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April 21, 2020

Harnessing long-read amplicon sequencing to uncover NRPS and Type I PKS gene sequence diversity in polar desert soils.

The severity of environmental conditions at Earth’s frigid zones present attractive opportunities for microbial biomining due to their heightened potential as reservoirs for novel secondary metabolites. Arid soil microbiomes within the Antarctic and Arctic circles are remarkably rich in Actinobacteria and Proteobacteria, bacterial phyla known to be prolific producers of natural products. Yet the diversity of secondary metabolite genes within these cold, extreme environments remain largely unknown. Here, we employed amplicon sequencing using PacBio RS II, a third generation long-read platform, to survey over 200 soils spanning twelve east Antarctic and high Arctic sites for natural product-encoding genes, specifically targeting non-ribosomal peptides (NRPS) and Type I polyketides (PKS). NRPS-encoding genes were more widespread across the Antarctic, whereas PKS genes were only recoverable from a handful of sites. Many recovered sequences were deemed novel due to their low amino acid sequence similarity to known protein sequences, particularly throughout the east Antarctic sites. Phylogenetic analysis revealed that a high proportion were most similar to antifungal and biosurfactant-type clusters. Multivariate analysis showed that soil fertility factors of carbon, nitrogen and moisture displayed significant negative relationships with natural product gene richness. Our combined results suggest that secondary metabolite production is likely to play an important physiological component of survival for microorganisms inhabiting arid, nutrient-starved soils. © FEMS 2019.

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April 21, 2020

A hybrid de novo assembly of the sea pansy (Renilla muelleri) genome.

More than 3,000 species of octocorals (Cnidaria, Anthozoa) inhabit an expansive range of environments, from shallow tropical seas to the deep-ocean floor. They are important foundation species that create coral “forests,” which provide unique niches and 3-dimensional living space for other organisms. The octocoral genus Renilla inhabits sandy, continental shelves in the subtropical and tropical Atlantic and eastern Pacific Oceans. Renilla is especially interesting because it produces secondary metabolites for defense, exhibits bioluminescence, and produces a luciferase that is widely used in dual-reporter assays in molecular biology. Although several anthozoan genomes are currently available, the majority of these are hexacorals. Here, we present a de novo assembly of an azooxanthellate shallow-water octocoral, Renilla muelleri.We generated a hybrid de novo assembly using MaSuRCA v.3.2.6. The final assembly included 4,825 scaffolds and a haploid genome size of 172 megabases (Mb). A BUSCO assessment found 88% of metazoan orthologs present in the genome. An Augustus ab initio gene prediction found 23,660 genes, of which 66% (15,635) had detectable similarity to annotated genes from the starlet sea anemone, Nematostella vectensis, or to the Uniprot database. Although the R. muelleri genome may be smaller (172 Mb minimum size) than other publicly available coral genomes (256-448 Mb), the R. muelleri genome is similar to other coral genomes in terms of the number of complete metazoan BUSCOs and predicted gene models.The R. muelleri hybrid genome provides a novel resource for researchers to investigate the evolution of genes and gene families within Octocorallia and more widely across Anthozoa. It will be a key resource for future comparative genomics with other corals and for understanding the genomic basis of coral diversity. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Divergent evolution in the genomes of closely related lacertids, Lacerta viridis and L. bilineata, and implications for speciation.

Lacerta viridis and Lacerta bilineata are sister species of European green lizards (eastern and western clades, respectively) that, until recently, were grouped together as the L. viridis complex. Genetic incompatibilities were observed between lacertid populations through crossing experiments, which led to the delineation of two separate species within the L. viridis complex. The population history of these sister species and processes driving divergence are unknown. We constructed the first high-quality de novo genome assemblies for both L. viridis and L. bilineata through Illumina and PacBio sequencing, with annotation support provided from transcriptome sequencing of several tissues. To estimate gene flow between the two species and identify factors involved in reproductive isolation, we studied their evolutionary history, identified genomic rearrangements, detected signatures of selection on non-coding RNA, and on protein-coding genes.Here we show that gene flow was primarily unidirectional from L. bilineata to L. viridis after their split at least 1.15 million years ago. We detected positive selection of the non-coding repertoire; mutations in transcription factors; accumulation of divergence through inversions; selection on genes involved in neural development, reproduction, and behavior, as well as in ultraviolet-response, possibly driven by sexual selection, whose contribution to reproductive isolation between these lacertid species needs to be further evaluated.The combination of short and long sequence reads resulted in one of the most complete lizard genome assemblies. The characterization of a diverse array of genomic features provided valuable insights into the demographic history of divergence among European green lizards, as well as key species differences, some of which are candidates that could have played a role in speciation. In addition, our study generated valuable genomic resources that can be used to address conservation-related issues in lacertids. © The Author(s) 2018. Published by Oxford University Press.

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April 21, 2020

Biodiversity seen through the perspective of insects: 10 simple rules on methodological choices and experimental design for genomic studies.

Massively parallel DNA sequencing opens up opportunities for bridging multiple temporal and spatial dimensions in biodiversity research, thanks to its efficiency to recover millions of nucleotide polymorphisms. Here, we identify the current status, discuss the main challenges, and look into future perspectives on biodiversity genomics focusing on insects, which arguably constitute the most diverse and ecologically important group among all animals. We suggest 10 simple rules that provide a succinct step-by-step guide and best-practices to anyone interested in biodiversity research through the study of insect genomics. To this end, we review relevant literature on biodiversity and evolutionary research in the field of entomology. Our compilation is targeted at researchers and students who may not yet be specialists in entomology or molecular biology. We foresee that the genomic revolution and its application to the study of non-model insect lineages will represent a major leap to our understanding of insect diversity.

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April 21, 2020

Hidden genomic evolution in a morphospecies-The landscape of rapidly evolving genes in Tetrahymena.

A morphospecies is defined as a taxonomic species based wholly on morphology, but often morphospecies consist of clusters of cryptic species that can be identified genetically or molecularly. The nature of the evolutionary novelty that accompanies speciation in a morphospecies is an intriguing question. Morphospecies are particularly common among ciliates, a group of unicellular eukaryotes that separates 2 kinds of nuclei-the silenced germline nucleus (micronucleus [MIC]) and the actively expressed somatic nucleus (macronucleus [MAC])-within a common cytoplasm. Because of their very similar morphologies, members of the Tetrahymena genus are considered a morphospecies. We explored the hidden genomic evolution within this genus by performing a comprehensive comparative analysis of the somatic genomes of 10 species and the germline genomes of 2 species of Tetrahymena. These species show high genetic divergence; phylogenomic analysis suggests that the genus originated about 300 million years ago (Mya). Seven universal protein domains are preferentially included among the species-specific (i.e., the youngest) Tetrahymena genes. In particular, leucine-rich repeat (LRR) genes make the largest contribution to the high level of genome divergence of the 10 species. LRR genes can be sorted into 3 different age groups. Parallel evolutionary trajectories have independently occurred among LRR genes in the different Tetrahymena species. Thousands of young LRR genes contain tandem arrays of exactly 90-bp exons. The introns separating these exons show a unique, extreme phase 2 bias, suggesting a clonal origin and successive expansions of 90-bp-exon LRR genes. Identifying LRR gene age groups allowed us to document a Tetrahymena intron length cycle. The youngest 90-bp exon LRR genes in T. thermophila are concentrated in pericentromeric and subtelomeric regions of the 5 micronuclear chromosomes, suggesting that these regions act as genome innovation centers. Copies of a Tetrahymena Long interspersed element (LINE)-like retrotransposon are very frequently found physically adjacent to 90-bp exon/intron repeat units of the youngest LRR genes. We propose that Tetrahymena species have used a massive exon-shuffling mechanism, involving unequal crossing over possibly in concert with retrotransposition, to create the unique 90-bp exon array LRR genes.

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April 21, 2020

Comparative genomic analysis of eight novel haloalkaliphilic bacteriophages from Lake Elmenteita, Kenya.

We report complete genome sequences of eight bacteriophages isolated from Haloalkaline Lake Elmenteita found on the floor of Kenyan Rift Valley. The bacteriophages were sequenced, annotated and a comparative genomic analysis using various Bioinformatics tools carried out to determine relatedness of the bacteriophages to each other, and to those in public databases. Basic genome properties like genome size, percentage coding density, number of open reading frames, percentage GC content and gene organizations revealed the bacteriophages had no relationship to each other. Comparison to other nucleotide sequences in GenBank database showed no significant similarities hence novel. At the amino acid level, phages of our study revealed mosaicism to genes with conserved domains to already described phages. Phylogenetic analyses of large terminase gene responsible for DNA packaging and DNA polymerase gene for replication further showed diversity among the bacteriophages. Our results give insight into diversity of bacteriophages in Lake Elmenteita and provide information on their evolution. By providing primary sequence information, this study not only provides novel sequences for biotechnological exploitation, but also sets stage for future studies aimed at better understanding of virus diversity and genomes from haloalkaline lakes in the Rift Valley.

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April 21, 2020

Complete Genome Sequence of Lactococcus lactis subsp. cremoris 3107, Host for the Model Lactococcal P335 Bacteriophage TP901-1.

The complete genome sequence of Lactococcus lactis subsp. cremoris 3107, a dairy starter strain and a host for the model lactococcal P335 bacteriophage TP901-1, is reported here. The circular chromosome of L. lactis subsp. cremoris 3107 is among the smallest genomes of currently sequenced lactococcal strains. L. lactis subsp. cremoris 3107 harbors a complement of six plasmids, which appears to be a reflection of its adaptation to the nutrient-rich dairy environment.

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April 21, 2020

Reconstruction of the genomes of drug-resistant pathogens for outbreak investigation through metagenomic sequencing

Culture-independent methods that target genome fragments have shown promise in identifying certain pathogens, but the holy grail of comprehensive pathogen genome detection from microbiologically complex samples for subsequent forensic analyses remains a challenge. In the context of an investigation of a nosocomial outbreak, we used shotgun metagenomic sequencing of a human fecal sample and a neural network algorithm based on tetranucleotide frequency profiling to reconstruct microbial genomes and tested the same approach using rectal swabs from a second patient. The approach rapidly and readily detected the genome of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in the patient fecal specimen and in the rectal swab sample, achieving a level of strain resolution that was sufficient for confident transmission inference during a highly clonal outbreak. The analysis also detected previously unrecognized colonization of the patient by vancomycin-resistant Enterococcus faecium, another multidrug-resistant bacterium.IMPORTANCE The study results reported here perfectly demonstrate the power and promise of clinical metagenomics to recover genome sequences of important drug-resistant bacteria and to rapidly provide rich data that inform outbreak investigations and treatment decisions, independently of the need to culture the organisms.

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April 21, 2020

The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications.

UNITE (https://unite.ut.ee/) is a web-based database and sequence management environment for the molecular identification of fungi. It targets the formal fungal barcode-the nuclear ribosomal internal transcribed spacer (ITS) region-and offers all ~1 000 000 public fungal ITS sequences for reference. These are clustered into ~459 000 species hypotheses and assigned digital object identifiers (DOIs) to promote unambiguous reference across studies. In-house and web-based third-party sequence curation and annotation have resulted in more than 275 000 improvements to the data over the past 15 years. UNITE serves as a data provider for a range of metabarcoding software pipelines and regularly exchanges data with all major fungal sequence databases and other community resources. Recent improvements include redesigned handling of unclassifiable species hypotheses, integration with the taxonomic backbone of the Global Biodiversity Information Facility, and support for an unlimited number of parallel taxonomic classification systems.

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April 21, 2020

Biphasic cellular adaptations and ecological implications of Alteromonas macleodii degrading a mixture of algal polysaccharides.

Algal polysaccharides are an important bacterial nutrient source and central component of marine food webs. However, cellular and ecological aspects concerning the bacterial degradation of polysaccharide mixtures, as presumably abundant in natural habitats, are poorly understood. Here, we contextualize marine polysaccharide mixtures and their bacterial utilization in several ways using the model bacterium Alteromonas macleodii 83-1, which can degrade multiple algal polysaccharides and contributes to polysaccharide degradation in the oceans. Transcriptomic, proteomic and exometabolomic profiling revealed cellular adaptations of A. macleodii 83-1 when degrading a mix of laminarin, alginate and pectin. Strain 83-1 exhibited substrate prioritization driven by catabolite repression, with initial laminarin utilization followed by simultaneous alginate/pectin utilization. This biphasic phenotype coincided with pronounced shifts in gene expression, protein abundance and metabolite secretion, mainly involving CAZymes/polysaccharide utilization loci but also other functional traits. Distinct temporal changes in exometabolome composition, including the alginate/pectin-specific secretion of pyrroloquinoline quinone, suggest that substrate-dependent adaptations influence chemical interactions within the community. The ecological relevance of cellular adaptations was underlined by molecular evidence that common marine macroalgae, in particular Saccharina and Fucus, release mixtures of alginate and pectin-like rhamnogalacturonan. Moreover, CAZyme microdiversity and the genomic predisposition towards polysaccharide mixtures among Alteromonas spp. suggest polysaccharide-related traits as an ecophysiological factor, potentially relating to distinct ‘carbohydrate utilization types’ with different ecological strategies. Considering the substantial primary productivity of algae on global scales, these insights contribute to the understanding of bacteria-algae interactions and the remineralization of chemically diverse polysaccharide pools, a key step in marine carbon cycling.

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April 21, 2020

Expedited assessment of terrestrial arthropod diversity by coupling Malaise traps with DNA barcoding 1.

Monitoring changes in terrestrial arthropod communities over space and time requires a dramatic increase in the speed and accuracy of processing samples that cannot be achieved with morphological approaches. The combination of DNA barcoding and Malaise traps allows expedited, comprehensive inventories of species abundance whose cost will rapidly decline as high-throughput sequencing technologies advance. Aside from detailing protocols from specimen sorting to data release, this paper describes their use in a survey of arthropod diversity in a national park that examined 21?194 specimens representing 2255 species. These protocols can support arthropod monitoring programs at regional, national, and continental scales.

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April 21, 2020

Interspecies association mapping links reduced CG to TG substitution rates to the loss of gene-body methylation.

Comparative genomics can unravel the genetic basis of species differences; however, successful reports on quantitative traits are still scarce. Here we present genome assemblies of 31 so-far unassembled Brassicaceae plant species and combine them with 16 previously published assemblies to establish the Brassicaceae Diversity Panel. Using a new interspecies association strategy for quantitative traits, we found a so-far unknown association between the unexpectedly high variation in CG to TG substitution rates in genes and the absence of CHROMOMETHYLASE3 (CMT3) orthologues. Low substitution rates were associated with the loss of CMT3, while species with conserved CMT3 orthologues showed high substitution rates. Species without CMT3 also lacked gene-body methylation (gbM), suggesting an evolutionary trade-off between the unknown function of gbM and low substitution rates in Brassicaceae, possibly due to low mutability of non-methylated cytosines.

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April 21, 2020

Genome of Crucihimalaya himalaica, a close relative of Arabidopsis, shows ecological adaptation to high altitude.

Crucihimalaya himalaica, a close relative of Arabidopsis and Capsella, grows on the Qinghai-Tibet Plateau (QTP) about 4,000 m above sea level and represents an attractive model system for studying speciation and ecological adaptation in extreme environments. We assembled a draft genome sequence of 234.72 Mb encoding 27,019 genes and investigated its origin and adaptive evolutionary mechanisms. Phylogenomic analyses based on 4,586 single-copy genes revealed that C. himalaica is most closely related to Capsella (estimated divergence 8.8 to 12.2 Mya), whereas both species form a sister clade to Arabidopsis thaliana and Arabidopsis lyrata, from which they diverged between 12.7 and 17.2 Mya. LTR retrotransposons in C. himalaica proliferated shortly after the dramatic uplift and climatic change of the Himalayas from the Late Pliocene to Pleistocene. Compared with closely related species, C. himalaica showed significant contraction and pseudogenization in gene families associated with disease resistance and also significant expansion in gene families associated with ubiquitin-mediated proteolysis and DNA repair. We identified hundreds of genes involved in DNA repair, ubiquitin-mediated proteolysis, and reproductive processes with signs of positive selection. Gene families showing dramatic changes in size and genes showing signs of positive selection are likely candidates for C. himalaica’s adaptation to intense radiation, low temperature, and pathogen-depauperate environments in the QTP. Loss of function at the S-locus, the reason for the transition to self-fertilization of C. himalaica, might have enabled its QTP occupation. Overall, the genome sequence of C. himalaica provides insights into the mechanisms of plant adaptation to extreme environments.Copyright © 2019 the Author(s). Published by PNAS.

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April 21, 2020

Megaphylogeny resolves global patterns of mushroom evolution.

Mushroom-forming fungi (Agaricomycetes) have the greatest morphological diversity and complexity of any group of fungi. They have radiated into most niches and fulfil diverse roles in the ecosystem, including wood decomposers, pathogens or mycorrhizal mutualists. Despite the importance of mushroom-forming fungi, large-scale patterns of their evolutionary history are poorly known, in part due to the lack of a comprehensive and dated molecular phylogeny. Here, using multigene and genome-based data, we assemble a 5,284-species phylogenetic tree and infer ages and broad patterns of speciation/extinction and morphological innovation in mushroom-forming fungi. Agaricomycetes started a rapid class-wide radiation in the Jurassic, coinciding with the spread of (sub)tropical coniferous forests and a warming climate. A possible mass extinction, several clade-specific adaptive radiations and morphological diversification of fruiting bodies followed during the Cretaceous and the Paleogene, convergently giving rise to the classic toadstool morphology, with a cap, stalk and gills (pileate-stipitate morphology). This morphology is associated with increased rates of lineage diversification, suggesting it represents a key innovation in the evolution of mushroom-forming fungi. The increase in mushroom diversity started during the Mesozoic-Cenozoic radiation event, an era of humid climate when terrestrial communities dominated by gymnosperms and reptiles were also expanding.

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