Ultra-deep sequencing of foraminiferal microbarcodes unveils hidden richness of early monothalamous lineages in deep-sea sediments

How to know the fungi: combining field inventories and DNA-barcoding to document fungal diversity.

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79-97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.

Extracellular DNA amplicon sequencing reveals high levels of benthic eukaryotic diversity in the central Red Sea

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.

Fungal community analysis in the deep-sea sediments of the Pacific Ocean assessed by comparison of ITS, 18S and 28S ribosomal DNA regions

Northern peatlands play a key role in the global carbon and water budget, but the bacterial diversity in these ecosystems remains poorly described. Here, we compared the bacterial community composition in the surface (0–5 cm depth) and subsurface (45–50 cm) peat layers of an acidic (pH 4.0) Sphagnum-dominated wetland, using pyrosequencing of 16S rRNA genes. The denoised sequences (37,229 reads, average length ∼430 bp) were affiliated with 27 bacterial phyla and corresponded to 1,269 operational taxonomic units (OTUs) determined at 97% sequence identity. Abundant OTUs were affiliated with the Acidobacteria (35.5±2.4% and 39.2±1.2% of all classified sequences in surface and subsurface peat, respectively), Alphaproteobacteria (15.9±1.7% and 25.8±1.4%), Actinobacteria (9.5±2.0% and 10.7±0.5%), Verrucomicrobia (8.5±1.4% and 0.6±0.2%), Planctomycetes (5.8±0.4% and 9.7±0.6%), Deltaproteobacteria (7.1±0.4% and 4.4%±0.3%), and Gammaproteobacteria (6.6±0.4% and 2.1±0.1%). The taxonomic patterns of the abundant OTUs were uniform across all the subsamples taken from each peat layer. In contrast, the taxonomic patterns of rare OTUs were different from those of the abundant OTUs and varied greatly among subsamples, in both surface and subsurface peat. In addition to the bacterial taxa listed above, rare OTUs represented the following groups: Armatimonadetes, Bacteroidetes, Chlamydia, Chloroflexi, Cyanobacteria, Elusimicrobia, Fibrobacteres, Firmicutes, Gemmatimonadetes, Spirochaetes, AD3, WS1, WS4, WS5, WYO, OD1, OP3, BRC1, TM6, TM7, WPS-2, and FCPU426. OTU richness was notably higher in the surface layer (882 OTUs) than in the anoxic subsurface peat (483 OTUs), with only 96 OTUs common to both data sets. Most members of poorly studied phyla, such as the Acidobacteria, Verrucomicrobia, Planctomycetes and the candidate division TM6, showed a clear preference for growth in either oxic or anoxic conditions. Apparently, the bacterial communities in surface and subsurface layers of northern peatlands are highly diverse and taxonomically distinct, reflecting the different abiotic conditions in microhabitats within the peat profile.

Dependence of bacterial magnetosome morphology on chemical conditions in deep-sea sediments

Here we investigated the diversity of bacterial communities from deep-sea surface sediments under influence of asphalt seeps at the Sao Paulo Plateau using next-generation sequencing method. Sampling was performed at North São Paulo Plateau using the human occupied vehicle Shinkai 6500 and her support vessel Yokosuka. The microbial diversity was studied at two surficial sediment layers (0-1 and 1-4cm) of five samples collected in cores in water depths ranging from 2456 to 2728m. Bacterial communities were studied through sequencing of 16S rRNA gene on the Ion Torrent platform and clustered in operational taxonomic units. We observed high diversity of bacterial sediment communities as previously described by other studies. When we considered community composition, the most abundant classes were Alphaproteobacteria (27.7%), Acidimicrobiia (20%), Gammaproteobacteria (11.3%) and Deltaproteobacteria (6.6%). Most abundant OTUs at family level were from two uncultured bacteria from Actinomarinales (5.95%) and Kiloniellaceae (3.17%). The unexpected high abundance of Alphaproteobacteria and Acidimicrobiia in our deep-sea microbial communities may be related to the presence of asphalt seep at North São Paulo Plateau, since these bacterial classes contain bacteria that possess the capability of metabolizing hydrocarbon compounds.

Sediment samples collected off the coast of San Diego were analyzed for actinomycete diversity using culture-independent techniques. Eight new operational taxonomic units (OTUs) in the Streptomycetaceae were identified as well as new diversity within previously cultured marine OTUs. Sequences belonging to the marine actinomycete genus Salinispora were also detected, despite the fact that this genus has only been reported from more tropical environments. Independent analyses of marine sediments from the Canary Basin (3814m) and the South Pacific Gyre (5126 and 5699m) also revealed Salinispora sequences providing further support for the occurrence of this genus in deep-sea sediments. Efforts to culture Salinispora spp. from these samples have yet to be successful. This is the first report of Salinispora spp. from marine sediments >1100m and suggests that the distribution of this genus is broader than previously believed.

BackgroundThe deep sea floor is considered one of the most diverse ecosystems on Earth. Recent environmental DNA surveys based on clone libraries of rRNA genes confirm this observation and reveal a high diversity of eukaryotes present in deep-sea sediment samples. However, environmental clone-library surveys yield only a modest number of sequences with which to evaluate the diversity of abyssal eukaryotes.Methodology/Principal FindingsHere, we examined the richness of eukaryotic DNA in deep Arctic and Southern Ocean samples using massively parallel sequencing of the 18S ribosomal RNA (rRNA) V9 hypervariable region. In very small volumes of sediments, ranging from 0.35 to 0.7 g, we recovered up to 7,499 unique sequences per sample. By clustering sequences having up to 3 differences, we observed from 942 to 1756 Operational Taxonomic Units (OTUs) per sample. Taxonomic analyses of these OTUs showed that DNA of all major groups of eukaryotes is represented at the deep-sea floor. The dinoflagellates, cercozoans, ciliates, and euglenozoans predominate, contributing to 17%, 16%, 10%, and 8% of all assigned OTUs, respectively. Interestingly, many sequences represent photosynthetic taxa or are similar to those reported from the environmental surveys of surface waters. Moreover, each sample contained from 31 to 71 different metazoan OTUs despite the small sample volume collected. This indicates that a significant faction of the eukaryotic DNA sequences likely do not belong to living organisms, but represent either free, extracellular DNA or remains and resting stages of planktonic species.Conclusions/SignificanceIn view of our study, the deep-sea floor appears as a global DNA repository, which preserves genetic information about organisms living in the sediment, as well as in the water column above it. This information can be used for future monitoring of past and present environmental changes.

The deep ocean floor covers more than 60% of the Earth’s surface, and hosts diverse bacterial communities with important functions in carbon and nutrient cycles. The identification of key bacterial members remains a challenge and their patterns of distribution in seafloor sediment yet remain poorly described. Previous studies were either regionally restricted or included few deep-sea sediments, and did not specifically test biogeographic patterns across the vast oligotrophic bathyal and abyssal seafloor. Here we define the composition of this deep seafloor microbiome by describing those bacterial operational taxonomic units (OTU) that are specifically associated with deep-sea surface sediments at water depths ranging from 1000–5300 m. We show that the microbiome of the surface seafloor is distinct from the subsurface seafloor. The cosmopolitan bacterial OTU were affiliated with the clades JTB255 (class Gammaproteobacteria, order Xanthomonadales) and OM1 (Actinobacteria, order Acidimicrobiales), comprising 21% and 7% of their respective clades, and about 1% of all sequences in the study. Overall, few sequence-abundant bacterial types were globally dispersed and displayed positive range-abundance relationships. Most bacterial populations were rare and exhibited a high degree of endemism, explaining the substantial differences in community composition observed over large spatial scales. Despite the relative physicochemical uniformity of deep-sea sediments, we identified indicators of productivity regimes, especially sediment organic matter content, as factors significantly associated with changes in bacterial community structure across the globe.

Fungal assemblages in live, newly shed and partly decomposed leaves of Camellia japonica were investigated with a clone library analysis to assess the fungal diversity and succession in a subtropical forest in southern Japan. Partly decomposed leaves were divided into bleached and adjacent nonbleached portions to estimate the fungi functionally associated with lignin decomposition in the bleached portions, with an emphasis on Coccomyces sinensis (Rhytismataceae, Ascomycota). From 144 cloned 28S ribosomal DNA (rDNA) sequences, 48 operational taxonomic units (OTUs) were defined based on a sequence similarity threshold of 98%. Forty-one (85%) of the 48 OTUs belonged to the Ascomycota and seven OTUs (15%) to the Basidiomycota. Twenty-six OTUs (54%) were detected only once (singletons). The number of OTUs and the diversity indices of the fungal assemblages in the different leaves were in this order: live leaves > newly shed leaves > bleached portions > nonbleached portions of partly decomposed leaves. The fungal assemblages were similar in newly shed leaves and the bleached portions of partly decomposed leaves. Ligninolytic fungi of the genera Coccomyces, Lophodermium and Xylaria were frequently detected in the bleached portions. OTU3, identified as Coccomyces sinensis, was detected in live and newly shed leaves and the bleached portions of partly decomposed leaves, suggesting that this fungus latently infects live leaves, persists after leaf fall and takes part in lignin decomposition.

ABSTRACTBenefiting from the rapid developments and wide applications of high-throughput sequencing, great advancements have been made in investigating microbiota, which are highly diverse and play key roles in both element cycling and the energy flow of ecosystems. There have been inherent limitations of amplicon sequencing that could introduce uncertainty and raise concerns about the accuracy and reproducibility of this technology. However, studies focusing on the reproducibility of amplicon sequencing are limited, especially in characterizing microbial communities in deep-sea sediments. To evaluate reproducibility, 118 deep-sea sediment samples were used for 16S rRNA gene sequencing in technical replicates (repeated measurements of the same sample) that demonstrate the variability of amplicon sequencing. The average occurrence-based overlaps were 35.98% and 27.02% between two and among three technical replicates, respectively, whereas their abundance-based overlaps reached 84.88% and 83.16%, respectively. Although variations of alpha and beta diversity indices were found between/among technical replicates, alpha diversity indices were similar across samples, and the average beta diversity indices were much smaller for technical replicates than among samples. Moreover, clustering methods (i.e., operational taxonomic units [OTUs] and amplicon sequence variants [ASVs]) were shown to have little impact on the alpha and beta diversity patterns of microbial communities. Taken together, although there are variations between/among technical replicates, amplicon sequencing is still a powerful tool with which to reveal diversity patterns of microbiota in deep-sea sediments.IMPORTANCE The reproducibility of amplicon sequencing is vital for whether the diversities of microbial communities could be accurately estimated. Thus, reproducibility influences the drawing of sound ecological conclusions. Nevertheless, few studies have focused on the reproducibility of microbial communities that are characterized by amplicon sequencing, and studies focusing on microbiota in deep-sea sediments have been especially lacking. In this study, we evaluated the reproducibility of amplicon sequencing targeting microbiota in deep-sea sediments of cold seep. Our results revealed that there were variations between/among technical replicates and that amplicon sequencing was still a powerful tool with which to characterize the diversities of microbial communities in deep-sea sediments. This study provides valuable guidelines for the reproducibility evaluation of future work in experimental design and interpretation.

Species prevalence across the landscape is related to their local abundance, which is a result of deterministic and stochastic processes that select organisms capable of recolonizing sites where they were once extinct, a process known as the rescue effect. The occupancy-frequency distribution (OFD) describes these patterns and has been extensively used to understand organism's distribution but has been poorly tested on microorganisms. In order to test OFD on freshwater bacteria, we collected data from 60 shallow lakes distributed across a wide area in southeastern Brazil, to determine the bacterial operational taxonomic units (OTUs) that were present in all sites (core) and at only one site (satellite). Then, we analyzed the spatial abundance distributions of individual OTUs to understand the influence of local abundances on regional occupancy patterns. Finally, we tested the environmental factors that influenced occupancy and abundance. We found a significant bimodal OFD for freshwater bacteria using both OTUs (97% clustering) and amplicon sequence variants (ASVs, unique sequences), with 13 core OTUs and 1169 satellite OTUs, but only three core ASVs. Core organisms had a bimodal or gamma abundance distribution. The main driver of the core community was pH, while nutrients were key when the core community was excluded and the rest of the community (mild and satellite taxa) was considered. This study demonstrates the close relationship between local environmental conditions and the abundance and dispersion of microorganisms, which shapes their distribution across the landscape.

Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing