Evidence for emergence of an amphibian iridoviral disease because of human-enhanced spread

Our understanding of origins and spread of emerging infectious diseases has increased dramatically because of recent applications of phylogenetic theory. Iridoviruses are emerging pathogens that cause global amphibian epizootics, including tiger salamander (Ambystoma tigrinum) die-offs throughout western North America. To explain phylogeographical relationships and potential causes for emergence of western North American salamander iridovirus strains, we sequenced major capsid protein and DNA methyltransferase genes, as well as two noncoding regions from 18 geographically widespread isolates. Phylogenetic analyses of sequence data from the capsid protein gene showed shallow genetic divergence (< 1%) among salamander iridovirus strains and monophyly relative to available fish, reptile, and other amphibian iridovirus strains from the genus Ranavirus, suggesting a single introduction and radiation. Analysis of capsid protein sequences also provided support for a closer relationship of tiger salamander virus strains to those isolated from sport fish (e.g. rainbow trout) than other amphibian isolates. Despite monophyly based on capsid protein sequences, there was low genetic divergence among all strains (< 1.1%) based on a supergene analysis of the capsid protein and the two noncoding regions. These analyses also showed polyphyly of strains from Arizona and Colorado, suggesting recent spread. Nested clade analyses indicated both range expansion and long-distance colonization in clades containing virus strains isolated from bait salamanders and the Indiana University axolotl (Ambystoma mexicanum) colony. Human enhancement of viral movement is a mechanism consistent with these results. These findings suggest North American salamander ranaviruses cause emerging disease, as evidenced by apparent recent spread over a broad geographical area.     

A test of alternative hypotheses for kin recognition in cannibalistic tiger salamanders

The function of kin recognition is controversial. We investigatedthe adaptive significance of kin discrimination in cannibalistictiger salamander larvae, Ambystoma tigrinum. Previous laboratoryexperiments show that cannibals preferentially consume lessrelated individuals. We hypothesized that this example of kinrecognition (1) is a laboratory artifact, (2) is a by-productof sibship-specific variation in escape responses, because cannibalsfrom families with rapid responses may be more likely to cannibalize slowlyescaping non-kin, (3) is an epiphenomenon of species recognition,(4) functions in disease avoidance, because kin may be moreinfectious than non-kin, or (5) is favored by kin selection.We evaluated these five hypotheses by using laboratory and fieldexperiments to test specific predictions made by each hypothesis.We rejected hypotheses 1-4 above because (1) kin recognitionwas expressed in the wild, (2) escape responses did not reliablypredict whether a cannibal would ingest kin or non-kin, (3)kin recognition was not most pronounced in populations wheretiger salamanders co-occur with other species of salamanders,and (4) non-kin prey were more likely than kin to transmit pathogensto cannibals. However, we established that the necessary conditionfor kin selection, Hamilton's rule, was met. Thus, our resultsimplicate kin selection as the overriding reason that cannibalistictiger salamanders discriminate kin.     

Habitat fragmentation as a result of biotic and abiotic factors controls pathogen transmission throughout a host population

1. The hypothesis that habitat fragmentation (biotic or abiotic) alters the transmission of disease within a population is explored using field data from a well-studied amphibian-pathogen system. 2. We used the Ambystoma tigrinum-A. tigrinum virus (ATV) model system to show how habitat fragmentation as a result of emergent vegetation and habitat management affects disease transmission dynamics in ponds across a landscape. 3. We quantified variation in ATV infection over time and across the landscape. ATV infection was significantly higher in ponds modified for livestock use (P = 0.032). Disease incidence decreased with increased amounts of emergent vegetation (P < 0.001). These factors appear to control disease transmission by altering the host contact rate and with it disease transmission. 4. A field experiment to test the effect of emergent vegetation on the distribution of larvae in ponds demonstrated a behavioural change in larvae found in sparsely vegetated ponds. Microhabitat choices resulted in larvae being concentrated at the pond edge resulting in a 'halo effect' in sparsely vegetated ponds, whereas larvae in heavily vegetated ponds were distributed more evenly throughout. Microhabitat choice affects the effective density that larvae experience. This 'halo effect' increases contact rates in the shallows of sparsely vegetated ponds and increases the transmission of a directly transmitted pathogen. 5. Despite recurrent epidemics of a lethal Ranavirus in tiger salamanders on the Kaibab Plateau, Arizona, USA, these populations persist. We discuss the implications of our results in the context of density-dependent transmission and homogeneous mixing, two increases key assumptions of epidemiological theory.     

Selective processes and adaptive evolution of the cytochrome b gene in salamanders of the genus Salamandrella

Sequence analysis of the cytochrome b gene fragment in the salamanders of the genus Salamandrella, Siberian salamander and Schrenk salamander was performed with the purpose to elucidate the effect of natural selection on the evolution of mitochondrial DNA (mtDNA) in these species. It was demonstrated that despite of notable influence of negative selection (expressed as very low dN/dS values), speciation and intraspecific divergence in salamanders was accompanied by the appearance of radical amino acid substitutions, caused by the influence of positive (directional) selection. To examine the evolutionary pattern of synonymous mtDNA sites, distribution of conservative and non-conservative substitutions was analyzed. The rates of conservative and non-conservative substitutions were nearly equal, pointing to neutrality of mutation process at synonymous mtDNA sites of salamanders. Analysis of conservative and non-conservative synonymous substitution distributions in different parts of phylogenetic trees showed that the differences between the synonymous groups compared were statistically significant only in one phylogenetic group of Siberian salamander (haplogroup C) (P = 0.02). In the group of single substitutions, located at terminal phylogenetic branches of Siberian salamanders from group C, increased rate of conservative substitutions was observed. Based on these findings, it was suggested that selective processes could have an influence on the formation of the synonymous substitution profile in the Siberian salamander mtDNA fragment examined.     

Evolutionary analyses on the HA gene ofpandemic H1N1/09: early findings

The HA protein is responsible for influenza virus attachment and the subsequent fusion of viral and cellular membranes. Antigenic drift is driven by an accumulation of point mutations in the HA. And, the receptor-binding specificity of HA is responsible for the host range restriction of the virus. In April 2009, large outbreaks of novel H1N1 influenza in human population were reported from North America. The pandemic H1N1 virus originated from swine influenza virus. Evolutionary process of the pandemic virus after its introduction to human population remains to be clarified. We conducted phylogenetic analyses constructing a phylogenetic tree for and calculating site-by-site selective pressures in the HA gene. Phylogenetic tree showed that pandemic viruses were not clustered clearly by their geographical location or isolation time in the phylogenetic tree. The virus has been circulating the globe extensively with multiple introductions into most geographical areas. We found 3 sites positively selected in the HA gene for pandemic H1N1 virus. Among them, position 206 is located in an antigenic site. We did not find significant negative selection on any of the receptor binding sites. The virus has been evolving under unique selective pressure.     

Mortality Rates Differ Among Amphibian Populations Exposed to Three Strains of a Lethal Ranavirus

Infectious diseases are a growing threat to biodiversity, in many cases because of synergistic effects with habitat loss, environmental contamination, and climate change. Emergence of pathogens as new threats to host populations can also arise when novel combinations of hosts and pathogens are unintentionally brought together, for example, via commercial trade or wildlife relocations and reintroductions. Chytrid fungus (Batrachochytrium dendrobatidis) and amphibian ranaviruses (family Iridoviridae) are pathogens implicated in global amphibian declines. The emergence of disease associated with these pathogens appears to be at least partly related to recent translocations over large geographic distances. We experimentally examined the outcomes of novel combinations of host populations and pathogen strains using the amphibian ranavirus Ambystoma tigrinum virus (ATV) and barred tiger salamanders (Ambystoma mavortium, formerly considered part of the Ambystoma tigrinum complex). One salamander population was highly resistant to lethal infections by all ATV strains, including its own strain, and mortality rates differed among ATV strains according to salamander population. Mortality rates in novel pairings of salamander population and ATV strain were not predictable based on knowledge of mortality rates when salamander populations were exposed to their own ATV strain. The underlying cause(s) for the differences in mortality rates are unknown, but local selection pressures on salamanders, viruses, or both, across the range of this widespread host–pathogen system are a plausible hypothesis. Our study highlights the need to minimize translocations of amphibian ranaviruses, even among conspecifc host populations, and the importance of considering intraspecific variation in endeavors to manage wildlife diseases.     

Concordance analysis in mitogenomic phylogenetics

Here I advocate the utility of Bayesian concordance analysis as a mechanism for exploring the magnitude and source of phylogenetic signal in concatenated mitogenomic phylogenetic studies. While typically applied to the study of independently evolving gene trees, Bayesian concordance analysis can also be applied to linked, but individually analyzed, gene regions using a prior probability that reflects the expectation of similar phylogenetic reconstructions. For true branches in the mitogenomic tree, concordance factors should represent the number of gene regions that contain phylogenetic signal for a particular clade. As a demonstration of the application of Bayesian concordance analysis to empirical data, I analyzed two different salamander (Hynobiidae and Plethodontidae) mitogenomic data sets using a gene-based partitioning strategy. The results revealed many strongly supported clades in the concatenated trees that have high concordance factors, permitting the inference that these are robustly resolved through phylogenetic signal distributed across the mitogenome. In contrast, a number of strongly supported clades in the concatenated tree received low concordance factors, indicating that their reconstruction is either driven primarily by phylogenetic signal in a small number of gene regions, or that they are inconsistent reconstructions influenced by properties of the data that can produce inaccurate trees (e.g., compositional bias, selection, etc.). Exploration of the Bayesian joint posterior distribution of trees highlighted partitions that contribute phylogenetic information to similar clade reconstructions. This approach was particularly insightful in the hynobiid data, where different combinations of genes were identified that support alternative tree reconstructions. Concatenated analysis of these different subsets of genes highlighted through Bayesian concordance analysis produced strongly supported and contrasting trees, demonstrating the potential for inconsistency in concatenated mitogenomic phylogenetics. The overall results presented here suggest that Bayesian concordance analysis can serve as an effective exploration of the influence of different gene regions in mitogenomic (and other organellar genomic) phylogenetic studies.     

Multiple nuclear gene sequences identify phylogenetic species boundaries in the rapidly radiating clade of Mexican ambystomatid salamanders

Delimiting the boundaries of species involved in radiations is critical to understanding the tempo and mode of lineage formation. Single locus gene trees may or may not reflect the underlying pattern of population divergence and lineage formation, yet they constitute the vast majority of the empirical data in species radiations. In this study we make use of an expressed sequence tag (EST) database to perform nuclear (nDNA) and mitochondrial (mtDNA) genealogical tests of species boundaries in Ambystoma ordinarium, a member of an adaptive radiation of metamorphic and paedomorphic salamanders (the Ambystoma tigrinum complex) that have diversified across terrestrial and aquatic environments. Gene tree comparisons demonstrate extensive nonmonophyly in the mtDNA genealogy of A. ordinarium, while seven of eight independent nuclear loci resolve the species as monophyletic or nearly so, and diagnose it as a well-resolved genealogical species. A differential introgression hypothesis is supported by the observation that western A. ordinarium localities contain mtDNA haplotypes that are identical or minimally diverged from haplotypes sampled from a nearby paedomorphic species, Ambystoma dumerilii, while most nDNA trees place these species in distant phylogenetic positions. These results provide a strong example of how historical introgression can lead to radical differences between gene trees and species histories, even among currently allopatric species with divergent life history adaptations and morphologies. They also demonstrate how EST-based nuclear resources can be used to more fully resolve the phylogenetic history of species radiations.     

DURATION AND CONSISTENCY OF HISTORICAL SELECTION ARE CORRELATED WITH ADAPTIVE TRAIT EVOLUTION IN THE STREAMSIDE SALAMANDER, AMBYSTOMA BARBOURI

A fundamental challenge in evolutionary biology concerns estimating the extent to which ecological trade-offs may impose constraints on adaptive evolution. Novel ecological stressors may limit adaptive evolution of naive lineages that have experienced historically different selective regimes. Regarded as recently derived from a pond-breeding ancestor, streamside salamanders face the novel and strong selection pressure of breeding in streams with fish predators. A statistical phylogenetic approach was used to test whether adaptive evolution of antipredator performance phenotypes in streamside salamanders was positively associated with: (1) estimated per-lineage duration of coexistence with predatory fish; and (2) consistency of this predator selective-regime within lineages. Average durations of fish contact were computed for each salamander lineage on a set of chronograms. Selection consistency was determined by estimating the number of ecological transitions between fish and fishless states using stochastic character mapping. Historical selection in streamside salamanders can be generally characterized as unstable, apparently punctuated by the stochastic loss and recolonization of predatory fish in most lineages. We found that the efficacy of antipredator phenotypes in salamanders is strongly related to historical duration, as well as consistency, of selection imposed by predatory fish.     

Landscape genetics of the blotched tiger salamander (Ambystoma tigrinum melanostictum)

The field of landscape genetics has great potential to identify habitat features that influence population genetic structure. To identify landscape correlates of genetic differentiation in a quantitative fashion, we developed a novel approach using geographical information systems analysis. We present data on blotched tiger salamanders (Ambystoma tigrinum melanostictum) from 10 sites across the northern range of Yellowstone National Park in Montana and Wyoming, USA. We used eight microsatellite loci to analyse population genetic structure. We tested whether landscape variables, including topographical distance, elevation, wetland likelihood, cover type and number of river and stream crossings, were correlated with genetic subdivision (F(ST)). We then compared five hypothetical dispersal routes with a straight-line distance model using two approaches: (i) partial Mantel tests using Akaike's information criterion scores to evaluate model robustness and (ii) the BIOENV procedure, which uses a Spearman rank correlation to determine the combination of environmental variables that best fits the genetic data. Overall, gene flow appears highly restricted among sites, with a global F(ST) of 0.24. While there is a significant isolation-by-distance pattern, incorporating landscape variables substantially improved the fit of the model (from an r2 of 0.3 to 0.8) explaining genetic differentiation. It appears that gene flow follows a straight-line topographic route, with river crossings and open shrub habitat correlated with lower F(ST) and thus, decreased differentiation, while distance and elevation difference appear to increase differentiation. This study demonstrates a general approach that can be used to determine the influence of landscape variables on population genetic structure.     

Phylogenetic Insight into Zika and Emerging Viruses for a Perspective on Potential Hosts

Global viral diversity is substantial, but viruses that contribute little to the public health burden or to agricultural damage receive minimal attention until a seemingly unimportant virus becomes a threat. The Zika virus (ZIKV) illustrated this, as there was limited information and awareness of the virus when it was identified as a public health emergency in February 2016. Predicting which virus may pose a future threat is difficult. This is in part because significant knowledge gaps in the basic biology and ecology of an emerging virus can impede policy development, delay decision making, and hinder public health action. We suggest using a phylogenetic framework of pathogens and their infected host species for insight into which animals may serve as reservoirs. For example, examining flaviviruses closely related to ZIKV, the phylogenetic framework indicates New World monkeys are the most likely candidates to be potential reservoirs for ZIKV. Secondarily, mammals that are in close proximity to humans should be considered because of the increased opportunity for pathogen exchange. The increase in human-mediated environmental change is accelerating the probability of another previously overlooked virus becoming a significant concern. By investing in basic science research and organizing our knowledge into an evolutionary framework, we will be better prepared to respond to the next emerging infectious disease.     

Evolutionary insights into the ecology of coronaviruses

Although many novel members of the Coronaviridae have recently been recognized in different species, the ecology of coronaviruses has not been established. Our study indicates that bats harbor a much wider diversity of coronaviruses than any other animal species. Dating of different coronavirus lineages suggests that bat coronaviruses are older than those recognized in other animals and that the human severe acute respiratory syndrome (SARS) coronavirus was directly derived from viruses from wild animals in wet markets of southern China. Furthermore, the most closely related bat and SARS coronaviruses diverged in 1986, an estimated divergence time of 17 years prior to the outbreak, suggesting that there may have been transmission via an unknown intermediate host. Analysis of lineage-specific selection pressure also indicated that only SARS coronaviruses in civets and humans were under significant positive selection, also demonstrating a recent interspecies transmission. Analysis of population dynamics revealed that coronavirus populations in bats have constant population growth, while viruses from all other hosts show epidemic-like increases in population. These results indicate that diverse coronaviruses are endemic in different bat species, with repeated introductions to other animals and occasional establishment in other species. Our findings suggest that bats are likely the natural hosts for all presently known coronavirus lineages and that all coronaviruses recognized in other species were derived from viruses residing in bats. Further surveillance of bat and other animal populations is needed to fully describe the ecology and evolution of this virus family.     

Phylogeographic concordance in the southeastern United States: the flatwoods salamander, Ambystoma cingulatum, as a test case

Well-supported, congruent phylogeographic and biogeographic patterns permit the development of a priori phylogeographic and distributional predictions. In the southeastern Coastal Plain of the United States, the common discovery of east-west disjunctions (phylogeographic breaks and species' distributional boundaries) suggests that similar disjunctions should occur in codistributed taxa. Despite the near ubiquity of these disjunctions, the most recent morphological analyses of the flatwoods salamander, Ambystoma cingulatum, indicate that none occur in this low-vagility, Coastal Plain endemic. We conducted molecular and morphological analyses to test whether the flatwoods salamander is an exception to this common biogeographic pattern. Assessing geographic variation in this species is also an important management tool for this threatened, declining amphibian. We demonstrate that flatwoods salamanders, as predicted by comparisons to codistributed taxa, are polytypic with a major disjunction at the Apalachicola River. This drainage is a common site for east-west phylogeographic breaks, probably because repeated marine embayments during the Pliocene and Pleistocene interglacials generated barriers to gene flow. Based on mitochondrial DNA, morphology, and allozymes, we recognize two species of flatwoods salamanders -- Ambystoma cingulatum to the east of the Apalachicola drainage and Ambystoma bishopi to the west. Given this increased diversity, the conservation status of these two taxa may warrant re-evaluation. More generally, these results emphasize that in the absence of taxon-specific data, established comparative patterns can provide strong expectations for designing management units for unstudied species of conservation concern.     

Tree shape‐based approaches for the comparative study of cophylogeny

Cophylogeny is the congruence of phylogenetic relationships between two different groups of organisms due to their long‐term interaction. We investigated the use of tree shape distance measures to quantify the degree of cophylogeny. We implemented a reverse‐time simulation model of pathogen phylogenies within a fixed host tree, given cospeciation probability, host switching, and pathogen speciation rates. We used this model to evaluate 18 distance measures between host and pathogen trees including two kernel distances that we developed for labeled and unlabeled trees, which use branch lengths and accommodate different size trees. Finally, we used these measures to revisit published cophylogenetic studies, where authors described the observed associations as representing a high or low degree of cophylogeny. Our simulations demonstrated that some measures are more informative than others with respect to specific coevolution parameters especially when these did not assume extreme values. For real datasets, trees’ associations projection revealed clustering of high concordance studies suggesting that investigators are describing it in a consistent way. Our results support the hypothesis that measures can be useful for quantifying cophylogeny. This motivates their usage in the field of coevolution and supports the development of simulation‐based methods, i.e., approximate Bayesian computation, to estimate the underlying coevolutionary parameters.     

Adaptive intraspecific divergence: an example using the animal cytochrome b gene

The topologies of phylogenetic trees characterized by a high level of intraspecific divergence between the phylogenetic DNA groups (clades) are often explained in terms of the theory of Pleistocene refugia. To elucidate the issue of the adaptive role of intraspecific divergence, the changes in the physicochemical properties of amino acids in the course ofcladogenesis (MM01 model of the TreeSAAP 3.2 package) were analyzed in this work using as an example the nucleotide sequences of the cytochrome b gene in some species of northern animals (lemmings, redbacked voles, chipmunks, flying squirrels, ermines). It was shown that the process of intraspecific divergence was rarely accompanied by radical amino acid substitutions in cytochrome b caused by adaptation (directional selection). In connection with this, the hypothesis is discussed according to which the adaptive variants formed in the species at the peak of cold were lost with climatic warming due to the drift or selection against individuals adapted to cold.     

Herpes simplex virus eliminates host mitochondrial DNA

Mitochondria have crucial roles in the life and death of mammalian cells, and help to orchestrate host antiviral defences. Here, we show that the ubiquitous human pathogen herpes simplex virus (HSV) induces rapid and complete degradation of host mitochondrial DNA during productive infection of cultured mammalian cells. The depletion of mitochondrial DNA requires the viral UL12 gene, which encodes a conserved nuclease with orthologues in all herpesviruses. We show that an amino-terminally truncated UL12 isoform-UL12.5-localizes to mitochondria and triggers mitochondrial DNA depletion in the absence of other HSV gene products. By contrast, full-length UL12, a nuclear protein, has little or no effect on mitochondrial DNA levels. Our data document that HSV inflicts massive genetic damage to a crucial host organelle and show a novel mechanism of virus-induced shutoff of host functions, which is likely to contribute to the cell death and tissue damage caused by this widespread human pathogen.     

Emergence of novel fungal pathogens by ecological speciation: importance of the reduced viability of immigrants

Expanding global trade and the domestication of ecosystems have greatly accelerated the rate of emerging infectious fungal diseases, and host-shift speciation appears to be a major route for disease emergence. There is therefore an increased interest in identifying the factors that drive the evolution of reproductive isolation between populations adapting to different hosts. Here, we used genetic markers and cross-inoculations to assess the level of gene flow and investigate barriers responsible for reproductive isolation between two sympatric populations of Venturia inaequalis, the fungal pathogen causing apple scab disease, one of the fungal populations causing a recent emerging disease on resistant varieties. Our results showed the maintenance over several years of strong and stable differentiation between the two populations in the same orchards, suggesting ongoing ecological divergence following a host shift. We identified strong selection against immigrants (i.e. host specificity) from different host varieties as the strongest and likely most efficient barrier to gene flow between local and emerging populations. Cross-variety disease transmission events were indeed rare in the field and cross-inoculation tests confirmed high host specificity. Because the fungus mates within its host after successful infection and because pathogenicity-related loci prevent infection of nonhost trees, adaptation to specific hosts may alone maintain both genetic differentiation between and adaptive allelic combinations within sympatric populations parasitizing different apple varieties, thus acting as a 'magic trait'. Additional intrinsic and extrinsic postzygotic barriers might complete reproductive isolation and explain why the rare migrants and F1 hybrids detected do not lead to pervasive gene flow across years.     

Pathogen evolution and disease emergence in carnivores

Emerging infectious diseases constitute some of the most pressing problems for both human and domestic animal health, and biodiversity conservation. Currently it is not clear whether the removal of past constraints on geographical distribution and transmission possibilities for pathogens alone are sufficient to give rise to novel host-pathogen combinations, or whether pathogen evolution is also generally required for establishment in novel hosts. Canine distemper virus (CDV) is a morbillivirus that is prevalent in the world dog population and poses an important conservation threat to a diverse range of carnivores. We performed an extensive phylogenetic and molecular evolution analysis on complete sequences of all CDV genes to assess the role of selection and recombination in shaping viral genetic diversity and driving the emergence of CDV in non-dog hosts. We tested the specific hypothesis that molecular adaptation at known receptor-binding sites of the haemagglutinin gene is associated with independent instances of the spread of CDV to novel non-dog hosts in the wild. This hypothesis was upheld, providing compelling evidence that repeated evolution at known functional sites (in this case residues 530 and 549 of the haemagglutinin molecule) is associated with multiple independent occurrences of disease emergence in a range of novel host species.     

Virus adaptation by manipulation of host's gene expression

Viruses adapt to their hosts by evading defense mechanisms and taking over cellular metabolism for their own benefit. Alterations in cell metabolism as well as side-effects of antiviral responses contribute to symptoms development and virulence. Sometimes, a virus may spill over from its usual host species into a novel one, where usually will fail to successfully infect and further transmit to new host. However, in some cases, the virus transmits and persists after fixing beneficial mutations that allow for a better exploitation of the new host. This situation would represent a case for a new emerging virus. Here we report results from an evolution experiment in which a plant virus was allowed to infect and evolve on a na?ve host. After 17 serial passages, the viral genome has accumulated only five changes, three of which were non-synonymous. An amino acid substitution in the viral VPg protein was responsible for the appearance of symptoms, whereas one substitution in the viral P3 protein the epistatically contributed to exacerbate severity. DNA microarray analyses show that the evolved and ancestral viruses affect the global patterns of host gene expression in radically different ways. A major difference is that genes involved in stress and pathogen response are not activated upon infection with the evolved virus, suggesting that selection has favored viral strategies to escape from host defenses.