Unveiling Crucivirus Diversity by Mining Metagenomic Data.

The discovery of cruciviruses revealed the most explicit example of a common protein homologue between DNA and RNA viruses to date. Cruciviruses are a novel group of circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) viruses that encode capsid proteins that are most closely related to those encoded by RNA viruses in the family Tombusviridae The apparent chimeric nature of the two core proteins encoded by crucivirus genomes suggests horizontal gene transfer of capsid genes between DNA and RNA viruses. Here, we identified and characterized 451 new crucivirus genomes and 10 capsid-encoding circular genetic elements through de novo assembly and mining of metagenomic data. These genomes are highly diverse, as demonstrated by sequence comparisons and phylogenetic analysis of subsets of the protein sequences they encode. Most of the variation is reflected in the replication-associated protein (Rep) sequences, and much of the sequence diversity appears to be due to recombination. Our results suggest that recombination tends to occur more frequently among groups of cruciviruses with relatively similar capsid proteins and that the exchange of Rep protein domains between cruciviruses is rarer than intergenic recombination. Additionally, we suggest members of the stramenopiles/alveolates/Rhizaria supergroup as possible crucivirus hosts. Altogether, we provide a comprehensive and descriptive characterization of cruciviruses.IMPORTANCE Viruses are the most abundant biological entities on Earth. In addition to their impact on animal and plant health, viruses have important roles in ecosystem dynamics as well as in the evolution of the biosphere. Circular Rep-encoding single-stranded (CRESS) DNA viruses are ubiquitous in nature, many are agriculturally important, and they appear to have multiple origins from prokaryotic plasmids. A subset of CRESS-DNA viruses, the cruciviruses, have homologues of capsid proteins encoded by RNA viruses. The genetic structure of cruciviruses attests to the transfer of capsid genes between disparate groups of viruses. However, the evolutionary history of cruciviruses is still unclear. By collecting and analyzing cruciviral sequence data, we provide a deeper insight into the evolutionary intricacies of cruciviruses. Our results reveal an unexpected diversity of this virus group, with frequent recombination as an important determinant of variability.

Flying south: Foraging locations of the Hutton's shearwater (Puffinus huttoni) revealed by Time-Depth Recorders and GPS tracking.

The Hutton's shearwater Puffinus huttoni is an endangered seabird endemic to Kaikoura, New Zealand, but the spatial and temporal aspects of its at-sea foraging behavior are not well known.To identify foraging areas and estimate trip durations, we deployed Global Positioning Systems (GPS) devices and Time-Depth Recorders (TDR) on 26 adult Hutton's shearwaters during the chick-rearing period in 2017 and 2018.We found Hutton's shearwaters traveled much further from their breeding grounds at Kaikoura than previously considered, with most individuals foraging in coastal and oceanic areas 125-365 km south and near Banks Peninsula. Trip durations varied from 1 to 15 days (mean = 5 days), and total track lengths varied from 264 to 2,157 km (mean = 1092.9 km).Although some diving occurred in near-shore waters near the breeding colony, most foraging was concentrated in four regions south of Kaikoura. Dive durations averaged 23.2 s (range 8.1 to 71.3 s) and dive depths averaged 7.1 m (range 1.5 to 30 m). Foraging locations had higher chlorophyll a levels and shallower water depths than nonforaging locations. Birds did not feed at night, but tended to raft in areas with deeper water than foraging locations.Mapping the spatial and temporal distribution of Hutton's shearwaters at sea will be fundamental to their conservation, as it can reveal potential areas of overlap with fisheries and other industrial users of the marine environment.

Chimerism and population dieback alter genetic inference related to invasion pathways and connectivity of biofouling populations on artificial substrata.

Disentangling pathways by which nonindigenous species expand and spread regionally remains challenging. Molecular ecology tools are often employed to determine the origins and spread of introduced species, but the complexities of some organisms may be reducing the efficacy of these tools. Some colonial species exhibit complexities by way of chimerism and winter colony regression, which may alter the genetic diversity of populations and mask the connectivity occurring among them. This study uses nuclear microsatellite data and simple GIS-based modeling to investigate the influence of chimerism and winter regression on the genetic diversity and patterns of genetic population connectivity among colonies of Didemnum vexillum on artificial substrates. Colonies sampled in summer were shown to form a metapopulation, with high levels of admixture, extreme outcrossing, and some substructure. These patterns were consistent within the subsampled winter colonies and with the inclusion of chimeric data. However, allelic richness and diversity were significantly different between winter and summer samples, altering interpretations relating to population connectivity and pelagic larval duration. This study demonstrates the importance of including seasonal sampling and imperative life history traits in genetic studies for clear interpretations and the successful management of introduced species.

Effects of waterborne cadmium on energy metabolism in the tropical sea cucumber, Stichopus horrens, and a comparison of tissue-specific cadmium accumulation with the temperate sea cucumber Australostichopus mollis.

The sea cucumber Stichopus horrens is an important component of near-shore ecosystems, and in the Kingdom of Tonga it also comprises an important commercial and subsistence fishery. To assess the sensitivity of this species to the toxic trace metal cadmium (Cd), adult S. horrens were exposed for 96h to an environmental (15µgL-1) or effect (765µgL-1) concentration of waterborne Cd. The respiratory tree and intestine accumulated higher concentrations of Cd than the muscle and body wall, but there were no effects of Cd on tissue ions (sodium, potassium, magnesium, calcium). For comparison, Cd accumulation was also examined in the Australasian sea cucumber Australostichopus mollis. This species displayed a similar pattern of tissue-specific accumulation to S. horrens, but exhibited lower tissue Cd burdens, likely a consequence of lower experimental temperature. Effects on gonad ion content were also seen in this species. At the highest Cd exposure concentration, S. horrens showed impaired ammonia excretion rates and an increased molar oxygen:nitrogen ratio (O:N), indicative of a decreased reliance on protein metabolism. Overall, this study suggests that S. horrens is relatively tolerant of Cd exposure, but raises concerns regarding the subsistence fishery practice of consuming the viscera of this species.

Evolutionary history of ssDNA bacilladnaviruses features horizontal acquisition of the capsid gene from ssRNA nodaviruses.

Bacilladnaviruses have single-stranded (ss) DNA genomes and infect diatoms, a major group of unicellular algae widespread in aquatic habitats. Despite their ecological importance, the provenance and relationships of bacilladnaviruses to other eukaryotic viruses remain unclear. Accordingly, they are currently classified into the 'floating' genus Bacilladnavirus. Here we present three new bacilladnavirus genomes recovered from a mollusc Amphibola crenata and benthic sediments from the Avon-Heathcote estuary in New Zealand. Our analysis shows that the rolling-circle replication-initiation proteins of bacilladnaviruses display unique conserved motifs and in phylogenetic trees form a monophyletic clade separated from other groups of ssDNA viruses. Unexpectedly, distant homology detection combined with structural modeling indicates that bacilladnavirus capsid proteins are homologous to those of ssRNA viruses from the Nodaviridae family. Considering the sequence diversity within the expanding Bacilladnavirus genus, we argue that classification of these viruses has to be revised and the current genus upgraded to the family level.

Factors influencing the en route survivorship and post-voyage growth of a common ship biofouling organism, Bugula neritina.

The likelihood that viable non-indigenous biofouling species will survive a voyage on a vessel is influenced by a range of factors, including the speed, duration, and route of the voyage and the amount of time the vessel spends in port. In this study, a land-based dynamic flow device was used to test the effect of recruit age, vessel speed and voyage duration on the survivorship and growth of the bryozoan Bugula neritina. In the experiment, one-week-old recruits had a higher likelihood (100%) of surviving voyages than older (one-month-old, 90%) or younger (one-day-old, 79%) recruits, but survival was not influenced by vessel speed (6 and 18 knots) or voyage duration (two and eight days). The results suggest that the non-indigenous species B. neritina can be effectively transferred at a range of ages but one-week-old recruits are more likely to survive the translocation process and survive in the recipient environment.

Metabarcoding improves detection of eukaryotes from early biofouling communities: implications for pest monitoring and pathway management.

In this experimental study the patterns in early marine biofouling communities and possible implications for surveillance and environmental management were explored using metabarcoding, viz. 18S ribosomal RNA gene barcoding in combination with high-throughput sequencing. The community structure of eukaryotic assemblages and the patterns of initial succession were assessed from settlement plates deployed in a busy port for one, five and 15 days. The metabarcoding results were verified with traditional morphological identification of taxa from selected experimental plates. Metabarcoding analysis identified > 400 taxa at a comparatively low taxonomic level and morphological analysis resulted in the detection of 25 taxa at varying levels of resolution. Despite the differences in resolution, data from both methods were consistent at high taxonomic levels and similar patterns in community shifts were observed. A high percentage of sequences belonging to genera known to contain non-indigenous species (NIS) were detected after exposure for only one day.

Phylogenetic affinities of the Fregetta storm-petrels are not black and white.

The Fregetta storm-petrels generally are regarded to comprise two species: black-bellied storm-petrels F. tropica (monotypic) breed at Antarctic and sub-Antarctic islands (46-63°S), and white-bellied storm-petrels F. grallaria breed at south temperate islands (28-37°S), with four recognized subspecies. Confusion surrounds the status of birds at Gough Island (40°S), central South Atlantic, which have been attributed usually to a white-bellied form of black-bellied storm-petrel F. t. melanoleuca. We use cytochrome b and nuclear ß-fibrinogen gene sequences to show that F. t. melanoleuca are present during the breeding season at Gough and islands in the nearby Tristan da Cunha archipelago (37°S), exhibiting limited divergence from F. t. tropica. We also show that there is greater diversity among F. grallaria populations, with eastern South Pacific F. g. segethi and F. g. titan differing by c. 0.011, and both differing from western South Pacific nominate F. g. grallaria by c. 0.059. The Tristan archipelago supports a population of F. grallaria closely allied to the nominate form, as well as a distinct form identified as F. g. leucogaster. Further research is needed to assess how F. grallaria and F. tropica segregate in sympatry at Tristan and Gough, and why this is the only location where both species have white-bellies.

Hybrid-Lambda: simulation of multiple merger and Kingman gene genealogies in species networks and species trees.

BACKGROUND: There has been increasing interest in coalescent models which admit multiple mergers of ancestral lineages; and to model hybridization and coalescence simultaneously. RESULTS: Hybrid-Lambda is a software package that simulates gene genealogies under multiple merger and Kingman's coalescent processes within species networks or species trees. Hybrid-Lambda allows different coalescent processes to be specified for different populations, and allows for time to be converted between generations and coalescent units, by specifying a population size for each population. In addition, Hybrid-Lambda can generate simulated datasets, assuming the infinitely many sites mutation model, and compute the F ST statistic. As an illustration, we apply Hybrid-Lambda to infer the time of subdivision of certain marine invertebrates under different coalescent processes. CONCLUSIONS: Hybrid-Lambda makes it possible to investigate biogeographic concordance among high fecundity species exhibiting skewed offspring distribution.

Diverse small circular DNA viruses circulating amongst estuarine molluscs.

Our understanding of the diversity and abundance of circular replication associated protein (Rep) - encoding single stranded (CRESS) DNA viruses has increased considerably over the last few years due to a combination of modern sequencing technologies and new molecular tools. Studies have used these to identify and recover CRESS DNA viruses from a range of different marine organisms, including copepods, shrimp and molluscs. In our study we identified 79 novel CRESS DNA viruses from three mollusc species (Austrovenus stutchburyi, Paphies subtriangulata and Amphibola crenata) and benthic sediments from the Avon-Heathcote estuary in Christchurch, New Zealand. The genomes recovered have varying genome architectures, with all encoding at least two major ORFs that have either unidirectional or bidirectional organisation. Analysis of the Reps of the viral genomes showed they are all highly diverse, with only one Rep sequence sharing 65% amino acid identity with the Rep of gastropod-associated circular DNA virus (GaCSV). Our study adds significantly to the wealth of CRESS DNA viruses recovered from freshwater and marine environments and extends our knowledge of the distribution of these viruses.

Identification of Starling Circovirus in an Estuarine Mollusc (Amphibola crenata) in New Zealand Using Metagenomic Approaches.

Two complete genomes of starling circovirus (StCV) were recovered from Amphibola crenata, an estuarine New Zealand mollusc. This is the first report of StCV outside Europe. The viral genomes were recovered from rolling circle-amplified enriched circular DNA followed by back-to-back primers and specific primer PCR amplification.

Using temporal sampling to improve attribution of source populations for invasive species.

Numerous studies have applied genetic tools to the identification of source populations and transport pathways for invasive species. However, there are many gaps in the knowledge obtained from such studies because comprehensive and meaningful spatial sampling to meet these goals is difficult to achieve. Sampling populations as they arrive at the border should fill the gaps in source population identification, but such an advance has not yet been achieved with genetic data. Here we use previously acquired genetic data to assign new incursions as they invade populations within New Zealand ports and marinas. We also investigated allelelic frequency change in these recently established populations over a two-year period, and assessed the effect of temporal genetic sampling on our ability to assign new incursions to their population of source. We observed shifts in the allele frequencies among populations, as well as the complete loss of some alleles and the addition of alleles novel to New Zealand, within these recently established populations. There was no significant level of genetic differentiation observed in our samples between years, and the use of these temporal data did alter the assignment probability of new incursions. Our study further suggests that new incursions can add genetic variation to the population in a single introduction event as the founders themselves are often more genetically diverse than theory initially predicted.

Novel ssDNA virus recovered from estuarine Mollusc (Amphibola crenata) whose replication associated protein (Rep) shares similarities with Rep-like sequences of bacterial origin.

Over the past couple of years highly diverse novel ssDNA viruses have been discovered. Here, we present the first ssDNA virus, Gastropod-associated circular ssDNA virus (GaCSV), recovered from a mollusc Amphibola crenata Martyn 1784, which is a deposit feeder that grazes micro-organisms and organic detritus on the surface of tidal mudflats. The GaCSV (2351 nt) genome contains two large bidirectionally transcribed ORFs. The smaller ORF (874 nt) has similarities to viral replication-associated protein (Rep) sequences of some bacteria and circoviruses, whereas the larger ORF (955 nt) does not relate to any sequences in public databases and we presume it potentially encodes the capsid protein. Phylogenetic analysis shows that the GaCSV Rep clusters with Rep-like sequences of bacterial origin, highlighting the role of ssDNA viruses in horizontal gene transfer. The occurrence of previously unknown viruses in organisms associated with human pollution is a relatively unexplored field.

When rediscovery is not enough: taxonomic uncertainty hinders conservation of a critically endangered bird.

In 2003, birds similar to the extinct New Zealand storm-petrel Oceanites maorianus were observed in Hauraki Gulf NZ, raising the possibility of rediscovery after 150 years. O. maorianus has and continues to be surrounded by taxonomic uncertainty, being variously described as a distinct genus, a distinct species, or merely a plumage variant. This uncertainty has hindered conservation planning and funding for the species. Here we examine the taxonomic identity of the rediscovered birds and museum specimens using phylogenetic analysis of mitochondrial (1143 bp cytochrome b) and nuclear (890 bp ß-fibrinogen) genes. Using cytochrome b sequence amplified from the 150+ year old specimens, we found that the extant and museum O. maorianus were the same taxon (0.01 genetic distance), with both differing from all other storm-petrel taxa. Using both genes, we examined the phylogenetic affinities of O. maorianus to the Oceanitinae and Hydrobatinae storm-petrels finding that O. maorianus was more closely aligned to Fregetta (0.08-0.09) than Oceanites (0.11-0.12), thereby confirming its status as a distinct taxon, not a plumage variant of O. oceanicus. Our analysis verifies that the previously presumed extinct New Zealand storm-petrel has been rediscovered and can now be assigned a conservation priority commensurate with its critically endangered status.

Global phylogeography of the widely introduced North West Pacific ascidian Styela clava.

The solitary ascidian Styela clava Herdman, 1882 is considered to be native to Japan, Korea, northern China and the Russian Federation in the NW Pacific, but it has spread globally over the last 80 years and is now established as an introduced species on the east and west coasts of North America, Europe, Australia and New Zealand. In eastern Canada it reaches sufficient density to be a serious pest to aquaculture concerns. We sequenced a fragment of the cytochrome oxidase subunit I mitochondrial gene (COI) from a total of 554 individuals to examine the genetic relationships of 20 S. clava populations sampled throughout the introduced and native ranges, in order to investigate invasive population characteristics. The data presented here show a moderate level of genetic diversity throughout the northern hemisphere. The southern hemisphere (particularly New Zealand) displays a greater amount of haplotype and nucleotide diversity in comparison. This species, like many other invasive species, shows a range of genetic diversities among introduced populations independent of the age of incursion. The successful establishment of this species appears to be associated with multiple incursions in many locations, while other locations appear to have experienced rapid expansion from a potentially small population with reduced genetic diversity. These contrasting patterns create difficulties when attempting to manage and mitigate a species that continues to spread among ports and marinas around the world.

Comparative phylogeography of coastal limpets across a marine disjunction in New Zealand.

Cook Strait, which separates the North and South Island of New Zealand, has been a transient, but re-occurring feature of the New Zealand land mass throughout the Pleistocene, maintaining its current width and depth for the past 5000 years. Historic land fragmentation coupled with the complex hydrography of the Greater Cook Strait region has created both biogeographic and phylogeographic disjunctions between the North and South Island in several marine species. Here we use mitochondrial cytochrome b DNA sequences of three endemic intertidal limpets, Cellana ornata, Cellana radians and Cellana flava to assess intraspecific phylogeographic patterns across Cook Strait and to look for interspecific concordance of ecological and evolutionary processes among closely related taxa. We sequenced 328-359 bp in 85-321 individuals from 8-31 populations spanning the biogeographic range of the three species. Intraspecific phylogeographic analyses show moderate to strong genetic discontinuity among North and South Island populations due to allopatric fragmentation. This pattern was broadly concordant across the three species and the observed divergence among this group of intertidal limpets (0.3-2.0%) is similar to that of previously studied subtidal organisms. For each species, divergence time calculations suggest contemporary North and South Island lineages diverged from their respective most recent common ancestor approximately 200 000 to 300 000 years before present (bp), significantly earlier than previous estimates in other coastal marine taxa that arose from a miscalculation of divergence time.