Using noninvasive metagenomics to characterize viral communities from wildlife

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.     

Population structure of two rabies hosts relative to the known distribution of rabies virus variants in Alaska

For pathogens that infect multiple species, the distinction between reservoir hosts and spillover hosts is often difficult. In Alaska, three variants of the arctic rabies virus exist with distinct spatial distributions. We tested the hypothesis that rabies virus variant distribution corresponds to the population structure of the primary rabies hosts in Alaska, arctic foxes (Vulpes lagopus) and red foxes (Vulpes vulpes) to possibly distinguish reservoir and spillover hosts. We used mitochondrial DNA (mtDNA) sequence and nine microsatellites to assess population structure in those two species. mtDNA structure did not correspond to rabies virus variant structure in either species. Microsatellite analyses gave varying results. Bayesian clustering found two groups of arctic foxes in the coastal tundra region, but for red foxes it identified tundra and boreal types. Spatial Bayesian clustering and spatial principal components analysis identified 3 and 4 groups of arctic foxes, respectively, closely matching the distribution of rabies virus variants in the state. Red foxes, conversely, showed eight clusters comprising two regions (boreal and tundra) with much admixture. These results run contrary to previous beliefs that arctic fox show no fine‐scale spatial population structure. While we cannot rule out that the red fox is part of the maintenance host community for rabies in Alaska, the distribution of virus variants appears to be driven primarily by the arctic fox. Therefore, we show that host population genetics can be utilized to distinguish between maintenance and spillover hosts when used in conjunction with other approaches.     

Genes on the edge: A framework to detect genetic diversity imperiled by climate change

Ongoing global warming is disrupting several ecological and evolutionary processes, spanning different levels of biological organization. Species are expected to shift their ranges as a response to climate change, with relevant implications to peripheral populations at the trailing and leading edges. Several studies have analyzed the exposure of species to climate change but few have explored exposure at the intraspecific level. We introduce a framework to forecast exposure to climate change at the intraspecific level. We build on existing methods by combining correlative species distribution models, a model of species range dynamics, and a model of phylogeographic interpolation. We demonstrate the framework by applying it to 20 Iberian amphibian and reptile species. Our aims were to: (a) identify which species and intraspecific lineages will be most exposed to future climate change; (b) test if nucleotide diversity at the edges of species ranges are significantly higher or lower than on the overall range; and (c) analyze if areas of higher species gain, loss, and turnover coincide with those predicted for lineages richness and nucleotide diversity. We found that about 80% of the studied species are predicted to contract their range. Within each species, some lineages were predicted to contract their range, while others were predicted to maintain or expand it. Therefore, estimating the impacts of climate change at the species level only can underestimate losses at the intraspecific level. Some species had significant high amount of nucleotide at the trailing or leading edge, or both, but we did not find a consistent pattern across species. Spatial patterns of species richness, gain, loss, and turnover were fairly concurrent with lineages richness and nucleotide diversity. Our results support the need for increased attention to intraspecific diversity regarding monitoring and conservation strategies under climate change.     

The quick and the dead: microbial demography at the yeast thermal limit

The niche of microorganisms is determined by where their populations can expand. Populations can fail to grow because of high death or low birth rates, but these are challenging to measure in microorganisms. We developed a novel technique that enables single‐cell measurement of age‐structured birth and death rates in the budding yeast, Saccharomyces cerevisiae, and used this method to study responses to heat stress in a genetically diverse panel of strains. We find that individual cells show significant heterogeneity in their rates of birth and death during heat stress. Genotype‐by‐environment effects on processes that regulate asymmetric cell division contribute to this heterogeneity. These lead to either premature senescence or early life mortality during heat stress, and we find that a mitochondrial inheritance defect explains the early life mortality phenotype of one of the strains we studied. This study demonstrates how the interplay of physiology, genetic variation and environmental variables influence where microbial populations survive and flourish.     

Demographic and environmental drivers of metagenomic viral diversity in vampire bats

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.     

Long‐term change in a behavioural trait: truncated spawning distribution and demography in Northeast Arctic cod

Harvesting may be a potent driver of demographic change and contemporary evolution, which both may have great impacts on animal populations. Research has focused on changes in phenotypic traits that are easily quantifiable and for which time series exist, such as size, age, sex, or gonad size, whereas potential changes in behavioural traits have been under‐studied. Here, we analyse potential drivers of long‐term changes in a behavioural trait for the Northeast Arctic stock of Atlantic cod Gadus morhua, namely choice of spawning location. For 104 years (1866–1969), commercial catches were recorded annually and reported by county along the Norwegian coast. During this time period, spawning ground distribution has fluctuated with a trend towards more northerly spawning. Spawning location is analysed against a suite of explanatory factors including climate, fishing pressure, density dependence, and demography. We find that demography (age or age at maturation) had the highest explanatory power for variation in spawning location, while climate had a limited effect below statistical significance. As to potential mechanisms, some effects of climate may act through demography, and explanatory variables for demography may also have absorbed direct evolutionary change in migration distance for which proxies were unavailable. Despite these caveats, we argue that fishing mortality, either through demographic or evolutionary change, has served as an effective driver for changing spawning locations in cod, and that additional explanatory factors related to climate add no significant information.     

Caldesmon affects actin organization at the leading edge and inhibits cell migration

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than a fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibers and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced more than 1,5-fold accumulation of stress fibers only in control cells, but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type, but not in the cells with low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.     

Precocious reproduction increases at the leading edge of a mangrove range expansion

Climate change‐driven shifts in species ranges are ongoing and expected to increase. However, life‐history traits may interact with climate to influence species ranges, potentially accelerating or slowing range shifts in response to climate change. Tropical mangroves have expanded their ranges poleward in the last three decades. Here, we report on a shift at the range edge in life‐history traits related to reproduction and dispersal. With a common garden experiment and field observations, we show that Rhizophora mangle individuals from northern populations reproduce at a younger age than those from southern populations. In a common garden at the northern range limit, 38% of individuals from the northernmost population were reproductive by age 2, but less than 10% of individuals from the southernmost population were reproductive by the same age, with intermediate amounts of reproduction from intermediate latitudes. Field observations show a similar pattern of younger reproductive individuals toward the northern range limit. We also demonstrate a shift toward larger propagule size in populations at the leading range edge, which may aid seedling growth. The substantial increase in precocious reproduction at the leading edge of the R. mangle range could accelerate population growth and hasten the expansion of mangroves into salt marshes.     

Habitat fragmentation is associated with dietary shifts and microbiota variability in common vampire bats

Host ecological factors and external environmental factors are known to influence the structure of gut microbial communities, but few studies have examined the impacts of environmental changes on microbiotas in free‐ranging animals. Rapid land‐use change has the potential to shift gut microbial communities in wildlife through exposure to novel bacteria and/or by changing the availability or quality of local food resources. The consequences of such changes to host health and fitness remain unknown and may have important implications for pathogen spillover between humans and wildlife. To better understand the consequences of land‐use change on wildlife microbiotas, we analyzed long‐term dietary trends, gut microbiota composition, and innate immune function in common vampire bats (Desmodus rotundus) in two nearby sites in Belize that vary in landscape structure. We found that vampire bats living in a small forest fragment had more homogenous diets indicative of feeding on livestock and shifts in microbiota heterogeneity, but not overall composition, compared to those living in an intact forest reserve. We also found that irrespective of sampling site, vampire bats which consumed relatively more livestock showed shifts in some core bacteria compared with vampire bats which consumed relatively less livestock. The relative abundance of some core microbiota members was associated with innate immune function, suggesting that future research should consider the role of the host microbiota in immune defense and its relationship to zoonotic infection dynamics. We suggest that subsequent homogenization of diet and habitat loss through livestock rearing in the Neotropics may lead to disruption to the microbiota that could have downstream impacts on host immunity and cross‐species pathogen transmission.     

Multilocus nuclear DNA markers reveal population structure and demography of Anopheles minimus

Utilization of multiple putatively neutral DNA markers for inferring evolutionary history of species population is considered to be the most robust approach. Molecular population genetic studies have been conducted in many species of Anopheles genus, but studies based on single nucleotide polymorphism (SNP) data are still very scarce. Anopheles minimus is one of the principal malaria vectors of Southeast (SE) Asia including the Northeastern (NE) India. Although population genetic studies with mitochondrial genetic variation data have been utilized to infer phylogeography of the SE Asian populations of this species, limited information on the population structure and demography of Indian An. minimus is available. We herewith have developed multilocus nuclear genetic approach with SNP markers located in X chromosome of An. minimus in eight Indian and two SE Asian population samples (121 individual mosquitoes in total) to infer population history and test several hypotheses on the phylogeography of this species. While the Thai population sample of An. minimus presented the highest nucleotide diversity, majority of the Indian samples were also fairly diverse. In general, An. minimus populations were moderately substructured in the distribution range covering SE Asia and NE India, largely falling under three distinct genetic clusters. Moreover, demographic expansion events could be detected in the majority of the presently studied populations of An. minimus. Additional DNA sequencing of the mitochondrial COII region in a subset of the samples (40 individual mosquitoes) corroborated the existing hypothesis of Indian An. minimus falling under the earlier reported mitochondrial lineage B.     

Phenotypic variability of rusty crayfish (Faxonius rusticus) at the leading edge of its riverine invasion

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Genetic differentiation of the pine processionary moth at the southern edge of its range: contrasting patterns between mitochondrial and nuclear markers

The pine processionary moth (Thaumetopoea pityocampa) is an important pest of coniferous forests at the southern edge of its range in Maghreb. Based on mitochondrial markers, a strong genetic differentiation was previously found in this species between western (pityocampa clade) and eastern Maghreb populations (ENA clade), with the contact zone between the clades located in Algeria. We focused on the moth range in Algeria, using both mitochondrial (a 648 bp fragment of the tRNA‐cox2) and nuclear (11 microsatellite loci) markers. A further analysis using a shorter mtDNA fragment and the same microsatellite loci was carried out on a transect in the contact zone between the mitochondrial clades. Mitochondrial diversity showed a strong geographical structure and a well‐defined contact zone between the two clades. In particular, in the pityocampa clade, two inner subclades were found whereas ENA did not show any further structure. Microsatellite analysis outlined a different pattern of differentiation, with two main groups not overlapping with the mitochondrial clades. The inconsistency between mitochondrial and nuclear markers is probably explained by sex‐biased dispersal and recent afforestation efforts that have bridged isolated populations.     

Genetic diversity and structuring across the range of a widely distributed ladybird: focus on rear‐edge populations phenotypically divergent

Population genetics and phenotypic structures are often predicted to vary along the geographic range of a species. This phenomenon would be accentuated for species with large range areas, with discontinuities and marginal populations. We herein compare the genetic patterns of central populations of Coccinella septempunctata L. with those of two phenotypically differentiated populations considered as rear‐edge populations and subspecies based on phenotype (Algeria and Japan). According to the central‐marginal model and expected characteristics of rear‐edge populations, we hypothesize that these rear‐edge populations have (1) a reduced genetic diversity, resulting from their relative isolation over long periods of time, (2) a higher population genetic differentiation, explained by low contemporary gene flow levels, and (3) a relationship between genetic diversity characteristics and phenotypes, due to historical isolation and/or local adaptation. Based on genotyping of 28 populations for 18 microsatellite markers, several levels of regional genetic diversity and differentiation are observed between and within populations, according to their localization: low within‐population genetic diversity and higher genetic differentiation of rear‐edge populations. The genetic structuring clearly dissociates the Algerian and Eastern Asia populations from the others. Geographical patterns of genetic diversity and differentiation support the hypothesis of the central‐marginal model. The pattern observed is in agreement with the phenotypic structure across species range. A clear genetic break between populations of Algeria, the Eastern Asia, and the remaining populations is a dominant feature of the data. Differential local adaptations, absence of gene flow between marginal and central populations, and/or incapacity to mate after colonization, have contributed to their distinct genotypic and phenotypic characteristics.     

Trapped within the city: integrating demography,time since isolation and population‐specific traits to assess the genetic effects of urbanization

Urbanization is a severe form of habitat fragmentation that can cause many species to be locally extirpated and many others to become trapped and isolated within an urban matrix. The role of drift in reducing genetic diversity and increasing genetic differentiation is well recognized in urban populations. However, explicit incorporation and analysis of the demographic and temporal factors promoting drift in urban environments are poorly studied. Here, we genotyped 15 microsatellites in 320 fire salamanders from the historical city of Oviedo (Est. 8th century) to assess the effects of time since isolation, demographic history (historical effective population size; N_e) and patch size on genetic diversity, population structure and contemporary N_e. Our results indicate that urban populations of fire salamanders are highly differentiated, most likely due to the recent N_e declines, as calculated in coalescence analyses, concomitant with the urban development of Oviedo. However, urbanization only caused a small loss of genetic diversity. Regression modelling showed that patch size was positively associated with contemporary N_e, while we found only moderate support for the effects of demographic history when excluding populations with unresolved history. This highlights the interplay between different factors in determining current genetic diversity and structure. Overall, the results of our study on urban populations of fire salamanders provide some of the very first insights into the mechanisms affecting changes in genetic diversity and population differentiation via drift in urban environments, a crucial subject in a world where increasing urbanization is forecasted.     

Genomics of clinal variation in Drosophila: disentangling the interactions of selection and demography

Clines in phenotypes and genotype frequencies across environmental gradients are commonly taken as evidence for spatially varying selection. Classical examples include the latitudinal clines in various species of Drosophila, which often occur in parallel fashion on multiple continents. Today, genomewide analysis of such clinal systems provides a fantastic opportunity for unravelling the genetics of adaptation, yet major challenges remain. A well‐known but often neglected problem is that demographic processes can also generate clinality, independent of or coincident with selection. A closely related issue is how to identify true genic targets of clinal selection. In this issue of Molecular Ecology, three studies illustrate these challenges and how they might be met. Bergland et al. report evidence suggesting that the well‐known parallel latitudinal clines in North American and Australian D. melanogaster are confounded by admixture from Africa and Europe, highlighting the importance of distinguishing demographic from adaptive clines. In a companion study, Machado et al. provide the first genomic comparison of latitudinal differentiation in D. melanogaster and its sister species D. simulans. While D. simulans is less clinal than D. melanogaster, a significant fraction of clinal genes is shared between both species, suggesting the existence of convergent adaptation to clinaly varying selection pressures. Finally, by drawing on several independent sources of evidence, Bo?i?evi? et al. identify a functional network of eight clinal genes that are likely involved in cold adaptation. Together, these studies remind us that clinality does not necessarily imply selection and that separating adaptive signal from demographic noise requires great effort and care.     

Genetic architecture of a body colour cline in Drosophila americana

Phenotypic variation within a species is often structured geographically in clines. In Drosophila americana, a longitudinal cline for body colour exists within North America that appears to be due to local adaptation. The tan and ebony genes have been hypothesized to contribute to this cline, with alleles of both genes that lighten body colour found in D. americana. These alleles are similar in sequence and function to the allele fixed in D. americana's more lightly pigmented sister species, Drosophila novamexicana. Here, we examine the frequency and geographic distribution of these D. novamexicana‐like alleles in D. americana. Among alleles from over 100 strains of D. americana isolated from 21 geographic locations, we failed to identify additional alleles of tan or ebony with as much sequence similarity to D. novamexicana as the D. novamexicana‐like alleles previously described. However, using genetic analysis of 51 D. americana strains derived from 20 geographic locations, we identified one new allele of ebony and one new allele of tan segregating in D. americana that are functionally equivalent to the D. novamexicana allele. An additional 5 alleles of tan also showed marginal evidence of functional similarity. Given the rarity of these alleles, however, we conclude that they are unlikely to be driving the pigmentation cline. Indeed, phenotypic distributions of the 51 backcross populations analysed indicate a more complex genetic architecture, with diversity in the number and effects of loci altering pigmentation observed both within and among populations of D. americana. This genetic heterogeneity poses a challenge to association studies and genomic scans for clinal variation, but might be common in natural populations.     

Low Genetic Diversity Suggests a Single Introduction and Recent Spread of Tomato chlorosis virus in Brazil

By comparing the partial nucleotide sequences of the heat shock protein HSP70 homologue gene, we assessed the genetic diversity of Brazilian tomato isolates of Tomato chlorosis virus (ToCV), as well as their relationship with other ToCV isolates found worldwide. The Brazilian ToCV isolates shared 99.9–100% nucleotide identity, which indicates low genetic diversity. Brazilian ToCV isolates showed a closer evolutionary relationship to those from Mediterranean countries. Based on these results, the origin of Brazilian ToCV isolates and the possible number of introductions of the virus into Brazil are discussed.     

Effects of cyanobacteria,fish kairomones,and the presence of ostracods on the demography of Simocephalus vetulus (Cladocera)

Simocephalus vetulus is a large (2.0–4.0 mm at maturity) cladoceran often found in the littoral region of lakes and ponds, and capable of moderate growth rates even on poor‐quality cyanobacterial diets. It frequently co‐occurs with fishes and similar sized ostracods such as Heterocypris incongruens, but little is known of its response to fish kairomones or its interactions with potential competitors. We studied the demographic responses of S. vetulus fed the green alga Scenedesmus acutus, Microcystis cf. aeruginosa strain A, Microcystis cf. aeruginosa strain B, or Limnothrix sp. Experiments were conducted separately and together in the presence of Heterocypris incongruens and cichlid fish (Oreochromis) kairomones. A diet of Limnothrix sp. resulted in the lowest population growth rate (0.21±0.023 d^?1), while on diets of S. acutus or Microcystis, population growth was higher (0.30±0.009 d^?1). The presence of ostracods resulted in significantly higher growth rates of S. vetulus fed Limnothrix (0.33±0.01 d^?1), but not Microcystis or S. acutus. Regardless of the diet, the presence of fish kairomones resulted in significantly higher growth rates as compared with controls, particularly when ostracods were also present. Coexistence with ostracods may be beneficial to S. vetulus, particularly when food quality is poor.