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Hepatitis delta virus (HDV) is an unusual RNA agent that replicates using host machinery but exploits hepatitis B virus (HBV) to mobilize its spread within and between hosts. In doing so, HDV enhances the virulence of HBV. How this seemingly improbable hyper-parasitic lifestyle emerged is unknown, but underpins the likelihood that HDV and related deltaviruses may alter other host-virus interactions. Here, we show that deltaviruses diversify by transmitting between mammalian species. Among 96,695 RNA sequence datasets, deltaviruses infected bats, rodents and an artiodactyl from the Americas, but were absent from geographically overrepresented Old World representatives of each mammalian order, suggesting a relatively recent diversification within the Americas. Consistent with diversification by host shifting, both bat and rodent-infecting deltaviruses were paraphyletic and co-evolutionary modeling rejected co-speciation with mammalian hosts. In addition, a two-year field study showed common vampire bats in Peru were infected by two divergent deltaviruses, indicating multiple introductions to a single host species. One vampire bat-associated deltavirus was detected in the saliva of up to 35% of individuals, formed phylogeographically compartmentalized clades, and infected a sympatric bat, illustrating horizontal transmission within and between species on ecological timescales. Consistent absence of HBV-like viruses in two deltavirus-infected bat species indicated acquisitions of novel viral associations during the divergence of bat and human-infecting deltaviruses. Our analyses support an American zoonotic origin of HDV and reveal prospects for future cross-species emergence of deltaviruses. Given their peculiar life history, deltavirus host shifts will have different constraints and disease outcomes compared to ordinary animal pathogens.

Bats host diverse coronaviruses, including taxa capable of pandemic spread in humans. We report the genome of an alphacoronavirus from a neotropical bat species (Desmodus rotundus) in Peru, which contributes to our understanding of bat coronaviruses in nature.

The Grallaria rufula complex is currently considered to consist of 2 species, G. rufula (Rufous Antpitta) and G. blakei (Chestnut Antpitta). However, it has been suggested that the complex, populations of which occur in humid montane forests from Venezuela to Bolivia, comprises a suite of vocally distinct yet morphologically cryptic species. We sequenced nuclear and mitochondrial DNA for 80 individuals from across the distribution of the complex to determine the extent of genetic variation between and within described taxa. Our results revealed 18 geographically coherent clades separated by substantial genetic divergence: 14 within rufula, 3 within blakei, and 1 corresponding to G. rufocinerea (Bicolored Antpitta), a species with distinctive plumage found to be nested within the complex. Neither G. rufula nor G. blakei as presently defined was monophyletic. Although 6 of the 7 recognized subspecies of G. rufula were monophyletic, several subspecies contained substantial genetic differentiation. Genetic variation was largely partitioned across recognized geographic barriers, especially across deep river valleys in Peru and Colombia. Coalescent modeling identified 17 of the 18 clades as significantly differentiated lineages, whereas analyses of vocalizations delineated 16 biological species within the complex. The G. rufula complex seems unusually diverse even among birds of the humid Andes, a prime location for cryptic speciation; however, the extent to which other dispersal-limited Andean species groups exhibit similar degrees of cryptic differentiation awaits further study.

The Mediterranean Basin Biodiversity Hotspot is among the most biologically diverse regions worldwide and renowned for its richness of endemic freshwater species. But the hotspot’s lakes, rivers and wetlands are seriously threatened by an unprecedented boom in hydropower development. As a result, aquatic ecosystems have become increasingly degraded due to construction of dams and other infrastructure, compounded by a range of other threats. Advocates of this flawed development model repeatedly overestimate economic benefits and ignore severe environmental consequences. In particular, the impacts of small (10 MW) hydropower plants are disproportionately high when offset against their minuscule contribution to global energy production. At least 5,269 hydropower plants are already operating in the region, with 202 currently under construction. A further 6,393 are proposed, including 5,962 small projects, while 1,579 exist and 2,091 are planned within the boundaries of national parks, biosphere reserves, and other protected areas. In order to anticipate the biodiversity impact of this widening hydropower infrastructure it is essential to understand where threatened species are distributed. This report presents detailed range maps for all 251 freshwater fish species presently recognised as threatened in the Mediterranean Basin Biodiversity Hotspot alongside an updated evaluation of their respective extinction risks. The results suggest that an alarming 63% of all threatened freshwater fish species have already been driven closer to extinction by existing hydropower plants, with 55% of all Critically Endangered species highly impacted. Should hydroelectric expansion in the region go ahead as planned, 74% of all threatened freshwater fish species will be negatively impacted, with 65% set to decline due to small projects alone. At least seven of these could become globally extinct in the wild as a result. Hydropower projects, particularly small schemes, are therefore the most important driver of potential fish species extinctions in the Mediterranean Basin Biodiversity Hotspot. Impoundments created by dams and other hydroelectric infrastructure are also the key pathway by which non-native fish species, another major cause of freshwater fish declines, enter fluvial ecosystems. Moreover, if the hydroelectric schemes planned in the Mediterranean Basin Biodiversity Hotspot are implemented, nations will be unable to comply with the United Nations 2030 Agenda for Sustainable Development or binding international biodiversity targets agreed by parties to the Convention on Biological Diversity and European Union member states. There is an urgent need to mitigate the escalating ecological damage triggered by the hydropower binge through preservation and restoration of free-flowing rivers. Rapid and significant action underpinned by sound scientific evidence is required to safeguard freshwater ecosystems and the incredibly rich biodiversity they support throughout the region. By improving the state of the natural environment, the impacts of ongoing climate change would also be reduced. It is hoped that this report will help inform nature conservation, governments and policy-makers to make the right political, financial and consumer choices to reverse the trend of freshwater biodiversity decline throughout the Mediterranean Basin Biodiversity Hotspot.

Microbial communities play an important role in organismal and ecosystem health. While high‐throughput metabarcoding has revolutionized the study of bacterial communities, generating comparable viral communities has proven elusive, particularly in wildlife samples where the diversity of viruses and limited quantities of viral nucleic acid present distinctive challenges. Metagenomic sequencing is a promising solution for studying viral communities, but the lack of standardized methods currently precludes comparisons across host taxa or localities. Here, we developed an untargeted shotgun metagenomic sequencing protocol to generate comparable viral communities from noninvasively collected faecal and oropharyngeal swabs. Using samples from common vampire bats (Desmodus rotundus), a key species for virus transmission to humans and domestic animals, we tested how different storage media, nucleic acid extraction procedures and enrichment steps affect viral community detection. Based on finding viral contamination in foetal bovine serum, we recommend storing swabs in RNAlater or another nonbiological medium. We recommend extracting nucleic acid directly from swabs rather than from supernatant or pelleted material, which had undetectable levels of viral RNA. Results from a low‐input RNA library preparation protocol suggest that ribosomal RNA depletion and light DNase treatment reduce host and bacterial nucleic acid, and improve virus detection. Finally, applying our approach to twelve pooled samples from seven localities in Peru, we showed that detected viral communities saturated at the attained sequencing depth, allowing unbiased comparisons of viral community composition. Future studies using the methods outlined here will elucidate the determinants of viral communities across host species, environments and time.

Microbial communities play important roles in organismal and ecosystem health. High throughput sequencing has revolutionized our understanding of host-associated microbial communities, but the viral component of these communities remains poorly characterized relative to microbes such as bacteria, particularly in non-human hosts. This knowledge gap has implications for global health, as viruses originating in wildlife are responsible for recent disease outbreaks in humans and domestic animals. Although studies have identified factors differentiating viral communities between species, we have little understanding of the variability of viral communities within species. Comparative studies of viral communities are therefore necessary to characterize novel taxa and to evaluate the ecological factors influencing intraspecific viral diversity and distribution. Bats are recognized as “special” reservoirs for viruses because they are associated with diverse viral communities and display deep evolutionary relationships with individual viral taxa. Common vampire bats (Desmodus rotundus) represent a particularly interesting system in which to investigate viral communities, as they are obligate blood feeders that interact ecologically with many different host species, providing opportunities for the acquisition of diverse viruses. The overall objective of this thesis was to advance our understanding of intraspecific wildlife-associated viral communities using an established field network of common vampire bat colonies across Peru. Specifically, I developed a novel method for comparative viral community studies, characterized the viral communities of vampire bats, and examined the ecological correlates of vampire bat viral diversity across Peru. Metagenomic sequencing is a promising technique for comparative studies of viral communities in wildlife, but there is a need to first develop standardized methods that can be applied to samples collected in the field. In Chapter 2 I developed a shotgun metagenomic sequencing approach to characterizing viral communities from non-invasive samples. Specifically, I optimized extraction and sequencing protocols using fecal and oropharyngeal swabs collected from common vampire bats in Peru. Two preliminary sequencing runs were performed, the results of which motivated four pilot studies in which I tested how different storage media, nucleic acid extraction procedures, and enrichment steps affect the viral community detected. Metagenomic sequencing revealed viral contamination of fetal bovine serum, a component of viral transport medium, suggesting that swabs should be stored in RNALater or another non-biological medium. Extraction and qPCR tests were performed on swabs inoculated with known concentrations of virus, which revealed that nucleic acid should be directly extracted from swabs rather than from supernatant or pelleted material. Metagenomic sequencing of paired samples was used to test enrichment by ribosomal RNA depletion and light DNAse treatment, which both reduced host and bacterial nucleic acid in samples and improved virus detection. A bioinformatic pipeline was developed specifically for processing vampire bat shotgun viral metagenomic data. Finally, the optimized protocol was applied to twelve pooled samples from seven localities in Peru, and read subsampling demonstrated that the viral communities detected were consistent at commonly attained depths of sequencing. The protocol developed in this chapter enables minimally biased comparative viral community studies in non-invasive samples collected from wildlife. Having a detailed understanding of viral diversity in key wildlife hosts is an important first step in evaluating the risk of zoonotic disease emergence, but we still lack a holistic view of viral communities in many species including vampire bats. In Chapter 3, I used the metagenomic sequencing protocol developed in Chapter 2 to thoroughly characterize viral communities in the saliva and feces of vampire bats captured across Peru. Viruses were detected from a range of natural host groups including vertebrate-associated taxa that were potentially infecting vampire bats, bacteriophages associated with gut bacteria, and plant- or insect-infecting viruses potentially acquired from the environment. There were broad differences between fecal and saliva viral communities, showing evidence of body habitat compartmentalization. Overall, results established that vampire bat viral communities differ between body habitats and suggested that, for the vertebrate-infecting families analyzed, novel viruses mostly fall within bat-specific clades, without evidence of livestock or humans acting as a major source of viral diversity in vampire bats. Interspecific differences in ecological and life history traits are known to impact viral richness in bats, but the factors structuring viral communities within bat species are less well understood. In Chapter 4, I examined the spatial, demographic and environmental correlates of intraspecific viral diversity in vampire bats. Three measures of viral diversity were calculated at the colony level: richness, a novel measure of taxonomic diversity, and community composition. Generalized linear models were then used to test the effects of broad scale and local ecological variables on saliva and fecal viral diversity. The results showed for the first time that ecological variables can influence intraspecific viral diversity. In summary, the work presented in this thesis advances our understanding of wildlife-associated viral communities in an ecologically important bat host. Future directions in comparative wildlife viral metagenomics, as discussed in Chapter 5, will include exploring the determinants of viral communities across host species, environments and time.

Viruses infect all forms of life and play critical roles as agents of disease, drivers of biochemical cycles and sources of genetic diversity for their hosts. Our understanding of viral diversity derives primarily from comparisons among host species, precluding insight into how intraspecific variation in host ecology affects viral communities or how predictable viral communities are across populations. Here we test spatial, demographic and environmental hypotheses explaining viral richness and community composition across populations of common vampire bats, which occur in diverse habitats of North, Central and South America. We demonstrate marked variation in viral communities that was not consistently predicted by a null model of declining community similarity with increasing spatial or genetic distances separating populations. We also find no evidence that larger bat colonies host greater viral diversity. Instead, viral diversity follows an elevational gradient, is enriched by juvenile‐biased age structure, and declines with local anthropogenic food resources as measured by livestock density. Our results establish the value of linking the modern influx of metagenomic sequence data with comparative ecology, reveal that snapshot views of viral diversity are unlikely to be representative at the species level, and affirm existing ecological theories that link host ecology not only to single pathogen dynamics but also to viral communities., https://doi.org/10.1111/mec.15306

The Mediterranean Basin Biodiversity Hotspot is among the most biologically diverse regions worldwide and renowned for its richness of endemic freshwater species. But the hotspot’s lakes, rivers and wetlands are seriously threatened by an unprecedented boom in hydropower development. As a result, aquatic ecosystems have become increasingly degraded due to construction of dams and other infrastructure, compounded by a range of other threats. Advocates of this flawed development model repeatedly overestimate economic benefits and ignore severe environmental consequences. In particular, the impacts of small (10 MW) hydropower plants are disproportionately high when offset against their minuscule contribution to global energy production. At least 5,269 hydropower plants are already operating in the region, with 202 currently under construction. A further 6,393 are proposed, including 5,962 small projects, while 1,579 exist and 2,091 are planned within the boundaries of national parks, biosphere reserves, and other protected areas. In order to anticipate the biodiversity impact of this widening hydropower infrastructure it is essential to understand where threatened species are distributed. This report presents detailed range maps for all 251 freshwater fish species presently recognised as threatened in the Mediterranean Basin Biodiversity Hotspot alongside an updated evaluation of their respective extinction risks. The results suggest that an alarming 63% of all threatened freshwater fish species have already been driven closer to extinction by existing hydropower plants, with 55% of all Critically Endangered species highly impacted. Should hydroelectric expansion in the region go ahead as planned, 74% of all threatened freshwater fish species will be negatively impacted, with 65% set to decline due to small projects alone. At least seven of these could become globally extinct in the wild as a result. Hydropower projects, particularly small schemes, are therefore the most important driver of potential fish species extinctions in the Mediterranean Basin Biodiversity Hotspot. Impoundments created by dams and other hydroelectric infrastructure are also the key pathway by which non-native fish species, another major cause of freshwater fish declines, enter fluvial ecosystems. Moreover, if the hydroelectric schemes planned in the Mediterranean Basin Biodiversity Hotspot are implemented, nations will be unable to comply with the United Nations 2030 Agenda for Sustainable Development or binding international biodiversity targets agreed by parties to the Convention on Biological Diversity and European Union member states. There is an urgent need to mitigate the escalating ecological damage triggered by the hydropower binge through preservation and restoration of free-flowing rivers. Rapid and significant action underpinned by sound scientific evidence is required to safeguard freshwater ecosystems and the incredibly rich biodiversity they support throughout the region. By improving the state of the natural environment, the impacts of ongoing climate change would also be reduced. It is hoped that this report will help inform nature conservation, governments and policy-makers to make the right political, financial and consumer choices to reverse the trend of freshwater biodiversity decline throughout the Mediterranean Basin Biodiversity Hotspot.