Prospecting for Zoonotic Pathogens by Using Targeted DNA Enrichment.

More than 60 zoonoses are linked to small mammals, including some of the most devastating pathogens in human history. Millions of museum-archived tissues are available to understand natural history of those pathogens. Our goal was to maximize the value of museum collections for pathogen-based research by using targeted sequence capture. We generated a probe panel that includes 39,916 80-bp RNA probes targeting 32 pathogen groups, including bacteria, helminths, fungi, and protozoans. Laboratory-generated, mock-control samples showed that we are capable of enriching targeted loci from pathogen DNA 2,882‒6,746-fold. We identified bacterial species in museum-archived samples, including Bartonella, a known human zoonosis. These results showed that probe-based enrichment of pathogens is a highly customizable and efficient method for identifying pathogens from museum-archived tissues.

Bridging the Research Gap between Live Collections in Zoos and Preserved Collections in Natural History Museums.

Zoos and natural history museums are both collections-based institutions with important missions in biodiversity research and education. Animals in zoos are a repository and living record of the world's biodiversity, whereas natural history museums are a permanent historical record of snapshots of biodiversity in time. Surprisingly, despite significant overlap in institutional missions, formal partnerships between these institution types are infrequent. Life history information, pedigrees, and medical records maintained at zoos should be seen as complementary to historical records of morphology, genetics, and distribution kept at museums. Through examining both institution types, we synthesize the benefits and challenges of cross-institutional exchanges and propose actions to increase the dialog between zoos and museums. With a growing recognition of the importance of collections to the advancement of scientific research and discovery, a transformational impact could be made with long-term investments in connecting the institutions that are caretakers of living and preserved animals.

Climatic refugia and reduced extinction correlate with underdispersion in mammals and birds in Africa.

Macroevolutionary patterns, often inferred from metrics of community relatedness, are often used to ascertain major evolutionary processes shaping communities. These patterns have been shown to be informative of biogeographic barriers, of habitat suitability and invasibility (especially with regard to environmental filtering), and of regions that function as evolutionary cradles (i.e., sources of diversification) or museums (i.e., regions of reduced extinction). Here, we analyzed continental datasets of mammal and bird distributions to identify primary drivers of community evolution on the African continent for mostly endothermic vertebrates. We find that underdispersion (i.e., relatively low phylogenetic diversity compared to species richness) closely correlates with specific ecoregions that have been identified as climatic refugia in the literature, regardless of whether these specific regions have been touted as cradles or museums. Using theoretical models of identical communities that differ only with respect to extinction rates, we find that even small suppressions of extinction rates can result in underdispersed communities, supporting the hypothesis that climatic stability can lead to underdispersion. We posit that large-scale patterns of under- and overdispersion between regions of similar species richness are more reflective of a particular region's extinction potential, and that the very nature of refugia can lead to underdispersion via the steady accumulation of species richness through diversification within the same ecoregion during climatic cycles. Thus, patterns of environmental filtering can be obfuscated by environments that coincide with biogeographic refugia, and considerations of regional biogeographic history are paramount for inferring macroevolutionary processes.

Strengthening global health security by improving disease surveillance in remote rural areas of low-income and middle-income countries.

The COVID-19 pandemic has underscored the need to strengthen national surveillance systems to protect a globally connected world. In low-income and middle-income countries, zoonotic disease surveillance has advanced considerably in the past two decades. However, surveillance efforts often prioritise urban and adjacent rural communities. Communities in remote rural areas have had far less support despite having routine exposure to zoonotic diseases due to frequent contact with domestic and wild animals, and restricted access to health care. Limited disease surveillance in remote rural areas is a crucial gap in global health security. Although this point has been made in the past, practical solutions on how to implement surveillance efficiently in these resource-limited and logistically challenging settings have yet to be discussed. We highlight why investing in disease surveillance in remote rural areas of low-income and middle-income countries will benefit the global community and review current approaches. Using semi-arid regions in Kenya as a case study, we provide a practical approach by which surveillance in remote rural areas can be strengthened and integrated into existing systems. This Viewpoint represents a transition from simply highlighting the need for a more holistic approach to disease surveillance to a solid plan for how this outcome might be achieved.

Phylogenomic systematics of the spotted skunks (Carnivora, Mephitidae, Spilogale): Additional species diversity and Pleistocene climate change as a major driver of diversification.

Four species of spotted skunks (Carnivora, Mephitidae, Spilogale) are currently recognized: Spilogale angustifrons, S. gracilis, S. putorius, and S. pygmaea. Understanding species boundaries within this group is critical for effective conservation given that regional populations or subspecies (e.g., S. p. interrupta) have experienced significant population declines. Further, there may be currently unrecognized diversity within this genus as some taxa (e.g., S. angustifrons) and geographic regions (e.g., Central America) never have been assessed using DNA sequence data. We analyzed species limits and diversification patterns in spotted skunks using multilocus nuclear (ultraconserved elements) and mitochondrial (whole mitogenomes and single gene analysis) data sets from broad geographic sampling representing all currently recognized species and subspecies. We found a high degree of genetic divergence among Spilogale that reflects seven distinct species and eight unique mitochondrial lineages. Initial divergence between S. pygmaea and all other Spilogale occurred in the Early Pliocene (~ 5.0 million years ago). Subsequent diversification of the remaining Spilogale into an "eastern" and a "western" lineage occurred during the Early Pleistocene (~1.5 million years ago). These two lineages experienced temporally coincident patterns of diversification at ~0.66 and ~0.35 million years ago into two and ultimately three distinct evolutionary units, respectively. Diversification was confined almost entirely within the Pleistocene during a timeframe characterized by alternating glacial-interglacial cycles, with the origin of this diversity occurring in northeastern Mexico and the southwestern United States of America. Mitochondrial-nuclear discordance was recovered across three lineages in geographic regions consistent with secondary contact, including a distinct mitochondrial lineage confined to the Sonoran Desert. Our results have direct consequences for conservation of threatened populations, or species, as well as for our understanding of the evolution of delayed implantation in this enigmatic group of small carnivores.

Preserve a Voucher Specimen! The Critical Need for Integrating Natural History Collections in Infectious Disease Studies.

Despite being nearly 10 months into the COVID-19 (coronavirus disease 2019) pandemic, the definitive animal host for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causal agent of COVID-19, remains unknown. Unfortunately, similar problems exist for other betacoronaviruses, and no vouchered specimens exist to corroborate host species identification for most of these pathogens. This most basic information is critical to the full understanding and mitigation of emerging zoonotic diseases. To overcome this hurdle, we recommend that host-pathogen researchers adopt vouchering practices and collaborate with natural history collections to permanently archive microbiological samples and host specimens. Vouchered specimens and associated samples provide both repeatability and extension to host-pathogen studies, and using them mobilizes a large workforce (i.e., biodiversity scientists) to assist in pandemic preparedness. We review several well-known examples that successfully integrate host-pathogen research with natural history collections (e.g., yellow fever, hantaviruses, helminths). However, vouchering remains an underutilized practice in such studies. Using an online survey, we assessed vouchering practices used by microbiologists (e.g., bacteriologists, parasitologists, virologists) in host-pathogen research. A much greater number of respondents permanently archive microbiological samples than archive host specimens, and less than half of respondents voucher host specimens from which microbiological samples were lethally collected. To foster collaborations between microbiologists and natural history collections, we provide recommendations for integrating vouchering techniques and archiving of microbiological samples into host-pathogen studies. This integrative approach exemplifies the premise underlying One Health initiatives, providing critical infrastructure for addressing related issues ranging from public health to global climate change and the biodiversity crisis.

Volunteer based approach to dog vaccination campaigns to eliminate human rabies: Lessons from Laikipia County, Kenya.

BACKGROUND: An estimated 59,000 people die from rabies annually, with 99% of those deaths attributable to bites from domestic dogs (Canis lupus familiaris). This preventable Neglected Tropical Disease has a large impact across continental Africa, especially for rural populations living in close contact with livestock and wildlife. Mass vaccinations of domestic dogs are effective at eliminating rabies but require large amounts of resources, planning, and political will to implement. Grassroots campaigns provide an alternative method to successful implementation of rabies control but remain understudied in their effectiveness to eliminate the disease from larger regions. METHODOLOGY/PRINCIPAL FINDINGS: We report on the development, implementation, and effectiveness of a grassroots mass dog rabies vaccination campaign in Kenya, the Laikipia Rabies Vaccination Campaign. During 2015-2017, a total of 13,155 domestic dogs were vaccinated against rabies in 17 communities covering approximately 1500 km2. Based on an estimated population size of 34,275 domestic dogs, percent coverages increased across years, from 2% in 2015 to 24% in 2017, with only 3 of 38 community-years of vaccination exceeding the 70% target. The average cost of vaccinating an animal was $3.44 USD with in-kind contributions and $7.44 USD without in-kind contributions. CONCLUSIONS/SIGNIFICANCE: The evolution of the Laikipia Rabies Vaccination Campaign from a localized volunteer-effort to a large-scale program attempting to eliminate rabies at the landscape scale provides a unique opportunity to examine successes, failures, and challenges facing grassroots campaigns. Success, in the form of vaccinating more dogs across the study area, was relatively straightforward to achieve. However, lack of effective post-vaccination monitoring and education programs, limited funding, and working in diverse community types appeared to hinder achievement of 70% coverage levels. These results indicate that grassroots campaigns will inevitably be faced with a philosophical question regarding the value of local impacts versus their contributions to a larger effort to eliminate rabies at the regional, country, or global scale.

Phylogeography of a widespread small carnivore, the western spotted skunk (Spilogale gracilis) reveals temporally variable signatures of isolation across western North America.

We analyzed phylogeographic patterns in the western spotted skunk, Spilogale gracilis Merriam, 1890 (Carnivora: Mephitidae) in relation to historical events associated with Pre-Pleistocene Divergence (PPD) and Quaternary climate change (QCC) using mitochondrial DNA from 97 individuals distributed across Western North America. Divergence times were generated using BEAST to estimate when isolation in putative refugia occurred. Patterns and timing of demographic expansion was performed using Bayesian skyline plot. Putative climatic refugia resulting from Quaternary climate change were identified using paleoecological niche modeling and divergence dates compared to major vicariant events associated with Pre-Pleistocene conditions. We recovered three major mitochondrial clades corresponding to western North America (California, Baja, and across the Great Basin), east-central North America (Texas, central Mexico, New Mexico), and southwestern Arizona/northwestern Mexico. Time to most recent common ancestor for S. gracilis occurred ~1.36 Ma. Divergence times for each major clade occurred between 0.25 and 0.12 Ma, with signature of population expansion occurring 0.15 and 0.10 Ma. Ecological niche models identified three potential climatic refugia during the Last Interglacial, (1) west coast of California and Oregon, (2) northwestern Mexico, and (3) southern Texas/northeastern Mexico as well as two refugia during the Last Glacial Maximum, (1) western USA and (2) southern Texas/northeastern Mexico. This study supports PPD in shaping species-level diversity compared to QCC-driven changes at the intraspecific level for Spilogale, similar to the patterns reported for other small mammals (e.g., rodents and bats). Phylogeographic patterns also appear to have been shaped by both habitat and river vicariance, especially across the desert southwest. Further, continuing climate change during the Holocene coupled with anthropogenic modifications during the Anthropocene appears to be removing both of these barriers to current dispersal of western spotted skunks.

Multilocus phylogeography of a widespread savanna-woodland-adapted rodent reveals the influence of Pleistocene geomorphology and climate change in Africa's Zambezi region.

Understanding historical influences of climate and physiographic barriers in shaping patterns of biodiversity remains limited for many regions of the world. For mammals of continental Africa, phylogeographic studies, particularly for West African lineages, implicate both geographic barriers and climate oscillations in shaping small mammal diversity. In contrast, studies for southern African species have revealed conflicting phylogenetic patterns for how mammalian lineages respond to both climate change and geologic events such as river formation, especially during the Pleistocene. However, these studies were often biased by limited geographic sampling or exclusively focused on large-bodied taxa. We exploited the broad southern African distribution of a savanna-woodland-adapted African rodent, Gerbilliscus leucogaster (bushveld gerbil) and generated mitochondrial, autosomal and sex chromosome data to quantify regional signatures of climatic and vicariant biogeographic phenomena. Results indicate the most recent common ancestor for all G. leucogaster lineages occurred during the early Pleistocene. We documented six divergent mitochondrial lineages that diverged ~0.270-0.100 mya, each of which was geographically isolated during periods characterized by alterations to the course of the Zambezi River and its tributaries as well as regional 'megadroughts'. Results demonstrate the presence of a widespread lineage exhibiting demographic expansion ~0.065-0.035 mya, a time that coincides with savanna-woodland expansion across southern Africa. A multilocus autosomal perspective revealed the influence of the Kafue River as a current barrier to gene flow and regions of secondary contact among divergent mitochondrial lineages. Our results demonstrate the importance of both climatic fluctuations and physiographic vicariance in shaping the distribution of southern African biodiversity.

Context-dependent effects of large-wildlife declines on small-mammal communities in central Kenya.

Many species of large wildlife have declined drastically worldwide. These reductions often lead to profound shifts in the ecology of entire communities and ecosystems. However, the effects of these large-wildlife declines on other taxa likely hinge upon both underlying abiotic properties of these systems and on the types of secondary anthropogenic changes associated with wildlife loss, making impacts difficult to predict. To better understand how these important contextual factors determine the consequences of large-wildlife declines on other animals in a community, we examined the effects of three common forms of large-wildlife loss (removal without replacement [using fences], removal followed by replacement with domestic stock, and removal accompanied by crop agricultural use) on small-mammal abundance, diversity, and community composition, in landscapes that varied in several abiotic attributes (rainfall, soil fertility, land-use intensity) in central Kenya. We found that small-mammal communities were indeed heavily impacted by all forms of large-wildlife decline, showing, on average: (1) higher densities, (2) lower species richness per site, and (3) different species assemblages in sites from which large wildlife were removed. However, the nature and magnitude of these effects were strongly context dependent. Rainfall, type of land-use change, and the interaction of these two factors were key predictors of both the magnitude and type of responses of small mammals. The strongest effects, particularly abundance responses, tended to be observed in low-rainfall areas. Whereas isolated wildlife removal primarily led to increased small-mammal abundance, wildlife removal associated with secondary uses (agriculture, domestic stock) had much more variable effects on abundance and stronger impacts on diversity and composition. Collectively, these results (1) highlight the importance of context in determining the impacts of large-wildlife decline on small-mammal communities, (2) emphasize the challenges in extrapolating results from controlled experimental studies to predict the effects of wildlife declines that are accompanied by secondary land-uses, and (3) suggest that, because of the context-dependent nature of the responses to large-wildlife decline, large-wildlife status alone cannot be reliably used to predict small-mammal community changes.

Mitochondrial DNA and karyotypic data confirm the presence of Mus indutus and Mus minutoides (Mammalia, Rodentia, Muridae, Nannomys) in Botswana.

We use a combination of cytochrome b sequence data and karyological evidence to confirm the presence of Mus indutus and Mus minutoides in Botswana. Our data include sampling from five localities from across the country, including one site in northwestern Botswana where both species were captured in syntopy. Additionally, we find evidence for two mitochondrial lineages of M. minutoides in northwestern Botswana that differ by 5% in sequence variation. Also, we report that M. minutoides in Botswana have the 2n=34 karyotype with the presence of a (X.1) sex-autosome translocation.

Microbiome analysis among bats describes influences of host phylogeny, life history, physiology and geography.

Metagenomic methods provide an experimental approach to inform the relationships between hosts and their microbial inhabitants. Previous studies have provided the conceptual realization that microbiomes are dynamic among hosts and the intimacy of relation between micro- and macroorganisms. Here, we present an intestinal microflora community analysis for members of the order Chiroptera and investigate the relative influence of variables in shaping observed microbiome relationships. The variables ranged from those considered to have ancient and long-term influences (host phylogeny and life history) to the relatively transient variable of host reproductive condition. In addition, collection locality data, representing the geographic variable, were included in analyses. Results indicate a complex influence of variables in shaping sample relationships in which signal for host phylogeny is recovered at broad taxonomic levels (family), whereas intrafamilial analyses disclosed various degrees of resolution for the remaining variables. Although cumulative probabilities of assignment indicated both reproductive condition and geography influenced relationships, comparison of ecological measures among groups revealed statistical differences between most variable classifications. For example, ranked ecological diversity was associated with host phylogeny (deeper coalescences among families were associated with more microfloral diversity), dietary strategy (herbivory generally retained higher diversity than carnivory) and reproductive condition (reproductively active females displayed more diverse microflora than nonreproductive conditions). Overall, the results of this study describe a complex process shaping microflora communities of wildlife species as well as provide avenues for future research that will further inform the nature of symbiosis between microflora communities and hosts.