Metagenome Analysis of Speleothem Microbiome from Subterranean Cave Reveals Insight into Community Structure, Metabolic Potential, and BGCs Diversity.

The Borra caves, the second largest subterranean karst cave ecosystem in the Indian sub-continent, are located at the Ananthagiri hills of Araku Valley in the Alluri district of Andhra Pradesh, India. The present investigation applied a shotgun metagenomic approach to gain insights into the microbial community structure, metabolic potential, and biosynthetic gene cluster (BGC) diversity of the microbes colonizing the surface of the speleothems from the aphotic zone of Borra caves. The taxonomic analysis of the metagenome data illustrated that the speleothem-colonizing core microbial community was dominated mainly by Alpha-, Beta-, and Gamma-Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The key energy metabolic pathways analysis provides strong evidence of chemolithoautotrophic and chemoheterotrophic modes of nutrition in the speleothem-colonizing microbial community. Metagenome data suggests that sulfur reducers and sulfur-disproportionating microbes might play a vital role in energy generation in this ecosystem. Our metagenome data also suggest that the dissimilatory nitrifiers and nitrifying denitrifiers might play an essential role in conserving nitrogen pools in the ecosystem. Furthermore, metagenome-wide BGCs mining retrieved 451 putative BGCs; NRPS was the most abundant (24%). Phylogenetic analysis of the C domain of NRPS showed that sequences were distributed across all six function categories of the known C domain, including several novel subclades. For example, a novel subclade had been recovered within the LCL domain clade as a sister subclade of immunosuppressant cyclosporin encoding C domain sequences. Our result suggested that subterranean cave microbiomes might be a potential reservoir of novel microbial metabolites.

A consensus secondary structure of ITS2 for the diatom Order Cymatosirales (Mediophyceae, Bacillariophyta) and reappraisal of the order based on DNA, morphology, and reproduction.

In 1983, Hasle and colleagues removed cymatosiroid diatoms from the pennates, and erected a new centric diatom family, the Cymatosiraceae, mainly to accommodate for their newly discovered mode of sexual reproduction. The new family consisted of two subfamilies differing in frustule structure. The family was later elevated to the rank of Order Cymatosirales Round and Crawford. We revisited intra-ordinal relationships within Cymatosirales using combined genetic (DNA sequences), morphological (valve and frustule structure), and reproductive (auxospore type) characters. In total, 36 cymatosiroid strains from 19 species representing 13 genera (80% of the total number of extant genera; nine of them represented by their generitypes) were used in this study. Instead of only the commonly used loci (18S rRNA and plastidal genes) to infer diatom phylogeny, we developed a consensus secondary structure model of the Internal Transcribed Spacer 2 (ITS2) for this order and applied it to aid in sequence alignments for ITS2. This improved the alignment and thus the robustness of the phylogenetic framework. The compensatory base changes (CBCs) found in ITS2 secondary structures were mapped onto the multi-gene (18S rRNA + ITS2 + rbcL) phylogenetic tree topology. In all these trees, all species grouped into two morphologically and genetically distinct clades. Each clade was supported by multiple CBCs, as did all the clades representing genera. However, these clades did not correspond to the previously established subfamilies. Consequently, we amend the Order Cymatosirales and family Cymatosiraceae, and propose a new family, the Leyanellaceae. The structure of the auxospore was an additional synapomorphic character for Cymatosirales. Overall, we demonstrate a novel approach to study diatom phylogeny across a broader taxonomic range using ITS2 secondary structural information. Our results suggest that this approach might be useful in establishing higher taxonomic relationships in other groups of diatoms.

Analysis of diversity of chromophytic phytoplankton in a mangrove ecosystem using rbcL gene sequencing.

Phytoplankton forms the basis of primary production in mangrove environments. The phylogeny and diversity based on the amplification and sequencing of rbcL, the large subunit encoding the key enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase was investigated for improved understanding of the community structure and temporal trends of chromophytic eukaryotic phytoplankton assemblages in Sundarbans, the world's largest continuous mangrove. Diatoms (Bacillariophyceae) were by far the most frequently detected group in clone libraries (485 out of 525 clones), consistent with their importance as a major bloom-forming group. Other major chromophytic algal groups including Cryptophyceae, Haptophyceae, Pelagophyceae, Eustigmatophyceae, and Raphidophyceae which are important component of the assemblages were detected for the first time from Sundarbans based on rbcL approach. Many of the sequences from Sundarbans rbcL clone libraries showed identity with key bloom forming diatom genera namely Thalassiosira, Skeletonema and Nitzschia. Similarly, several rbcL sequences which were diatom-like were also detected highlighting the need to explore diatom communities from the study area. Some of the rbcL sequences detected from Sundarbans were ubiquitous in distribution showing 100% identities with uncultured rbcL sequences targeted previously from the Gulf of Mexico and California upwelling system that are geographically separated from study area. Novel rbcL lineages were also detected highlighting the need to culture and sequence phytoplankton from the ecoregion. Principal component analysis revealed that nitrate is an important variable that is associated with observed variation in phytoplankton assemblages (operational taxonomic units). This study applied molecular tools to highlight the ecological significance of diatoms, in addition to other chromophytic algal groups in Sundarbans.

Structural evolution of SLA promoter in mosquito-borne flaviviruses: A sequence-structure based phylogenetic framework.

All the flaviviruses have a Y-shaped stem-loop secondary structure known as the SLA element, and the structural features of this element are crucial to initiating the infection cycle. The present study particularly investigated how flaviviruses retained the common core SLA element secondary structure during the species evolution by selecting mosquito-borne flaviviruses (MBFVs) as a case study. The detailed search of nucleotide substitutions in species-wise consensus SLA secondary structure models suggested that the compensatory and hemi-compensatory base changes in the helices are crucial to preserving the common core secondary structure. In contrast to the coding region-based phylogeny, the SLA sequence-structure-based phylogenetic tree revealed an intriguing evolutionary relationship among MBFVs. Overall, this paper demonstrated for the first time the efficacy of RNA secondary structures as a phylogenetic marker to study the RNA virus evolution.

Study of diatom assemblages in Sundarbans mangrove water based on light microscopy and rbcL gene sequencing.

Sundarbans, the world's largest mangrove deltaic region, is one of the most productive ecosystems in tropical and subtropical latitudes and also serve as a nursery ground for rich coastal fisheries. In this study, we highlighted diatom assemblages from the Indian part of Sundarbans Biosphere Reserve (SBR) area for the first time based on light microscopy and rbcL gene sequencing and phylogeny. In total, 15 diatom species (11 centric forms and 4 pennate forms) were documented using light microscopy, and 3 major clades of diatoms were detected in rbcL phylogeny. Out of 15 diatom species, 7 were the first record from Sundarbans mangrove water. One of the species, Thalassiosira ferelineata Hasle and Fryxell, was reported for the first time in an Asian mangrove ecosystem based on light microscopy. Our study suggests the importance of establishing cultures and their polyphasic taxonomy are the future necessity to create an authenticated diatom database from mangrove water, which is still overlooked globally.

Gametogenesis and Auxospore Development in the Bipolar Centric Diatom Brockmanniella brockmannii (Family Cymatosiraceae).

We examined gametogenesis and auxospore development in the cymatosiroid diatom Brockmanniella brockmannii. Our mating experiments demonstrated that the clones were homothallic, self-compatible, produced two eggs per oogonium and four hologenous uniflagellate male gametes per spermatogonium. Auxospores grew free in the culture medium and the most external layer of the auxospore wall was made up of spinescent scaly elements in their early globular stage of development. The anisodiametric expansion of the globular auxospore was rather limited, at best proceeding only to a sub-globular stage. Our data suggest that the initial epivalves, nearly pennate-like in outline, developed within such auxospores curled up against the ventral side of the auxospore wall, not across its equatorial plane, and apparently without the help of rings, hoops or bands characteristic of pennate-type transverse perizonia. Thus, B. brockmannii showed a mode of initial epivalve development thus far unknown among diatoms. We propose that progressive silicification of the initial epivalve facilitated the breakdown of the sub-globular auxospore dorsal wall to accommodate for its straightening. The structural features of the spinescent scales on the auxospore wall suggest that Cymatosiraceae could be related to the mediophycean family Eupodiscaceae, although most current molecular phylogenies do not support a direct relationship.

A comprehensive framework for functional diversity patterns of marine chromophytic phytoplankton using rbcL phylogeny.

Marine chromophytes are taxonomically diverse group of algae and contribute approximately half of the total oceanic primary production. To understand the global patterns of functional diversity of chromophytic phytoplankton, robust bioinformatics and statistical analyses including deep phylogeny based on 2476 form ID rbcL gene sequences representing seven ecologically significant oceanographic ecoregions were undertaken. In addition, 12 form ID rbcL clone libraries were generated and analyzed (148 sequences) from Sundarbans Biosphere Reserve representing the world's largest mangrove ecosystem as part of this study. Global phylogenetic analyses recovered 11 major clades of chromophytic phytoplankton in varying proportions with several novel rbcL sequences in each of the seven targeted ecoregions. Majority of OTUs was found to be exclusive to each ecoregion, whereas some were shared by two or more ecoregions based on beta-diversity analysis. Present phylogenetic and bioinformatics analyses provide a strong statistical support for the hypothesis that different oceanographic regimes harbor distinct and coherent groups of chromophytic phytoplankton. It has been also shown as part of this study that varying natural selection pressure on form ID rbcL gene under different environmental conditions could lead to functional differences and overall fitness of chromophytic phytoplankton populations.