DNA Sequence Datasets Research Articles

(Invited) Machine Learning for DNA/SWCNT Based Molecular Perceptron: Finding Sequences and Training Sensor Arrays

Single wall carbon nanotube (SWCNT) based biosensors provide opportunities for building an ultra-sensitive biosensing system due to their unique optical properties and strong sensitivity to changes in the local environment. Consequently, much effort has been made to develop SWCNT-based sensors. However, the usual method is based on one-to-one recognition which is a difficult way to detect a variety of molecules. In this study, we describe a sensing system that uses an array of weakly-specific sensors combined with a machine learning model, which we call the Molecular Perceptron. We show how machine learning algorithms, along with choice of feature representation, can be used both for discovery of special resolving DNA sequences and to predict presence and concentration of biomarkers. DNA/SWCNT hybrids were utilized to optically detect biomarker analytes by observing changes in the fluorescence spectra of each SWCNT. Using the experimental data, machine learning models were trained using three different algorithms: Support Vector Machine, Random Forest, and Artificial Neural Network. We also demonstrate that machine learning models trained on relatively small DNA sequence data sets can very accurately predict new resolving DNA sequences

<strong><em>Impatiens bullatisepala</em> (Balsaminaceae), a new species from Guizhou, China</strong>

Impatiens bullatisepala (Balsaminaceae), a new species supported by morphological and phylogenetic evidence from Fanjing Mountain, Guizhou province in China, is described here. It is morphologically similar to I. davidii but can be distinguished by its dorsally ridged lateral sepals with sunk reticulate veins and bullate projections on abaxial surface, 2–2.5 cm deep saccate lower sepal with ca. 0.8 cm long narrowly triangular tip at the mouth, and broadly ovate dorsal petal. Phylogenetic analyses of a combined dataset of nuclear ITS and plastid atpB-rbcL intergenic spacer DNA sequences furtherly confirmed its novelty.

Comprehensive discovery of novel structured noncoding RNAs in 26 bacterial genomes

ABSTRACT Comparative sequence analysis methods are highly effective for uncovering novel classes of structured noncoding RNAs (ncRNAs) from bacterial genomic DNA sequence datasets. Previously, we developed a computational pipeline to more comprehensively identify structured ncRNA representatives from individual bacterial genomes. This search process exploits the fact that genomic regions serving as templates for the transcription of structured RNAs tend to be present in longer than average noncoding ‘intergenic regions’ (IGRs) that are enriched in G and C nucleotides compared to the remainder of the genome. In the present study, we apply this computational pipeline to identify structured ncRNA candidates from 26 diverse bacterial species. Numerous novel structured ncRNA motifs were discovered, including several riboswitch candidates, one whose ligand has been identified and others that have yet to be experimentally validated. Our findings support recent predictions that hundreds of novel ribo-switch classes and other ncRNAs remain undiscovered among the limited number of bacterial species whose genomes have been completely sequenced.

<p><strong>The taxonomy and phylogeny of <em>Austropleospora ochracea</em> sp. nov. (Didymosphaeriaceae) from Guizhou, China</strong></p>

Austropleospora is a genus of Didymosphaeriaceae with only three extant species. A survey of saprobic ascomycetes in Guizhou Province, China, discovered a new Austropleospora species on dead twigs in a terrestrial habitat. The molecular phylogeny based on a combined SSU-LSU-tef1-ITS DNA sequence dataset confirmed the new species’ taxonomic position in Austropleospora. Austropleospora ochracea sp. nov. is characterized by globose to subglobose and uni-loculate ascoma with a centric short papilla, brown to dark brown peridium with cells of textura angularis to textura prismatica and conical, comparatively smaller ascospores with narrowly rounded polar ends. The new taxon is compared with similar species in Austropleospora, and its taxonomic status is briefly discussed.

Phylogeny and time scale of diversification in the fossil-rich sunfishes and black basses (Teleostei: Percomorpha: Centrarchidae)

Species of the North American freshwater fish lineage Centrarchidae are apex predators in their habitats and are among the world’s most popular sport fishes. Centrarchids boast a rich fossil record that extends from the latest Eocene to the Pleistocene. To investigate the phylogeny and timing of diversification of Centrarchidae, we deploy a dataset of DNA sequences of 16 nuclear genes sampled from nearly all of the recognized and undescribed species. We also utilize previously published morphological datasets to assess the phylogenetic placement of one of the oldest known centrarchid fossils, †Plioplarchus whitei. A Bayesian multispecies coalescent species tree analysis provides insight on relationships that evaded resolution in earlier studies, such as the relationships of Acantharchus pomotis, the resolution of a clade consisting of species previously synonymized under the Spotted Bass, Micropterus punctulatus, and a clade of recently described species previously considered populations of the Redeye Bass, Micropterus coosae. This new molecular phylogeny and the inclusion of †P. whitei and other centrarchid fossils in the tip-dated fossilized birth–death analysis results in a new hypothesis of the timing of diversification in Centrarchidae that contextualizes the ages of centrarchid fossils to the timing of speciation among the extant species. In addition to providing new temporal perspectives on the diversification of freshwater fishes in North America, this study may close of the chapter of centrarchid phylogeny inferred using Sanger-sequenced genes, as the use of genomic-scale datasets becomes mainstream in the phylogenetics of fishes.

Molecular phylogenetics of sub-Saharan African natricine snakes, and the biogeographic origins of the Seychelles endemic Lycognathophis seychellensis

Phylogenetic relationships of sub-Saharan African natricine snakes are understudied and poorly understood, which in turn has precluded analyses of the historical biogeography of the Seychelles endemic Lycognathophis seychellensis. We inferred the phylogenetic relationships of Seychelles and mainland sub-Saharan natricines by analysing a multilocus DNA sequence dataset for three mitochondrial (mt) and four nuclear (nu) genes. The mainland sub-Saharan natricines and L. seychellensis comprise a well-supported clade. Two maximally supported sets of relationships within this clade are (Limnophis,Natriciteres) and (Afronatrix,(Hydraethiops,Helophis)). The relationships of L. seychellensis with respect to these two lineages are not clearly resolved by analysing concatenated mt and nu data. Analysed separately, nu data best support a sister relationship of L. seychellensis with (Afronatrix,(Hydraethiops,Helophis)) and mt data best support a sister relationship with all mainland sub-Saharan natricines. Methods designed to cope with incomplete lineage sorting strongly favour the former hypothesis. Genetic variation among up to 33 L. seychellensis from five Seychelles islands is low. Fossil calibrated divergence time estimates support an overseas dispersal of the L. seychellensis lineage to the Seychelles from mainland Africa ca. 43–25 million years before present (Ma), rather than this taxon being a Gondwanan relic.

Taxonomic realignment of Senecio glaucophyllus (Asteraceae; Senecioneae) necessitates a new name for a widespread New Zealand species

ABSTRACT As presently delimited, the endemic New Zealand species Senecio glaucophyllus Cheeseman shows considerable morphological and ecological diversity. Two morphologically similar forms associated with this taxon are found in northwest Nelson (South Island), of which one is often filed in herbaria as S. glaucophyllus subsp. glaucophyllus. The other form has been informally recognised as S. aff. glaucophyllus. The present study aimed to determine if S. aff. glaucophyllus merits taxonomic recognition by assessing if it is morphologically and genetically distinct from S. glaucophyllus subsp. glaucophyllus and, if so, to identify its diagnostic morphological characters. The results of Principal Coordinate Analyses of a morphometric data set and phylogenetic studies of an ITS DNA sequence data set show that the two taxa are morphologically and genetically distinct and only distantly related to each other. Random Forest analyses resulted in the discovery of several morphological characters that can be used to distinguish the taxa. The inclusion of data of type specimens of S. glaucophyllus in the PCoAs showed that specimens of S. aff. glaucophyllus are conspecific with S. glaucophyllus whereas most of S. glaucophyllus in its current delimitation belong to a taxon that is unnamed at the species level. We here provide a new name at species rank for this taxon: Senecio matatini. This taxonomic realignment reinstates Cheeseman’s original concept of S. glaucophyllus as a species that is restricted to northwest Nelson. Senecio matatini has a much wider distribution and is found throughout much of the South Island and the southern and central parts of the North Island. Because our results indicate that S. glaucophyllus subsp. basinudus, S. glaucophyllus subsp. discoideus, and S. glaucophyllus subsp. toa are currently best considered as subspecies of S. matatini, new combinations for these three taxa are also provided. In addition, we lectotypify the name Senecio lautus var. montanus Cheeseman.

Efficient DNA sequence compression with neural networks.

BackgroundThe increasing production of genomic data has led to an intensified need for models that can cope efficiently with the lossless compression of DNA sequences. Important applications include long-term storage and compression-based data analysis. In the literature, only a few recent articles propose the use of neural networks for DNA sequence compression. However, they fall short when compared with specific DNA compression tools, such as GeCo2. This limitation is due to the absence of models specifically designed for DNA sequences. In this work, we combine the power of neural networks with specific DNA models. For this purpose, we created GeCo3, a new genomic sequence compressor that uses neural networks for mixing multiple context and substitution-tolerant context models.FindingsWe benchmark GeCo3 as a reference-free DNA compressor in 5 datasets, including a balanced and comprehensive dataset of DNA sequences, the Y-chromosome and human mitogenome, 2 compilations of archaeal and virus genomes, 4 whole genomes, and 2 collections of FASTQ data of a human virome and ancient DNA. GeCo3 achieves a solid improvement in compression over the previous version (GeCo2) of n}{}2.4%, n}{}7.1%, n}{}6.1%, n}{}5.8%, and n}{}6.0%, respectively. To test its performance as a reference-based DNA compressor, we benchmark GeCo3 in 4 datasets constituted by the pairwise compression of the chromosomes of the genomes of several primates. GeCo3 improves the compression in n}{}12.4%, n}{}11.7%, n}{}10.8%, and n}{}10.1% over the state of the art. The cost of this compression improvement is some additional computational time (1.7–3 times slower than GeCo2). The RAM use is constant, and the tool scales efficiently, independently of the sequence size. Overall, these values outperform the state of the art.ConclusionsGeCo3 is a genomic sequence compressor with a neural network mixing approach that provides additional gains over top specific genomic compressors. The proposed mixing method is portable, requiring only the probabilities of the models as inputs, providing easy adaptation to other data compressors or compression-based data analysis tools. GeCo3 is released under GPLv3 and is available for free download at https://github.com/cobilab/geco3.

Climatic oscillations during the Mio/Pliocene epochs induced cladogenesis in the terrestrial snail genus Gittenedouardia (Mollusca: Gastropoda: Cerastidae) from South Africa

In South Africa, the terrestrial snail genus Gittenedouardia is the most species-rich member of the Cerastidae, where it is primarily distributed in the highly fragmented Afrotemperate and Indian Ocean coastal belt (IOCB) forest biomes. Phylogenetic relationships and cladogenetic events within the genus remain unstudied. In this respect, we reconstructed a dated phylogeny for eight Gittenedouardia species, and two populations identified to genus level using a combined mitochondrial (16S rRNA and COI) DNA sequencing dataset analysed using Bayesian inference and Maximum Likelihood framework. Furthermore, we investigated the population genetic substructure of the three widely distributed species (Gittenedouardia spadicea, G. natalensis and G. arenicola) for the COI locus, while also subsampling these species using the nuclear DNA ITS-2 locus. Phylogenetic results based on the combined mtDNA dataset supported the monophyly of Gittenedouardia and revealed three major clades and deep genetic structure among the three widely distributed species. Divergence-time estimates suggest that diversification within Gittenedouardia occurred during the middle Miocene/late Pliocene, a period characterised by a decrease in precipitation and the contraction of the Afrotemperate and IOCB forest biomes. We used two species delimitation methods, (PTP and STACEY) to infer putative species in G. spadicea, G. natalensis and G. arenicola. The two methods recovered a large number of evolutionary distinct units, with minimal consensus in the exact number of lineages. Our findings suggest the presence of undescribed diversity, necessitating the need for taxonomic revisionary work on Gittenedouardia. We discuss the climatic factors which may have contributed to the observed cladogenesis and compare our results with other studies of forest dwelling faunal taxa.

Are population isolations and declines a threat to island endemic water striders? A lesson from demographic and niche modelling of Metrocoris esakii (Hemiptera: Gerridae).

Genetic stochasticity and bottlenecking in the course of Pleistocene glaciations have been identified as threatening the survival of local endemics. However, the mechanisms by which local endemic species balance the influences of these two events remain poorly understood. Here, we generated a double-digest restriction site-associated DNA sequencing (ddRAD-seq) data set, mined mitochondrial sequences and constructed ecological niche models for the island endemic water strider Metrocoris esakii (Hemiptera: Gerridae). We found that M. esakii comprised three divergent lineages (i.e., north, central and south) isolated by geographical barriers and generally experienced population declines with the constriction of suitable areas during the Last Glacial Maximum (LGM). Further demographic model testing and stairway plots revealed a history of recent gene flow among the neighbouring lineages and rapid recovery at the end of the LGM, indicating that M. esakii at least had the potential for an adaptive response to population fragmentation and bottlenecking. The northern lineage did not show genetic bottlenecking during the LGM, which was probably due to its large effective population size (Ne ) from migration, which improved its adaptive potential. Relative to the ddRAD-seq data set, the demographic results based on mitochondrial sequences were less conclusive, showing weak differentiation and oversimplified demographic trajectories for the three genetic lineages. Overall, this study provides some degree of optimism for the survival of island endemic water striders from a demographic perspective, but further evaluation of their extinction risk under the impacts of human activities is required.

The fruticose genera in the Ramalinaceae (Ascomycota, Lecanoromycetes): their diversity and evolutionary history.

We present phylogenetic analyses of the fruticose Ramalinaceae based on extensive collections from many parts of the world, with a special focus on the Vizcaíno deserts in north-western Mexico and the coastal desert in Namibia. We generate a four-locus DNA sequence dataset for accessions of Ramalina and two additional loci for Niebla and Vermilacinia. Four genera are strongly supported: the subcosmopolitan Ramalina, the new genus Namibialina endemic to SW Africa, and a duo formed by Niebla and Vermilacinia, endemic to the New World except the sorediate V. zebrina that disjunctly occurs in Namibia. The latter three genera are restricted to coastal desert and chaparral where vegetation depends on moisture from ocean fog. Ramalina is subcosmopolitan and much more diverse in its ecology.We show that Ramalina and its sister genus Namibialina diverged from each other at c. 48 Myrs, whereas Vermilacinia and Niebla split at c. 30 Myrs. The phylogeny of the fruticose genera remains unresolved to their ancestral crustose genera.Species delimitation within Namibialina and Ramalina is rather straightforward. The phylogeny and taxonomy of Vermilacinia are fully resolved, except for the two youngest clades of corticolous taxa, and support current taxonomy, including four new taxa described here. Secondary metabolite variation in Niebla generally coincides with major clades which are comprised of species complexes with still unresolved phylogenetic relationships. A micro-endemism pattern of allopatric species is strongly suspected for both genera, except for the corticolous taxa within Vermilacinia. Both Niebla and saxicolous Vermilacinia have chemotypes unique to species clades that are largely endemic to the Vizcaíno deserts.The following new taxa are described: Namibialinagen. nov. with N. melanothrix (comb. nov.) as type species, a single new species of Ramalina (R. krogiae) and four new species of Vermilacinia (V. breviloba, V. lacunosa, V. pustulata and V. reticulata). The new combination V. granulans is introduced. Two epithets are re-introduced for European Ramalina species: R. crispans (= R. peruviana auct. eur.) and R. rosacea (= R. bourgeana auct. p.p). A lectotype is designated for Vermilacinia procera. A key to saxicolous species of Vermilacinia is presented.

Abstract 5120: Surveying the AML surfaceome for novel immunotherapeutic targets

Abstract Acute myeloid leukemia (AML) therapy requires very intensive chemotherapy/stem cell transplant that cures only 60% of children and 25% of adults with AML. The development of chimeric antigen receptor (CAR) T cells for AML has been hampered by the lack of optimal surface antigens. We hypothesized that unbiased identification of AML cell surface proteins will enable creation of novel CAR T cell immunotherapies and that integration of AML surfaceome data with AML RNA and DNA sequencing data will facilitate identification of new therapeutic targets. We also hypothesized that pediatric and adult surfaceomes are overlapping but distinguishable. Sucrose gradient ultracentrifugation was used for surfaceome enrichment of patient leukemia and normal bone marrow samples without chemical labeling or affinity purification followed by GeLC-MS. The MOLM-14 human AML cell line served as internal control. Mass spectrometry data were processed with MaxQuant, and 9 GO terms were used to define putative cell surface proteins. Samples with at least 6 of 12 known AML surface markers detected were further analyzed. RNA and DNA were extracted for a subset of samples and sequenced. To date, we have surfaceome data for 25 pediatric AML, 12 pediatric normal bone marrow, 9 adult AML, and 9 MOLM-14 samples. A median of 2050 ± 319 surface proteins were detected in the MOLM-14 samples. The mean Pearson correlation coefficient for these replicates was 0.82, confirming the robustness of our workflow. In addition, we found a high representation of known AML cell surface proteins among the captured surface proteins, such as CD33, CD123 and FLT3. A median 1269 ± 474 and 1994 ± 480 surface proteins were found in the pediatric primary AML and normal control samples, respectively and 1790 ± 553 proteins were identified in adult primary AML samples. The average correlation coefficients for the pediatric AML, pediatric control, and adult AML samples were 0.63, 0.75, and 0.63, respectively. Comparison of the pediatric AML and control samples revealed 208 unique and 19 differentially expressed (>2-fold, p<0.05) surface proteins in the patient samples. Similarly, comparison of pediatric and adult AML proteomes identified 210 unique and 23 differentially expressed surface proteins in the pediatric samples. RNA and DNA sequencing has also been performed on a subset of the pediatric and adult primary AML samples, and proteogenomic integration of the different datasets is in progress. We demonstrate that the surfaceome of primary pediatric and adult AML patient samples can be surveyed at depth for the discovery of new potential immunotherapeutic targets. The presence of unique or differentially expressed surface proteins increases our optimism that targeting AML with potent immunotherapies may become clinically feasible. A proteogenomic integration of these AML surfaceome and RNA and DNA sequencing datasets may illuminate AML biology as well as present novel therapeutic targets. Citation Format: Tina Glisovic-Aplenc, Kevin Nestler, Congcong Lu, Lusha Cao, Joseph Cesare, Hyoungjoo Lee, Mitchelle Matesva, Terzah Horton, Sarah Tasian, John Shern, Saar Gill, Benjamin Garcia, Richard Aplenc. Surveying the AML surfaceome for novel immunotherapeutic targets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5120.

Changes in Fungal Community Structure in Freshwater Canals across a Gradient of Urbanization

Fungi are an important, yet often, neglected component of the aquatic microflora, and is responsible for primary decomposition and further processing of organic matter. By comparison, the ecological roles of terrestrial fungi have been well-studied, but the diversity and function of fungi that populate aquatic environments remain poorly understood. Here, the impact of urbanization on fungal diversity and community composition in the canal system of Suzhou was assessed by sequencing the internal transcribed spacer 1 (ITS1) region of the rRNA operon. It was amplified from environmental DNA that has been extracted from water samples and pre-deployed decomposing leaves collected from nine sampling locations (high, medium and low urbanization) over two seasons. The fungal diversity and community composition were determined by bioinformatic analysis of the large DNA sequence datasets generated to identify operational taxonomic units (OTUs) for phylogenetic assignment; over 1 million amplicons were sequenced from 36 samples. The alpha-diversity estimates showed high differences in fungal diversity between water and leaf samples, and winter versus summer. Higher numbers of fungal OTUs were identified in both water and leaf samples collected in the summer, and fungal diversity was also generally higher in water than on colonized leaves in both seasons. The fungal community on leaves was usually dominated by Ascomycetes, especially in winter, while water samples contained more diversity at phylum level with Chytridiomycetes often prominent, particularly in summer. At a genus level, a very high relative abundance of Alternaria on leaves was observed in winter at all locations, in contrast to very low abundance of this genus across all water samples. Fungal community composition also varied between sampling locations (i.e., urbanization); in cluster analysis, samples from high urbanization locations formed a distinct cluster, with medium and low urbanization samples clustering together or in some instances, separately. Redundancy analysis shed further light on the relationships between variation in fungal community composition and water physico-chemical properties. Fungal community diversity variation and correlation with different parameters is discussed in detail, but overall, the influence of season outweighed that of urbanization. This study is significant in cataloguing the impact of urbanization on fungal diversity to inform future restoration of urban canal systems on the importance of protecting the natural aquatic fungal flora.

Sequence Compression Benchmark (SCB) database-A comprehensive evaluation of reference-free compressors for FASTA-formatted sequences.

BackgroundNearly all molecular sequence databases currently use gzip for data compression. Ongoing rapid accumulation of stored data calls for a more efficient compression tool. Although numerous compressors exist, both specialized and general-purpose, choosing one of them was difficult because no comprehensive analysis of their comparative advantages for sequence compression was available.FindingsWe systematically benchmarked 430 settings of 48 compressors (including 29 specialized sequence compressors and 19 general-purpose compressors) on representative FASTA-formatted datasets of DNA, RNA, and protein sequences. Each compressor was evaluated on 17 performance measures, including compression strength, as well as time and memory required for compression and decompression. We used 27 test datasets including individual genomes of various sizes, DNA and RNA datasets, and standard protein datasets. We summarized the results as the Sequence Compression Benchmark database (SCB database, http://kirr.dyndns.org/sequence-compression-benchmark/), which allows custom visualizations to be built for selected subsets of benchmark results.ConclusionWe found that modern compressors offer a large improvement in compactness and speed compared to gzip. Our benchmark allows compressors and their settings to be compared using a variety of performance measures, offering the opportunity to select the optimal compressor on the basis of the data type and usage scenario specific to a particular application.

SCSIM: Jointly simulating correlated single-cell and bulk next-generation DNA sequencing data

BackgroundRecently, it has become possible to collect next-generation DNA sequencing data sets that are composed of multiple samples from multiple biological units where each of these samples may be from a single cell or bulk tissue. Yet, there does not yet exist a tool for simulating DNA sequencing data from such a nested sampling arrangement with single-cell and bulk samples so that developers of analysis methods can assess accuracy and precision.ResultsWe have developed a tool that simulates DNA sequencing data from hierarchically grouped (correlated) samples where each sample is designated bulk or single-cell. Our tool uses a simple configuration file to define the experimental arrangement and can be integrated into software pipelines for testing of variant callers or other genomic tools.ConclusionsThe DNA sequencing data generated by our simulator is representative of real data and integrates seamlessly with standard downstream analysis tools.

Exploring the prevalence and diversity of bovine ticks in five agro-ecological zones of Pakistan using phenetic and genetic tools

Tick infestation is a leading cause of tick-worry and tick-borne diseases in livestock and associated economic losses in tropical and subtropical regions of the world. The cattle and buffalo populations in Pakistan are exposed to tick infestation throughout the year, but very little is known about the biology, diversity and distribution of tick species across different agro-ecological zones (AEZ) of the country. The present study aimed to investigate the prevalence (number of bovines infested with ticks out of the investigated population) and diversity of hard ticks infesting bovines in 30 villages located in five distinct AEZs (i.e. Arid, Indus delta, Northern irrigated plain, Sandy desert and Southern irrigated plain). We collected a total of 774 ticks (adult and nymphs) from cattle (n = 116) and water buffaloes (n = 88) on small-holder dairy farms (with <10 bovids per establishment) from September to November 2017. The overall tick prevalence was 46.1% (cattle: 47.9%; buffaloes: 44%), which varied significantly from 22.2% in the Indus delta to 70.5% in the Sandy desert. Tick prevalence was slightly higher in female (46.5%) than male animals (45%), and higher in calves (i.e. ≤ 1 year of age) (55%) than in young animals (i.e. up to 3 years of age) (39%) and adults (48%). Five tick species - Hyalomma anatolicum, Hyalomma hussaini, Hyalomma scupense, Rhipicephalus microplus and Rhipicephalus annulatus - were identified morphologically and then genetically. Genetic identification, achieved using the sequences of two mitochondrial (cytochrome c oxidase subunit 1 and 16S) and one nuclear ribosomal (second internal transcribed spacer) regions, was consistent with the morphological findings. Phylogenetic analyses of the DNA sequence data sets showed that the five species of tick identified here were closely related to the same species or closely related species from within and outside of Pakistan. Of five presently recognised taxa within the R. microplus complex, two were identified herein, including the R. microplus clade C and R. annulatus. This investigation provides the first genetic evidence of the occurrence of R. annulatus in Pakistan as well as Hy. hussaini and Hy. scupense in bovines specifically in the provinces of Sindh and Punjab, respectively. The present findings emphasise the importance of combining morphological and molecular approaches to study the diversity of ticks. Further longitudinal studies are required to establish seasonal variations in the prevalence and distribution of bovine ticks in different AEZs of Pakistan.

Phylogenetic lineages and the role of hybridization as driving force of evolution in grass supertribe Poodae

AbstractTo investigate the evolutionary diversification and morphological evolution of grass supertribe Poodae (subfam. Pooideae, Poaceae) we conducted a comprehensive molecular phylogenetic analysis including representatives from most of its accepted genera. We focused on generating a DNA sequence dataset of plastid matK gene–3′trnK exon and trnL‐trnF regions and nuclear ribosomal (nr) ITS1–5.8S gene–ITS2 and ETS that was taxonomically overlapping as completely as possible (altogether 257 species). The idea was to infer whether phylogenetic trees or certain clades based on plastid and nrDNA data correspond with each other or discord, revealing signatures of past hybridization. The datasets were analysed separately, in combination, by excluding taxa with discordant placements in the individual gene trees and with duplication of these taxa in a way that each duplicate has only one data partition (plastid or nrDNA). We used maximum likelihood, maximum parsimony and Bayesian approaches. Instances of severe conflicts between the phylogenetic trees derived from both datasets, some of which have been noted earlier, point to hybrid origin of several lineages such as the ABCV clade encompassing several subtribes and subordinate clades, subtribes Airinae, Anthoxanthinae, Antinoriinae, subtr. nov., Aristaveninae, Avenulinae, subtr. nov., Helictochloinae, subtr. nov., Holcinae, Phalaridinae, Scolochloinae, Sesleriinae, Torreyochloinae and genera Arctopoa, Castellia, Graphephorum, Hyalopodium, Lagurus, Macrobriza, Puccinellia plus Sclerochloa, Sesleria, Tricholemma, Tzveleviochloa, etc. ‘Calamagrostis’ flavens appears to be an intergeneric hybrid between Agrostis and Calamagrostis. Analyses excluding all lineages with demonstrably cytonuclear discordance revealed three supported main clades within Poodae that were present in both the plastid and nrDNA trees. They fully corresponded in their delineation but were phylogenetically differently arranged, pointing to hybrid origin of one of them. We propose to consider these main clades in classification as separate tribes Aveneae, Poeae s.str. and Festuceae with a phylogenetic arrangement of Aveneae(Poeae,Festuceae) in plastid versus Festuceae(Aveneae,Poeae) in nrDNA trees. Phylogenetic incongruence of the plastid and nuclear markers extends across all hierarchical taxonomic levels of Poodae, ranging from species (not studied here) to genera, subtribes and tribes, therefore the deepest taxonomic levels, emphasizing the enormous significance of reticulate evolution in this large group of grasses. A partly revised classification is presented, including the introduction of a new tribe Festuceae and a re‐instatement of tribe Aveneae. Following a comparatively narrow delineation of preferably monophyletic subtribes, Antinoriinae, Avenulinae, Brizochloinae, Helictochloinae and Hypseochloinae are described as new. New genera are Arctohyalopoa and Hyalopodium. New combinations are Anthoxanthum glabrum subsp. sibiricum, A. nitens subsp. kolymense, Arctohyalopoa ivanovae, A. jurtzevii, A. lanatiflora, A. momica, Colpodium biebersteinianum, C. kochii, C. pisidicum, C. trichopodum, C. verticillatum, Dupontia fulva, Festuca masafuerana, F. robinsoniana, Graphephorum canescens, G. cernuum, Hyalopodium araraticum, Paracolpodium baltistanicum, Parapholis cylindrica, P. ×pauneroi. Festuca dolichathera and F. masatierrae are new names.

Structural and Functional Characteristics of the Microbiome in Deep-Dentin Caries

Dental caries is a cariogenic bacteria-mediated, fermentable carbohydrate-driven dynamic disease. The new ecological hypothesis for dentin caries suggests that an alteration in the microbial community and the presence of specific metabolic pathway genes contribute to the initiation and progression of caries. This study aimed to determine the structural and functional characteristics of a microbial community of human deep-dentin carious lesions. Sixteen deep-dentin carious lesions were obtained from the first permanent molars of 8 patients aged 9 to 18 y. Shotgun metagenomic sequencing was used to measure the microbial composition and abundance at the phylum, class, order, family, genus, and species levels. Functional analysis of the DNA sequencing data set was also performed and compared among different layers of the lesions using DIAMOND software against the Kyoto Encyclopedia of Genes and Genomes database. This study found that in the deep-dentin carious lesions, Actinobacteria (35.8%) and Firmicutes (31.2%) were the most prevalent phyla, followed by Bacteroidetes (13.6%), Proteobacteria (3.6%), and Fusobacteria (2.5%). The microbial composition varied among the individuals, but there were no significant differences in the distribution of the relative microbial abundance between the superficial layers and the deep layers. Although 14.5% of the top 10 taxa were identified as Lactobacillus at the genus level, only 25% of the deep-dentin carious samples showed Lactobacillus as the most abundant genus. Other abundant taxa included Actinomyces (10.5%), Olsenella (9.4%), Prevotella (8.8%), Propionibacterium (7.2%), Streptococcus (3.9%), Selenomonas (3.7%), Corynebacterium (1.9%), Leptotrichia (1.4%), and Parascardovia (1.1%). The most abundant pathway identified in the KEGG database was the metabolic pathway containing 101,427 annotated genes, which consisted of 51.4% of all annotated genes. The carbohydrate metabolism pathway, amino acid metabolism, and membrane transport were the functional traits of the level 2 pathways. These findings suggest that the potent interaction within the microbial communities in deep-dentin carious lesions may play a fundamental role in caries etiology.

A Guide to Carrying Out a Phylogenomic Target Sequence Capture Project.

High-throughput DNA sequencing techniques enable time- and cost-effective sequencing of large portions of the genome. Instead of sequencing and annotating whole genomes, many phylogenetic studies focus sequencing effort on large sets of pre-selected loci, which further reduces costs and bioinformatic challenges while increasing coverage. One common approach that enriches loci before sequencing is often referred to as target sequence capture. This technique has been shown to be applicable to phylogenetic studies of greatly varying evolutionary depth. Moreover, it has proven to produce powerful, large multi-locus DNA sequence datasets suitable for phylogenetic analyses. However, target capture requires careful considerations, which may greatly affect the success of experiments. Here we provide a simple flowchart for designing phylogenomic target capture experiments. We discuss necessary decisions from the identification of target loci to the final bioinformatic processing of sequence data. We outline challenges and solutions related to the taxonomic scope, sample quality, and available genomic resources of target capture projects. We hope this review will serve as a useful roadmap for designing and carrying out successful phylogenetic target capture studies.

Multigene phylogeny and taxonomy of Dendryphion hydeiand Torula hydeispp. nov. from herbaceous litter in northern Thailand.

During our studies on asexual fungi colonizing herbaceous litter in northern Thailand, we discovered two new fungal species, viz. Dendryphion hydei and Torula hydei spp. nov. The latter are examined, and their morphological characters are described as well as their DNA sequences from ribosomal and protein coding genes are analysed to infer their phylogenetic relationships with extant fungi. Torula hydei is different from other similar Torula species in having tiny and catenate conidia. Dendryphion hydei can be distinguished from other similar Dendryphion species in having large conidiophores and subhyaline to pale olivaceous brown, 2-4(-5)-septate conidia. Multigene phylogenetic analyses of a combined LSU, SSU, TEF1-α, RPB2 and ITS DNA sequence dataset generated from maximum likelihood and Bayesian inference analyses indicate that T. hydei forms a distinct lineage and basal to T. fici. Dendryphion hydei forms a distinct lineage and basal to D. europaeum, D. comosum, D. aquaticum and D. fluminicola within Torulaceae (Pleosporales, Dothideomycetes).