Nanopore-Based Surveillance of Zoonotic Bacterial Pathogens in Farm-Dwelling Peridomestic Rodents.

The effective control of rodent populations on farms is crucial for food safety, as rodents are reservoirs and vectors for several zoonotic pathogens. Clear links have been identified between rodents and farm-level outbreaks of pathogens throughout Europe and Asia; however, comparatively little research has been devoted to studying the rodent-agricultural interface in the USA. Here, we address this knowledge gap by metabarcoding bacterial communities of rodent pests collected from Minnesota and Wisconsin food animal farms. We leveraged the Oxford Nanopore MinION sequencer to provide a rapid real-time survey of putative zoonotic foodborne pathogens, among others. Rodents were live trapped (n = 90) from three dairy and mixed animal farms. DNA extraction was performed on 63 rodent colons along with 2 shrew colons included as outgroups in the study. Full-length 16S amplicon sequencing was performed. Our farm-level rodent-metabarcoding data indicate the presence of multiple foodborne pathogens, including Salmonella spp., Campylobacter spp., Staphylococcus aureus, and Clostridium spp., along with many mastitis pathogens circulating within five rodent species (Microtus pennsylvanicus, Mus musculus, Peromyscus leucopus, Peromyscus maniculatus, and Rattus norvegicus) and a shrew (Blarina brevicauda). Interestingly, we observed a higher abundance of enteric pathogens (e.g., Salmonella) in shrew feces compared to the rodents analyzed in our study. Knowledge gained from our research efforts will directly inform and improve farm-level biosecurity efforts and public health interventions to reduce future outbreaks of foodborne and zoonotic disease.

The complexities of female mate choice and male polymorphisms: Elucidating the role of genetics, age, and mate-choice copying.

Genetic, life history, and environmental factors dictate patterns of variation in sexual traits within and across populations, and thus the action and outcome of sexual selection. This study explores patterns of inheritance, diet, age, and mate-choice copying on the expression of male sexual signals and associated female mate choice in a phenotypically diverse group of Schizocosa wolf spiders. Focal spiders exhibit one of two male phenotypes: 'ornamented' males possess large black brushes on their forelegs, and 'non-ornamented' males possess no brushes. Using a quantitative genetics breeding design in a mixed population of ornamented/non-ornamented males, we found a strong genetic basis to male phenotype and female choice. We also found that some ornamented males produced some sons with large brushes and others with barely visible brushes. Results of diet manipulations and behavioral mating trials showed no influence of diet on male phenotype or female mate choice. Age post maturation, however, influenced mate choice, with younger females being more likely to mate with ornamented males. A mate-choice copying experiment found that, following observations of another female's mate choice/copulation, virgin mature females tended to match the mate choice (ornamented vs. non-ornamented males) of the females they observed. Finally, analyses of genetic variation across phenotypically pure (only one male phenotype present) vs. mixed (both phenotypes present) populations revealed genetic distinction between phenotypes in phenotypically-pure populations, but no distinctionin phenotypically-mixed populations. The difference in patterns of genetic differentiation and mating across geographic locations suggests a complex network of factors contributing to the outcome of sexual selection.

Genetic differences in hemoglobin function between highland and lowland deer mice.

In high-altitude vertebrates, adaptive changes in blood-O(2) affinity may be mediated by modifications of hemoglobin (Hb) structure that affect intrinsic O(2) affinity and/or responsiveness to allosteric effectors that modulate Hb-O(2) affinity. This mode of genotypic specialization is considered typical of mammalian species that are high-altitude natives. Here we investigated genetically based differences in Hb-O(2) affinity between highland and lowland populations of the deer mouse (Peromyscus maniculatus), a generalist species that has the broadest altitudinal distribution of any North American mammal. The results of a combined genetic and proteomic analysis revealed that deer mice harbor a high level of Hb isoform diversity that is attributable to allelic polymorphism at two tandemly duplicated alpha-globin genes and two tandemly duplicated beta-globin genes. This high level of isoHb diversity translates into a correspondingly high level of interindividual variation in Hb functional properties. O(2) equilibrium experiments revealed that the Hbs of highland mice exhibit slightly higher intrinsic O(2) affinities and significantly lower Cl(-) sensitivities relative to the Hbs of lowland mice. The experiments also revealed distinct biochemical properties of deer mouse Hb related to the anion-dependent allosteric regulation of O(2) affinity. In conjunction with previous findings, our results demonstrate that modifications of Hb structure that alter allosteric anion sensitivity play an important role in the adaptive fine-tuning of blood-O(2) affinity.

Evolutionary and functional properties of a two-locus beta-globin polymorphism in Indian house mice.

Electrophoretic surveys of hemoglobin (Hb) polymorphism in house mice from South Asia and the Middle East have revealed that two alternative beta-globin haplotypes, Hbb(d) and Hbb(p), are often present at intermediate frequencies in geographically disparate populations. Both haplotypes harbor two functionally distinct beta-globin paralogs, HBB-T1 (which encodes the beta-chain subunits of the major Hb isoform) and HBB-T2 (which encodes the beta-chains of the minor Hb isoform). The Hbb(d) and Hbb(p) haplotypes share identical HBB-T1 alleles, but products of the alternative HBB-T2 alleles (d(minor) and p(minor)) are distinguished by two amino acid substitutions. To investigate the possible adaptive significance of the Hbb(d)/Hbb(p) polymorphism we conducted a population genetic analysis of the duplicated beta-globin genes of Indian house mice (Mus castaneus) in conjunction with experimental studies of Hb function in inbred strains of mice that carry the alternative Hbb(d) and Hbb(p) haplotypes. The main objectives of this study were (i) to characterize patterns of nucleotide polymorphism and linkage disequilibrium in the duplicated beta-globin genes of M. castaneus, (ii) to test the hypothesis that the Hbb(d) and Hbb(p) haplotypes are maintained as a balanced polymorphism, and (iii) to assess whether allelic differences in the alternative minor Hb isoforms (d(minor) and p(minor)) are associated with different O(2)-binding properties. A multilocus analysis of polymorphism and divergence revealed that levels of diversity at the HBB-T2 gene exceeded neutral expectations, and reconstructed haplotype networks for both beta-globin paralogs revealed extensive allele sharing with several other closely related species of Mus. However, despite this suggestive evidence for balancing selection, O(2)-equilibrium curves revealed no discernible functional differences between red cell lysates containing the d(minor) and p(minor) Hb isoforms. If the d(minor) and p(minor) alleles are maintained as a balanced polymorphism, our results indicate that the associated fitness variance is not directly related to respiratory functions of Hb.

Evolutionary and functional insights into the mechanism underlying high-altitude adaptation of deer mouse hemoglobin.

Adaptive modifications of heteromeric proteins may involve genetically based changes in single subunit polypeptides or parallel changes in multiple genes that encode distinct, interacting subunits. Here we investigate these possibilities by conducting a combined evolutionary and functional analysis of duplicated globin genes in natural populations of deer mice (Peromyscus maniculatus) that are adapted to different elevational zones. A multilocus analysis of nucleotide polymorphism and linkage disequilibrium revealed that high-altitude adaptation of deer mouse hemoglobin involves parallel functional differentiation at multiple unlinked gene duplicates: two alpha-globin paralogs on chromosome 8 and two beta-globin paralogs on chromosome 1. Differences in O(2)-binding affinity of the alternative beta-chain hemoglobin isoforms were entirely attributable to allelic differences in sensitivity to 2,3-diphosphoglycerate (DPG), an allosteric cofactor that stabilizes the low-affinity, deoxygenated conformation of the hemoglobin tetramer. The two-locus beta-globin haplotype that predominates at high altitude is associated with suppressed DPG-sensitivity (and hence, increased hemoglobin-O(2) affinity), which enhances pulmonary O(2) loading under hypoxia. The discovery that allelic differences in DPG-sensitivity contribute to adaptive variation in hemoglobin-O(2) affinity illustrates the value of integrating evolutionary analyses of sequence variation with mechanistic appraisals of protein function. Investigation into the functional significance of the deer mouse beta-globin polymorphism was motivated by the results of population genetic analyses which revealed evidence for a history of divergent selection between elevational zones. The experimental measures of O(2)-binding properties corroborated the tests of selection by demonstrating a functional difference between the products of alternative alleles.

Evolution of duplicated beta-globin genes and the structural basis of hemoglobin isoform differentiation in Mus.

The functional diversification of multigene families may be strongly influenced by mechanisms of concerted evolution such as interparalog gene conversion. The beta-globin gene family of house mice (genus Mus) represents an especially promising system for evaluating the effects of gene conversion on the functional divergence of duplicated genes. Whereas the majority of mammalian species possess tandemly duplicated copies of the adult beta-globin gene that are identical in sequence, natural populations of house mice are often polymorphic for distinct two-locus haplotypes that differ in levels of functional divergence between duplicated beta-globin genes, HBB-T1 and HBB-T2. Here, we use a phylogenetic approach to unravel the complex evolutionary history of the HBB-T1 and HBB-T2 paralogs in a taxonomically diverse set of species in the genus Mus. The main objectives of this study were 1) to reconstruct the evolutionary history of the different HBB haplotypes of house mice, 2) to assess the role of recombinational exchange between HBB-T1 and HBB-T2 in promoting concerted evolution, 3) to assess the role of recombinational exchange between HBB-T1 and HBB-T2 in creating chimeric genes, and 4) to assess the structural basis of hemoglobin isoform differentiation in species that possess distinct HBB paralogs. Results of our phylogenetic survey revealed that the HBB-T1 and HBB-T2 genes in different species of Mus exhibit the full range of evolutionary outcomes with respect to levels of interparalog divergence. At one end of the spectrum, the two identical HBB paralogs on the Hbb(s) haplotype (shared by Mus domesticus, Mus musculus, and Mus spretus) represent a classic example of concerted evolution. At the other end of the spectrum, the two distinct HBB paralogs on the Hbb(d), Hbb(p), Hbb(w1), and Hbb(w2) haplotypes (shared by multiple species in the subgenus Mus) show no trace of gene conversion and are distinguished by a number of functionally important amino acid substitutions. Because the possession of distinct HBB paralogs expands the repertoire of functionally distinct hemoglobin isoforms that can be synthesized during fetal development and postnatal life, variation in the level of functional divergence between HBB-T1 and HBB-T2 may underlie important physiological variation within and among species.

Historic hybridization and persistence of a novel mito-nuclear combination in red-backed voles (genus Myodes).

BACKGROUND: The role of hybridization in generating diversity in animals is an active area of discovery and debate. We assess hybridization across a contact zone of northern (Myodes rutilus) and southern (M. gapperi) red-backed voles using variation in skeletal features and both mitochondrial and nuclear loci. This transect extends approximately 550 km along the North Pacific Coast of North America and encompasses 26 populations (n = 485). We establish the history, geographic extent and directionality of hybridization, determine whether hybridization is ongoing, and assess the evolutionary stability of novel genomic combinations. RESULTS: Identification of M. rutilus and M. gapperi based on the degree of closure of the post-palatal bridge was concordant with the distribution of diagnostic nuclear MYH6 alleles; however, an 80 km zone of introgressed populations was identified. The introgressant form is characterized by having mitochondrial haplotypes closely related to the northern M. rutilus on a nuclear background and morphological characteristics of southern M. gapperi. CONCLUSION: Introgression appears to have been historic as pure populations of M. rutilus are now isolated to the north from introgressants or pure M. gapperi by the LeConte Glacier. As we do not find pure M. rutilus or M. gapperi individuals throughout the distribution of the introgressant form, it appears that the introgressants are a self-sustaining entity not requiring continued hybridization between pure parental forms to generate this novel combination of characters.

Postglacial expansion of the southern red-backed vole (Clethrionomys gapperi) in North America.

Dynamic climatic oscillations of the Pleistocene dramatically changed the distributions of high latitude species. Molecular investigations of a variety of organisms show that processes of postglacial colonization of boreal regions were more complex than initially thought. Phylogeographical and coalescent analyses were conducted on partial sequences of the cytochrome b gene (600 bp) from 64 individuals of Clethrionomys gapperi from North Carolina, Pennsylvania, Minnesota, Idaho, Washington, British Columbia, Northwest Territories, and Alaska to test hypotheses relating to Pleistocene refugia and postglacial colonization routes. Three divergent clades (east, west, central) were identified with highest net divergence (dA = 5.2%) between the eastern and western clades. Populations from the recently deglaciated higher latitudes of Canada and Alaska are closely related to lower latitude populations of the central clade (dA = 1.2%) suggesting recent expansion from this midwestern region. No representatives from the east or west clade were found at latitudes higher than 50 degrees N, indicating that postglacial colonization occurred through a midcontinental route. The high latitude population from the Northwest Territories exhibited demographic patterns and genetic diversity consistent with a stable noncolonizing population. This population is found near the Mackenzie range, where the two continental ice sheets were believed to have coalesced. Molecular variation observed in this population may be the result of leading edge population diversifying in the continental corridor or may reflect the signal of a high latitude refugial population.

Historical biogeography at the crossroads of the northern continents: molecular phylogenetics of red-backed voles (Rodentia: Arvicolinae).

Evolutionary relationships of red-backed voles and their relatives were examined and used to test biogeographic hypotheses. Sequences of the mitochondrial cytochrome b gene were obtained for 25 individuals representing Alticola macrotis, Clethrionomys californicus, C. gapperi, C. glareolus, C. rutilus, and C. rufocanus. These were combined with 21 partial sequences from GenBank for C. regulus, C. rex, C. rufocanus, C. rutilus, Eothenomys imaizumii, E. melanogaster, Phaulomys andersoni, and P. smithii. Complete sequences of three species of Microtus (M. montanus, M. oeconomus, and M. pennsylvanicus), representative species of other arvicoline genera (Myopus, Synaptomys, Arvicola, Ellobius, Ondatra, Lemmus, Dicrostonyx, and Phenacomys), and a sigmodontine representative (Peromyscus) were included as outgroups. We used maximum parsimony, maximum likelihood, distance, and Bayesian based methods and conducted statistical tests on proposed hypotheses of phylogenetic relationships and biogeographic histories. A close relationship of species representing the genera Alticola, Clethrionomys, and Eothenomys was supported (Clethrionomyini); however, the genus Clethrionomys was paraphyletic with respect to both Alticola and Eothenomys. Three major clades were identified as Asian (Eothenomys andersoni, E. smithii, C. rex, C. regulus, and C. rufocanus), Trans-beringian (Alticola macrotis, C. californicus, C. gapperi, C. glarelolus, and C. rutilus), and Taiwanese (E. melanogaster). These results are consistent with the fossil record which indicates an initial diversification in Asia followed by colonization of the Nearctic on at least two occasions. The holarctic species, C. rutilus, appears to have either reinvaded Asia from North America or colonized North America more recently (late Pleistocene) than the two species of Clethrionomys (C. gapperi and C. californicus) that are endemic to North America (early to mid-Pleistocene). Finally, C. gapperi, appears to be comprised of an eastern and a western species, the former with affinities to the Asian C. glareolus and the latter more closely related to C. californicus.