Evolutionarily Young African Rhinoceros Gammaretroviruses.

High-throughput sequences were generated from DNA and cDNA from four Southern white rhinoceros (Ceratotherium simum simum) located in the Taronga Western Plain Zoo in Australia. Virome analysis identified reads that were similar to Mus caroli endogenous gammaretrovirus (McERV). Previous analysis of perissodactyl genomes did not recover gammaretroviruses. Our analysis, including the screening of the updated white rhinoceros (Ceratotherium simum) and black rhinoceros (Diceros bicornis) draft genomes identified high-copy orthologous gammaretroviral ERVs. Screening of Asian rhinoceros, extinct rhinoceros, domestic horse, and tapir genomes did not identify related gammaretroviral sequences in these species. The newly identified proviral sequences were designated SimumERV and DicerosERV for the white and black rhinoceros retroviruses, respectively. Two long terminal repeat (LTR) variants (LTR-A and LTR-B) were identified in the black rhinoceros, with different copy numbers associated with each (n = 101 and 373, respectively). Only the LTR-A lineage (n = 467) was found in the white rhinoceros. The African and Asian rhinoceros lineages diverged approximately 16 million years ago. Divergence age estimation of the identified proviruses suggests that the exogenous retroviral ancestor of the African rhinoceros ERVs colonized their genomes within the last 8 million years, a result consistent with the absence of these gammaretroviruses from Asian rhinoceros and other perissodactyls. The black rhinoceros germ line was colonized by two lineages of closely related retroviruses and white rhinoceros by one. Phylogenetic analysis indicates a close evolutionary relationship with ERVs of rodents including sympatric African rats, suggesting a possible African origin of the identified rhinoceros gammaretroviruses. IMPORTANCE Rhinoceros genomes were thought to be devoid of gammaretroviruses, as has been determined for other perissodactyls (horses, tapirs, and rhinoceros). While this may be true of most rhinoceros, the African white and black rhinoceros genomes have been colonized by evolutionarily young gammaretroviruses (SimumERV and DicerosERV for the white and black rhinoceros, respectively). These high-copy endogenous retroviruses (ERVs) may have expanded in multiple waves. The closest relative of SimumERV and DicerosERV is found in rodents, including African endemic species. Restriction of the ERVs to African rhinoceros suggests an African origin for the rhinoceros gammaretroviruses.

Degradation and remobilization of endogenous retroviruses by recombination during the earliest stages of a germ-line invasion.

Endogenous retroviruses (ERVs) are proviral sequences that result from colonization of the host germ line by exogenous retroviruses. The majority of ERVs represent defective retroviral copies. However, for most ERVs, endogenization occurred millions of years ago, obscuring the stages by which ERVs become defective and the changes in both virus and host important to the process. The koala retrovirus, KoRV, only recently began invading the germ line of the koala (Phascolarctos cinereus), permitting analysis of retroviral endogenization on a prospective basis. Here, we report that recombination with host genomic elements disrupts retroviruses during the earliest stages of germ-line invasion. One type of recombinant, designated recKoRV1, was formed by recombination of KoRV with an older degraded retroelement. Many genomic copies of recKoRV1 were detected across koalas. The prevalence of recKoRV1 was higher in northern than in southern Australian koalas, as is the case for KoRV, with differences in recKoRV1 prevalence, but not KoRV prevalence, between inland and coastal New South Wales. At least 15 additional different recombination events between KoRV and the older endogenous retroelement generated distinct recKoRVs with different geographic distributions. All of the identified recombinant viruses appear to have arisen independently and have highly disrupted ORFs, which suggests that recombination with existing degraded endogenous retroelements may be a means by which replication-competent ERVs that enter the germ line are degraded.

Screening for depressive symptoms in cardiovascular patients at a tertiary centre in Trinidad and Tobago: investigation of correlates in the SAD CAT study.

BACKGROUND: This study aimed to screen cardiovascular patients for depressive symptoms at a tertiary centre in Trinidad and Tobago; and to determine any significant associations amongst patients' demographics, comorbidities, and cardiovascular medications with depressive symptoms. METHODS: In this observational, cross-sectional study, patients (n = 1203) were randomly selected from the cardiology outpatient clinics at the Eric Williams Medical Sciences Complex. After meeting selection criteria, informed consent was obtained, and patients were administered a case report form, which included the Patient Health Questionnaire-9 (PHQ-9). Descriptive analyses included frequency, percentage and summary statistics. Inferential analyses included 95% confidence intervals (CIs), independent sample t-test, Fisher's exact test, Chi-square test, and multivariate logistic regression. RESULTS: The study had a 96% respondent rate, whereby the average age was 62 years old. Slightly less than half were male, and 52.5% were female. Over 90 % of the sample had cardiovascular disease (CVD). One-quarter of the sample had a PHQ-9 score of ≥10, with almost one-fifth having no depressive symptoms. Females, lower levels of education and income were all found to be statistically significant at risk for depressive symptoms (all p-values < 0.001). Comorbidities associated with depressive symptoms included hypertension, prior cerebrovascular events, chronic kidney disease, and chronic obstructive pulmonary disease with odds ratios (ORs) and 95% confidence intervals (CIs) of OR 1.988 (CI 1.414-2.797), OR 1.847 (CI 1.251-2.728), OR 1.872 (CI 1.207-2.902) and OR 1.703 (CI 1.009-2.876) respectively. Only the cardiovascular medication of ticagrelor was found to be significantly associated with depressive symptoms (p-value < 0.001). CONCLUSIONS: Twenty-five percent of screened cardiovascular patients displayed significant depressive symptoms with a PHQ-9 ≥ 10. This study also highlights the importance of implementing a multidisciplinary approach to managing cardiovascular disease and screening for depressive symptoms in this subpopulation. Further studies are required to validate these findings. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT03863262 . This trial was retrospectively registered on 20th February 2019.

Noninvasive Detection of Equid Herpesviruses in Fecal Samples.

Equid herpesviruses (EHVs) are pathogens of equid and nonequid hosts that can cause disease and fatalities in captivity and in the wild. EHVs establish latent infections but can reactivate, and most EHVs are shed via the nasal passage. Therefore, nasal swabs are generally used for EHV monitoring. However, invasive sampling of wild equids is difficult. While feces is a commonly used substrate for detecting other pathogens, to our knowledge, EHVs have never been detected in feces of naturally infected equids. We systematically tested zebra feces for EHV presence by (i) establishing nested PCR conditions for fecal DNA extracts, (ii) controlling for environmental EHV contamination, and (iii) large-scale testing on a free-ranging zebra population. A dilution minimizing inhibition while maximizing viral DNA concentrations was determined in captive Grévy's zebra (Equus grevyi) fecal samples from individuals shedding EHV nasally. Sixteen of 42 fecal samples (38%) were EHV positive. To demonstrate that the EHV positivity was not a result of environmental contamination, rectal swabs of wild zebras were screened (n = 18 [Equusquagga and E. zebra]), and 50% were EHV positive, indicating that the source of EHV in feces is likely the intestinal mucosa and not postdefecation contamination. Out of 270 fecal samples of wild zebras, 26% were EHV positive. Quantitative PCRs showed that the amount of virus DNA in feces was not significantly smaller than that in other samples. In summary, fecal sampling facilitates large-scale screening and may be useful to noninvasively investigate phylogenetic EHV diversity in wild and domestic equids.IMPORTANCE Equid herpesviruses (EHVs) establish latent infections, and many EHVs are shed and transmitted via nasal discharge primarily through droplet and aerosol infection. Obtaining nasal swabs and other invasive samples from wildlife is often not possible without capture and physical restraint of individuals, which are resource intensive and a health risk for the captured animals. Fecal EHV shedding has never been demonstrated for naturally infected equids. We established the conditions for fecal EHV screening, and our results suggest that testing fecal samples is an effective noninvasive approach for monitoring acute EHV shedding in equids.

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.

High global diversity of cycloviruses amongst dragonflies.

Members of the family Circoviridae, specifically the genus Circovirus, were thought to infect only vertebrates; however, members of a sister group under the same family, the proposed genus Cyclovirus, have been detected recently in insects. In an effort to explore the diversity of cycloviruses and better understand the evolution of these novel ssDNA viruses, here we present five cycloviruses isolated from three dragonfly species (Orthetrum sabina, Xanthocnemis zealandica and Rhionaeschna multicolor) collected in Australia, New Zealand and the USA, respectively. The genomes of these five viruses share similar genome structure to other cycloviruses, with a circular ~1.7 kb genome and two major bidirectionally transcribed ORFs. The genomic sequence data gathered during this study were combined with all cyclovirus genomes available in public databases to identify conserved motifs and regulatory elements in the intergenic regions, as well as determine diversity and recombinant regions within their genomes. The genomes reported here represent four different cyclovirus species, three of which are novel. Our results confirm that cycloviruses circulate widely in winged-insect populations; in eight different cyclovirus species identified in dragonflies to date, some of these exhibit a broad geographical distribution. Recombination analysis revealed both intra- and inter-species recombination events amongst cycloviruses, including genomes recovered from disparate sources (e.g. goat meat and human faeces). Similar to other well-characterized circular ssDNA viruses, recombination may play an important role in cyclovirus evolution.

Novel ssDNA viruses discovered in yellow-crowned parakeet (Cyanoramphus auriceps) nesting material.

During routine monitoring of yellow-crowned parakeets in the Poulter Valley of the South Island of New Zealand, a dead parakeet chick was discovered in a nest. Known parrot-infecting viruses, such as beak and feather disease virus (BFDV), avian polyomavirus (APV), and parrot hepatitis B virus (PHBV), were not detected in the nesting material. However, we recovered two novel single-stranded DNA viruses (ssDNA), CynNCXV (2308 nt) and CynNCKV (2087 nt), which have genome architectures similar to those of circoviruses, characterised by circular genomes with two large bidirectional open reading frames (ORFs). Both contain a stem-loop element with a conserved nonanucleotide motif, known to be required for rolling-circle replication. The full genomes had no BLASTn similarity to known ssDNA viruses. However, in both genomes the larger ORFs have BLAST similarity to known replication-associated proteins (Reps). CynNCKV has 30 % similarity to picobiliphyte nano-like virus (Picobiliphyte M5584-5) with 66-88 % coverage (e-value of 5×10(-33)), whereas CynNCXV has 33 % similarity to rodent stool-associated virus (RodSCV M-45) with 92-94 % coverage (e-value of 5 × 10(-31)). Found within these ORFs were the rolling-circle replication motifs I, II, III and the helicase motifs Walker A and Walker B. Maximum-likelihood phylogenetic analysis of the Reps reveals that these are two novel ssDNA viruses. At this point, we are unable to attribute the death of the parakeet to these two new novel ssDNA viruses.

Discovery of a novel circular single-stranded DNA virus from porcine faeces.

A large number of novel single-stranded DNA (ssDNA) viruses have been characterised from various environmental sources in the last 5 years. The bulk of these have been from faecal sources, and faecal sampling is an ideal non-invasive pathogen sampling method. We characterised a novel ssDNA from a porcine faecal sample from Cass Basin of the South Island of New Zealand. The novel viral genome has two large open reading frames (ORFs), which are bidirectionally transcribed and separated by intergenic regions. The largest ORF has some degree of similarity (<30 %) to the putative capsid protein of chimpanzee stool-associated circular ssDNA virus (ChiSCV) and pig stool-associated single-stranded DNA virus (PigSCV), whereas the second-largest ORF has high similarity to the putative replication-associated protein (Rep) of ChiSCV (~50 %) and bovine stool-associated circular DNA virus (BoSCV; ~30 %). Based on genome architecture, location of putative stem-loop like elements, and maximum-likelihood phylogenetic analysis of the gene encoding the Rep protein, the novel isolate belongs to the same family of ssDNA viruses as ChiSCV and BoSCV.

Diverse circular ssDNA viruses discovered in dragonflies (Odonata: Epiprocta).

Viruses with circular ssDNA genomes that encode a replication initiator protein (Rep) are among the smallest viruses known to infect both eukaryotic and prokaryotic organisms. In the past few years an overwhelming diversity of novel circular Rep-encoding ssDNA (CRESS-DNA) viruses has been unearthed from various hosts and environmental sources. Since there is limited information regarding CRESS-DNA viruses in invertebrates, this study explored the diversity of CRESS-DNA viruses circulating among insect populations by targeting dragonflies (Epiprocta), top insect predators that accumulate viruses from their insect prey over space and time. Using degenerate PCR and rolling circle amplification coupled with restriction digestion, 17 CRESS-DNA viral genomes were recovered from eight different dragonfly species collected in tropical and temperate regions. Nine of the genomes are similar to cycloviruses and represent five species within this genus, suggesting that cycloviruses are commonly associated with insects. Three of the CRESS-DNA viruses share conserved genomic features with recently described viruses similar to the mycovirus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, leading to the proposal of the genus Gemycircularvirus. The remaining viruses are divergent species representing four novel CRESS-DNA viral genera, including a gokushovirus-like prokaryotic virus (microphage) and three eukaryotic viruses with Reps similar to circoviruses. The novelty of CRESS-DNA viruses identified in dragonflies using simple molecular techniques indicates that there is an unprecedented diversity of ssDNA viruses among insect populations.

Environmental Detection and Potential Transmission of Equine Herpesviruses.

Equine herpesviruses (EHV) are a major health concern for domestic and wild equids and represent one of the most economically important disease agents of horses. Most known EHVs are transmitted directly between individuals as a result of direct exposure to exudates and aerosols. However, accumulating evidence suggests that environmental transmission may play a role including air, water, and fomites. Here, we reviewed studies on environmental stability and transmission of EHVs, which may influence viral dynamics and the use of environmental samples for monitoring EHV shedding.

Seasonal host and ecological drivers may promote restricted water as a viral vector.

In climates with seasonally limited precipitation, terrestrial animals congregate at high densities at scarce water sources. We hypothesize that viruses can exploit the recurrence of these diverse animal congregations to spread. In this study, we test the central prediction of this hypothesis - that viruses employing this transmission strategy remain stable and infectious in water. Equid herpesviruses (EHVs) were chosen as a model as they have been shown to remain stable and infectious in water for weeks under laboratory conditions. Using fecal data from wild equids from a previous study, we establish that EHVs are shed more frequently by their hosts during the dry season, increasing the probability of water source contamination with EHV. We document the presence of several strains of EHVs present in high genome copy number from the surface water and sediments of waterholes sampled across a variety of mammalian assemblages, locations, temperatures and pH. Phylogenetic analysis reveals that the different EHV strains found exhibit little divergence despite representing ancient lineages. We employed molecular approaches to show that EHVs shed remain stable in waterholes with detection decreasing with increasing temperature in sediments. Infectivity experiments using cell culture reveals that EHVs remain infectious in water derived from waterholes. The results are supportive of water as an abiotic viral vector for EHV.

Retroviral integrations contribute to elevated host cancer rates during germline invasion.

Repeated retroviral infections of vertebrate germlines have made endogenous retroviruses ubiquitous features of mammalian genomes. However, millions of years of evolution obscure many of the immediate repercussions of retroviral endogenisation on host health. Here we examine retroviral endogenisation during its earliest stages in the koala (Phascolarctos cinereus), a species undergoing germline invasion by koala retrovirus (KoRV) and affected by high cancer prevalence. We characterise KoRV integration sites (IS) in tumour and healthy tissues from 10 koalas, detecting 1002 unique IS, with hotspots of integration occurring in the vicinity of known cancer genes. We find that tumours accumulate novel IS, with proximate genes over-represented for cancer associations. We detect dysregulation of genes containing IS and identify a highly-expressed transduced oncogene. Our data provide insights into the tremendous mutational load suffered by the host during active retroviral germline invasion, a process repeatedly experienced and overcome during the evolution of vertebrate lineages.

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.

How Host Specific Are Herpesviruses? Lessons from Herpesviruses Infecting Wild and Endangered Mammals.

Herpesviruses are ubiquitous and can cause disease in all classes of vertebrates but also in animals of lower taxa, including molluscs. It is generally accepted that herpesviruses are primarily species specific, although a species can be infected by different herpesviruses. Species specificity is thought to result from host-virus coevolutionary processes over the long term. Even with this general concept in mind, investigators have recognized interspecies transmission of several members of the Herpesviridae family, often with fatal outcomes in non-definitive hosts-that is, animals that have no or only a limited role in virus transmission. We here summarize herpesvirus infections in wild mammals that in many cases are endangered, in both natural and captive settings. Some infections result from herpesviruses that are endemic in the species that is primarily affected, and some result from herpesviruses that cause fatal disease after infection of non-definitive hosts. We discuss the challenges of such infections in several endangered species in the absence of efficient immunization or therapeutic options.

Novel Divergent Polar Bear-Associated Mastadenovirus Recovered from a Deceased Juvenile Polar Bear.

Polar bears in captivity can be exposed to opportunistic pathogens not present in their natural environments. A 4-month-old polar bear (Ursus maritimus) living in an isolated enclosure with his mother in the Tierpark Berlin, Berlin, Germany, was suffering from severe abdominal pain, mild diarrhea, and loss of appetite and died in early 2017. Histopathology revealed severe hepatic degeneration and necrosis without evidence of inflammation or inclusion bodies, although a viral infection had been suspected on the basis of the clinical signs. We searched for nucleic acids of pathogens by shotgun high-throughput sequencing (HTS) from genomic DNA and cDNA extracted from tissue and blood. We identified a novel Mastadenovirus and assembled a nearly complete genome from the shotgun sequences. Quantitative PCR (qPCR) revealed that viral DNA was present in various concentrations in all tissues examined and that the highest concentrations were found in blood. Viral culture did not yield cytopathic effects, but qPCR suggested that virus replication was sustained for up to three passages. Positive immunofluorescence staining confirmed that the virus was able to replicate in the cells during early passage. Phylogenetic analysis demonstrated that the virus is highly divergent compared to other previously identified Mastadenovirus members and basal to most known viral clades. The virus was found only in the 4-month-old bear and not in other captive polar bears tested. We surmised, therefore, that the polar bear was infected from an unknown reservoir, illustrating that adenoviral diversity remains underestimated and that cross-species transmission of viruses can occur even under conditions of relative isolation.IMPORTANCE Cross-species transmission of viral pathogens is becoming an increasing problem for captive-animal facilities. This study highlights how animals in captivity are vulnerable to novel opportunistic pathogens, many of which do not result in straightforward diagnosis from symptoms and histopathology. In this study, a novel pathogen was suspected to have contributed to the death of a juvenile polar bear. HTS techniques were employed, and a novel Mastadenovirus was isolated. The virus was present in both the tissue and blood samples. Phylogenetic analysis of the virus at both the gene and genome levels revealed that it is highly divergent to other known mastadenoviruses. Overall, this study shows that animals in isolated conditions still come into contact with novel pathogens, and for many of these pathogens, the host reservoir and mode of transmission are yet to be determined.

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.

Long term stability and infectivity of herpesviruses in water.

For viruses to utilize environmental vectors (hard surfaces, soil, water) for transmission, physical and chemical stability is a prerequisite. There are many factors including pH, salinity, temperature, and turbidity that are known to contribute to the ability of viruses to persist in water. Equine herpesvirus type-1 (EHV-1) is a pathogenic alphaherpesvirus associated with domestic horses and wild equids. EHV-1 and recombinants of EHV-1 and EHV-9 are able to cause infections in non-equid animal species, particularly in captive settings. Many of the captive non-equid mammals are not naturally sympatric with equids and do not share enclosures, however, in many cases water sources may overlap. Similarly, in the wild, equids encounter many species at waterholes in times of seasonal drought. Therefore, we hypothesized that EHV-1 is stable in water and that water may act as a vector for EHV-1. In order to establish the conditions promoting or hindering EHV-1 longevity, infectivity and genomic stability in water; we exposed EHV-1 to varied water environments (pH, salinity, temperature, and turbidity) in controlled experiments over 21 days. The presence and infectivity of the virus was confirmed by both qPCR and cell culture experiments. Our results show that EHV-1 remains stable and infectious under many conditions in water for up to three weeks.

Geometric morphometrics and molecular systematics of Xanthocnemis sobrina (McLachlan, 1873) (Odonata: Coenagrionidae) and comparison to its congeners.

The taxonomy of the damselfly genus Xanthocnemis is revised, with particular focus on populations inhabiting the North Island of New Zealand. Earlier studies revealed two species: X. sobrina, restricted to cool, shaded streams in kauri forests and other forested areas, and X. zealandica, a common species throughout New Zealand except the Chatham and subantarctic islands. A field study encompassing aquatic habitats throughout the whole North Island was carried out to establish the relationship between morphological variation (body size and various morphological traits over the entire body) observed by previous researchers with ecological conditions and/or geographical location. The main aim was to propose reliable diagnostic features that could be used in future studies. Morphological and molecular variation was assessed. Morphological examination included assigning landmarks for all body parts corresponding to the external morphological features that are usually used in Odonata taxonomy. Molecular analysis targeted fragments of the 28S and 16S rRNA genes. Congruence was sought between both types of data, statistical support for two morphological types previously described as different species and a maximum likelihood phylogenetic tree in conjunction with a pairwise genetic distance matrix constructed from the DNA sequences obtained from the sampled specimens. Geometric morphometrics revealed statistically significant differentiation between specimens identified as X. zealandica and X. sobrina for four traits: (1) dorsal view of the head for both sexes as well as male appendages from (2) dorsal, (3) ventral and (4) lateral views. Wings appeared different when analysed for males only. Molecular analysis, however, grouped all specimens into a single undifferentiated cluster with very low mean pairwise distance (<0.01) between them showing almost no variation at the molecular level among the sampled populations on the North Island. Therefore, an additional analysis of the mitochondrial cytochrome c-oxidase I gene was carried out comparing randomly selected North Island specimens to Xanthocnemis specimens targeted in other molecular studies (Nolan et al. 2007, Amaya-Perilla et al. 2014). The analysis of the COI gene confirmed that all North and South Island isolates of Xanthocnemis cluster together in a well-supported clade with pairwise identity >96% and ~93% pairwise identity with X. tuanuii sequences obtained from the Chatham Island specimens. A careful investigation of the thin plate spline deformations generated for the geometric morphometric landmarks showed that the significant variations in the appendages of the Xanthocnemis specimens appeared to be the result of size, rather than shape, differences. Therefore, X. sobrina is proposed as a synonym of X. zealandica. Recently Amaya-Perilla et al. (2014) synonymised X. sinclairi with X. zealandica and confirmed the status of the Chatham Island X. tuanuii as a distinct species. It is therefore proposed that the genus Xanthocnemis consists of two species only: zealandica occurring all over the North, South and Stewart Islands, and tuanuii, endemic to Chatham and Pitt islands. Considering several statistical tests involving body measurements and ecological variables recorded during the field study, as well as various discussion points from similar studies of other species of Odonata, two alternative hypotheses are proposed for future testing. The first hypothesis synonymises X. sobrina with X. zealandica and suggests a possible explanation for the evolution of the two morphological traits that have previously been considered diagnostic for these species. The second hypothesis suggests that as typical X. sobrina were not sampled during this study this could represent a species that is now extinct, unless future studies prove it otherwise.

Diverse circular replication-associated protein encoding viruses circulating in invertebrates within a lake ecosystem.

Over the last five years next-generation sequencing has become a cost effective and efficient method for identifying known and unknown microorganisms. Access to this technique has dramatically changed the field of virology, enabling a wide range of environmental viral metagenome studies to be undertaken of organisms and environmental samples from polar to tropical regions. These studies have led to the discovery of hundreds of highly divergent single stranded DNA (ssDNA) virus-like sequences encoding replication-associated proteins. Yet, few studies have explored how viruses might be shared in an ecosystem through feeding relationships. Here we identify 169 circular molecules (160 CRESS DNA molecules, nine circular molecules) recovered from a New Zealand freshwater lake, that we have tentatively classified into 51 putatively novel species and five previously described species (DflaCV-3, -5, -6, -8, -10). The CRESS DNA viruses identified in this study were recovered from molluscs (Echyridella menzeisii, Musculium novaezelandiae, Potamopyrgus antipodarum and Physella acuta) and insect larvae (Procordulia grayi, Xanthocnemis zealandica, and Chironomus zealandicus) collected from Lake Sarah, as well as from the lake water and benthic sediments. Extensive diversity was observed across most CRESS DNA molecules recovered. The putative capsid protein of one viral species was found to be most similar to those of members of the Tombusviridae family, thus expanding the number of known RNA-DNA hybrid viruses in nature. We noted a strong association between the CRESS DNA viruses and circular molecules identified in the water and browser organisms (C. zealandicus, P. antipodarum and P. acuta), and between water sediments and undefended prey species (C. zealandicus). However, we were unable to find any significant correlation of viral assemblages to the potential feeding relationships of the host aquatic invertebrates.

Circular replication-associated protein encoding DNA viruses identified in the faecal matter of various animals in New Zealand.

In recent years, innovations in molecular techniques and sequencing technologies have resulted in a rapid expansion in the number of known viral sequences, in particular those with circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA genomes. CRESS DNA viruses are present in the virome of many ecosystems and are known to infect a wide range of organisms. A large number of the recently identified CRESS DNA viruses cannot be classified into any known viral families, indicating that the current view of CRESS DNA viral sequence space is greatly underestimated. Animal faecal matter has proven to be a particularly useful source for sampling CRESS DNA viruses in an ecosystem, as it is cost-effective and non-invasive. In this study a viral metagenomic approach was used to explore the diversity of CRESS DNA viruses present in the faeces of domesticated and wild animals in New Zealand. Thirty-eight complete CRESS DNA viral genomes and two circular molecules (that may be defective molecules or single components of multicomponent genomes) were identified from forty-nine individual animal faecal samples. Based on shared genome organisations and sequence similarities, eighteen of the isolates were classified as gemycircularviruses and twelve isolates were classified as smacoviruses. The remaining eight isolates lack significant sequence similarity with any members of known CRESS DNA virus groups. This research adds significantly to our knowledge of CRESS DNA viral diversity in New Zealand, emphasising the prevalence of CRESS DNA viruses in nature, and reinforcing the suggestion that a large proportion of CRESS DNA viruses are yet to be identified.