Identification and Distribution of Novel Cressdnaviruses and Circular molecules in Four Penguin Species in South Georgia and the Antarctic Peninsula.

There is growing interest in uncovering the viral diversity present in wild animal species. The remote Antarctic region is home to a wealth of uncovered microbial diversity, some of which is associated with its megafauna, including penguin species, the dominant avian biota. Penguins interface with a number of other biota in their roles as marine mesopredators and several species overlap in their ranges and habitats. To characterize the circular single-stranded viruses related to those in the phylum Cressdnaviricota from these environmental sentinel species, cloacal swabs (n = 95) were obtained from King Penguins in South Georgia, and congeneric Adélie Penguins, Chinstrap Penguins, and Gentoo Penguins across the South Shetland Islands and Antarctic Peninsula. Using a combination of high-throughput sequencing, abutting primers-based PCR recovery of circular genomic elements, cloning, and Sanger sequencing, we detected 97 novel sequences comprising 40 ssDNA viral genomes and 57 viral-like circular molecules from 45 individual penguins. We present their detection patterns, with Chinstrap Penguins harboring the highest number of new sequences. The novel Antarctic viruses identified appear to be host-specific, while one circular molecule was shared between sympatric Chinstrap and Gentoo Penguins. We also report viral genotype sharing between three adult-chick pairs, one in each Pygoscelid species. Sequence similarity network approaches coupled with Maximum likelihood phylogenies of the clusters indicate the 40 novel viral genomes do not fall within any known viral families and likely fall within the recently established phylum Cressdnaviricota based on their replication-associated protein sequences. Similarly, 83 capsid protein sequences encoded by the viruses or viral-like circular molecules identified in this study do not cluster with any of those encoded by classified viral groups. Further research is warranted to expand knowledge of the Antarctic virome and would help elucidate the importance of viral-like molecules in vertebrate host evolution.

Identification of Circovirus Genome in a Chinstrap Penguin (Pygoscelis antarcticus) and Adélie Penguin (Pygoscelis adeliae) on the Antarctic Peninsula.

Circoviruses infect a variety of animal species and have small (~1.8-2.2 kb) circular single-stranded DNA genomes. Recently a penguin circovirus (PenCV) was identified associated with an Adélie Penguin (Pygoscelis adeliae) with feather disorder and in the cloacal swabs of three asymptomatic Adélie Penguins at Cape Crozier, Antarctica. A total of 75 cloacal swab samples obtained from adults and chicks of three species of penguin (genus: Pygoscelis) from seven Antarctic breeding colonies (South Shetland Islands and Western Antarctic Peninsula) in the 2015-2016 breeding season were screened for PenCV. We identified new variants of PenCV in one Adélie Penguin and one Chinstrap Penguin (Pygoscelis antarcticus) from Port Charcot, Booth Island, Western Antarctic Peninsula, a site home to all three species of Pygoscelid penguins. These two PenCV genomes (length of 1986 nucleotides) share > 99% genome-wide nucleotide identity with each other and share ~87% genome-wide nucleotide identity with the PenCV sequences described from Adélie Penguins at Cape Crozier ~4400 km away in East Antarctica. We did not find any evidence of recombination among PenCV sequences. This is the first report of PenCV in Chinstrap Penguins and the first detection outside of Ross Island, East Antarctica. Given the limited knowledge on Antarctic animal viral diversity, future samples from Antarctic wildlife should be screened for these and other viruses to determine the prevalence and potential impact of viral infections.

Environmental DNA reveals seasonal shifts and potential interactions in a marine community.

Environmental DNA (eDNA) analysis allows the simultaneous examination of organisms across multiple trophic levels and domains of life, providing critical information about the complex biotic interactions related to ecosystem change. Here we used multilocus amplicon sequencing of eDNA to survey biodiversity from an eighteen-month (2015-2016) time-series of seawater samples from Monterey Bay, California. The resulting dataset encompasses 663 taxonomic groups (at Family or higher taxonomic rank) ranging from microorganisms to mammals. We inferred changes in the composition of communities, revealing putative interactions among taxa and identifying correlations between these communities and environmental properties over time. Community network analysis provided evidence of expected predator-prey relationships, trophic linkages, and seasonal shifts across all domains of life. We conclude that eDNA-based analyses can provide detailed information about marine ecosystem dynamics and identify sensitive biological indicators that can suggest ecosystem changes and inform conservation strategies.

Assessing eukaryotic biodiversity in the Florida Keys National Marine Sanctuary through environmental DNA metabarcoding.

Environmental DNA (eDNA) is the DNA suspended in the environment (e.g., water column), which includes cells, gametes, and other material derived from but not limited to shedding of tissue, scales, mucus, and fecal matter. Amplifying and sequencing marker genes (i.e., metabarcoding) from eDNA can reveal the wide range of taxa present in an ecosystem through analysis of a single water sample. Metabarcoding of eDNA provides higher resolution data than visual surveys, aiding in assessments of ecosystem health. This study conducted eDNA metabarcoding of two molecular markers (cytochrome c oxidase I (COI) and 18S ribosomal RNA (rRNA) genes) to survey eukaryotic diversity across multiple trophic levels in surface water samples collected at three sites along the coral reef tract within the Florida Keys National Marine Sanctuary (FKNMS) during four research cruises in 2015. The 18S rRNA gene sequences recovered 785 genera while the COI gene sequences recovered 115 genera, with only 33 genera shared between the two datasets, emphasizing the complementarity of these marker genes. Community composition for both genetic markers clustered by month of sample collection, suggesting that temporal variation has a larger effect on biodiversity than spatial variability in the FKNMS surface waters. Sequences from both marker genes were dominated by copepods, but each marker recovered distinct phytoplankton groups, with 18S rRNA gene sequences dominated by dinoflagellates and COI sequences dominated by coccolithophores. Although eDNA samples were collected from surface waters, many benthic species such as sponges, crustaceans, and corals were identified. These results show the utility of eDNA metabarcoding for cataloging biodiversity to establish an ecosystem baseline against which future samples can be compared in order to monitor community changes.

Evaluation of marine zooplankton community structure through environmental DNA metabarcoding.

Zooplankton dominate the abundance and biomass of multicellular animals in pelagic marine environments; however, traditional methods to characterize zooplankton communities are invasive and laborious. This study compares zooplankton taxonomic composition revealed through metabarcoding of the cytochrome oxidase I (COI) and 18S rRNA genes to traditional morphological identification by microscopy. Triplicates of three different sample types were collected from three coral reef sites in the Florida Keys National Marine Sanctuary: (1) 1 L surface seawater samples prefiltered through 3 µm filters and subsequently collected on 0.22 µm filters for eDNA (PF-eDNA); (2) 1 L surface seawater samples filtered on 0.22 µm pore-size filters (environmental DNA; eDNA), and (3) zooplankton tissue samples from 64 µm, 200 µm, and 500 µm mesh size net tows. The zooplankton tissue samples were split, with half identified morphologically and tissue DNA (T-DNA) extracted from the other half. The COI and 18S rRNA gene metabarcoding of PF-eDNA, eDNA, and T-DNA samples was performed using Illumina MiSeq. Of the families detected with COI and 18S rRNA gene metabarcoding, 40% and 32%, respectively, were also identified through morphological assessments. Significant differences in taxonomic composition were observed between PF-DNA, eDNA, and T-DNA with both genetic markers. PF-eDNA resulted in detection of fewer taxa than the other two sample types; thus, prefiltering is not recommended. All dominant copepod taxa (> 5% of total abundance) were detected with eDNA, T-DNA, and morphological assessments, demonstrating that eDNA metabarcoding is a promising technique for future biodiversity assessments of pelagic zooplankton in marine systems.

The gut virome of the protochordate model organism, Ciona intestinalis subtype A.

The identification of host-specific bacterial and viral communities associated with diverse animals has led to the concept of the metaorganism, which defines the animal and all of its associated microbes as a single unit. Here we sequence the viruses found in the gut (i.e., the gut virome) of the marine invertebrate model system, Ciona intestinalis subtype A, in samples collected one year apart. We present evidence for a host-associated virome that is distinct from the surrounding seawater and contains some temporally-stable members. Comparison of gut tissues before and after starvation in virus-free water enabled the differentiation between the Ciona-specific virome and transient viral communities associated with dietary sources. The Ciona gut viromes were dominated by double-stranded DNA tailed phages (Order Caudovirales) and sequence assembly yielded a number of complete circular phage genomes, most of which were highly divergent from known genomes. Unique viral communities were found in distinct gut niches (stomach, midgut and hindgut), paralleling the compartmentalization of bacterial communities. Additionally, integrase and excisionase genes, including many that are similar to prophage sequences within the genomes of bacterial genera belonging to the Ciona core microbiome, were prevalent in the viromes, indicating the active induction of prophages within the gut ecosystem. Knowledge of the gut virome of this model organism lays the foundation for studying the interactions between viruses, bacteria, and host immunity.

Cutting through the smoke: the diversity of microorganisms in deep-sea hydrothermal plumes.

There are still notable gaps regarding the detailed distribution of microorganisms between and within insular habitats such as deep-sea hydrothermal vents. This study investigates the community composition of black smoker vent microorganisms in the Southern Hemisphere, and changes thereof along a spatial and chemical gradient ranging from the vent plume to surrounding waters. We sampled two hydrothermal vent fields, one at the South West Indian Ridge (SWIR), the other at the East Scotia Ridge (ESR). Samples were collected across vent fields at varying vertical distances from the origin of the plumes. The microbial data were sequenced on an Illumina MiSeq platform for the 16SrRNA gene. A substantial amount of vent-specific putative chemosynthetic microorganisms were found, particularly in samples from focused hydrothermal venting. Common vent-specific organisms from both vent fields were the genera Arcobacter, Caminibacter and Sulfurimonas from the Epsilonproteobacteria and the SUP05 group from the Gammaproteobacteria. There were no major differences in microbial composition between SWIR and ESR for focused plume samples. However, within the ESR the diffuse flow and focused samples differed significantly in microbial community composition and relative abundance. For Epsilonproteobacteria, we found evidence of niche-specificity to hydrothermal vent environments. This taxon decreased in abundance by three orders of magnitude from the vent orifice to background water. Epsilonproteobacteria distribution followed a distance-decay relationship as vent-effluents mixed with the surrounding seawater. This study demonstrates strong habitat affinity of vent microorganisms on a metre scale with distinct environmental selection.

Microbe biogeography tracks water masses in a dynamic oceanic frontal system.

Dispersal limitation, not just environmental selection, plays an important role in microbial biogeography. The distance-decay relationship is thought to be weak in habitats where dispersal is high, such as in the pelagic environment, where ocean currents facilitate microbial dispersal. Most studies of microbial community composition to date have observed little geographical heterogeneity on a regional scale (100 km). We present a study of microbial communities across a dynamic frontal zone in the southwest Indian Ocean and investigate the spatial structure of the microbes with respect to the different water masses separated by these fronts. We collected 153 samples of free-living microorganisms from five seamounts located along a gradient from subtropical to subantarctic waters and across three depth layers: (i) the sub-surface chlorophyll maximum (approx. 40 m), (ii) the bottom of the euphotic zone (approx. 200 m), and (iii) the benthic boundary layer (300-2000 m). Diversity and abundance of microbial operational taxonomic units (OTUs) were assessed by amplification and sequencing of the 16S rRNA gene on an Illumina MiSeq platform. Multivariate analyses showed that microbial communities were structured more strongly by depth than by latitude, with similar phyla occurring within each depth stratum across seamounts. The deep layer was homogeneous across the entire survey area, corresponding to the spread of Antarctic intermediate water. However, within both the sub-surface layer and the intermediate depth stratum there was evidence for OTU turnover across fronts. The microbiome of these layers appears to be divided into three distinct biological regimes corresponding to the subantarctic surface water, the convergence zone and subtropical. We show that microbial biogeography across depth and latitudinal gradients is linked to the water masses the microbes persist in, resulting in regional patterns of microbial biogeography that correspond to the regional scale physical oceanography.