludicola Hallez, 1892, Terricola Hallez, 1892, and Maricola Hallez, 1892. The scarce representation of the group and the peculiarities of the morphological features of the species, including several described more recently, have complicated new tests of the monophyly of the Cavernicola, the assessment of its taxonomic status, as well as the resolution of its internal relationships. Here we present the first molecular study including all genera currently known for the group, excepting one. We analysed newly generated 18S and 28S rDNA data for these species, together with a broad representation of other triclad flatworms. The resulting phylogenetic trees supported the monophyly of the Cavernicola, as well as its sister-group relationship to the Maricola. The sister-group relationship to the Maricola and affinities within the Cavernicola falsify the morphology-based phylogeny of the latter that was proposed previously. The relatively high diversity of some cavernicolan genera suggests that the presumed rarity of the group actually may in part be due to a collecting artefact. Ancestral state reconstruction analyses suggest that the ancestral habitat of the group concerned epigean freshwater conditions. Our results point to an evolutionary scenario in which the Cavernicola (a) originated in a freshwater habitat, (b) as the sister clade of the marine triclads, and (c) subsequently radiated and colonized both epigean and hypogean environments. Competition with other planarians, notably members of the Continenticola, or changes in epigean habitat conditions are two possible explanations -still to be tested- for the loss of most epigean diversity of the Cavernicola, which is currently reflected in their highly disjunct distributions.

The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA.

RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

Phylogenetic and secondary RNA structure analysis of monogenean gill ectoparasites(Dactylogyrus spp.) parasitizing certain freshwater fishes.

The current study is the first phylogenetic and secondary RNA structure analysis of Dactylogyrus species parasitising gill filaments of Iraqi cyprinid fishes. Most previous phylogenetic studies have targeted on primary DNA sequence data. Nevertheless, RNA secondary configuration is principally helpful in systematics since they comprise features that do not appear in the primary sequence and provide morphological information. The primary objective was molecular-based identification of Dactylogyrids species using evolutionary tree and secondary RNA structure prediction. A total of 681 fish were collected from the Lesser Zab River in the northeast of Iraq in the sub-district of Altun-Kopru from August 2016 to September 2017 and brought to the Zoology Research Laboratory, Salahaddin University-Erbil, Iraq. All fish were classified as 18 cyprinid species. The species of Dactylogyrus were identified by the 28S rDNA subunit using PCR and sequencing methods, and the obtained nucleotide sequences were then compared with the available GenBank sequences. Phylogenetic relationships were concluded using Neighbour-Joining (NJ), Maximum Likelihood (ML), and Minimum Evolution (ME) methods. The results justify the validation of 11 Dactylogyrus species (three of them were newly recorded in Iraq). Additionally, out of nine infected fish species, seven of them were regarded as a new host for Dactylogyrus species. Secondary RNA configuration prediction using minimum free energy was considered as a hopeful tool for species identification. This was considered the first comprehensive phylogenetic study in the area. It was concluded that PCR sequencing, phylogenetic and secondary RNA analysis were proper molecular methods for identifying Dactylogyrids species on the gills of fishes.

SHAPE Probing Reveals Human rRNAs Are Largely Unfolded in Solution.

Chemical probing experiments, coupled with empirically determined free energy change relationships, can enable accurate modeling of the secondary structures of diverse and complex RNAs. A current frontier lies in modeling large and structurally heterogeneous transcripts, including complex eukaryotic RNAs. To validate and improve on experimentally driven approaches for modeling large transcripts, we obtained high-quality SHAPE data for the protein-free human 18S and 28S ribosomal RNAs (rRNAs). To our surprise, SHAPE-directed structure models for the human rRNAs poorly matched accepted structures. Analysis of predicted rRNA structures based on low-SHAPE and low-entropy (lowSS) metrics revealed that, whereas ∼75% of Escherichia coli rRNA sequences form well-determined lowSS secondary structure, only ∼40% of the human rRNAs do. Critically, regions of the human rRNAs that specifically fold into well-determined lowSS structures were modeled to high accuracy using SHAPE data. This work reveals that eukaryotic rRNAs are more unfolded than are those of prokaryotic rRNAs and indeed are largely unfolded overall, likely reflecting increased protein dependence for eukaryotic ribosome structure. In addition, those regions and substructures that are well-determined can be identified de novo and successfully modeled by SHAPE-directed folding.

Inhibition of ricin A-chain (RTA) catalytic activity by a viral genome-linked protein (VPg).

Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A-chain (RTA) and a viral genome-linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N-terminal truncated variant, VPg1-110, bind to RTA and abolish ricin's catalytic depurination of 28S rRNA in vitro and in a cell-free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten-fold as temperature elevates (5 °C to 37 °C). RTA-VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = -136.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA-VPg complex, Ea = -62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.

Pleurotus opuntiae revisited - An insight to the phylogeny of dimitic Pleurotus species with emphasis on the P. djamor complex.

The name Pleurotus opuntiae is indiscriminately used for describing mushrooms with white to off-white to white-grey pilei with short or absent stipe and dimitic hyphal system, which grow on plants of the genera Opuntia, Yucca, Agave, Phytolacca etc. However, the outcome of the present study evidences that this name should be reserved for specimens deriving from the Mediterranean area only; an epitype originating from Italy on Opuntia ficus-indica is designated. Pertinent material was sequenced by using the internal transcribed spacer region (ITS) and found to be phylogenetically related to P. djamor from Kenya and Nigeria, while members of the P. djamor complex from other continents were clearly more distant. Results were further corroborated by examining the large subunit of nuclear ribosomal DNA (LSU) and the second subunit of RNA polymerase II (RPB2). The P. djamor complex shows high intraspecific polymorphism evidenced by sequence divergence and genetic distance values, presents a cosmopolitan distribution and also comprises material initially identified as P. flabellatus, P. opuntiae, P. ostreatoroseus, P. parsonsiae and P. salmoneostramineus. An ITS tree including representative specimens from all major Pleurotus species is provided for the first time and ambiguous taxa are discussed in the context of new findings.

BisAMP: A web-based pipeline for targeted RNA cytosine-5 methylation analysis.

RNA cytosine-5 methylation (m5C) has emerged as a key epitranscriptomic mark, which fulfills multiple roles in structural modulation, stress signaling and the regulation of protein translation. Bisulfite sequencing is currently the most accurate and reliable method to detect m5C marks at nucleotide resolution. Targeted bisulfite sequencing allows m5C detection at single base resolution, by combining the use of tailored primers with bisulfite treatment. A number of computational tools currently exist to analyse m5C marks in DNA bisulfite sequencing. However, these methods are not directly applicable to the analysis of RNA m5C marks, because DNA analysis focuses on CpG methylation, and because artifactual unconversion and misamplification in RNA can obscure actual methylation signals. We describe a pipeline designed specifically for RNA cytosine-5 methylation analysis in targeted bisulfite sequencing experiments. The pipeline is directly applicable to Illumina MiSeq (or equivalent) sequencing datasets using a web interface (https://bisamp.dkfz.de), and is defined by optimized mapping parameters and the application of tailored filters for the removal of artifacts. We provide examples for the application of this pipeline in the unambiguous detection of m5C marks in tRNAs from mouse embryonic stem cells and neuron-differentiated stem cells as well as in 28S rRNA from human fibroblasts. Finally, we also discuss the adaptability of BisAMP to the analysis of DNA methylation. Our pipeline provides an accurate, fast and user-friendly framework for the analysis of cytosine-5 methylation in amplicons from bisulfite-treated RNA.

Landscape of the complete RNA chemical modifications in the human 80S ribosome.

During ribosome biogenesis, ribosomal RNAs acquire various chemical modifications that ensure the fidelity of translation, and dysregulation of the modification processes can cause proteome changes as observed in cancer and inherited human disorders. Here, we report the complete chemical modifications of all RNAs of the human 80S ribosome as determined with quantitative mass spectrometry. We assigned 228 sites with 14 different post-transcriptional modifications, most of which are located in functional regions of the ribosome. All modifications detected are typical of eukaryotic ribosomal RNAs, and no human-specific modifications were observed, in contrast to a recently reported cryo-electron microscopy analysis. While human ribosomal RNAs appeared to have little polymorphism regarding the post-transcriptional modifications, we found that pseudouridylation at two specific sites in 28S ribosomal RNA are significantly reduced in ribosomes of patients with familial dyskeratosis congenita, a genetic disease caused by a point mutation in the pseudouridine synthase gene DKC1. The landscape of the entire epitranscriptomic ribosomal RNA modifications provides a firm basis for understanding ribosome function and dysfunction associated with human disease.

A ruthenium-platinum metal complex that binds to sarcin ricin loop RNA and lowers mRNA expression.

IT127 is a dinuclear transition metal complex that contains a Pt(ii) and a Ru(iii) metal center. We have shown that IT127 is significantly more effective in binding the 29-base sarcin ricin loop (SRL) RNA in comparison to Cisplatin, a hallmark anticancer agent. Binding site analysis shows that IT127 prefers purine bases and the GAGA tetraloop region of SRL RNA. Our results with a dihydrofolate reductase (DHFR) model system reveal that IT127 binding to mRNA reduces translation of DHFR enzyme and that the Ru(iii) and Pt(ii) centers in IT127 appear to work in a synergistic manner.

The Expansion Segments of Human 28S rRNA Match MicroRNAs Much Above 18S rRNA or Core Segments.

BACKGROUND: The size of eukaryotic 25-28S rRNAs shows a progressive phylogenetically linked increase which is pronounced in mammals, and especially in hominids. The increase is confined to specific expansion segments, inserted at points that are highly conserved from yeast to man. These segments also show a progressive increase in nucleotide bias, mostly the GC bias. Substantial parts of the large expansion segments 7, 15 and 27 of 28S rRNA are known to be exposed at the ribosome surface, with no clear association with ribosomal proteins. These segments could bind extraneous RNAs and proteins to support regulatory events. METHODS: This study examined the possible canonical matching of human 28S rRNA and 18S rRNA segments with 2586 human microRNAs. This was compared with matching of the microRNAs to sectors of 18810 human mRNAs. RESULTS: The overall matching was rather similar across 18S rRNA segments and core segments of 28S rRNA. However, the expansion segments of 28S rRNA (abbreviated ESL) collectively have a much higher (up to two-fold) capacity for the canonical association with microRNAs. This is pronounced in large ESL, and is found to strongly relate to the GC content of microRNAs. CONCLUSION: Oligonucleotides and microRNAs of high GC content through a strong canonical hydrogen bonding could have large activity in regulation of subcellular RNAs. In view of the considerable abundance of ribosomal RNAs in many mammalian tissues, ESL could constitute an important component of microRNA balance, possibly serving to lower the availability of GC-rich microRNAs (and thereby help conservation of GC-rich mRNAs).

Species diversity in the marine microturbellarian Astrotorhynchus bifidus sensu lato (Platyhelminthes: Rhabdocoela) from the Northeast Pacific Ocean.

Increasing evidence suggests that many widespread species of meiofauna are in fact regional complexes of (pseudo-)cryptic species. This knowledge has challenged the 'Everything is Everywhere' hypothesis and also partly explains the meiofauna paradox of widespread nominal species with limited dispersal abilities. Here, we investigated species diversity within the marine microturbellarian Astrotorhynchus bifidus sensu lato in the Northeast Pacific Ocean. We used a multiple-evidence approach combining multi-gene (18S, 28S, COI) phylogenetic analyses, several single-gene and multi-gene species delimitation methods, haplotype networks and conventional taxonomy to designate Primary Species Hypotheses (PSHs). This included the development of rhabdocoel-specific COI barcode primers, which also have the potential to aid in species identification and delimitation in other rhabdocoels. Secondary Species Hypotheses (SSHs) corresponding to morphospecies and pseudo-cryptic species were then proposed based on the minimum consensus of different PSHs. Our results showed that (a) there are at least five species in the A. bifidus complex in the Northeast Pacific Ocean, four of which can be diagnosed based on stylet morphology, (b) the A. bifidus complex is a mixture of sympatric and allopatric species with regional and/or subglobal distributions, (c) sympatry occurs on local (sample sites), regional (Northeastern Pacific) and subglobal (Northern Atlantic, Arctic, Northeastern Pacific) scales. Mechanisms for this co-occurrence are still poorly understood, but we hypothesize they could include habitat differentiation (spatial and/or seasonal) and life history characteristics such as sexual selection and dispersal abilities. Our results also suggest the need for improved sampling and exploration of molecular markers to accurately map gene flow and broaden our understanding of species diversity and distribution of microturbellarians in particular and meiofauna in general.

Molecular phylogeny of Candidula (Geomitridae) land snails inferred from mitochondrial and nuclear markers reveals the polyphyly of the genus.

The genus Candidula (Geomitridae), consisting of 28 species in Western Europe as currently described, has a disjunct distribution in the Iberian Peninsula, Italy, the Balkans, the Aegean Islands, and one species on the Canary Islands. Although the genus is seemingly well defined by characters of the reproductive system, the relationships within the genus are still unclear and some authors have indicated a possible subgeneric division based on the internal morphology of the dart sac. Despite substantial phylogenetic incongruence, we present a well-resolved molecular phylogeny of Candidula based on two mitochondrial genes (COI and 16S rRNA), the nuclear rDNA region (5.8S rNRA + ITS2 + 28S rRNA) and seven additional nuclear DNA regions developed specifically for this genus (60SL13, 60SL17, 60SL7, RPL14, 40SS6, 60SL9, 60SL13a), in total 5595 bp. Six reciprocally monophyletic entities including Candidula species were recovered, grouping into two major clades. The incorporation of additional geomitrid genera allowed us to unequivocally demonstrate the polyphyly of the genus Candidula. One major clade grouped species from southern France and Italy with the widely distributed species C. unifasciata. The second major clade grouped all the species from the Iberian Peninsula, including C. intersecta and C. gigaxii. Candidula ultima from the Canary Islands was recovered as separated lineage within the latter clade and related to African taxa. The six monophyla were defined as six new genera belonging to different tribes within the Helicellinae. Thus, we could show that similar structures of the stimulatory apparatus of the genital system in different taxa do not necessarily indicate a close phylogenetic relationship in the Geomitridae. More genera of the family are needed to clarify their evolutionary relationships, and to fully understand the evolution of the stimulatory apparatus of the genital system within the Geomitridae.

Cryptic elevational zonation in trapdoor spiders (Araneae, Antrodiaetidae, Aliatypus janus complex) from the California southern Sierra Nevada.

The relative roles of ecological niche conservatism versus niche divergence in promoting montane speciation remains an important topic in biogeography. Here, our aim was to test whether lineage diversification in a species complex of trapdoor spiders corresponds with riverine barriers or with an ecological gradient associated with elevational tiering. Aliatypus janus was sampled from throughout its range, with emphasis on populations in the southern Sierra Nevada Mountains of California. We collected multi-locus genetic data to generate a species tree for A. janus and its close relatives. Coalescent based hypothesis tests were conducted to determine if genetic breaks within A. janus conform to riverine barriers. Ecological niche models (ENM) under current and Last Glacial Maximum (LGM) conditions were generated and hypothesis tests of niche conservatism and divergence were performed. Coalescent analyses reveal deeply divergent genetic lineages within A. janus, likely corresponding to cryptic species. Two primary lineages meet along an elevational gradient on the western slopes of the southern Sierra Nevada Mountains. ENMs under both current and LGM conditions indicate that these groups occupy largely non-overlapping niches. ENM hypothesis testing rejected niche identity between the two groups, and supported a sharp ecological gradient occurring where the groups meet. However, the niche similarity test indicated that the two groups may not inhabit different background niches. The Sierra Nevada Mountains provide a natural laboratory for simultaneously testing ecological niche divergence and conservatism and their role in speciation across a diverse range of taxa. Aliatypus janus represents a species complex with cryptic lineages that may have diverged due to parapatric speciation along an ecological gradient, or been maintained by the evolution of ecological niche differences following allopatric speciation.

Visualization of conformational variability in the domains of long single-stranded RNA molecules.

We demonstrate an application of atomic force microscopy (AFM) for the structural analysis of long single-stranded RNA (>1 kb), focusing on 28S ribosomal RNA (rRNA). Generally, optimization of the conditions required to obtain three-dimensional (3D) structures of long RNA molecules is a challenging or nearly impossible process. In this study, we overcome these limitations by developing a method using AFM imaging combined with automated, MATLAB-based image analysis algorithms for extracting information about the domain organization of single RNA molecules. We examined the 5 kb human 28S rRNA since it is the largest RNA molecule for which a 3D structure is available. As a proof of concept, we determined a domain structure that is in accordance with previously described secondary structural models. Importantly, we identified four additional small (200-300 nt), previously unreported domains present in these molecules. Moreover, the single-molecule nature of our method enabled us to report on the relative conformational variability of each domain structure identified, and inter-domain associations within subsets of molecules leading to molecular compaction, which may shed light on the process of how these molecules fold into the final tertiary structure.

The nuclear 28S gene fragment D3 as species marker in oribatid mites (Acari, Oribatida) from German peatlands.

To make oribatid mites an applicable tool in monitoring programs it is necessary to find a molecular species marker that allows distinct, rapid and easy species identification. In previous studies, the common barcoding sequence COI showed to be too variable to serve as species marker in oribatid mites. The aim of the present study is to evaluate the potential use of the D3 region of the nuclear 28S rDNA gene for species identification. Therefore, we generated a reference DNA library of 28S D3 to identify specimens of the Oribatida from Germany, with focus on species occurring in peatlands being one of the most endangered habitats in Europe. New DNA sequences were obtained from 325 individuals and 64 species (58 genera, 34 families). By adding 28S D3-sequences from GenBank we altogether analysed 385 sequences from 89 German species, 32 of them restricted to peatlands and further 42 occurring in peatlands occasionally, representing 46 and 33% of the oribatids in German peatlands, respectively. P-distances were measured between species within families as well as for intraspecific divergence. 28S D3 showed low intraspecific genetic p-distances between 0 and 0.5%, interspecific distances within families varied between 0 and 9.7%. Most species pairs within families were further separated by one to four indels in addition to substitutions. Altogether, 93% of all analysed species are clearly delineated by 28S D3. Our study emphasises that 28S D3 rDNA is a useful barcode for the identification of oribatid mite specimens and represents an important step in building-up a comprehensive barcode library to allow metabarcoding analyses of environmental peatland samples for Oribatida in Germany as well as in Central Europe.