PhyloHerb: A phylogenomic pipeline for processing genome skimming data for plants

Premise of the study: The application of high throughput sequencing, especially to herbarium specimens, is greatly accelerating biodiversity research. Among various techniques, low coverage Illumina sequencing of total genomic DNA (genome skimming) can simultaneously recover the plastid, mitochondrial, and nuclear ribosomal regions across hundreds of species. Here, we introduce PhyloHerb -- a bioinformatic pipeline to efficiently and effectively assemble phylogenomic datasets derived from genome skimming. Methods and Results: PhyloHerb uses either a built-in database or user-specified references to extract orthologous sequences using BLAST search. It outputs FASTA files and offers a suite of utility functions to assist with alignment, data partitioning, concatenation, and phylogeny inference. The program is freely available at https://github.com/lmcai/PhyloHerb/. Conclusions: Using published data from Clusiaceae, we demonstrated that PhyloHerb can accurately identify genes using highly fragmented assemblies derived from sequencing older herbarium specimens. Our approach is effective at all taxonomic depths and is scalable to thousands of species.

Molecular Characterization of the First Alternavirus Identified in Fusarium oxysporum

A novel mycovirus named Fusarium oxysporum alternavirus 1(FoAV1) was identified as infecting Fusarium oxysporum strain BH19, which was isolated from a fusarium wilt diseased stem of Lilium brownii. The genome of FoAV1 contains four double-stranded RNA (dsRNA) segments (dsRNA1, dsRNA 2, dsRNA 3 and dsRNA 4, with lengths of 3.3, 2.6, 2.3 and 1.8 kbp, respectively). Additionally, dsRNA1 encodes RNA-dependent RNA polymerase (RdRp), and dsRNA2- dsRNA3- and dsRNA4-encoded hypothetical proteins (ORF2, ORF3 and ORF4), respectively. A homology BLAST search, along with multiple alignments based on RdRp, ORF2 and ORF3 sequences, identified FoAV1 as a novel member of the proposed family “Alternaviridae”. Evolutionary relation analyses indicated that FoAV1 may be related to alternaviruses, thus dividing the family “Alternaviridae” members into four clades. In addition, we determined that dsRNA4 was dispensable for replication and may be a satellite-like RNA of FoAV1—and could perhaps play a role in the evolution of alternaviruses. Our results provided evidence for potential genera establishment within the proposed family “Alternaviridae”. Additionally, FoAV1 exhibited biological control of Fusarium wilt. Our results also laid the foundations for the further study of mycoviruses within the family “Alternaviridae”, and provide a potential agent for the biocontrol of diseases caused by F. oxysporum.

Transposable element landscapes illuminate past evolutionary events in the endangered fern Vandenboschia speciosa

Vandenboschia speciosa is an endangered tetraploid fern species with a large genome (10.5 Gb). Its geographical distribution is characterized by disjoined tertiary flora refuges, with relict populations that survived past climate crises. Here we analyze the transposable elements (TEs) and found that they comprise about 76% of the V. speciosa genome, thus being the most abundant kind of DNA sequences in this gigantic genome. V. speciosa genome is composed of 51% and 5.6% of Class I and Class II elements, respectively. LTR retrotransposons were the most abundant TEs in this species (at least 42% of the genome), followed by non-LTR retrotransposons that constituted at least 8.7% of the genome of this species. We introduce an additional analysis to identify the nature of non-annotated elements (19% of the genome). A BLAST search of the non-annotated contigs against the V. speciosa TE database allowed determining the identity of almost half of them, which were most likely diverged sequence variants of the annotated TEs. In general, TE composition in V. speciosa resembles TE composition in seed plants. In addition, repeat landscapes revealed three episodes of amplification for all TEs, most likely due to demographic changes associated to past climate crises.

Freshwater Sordariomycetes: new species and new records in Pleurotheciaceae, Pleurotheciales

Eight freshwater asexual strains collected from China and Thailand were subjected to morpho-molecular analyses. BLAST search results showed that the new strains belonged to Pleurotheciales, Sordariomycetes. Phylogenetic analyses based on a combined LSU, SSU, ITS and RPB2 sequence dataset are used to clarify the placement of the new strains within Pleurotheciales. Pleurothecium guttulatum and Pleurotheciella sympodia are introduced as two new species from freshwater habitats. New geographical record of Pleurotheciella nilotica from Asia (Thailand and China) is reported. New habitat and geographical records of Phaeoisaria annesophieae (from freshwater habitats in Asia) are reported. Descriptions and illustrations of the new species and records, as well as known species Rhexoacrodictys erecta and Phaeoisaria annesophieae, are provided. A key to Pleurotheciella species is provided.

Morphological descriptions and DNA barcodes of Oziella viscida n. sp. (Eriophyoidea, Phytoptidae) and two infrequently reported Trisetacus species (Nalepellidae) from Crimea

We report on a new phytoptid mite species, Oziella viscida n. sp., collected in Western Crimea from sea rush, Juncus maritimus (Juncaceae), and give supplementary descriptions of two rarely encountered nalepellid species of the genus Trisetacus from pines: T. confusus Livshits & Vasilieva, 1982 (in Vasilieva et al. 1982) from needle sheaths of Pinus nigra ssp. pallasiana (Pinaceae), an endemic subspecies restricted to Crimea, and T. brevisetus Livshits & Sekerskaya, 1982 (in Vasilieva et al. 1982) from needle sheaths of Pinus brutia ssp. pityusa (Steven) Silba, a relatively isolated subspecies of Turkish pine (P. brutia Tenore) growing in Georgia, Caucasus and Crimea. Oziella viscida n. sp. is remarkable in that most specimens were found inhabiting the basal part of leaves and stems of J. maritimus, an area covered by a transparent, sticky exudate apparently secreted by the plant epidermis. The mites were completely embedded in this substance and, rather than crawling with their legs, were observed moving through the viscous material while bending their opisthosoma in a serpentine or wormlike manner—an adaptation that appears to be currently unreported in eriophyoids and possibly reminiscent of locomotion of ancestral “protoeriophyoids” associated with soil. In comparison to females, males of O. viscida n. sp. and T. confusus have a more distinct prodorsal shield pattern consisting of a larger number of longer lines. Three new barcode gene sequences were obtained: MZ220550 (Cox1, O. viscida n. sp., 1159 bp), MZ224497 (18S, 2012 bp, T. brevisetus), and MZ224498 (18S, 2013 bp T. confusus). A BLAST search of the 18S sequences of T. brevisetus and T. confusus shows them as slightly closer to other 18S sequences of Trisetacus from Pinaceae (95.5%–96.3% identity) than to Trisetacus from Cupressaceae (93.6%–94.0% identity). Comparison of sequences of nalepellids currently present in GenBank suggest that a complete 18S sequence KJ841938.1 (2252 bp) from China belongs to an identified Trisetacus from Pinaceae rather than to Setoptus koraiensis as labelled, highlighting the necessity to review carefully the sequences of Eriophyoidea prior to using them in phylogenetic analyses, as well as the need to recollect and resequence S. koraiensis to clarify the nature of the problematic data from GenBank assigned to this species.

Keqinzhangia Aquatica Gen. et sp. nov. And Pseudocoronospora Hainanense gen. et sp. nov., Isolated From Freshwater In Southern China

During the investigation of the diversity of aquatic hyphomycetes from southern China, two interesting isolates were collected. Then, the two isolates were cultured and sequenced, and a BLAST search of its LSU sequences against data in GenBank revealed that the closest related taxa in the genus Microthyrium. Phylogenetic analyses, based on the combined sequence data from the small and large nuclear subunit ribosomal DNA (SSU and LSU), revealed that our isolates belong to the Microthyriaceae. Combined morphological characters, we finally described our isolates as two new genera and species in Microthyriaceae, named as: Keqinzhangia aquatica and Pseudocoronospora hainanense. The full descriptions, illustrations and a phylogenetic tree showing the position of the two new genera were provided in this paper.

iBLAST: Incremental BLAST of new sequences via automated e-value correction

Search results from local alignment search tools use statistical scores that are sensitive to the size of the database to report the quality of the result. For example, NCBI BLAST reports the best matches using similarity scores and expect values (i.e., e-values) calculated against the database size. Given the astronomical growth in genomics data throughout a genomic research investigation, sequence databases grow as new sequences are continuously being added to these databases. As a consequence, the results (e.g., best hits) and associated statistics (e.g., e-values) for a specific set of queries may change over the course of a genomic investigation. Thus, to update the results of a previously conducted BLAST search to find the best matches on an updated database, scientists must currently rerun the BLAST search against the entire updated database, which translates into irrecoverable and, in turn, wasted execution time, money, and computational resources. To address this issue, we devise a novel and efficient method to redeem past BLAST searches by introducing iBLAST. iBLAST leverages previous BLAST search results to conduct the same query search but only on the incremental (i.e., newly added) part of the database, recomputes the associated critical statistics such as e-values, and combines these results to produce updated search results. Our experimental results and fidelity analyses show that iBLAST delivers search results that are identical to NCBI BLAST at a substantially reduced computational cost, i.e., iBLAST performs (1 + δ)/δ times faster than NCBI BLAST, where δ represents the fraction of database growth. We then present three different use cases to demonstrate that iBLAST can enable efficient biological discovery at a much faster speed with a substantially reduced computational cost.

Photophore Distribution and Enzymatic Diversity Within the Photogenic Integument of the Cookie-Cutter Shark Isistius brasiliensis (Chondrichthyes: Dalatiidae)

The cookie-cutter shark Isistius brasiliensis (Squaliformes: Dalatiidae) is a deep-sea species that emits a blue luminescence ventrally, except at the level of a black band located beneath the jaw. This study aims to (i) investigate the distribution and histology of the photophores (i.e., light-emitting organs) along the shark body, (ii) describe the tissue-specific transcriptomes of the black band integument region (i.e., non-photogenic) and the ventral integument region (i.e., photogenic), (iii) describe the repertoire of enzyme-coding transcripts expressed the two integument regions, and (iv) analyze the potential expression of transcripts coding for luciferase-like enzymes (i.e., close homologs of known luciferases involved in the bioluminescence of other organisms). Our analyses confirm the black band’s non-photogenic status and photophore absence within this region. The sub-rostral area is the region where the photophore density is the highest. In parallel, paired-end Illumina sequencing has been used to generate two pilot transcriptomes, from the black band and the ventral integument tissues of one individual. In total, 68,943 predicted unigenes have been obtained (i.e., 64,606 for the black band transcriptome, 43,996 for the ventral integument transcriptome) with 43,473 unigenes showing significant similarities to known sequences from public databases. BLAST search analyses of known luciferases, coupled with comparative predicted gene expression (i.e., photogenic versus non-photogenic), support the hypothesis that the species uses an unknown luciferase system. An enzymatic repertoire was predicted based on the PRIAM database, and Enzyme Commission numbers were assigned for all detected enzyme-coding unigenes. These pilot transcriptomes based on a single specimen, and the predicted enzyme repertoire, constitute a valuable resource for future investigations on the biology of this enigmatic luminous shark.

Fast Location and Isolation of Phenanthrene-degrading Halophilic Microbial Community and its Degradation Pathway

Microbial consortium WZ-4, which could degrade phenanthrene (PHE) as the main carbon and energy source, was isolated from the aerobic sludge of Weizhou wastewater treatment plant. Under the condition of high salinity (3%), the degradation of PHE (100 mg/L) was 87.76% in 7d. Its metabolites, genome sequence and biodegradation pathway were studied. The main metabolites include 1,2-dihydroxynaphthalene, 1-hydroxy-2-naphthalene, 5,6-benzocoumarin and phthalic acid. 12 PHE degrading enzyme genes appeared in the metagenome sequencing of WZ-4, and the genes involved in PHE degradation were included phdE, phdF, phdG, and pcaL. Based on the metabolites detected by GC-MS and the potential PHE-degrading genes identified by BLAST search, biodegradation pathway of PHE by WZ-4 was predicted.

The association of group IIB intron with integrons in hypersaline environments

Background Group II introns are mobile genetic elements used as efficient gene targeting tools. They function as both ribozymes and retroelements. Group IIC introns are the only class reported so far to be associated with integrons. In order to identify group II introns linked with integrons and CALINS (cluster of attC sites lacking a neighboring integron integrase) within halophiles, we mined for integrons in 28 assembled metagenomes from hypersaline environments and publically available 104 halophilic genomes using Integron Finder followed by blast search for group II intron reverse transcriptases (RT)s. Results We report the presence of different group II introns associated with integrons and integron-related sequences denoted by UHB.F1, UHB.I2, H.ha.F1 and H.ha.F2. The first two were identified within putative integrons in the metagenome of Tanatar-5 hypersaline soda lake, belonging to IIC and IIB intron classes, respectively at which the first was a truncated intron. Other truncated introns H.ha.F1 and H.ha.F2 were also detected in a CALIN within the extreme halophile Halorhodospira halochloris, both belonging to group IIB introns. The intron-encoded proteins (IEP) s identified within group IIB introns belonged to different classes: CL1 class in UHB.I2 and bacterial class E in H.ha.Fa1 and H.ha.F2. A newly identified insertion sequence (ISHahl1) of IS200/605 superfamily was also identified adjacent to H. halochloris CALIN. Finally, an abundance of toxin-antitoxin (TA) systems was observed within the identified integrons. Conclusion So far, this is the first investigation of group II introns within integrons in halophilic genomes and metagenomes from hypersaline environments. We report the presence of group IIB introns associated with integrons or CALINs. This study provides the basis for understanding the role of group IIB introns in the evolution of halophiles and their potential biotechnological role.