Caleb D. Phillips

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.

Bayesian inference of a historical bottleneck in a heavily exploited marine mammal

Emerging Bayesian analytical approaches offer increasingly sophisticated means of reconstructing historical population dynamics from genetic data, but have been little applied to scenarios involving demographic bottlenecks. Consequently, we analysed a large mitochondrial and microsatellite dataset from the Antarctic fur seal Arctocephalus gazella, a species subjected to one of the most extreme examples of uncontrolled exploitation in history when it was reduced to the brink of extinction by the sealing industry during the late eighteenth and nineteenth centuries. Classical bottleneck tests, which exploit the fact that rare alleles are rapidly lost during demographic reduction, yielded ambiguous results. In contrast, a strong signal of recent demographic decline was detected using both Bayesian skyline plots and Approximate Bayesian Computation, the latter also allowing derivation of posterior parameter estimates that were remarkably consistent with historical observations. This was achieved using only contemporary samples, further emphasizing the potential of Bayesian approaches to address important problems in conservation and evolutionary biology.

Analysis of the chronic wound microbiota of 2,963 patients by 16S rDNA pyrosequencing

The extent to which microorganisms impair wound healing is an ongoing controversy in the management of chronic wounds. Because the high diversity and extreme variability of the microbiota between individual chronic wounds lead to inconsistent findings in small cohort studies, evaluation of a large number of chronic wounds using identical sequencing and bioinformatics methods is necessary for clinicians to be able to select appropriate empiric therapies. In this study, we utilized 16S rDNA pyrosequencing to analyze the composition of the bacterial communities present in samples obtained from patients with chronic diabetic foot ulcers (N = 910), venous leg ulcers (N = 916), decubitus ulcers (N = 767), and nonhealing surgical wounds (N = 370). The wound samples contained a high proportion of Staphylococcus and Pseudomonas species in 63 and 25% of all wounds, respectively; however, a high prevalence of anaerobic bacteria and bacteria traditionally considered commensalistic was also observed. Our results suggest that neither patient demographics nor wound type influenced the bacterial composition of the chronic wound microbiome. Collectively, these findings indicate that empiric antibiotic selection need not be based on nor altered for wound type. Furthermore, the results provide a much clearer understanding of chronic wound microbiota in general; clinical application of this new knowledge over time may help in its translation to improved wound healing outcomes.

Rolling-circle transposons catalyze genomic innovation in a mammalian lineage.

Rolling-circle transposons (Helitrons) are a newly discovered group of mobile DNA widespread in plant and invertebrate genomes but limited to the bat family Vespertilionidae among mammals. Little is known about the long-term impact of Helitron activity because the genomes where Helitron activity has been extensively studied are predominated by young families. Here, we report a comprehensive catalog of vetted Helitrons from the 7× Myotis lucifugus genome assembly. To estimate the timing of transposition, we scored presence/absence across related vespertilionid genome sequences with estimated divergence times. This analysis revealed that the Helibat family has been a persistent source of genomic innovation throughout the vespertilionid diversification from approximately 30–36 Ma to as recently as approximately 1.8–6 Ma. This is the first report of persistent Helitron transposition over an extended evolutionary timeframe. These findings illustrate that the pattern of Helitron activity is akin to the vertical persistence of LINE retrotransposons in primates and other mammalian lineages. Like retrotransposition in primates, rolling-circle transposition has generated lineage-specific variation and accounts for approximately 110 Mb, approximately 6% of the genome of M. lucifugus. The Helitrons carry a heterogeneous assortment of host sequence including retroposed messenger RNAs, retrotransposons, DNA transposons, as well as introns, exons and regulatory regions (promoters, 5′-untranslated regions [UTRs], and 3′-UTRs) of which some are evolving in a pattern suggestive of purifying selection. Evidence that Helitrons have contributed putative promoters, exons, splice sites, polyadenylation sites, and microRNA-binding sites to transcripts otherwise conserved across mammals is presented, and the implication of Helitron activity to innovation in these unique mammals is discussed.

Global population structure and demographic history of the grey seal.

Although the grey seal Halichoerus grypus is one of the most familiar and intensively studied of all pinniped species, its global population structure remains to be elucidated. Little is also known about how the species as a whole may have historically responded to climate‐driven changes in habitat availability and anthropogenic exploitation. We therefore analysed samples from over 1500 individuals collected from 22 colonies spanning the Western and Eastern Atlantic and the Baltic Sea regions, represented by 350 bp of the mitochondrial hypervariable region and up to nine microsatellites. Strong population structure was observed at both types of marker, and highly asymmetrical patterns of gene flow were also inferred, with the Orkney Islands being identified as a source of emigrants to other areas in the Eastern Atlantic. The Baltic and Eastern Atlantic regions were estimated to have diverged a little over 10 000 years ago, consistent with the last proposed isolation of the Baltic Sea. Approximate Bayesian computation also identified genetic signals consistent with postglacial population expansion across much of the species range, suggesting that grey seals are highly responsive to changes in habitat availability.

Contrasting Evolutionary Dynamics of the Developmental Regulator PAX9, among Bats, with Evidence for a Novel Post-Transcriptional Regulatory Mechanism

Morphological evolution can be the result of natural selection favoring modification of developmental signaling pathways. However, little is known about the genetic basis of such phenotypic diversity. Understanding these mechanisms is difficult for numerous reasons, yet studies in model organisms often provide clues about the major developmental pathways involved. The paired-domain gene, PAX9, is known to be a key regulator of development, particularly of the face and teeth. In this study, using a comparative genetics approach, we investigate PAX9 molecular evolution among mammals, focusing on craniofacially diversified (Phyllostomidae) and conserved (Vespertilionidae) bat families, and extend our comparison to other orders of mammal. Open-reading frame analysis disclosed signatures of selection, in which a small percentage of residues vary, and lineages acquire different combinations of variation through recurrent substitution and lineage specific changes. A few instances of convergence for specific residues were observed between morphologically convergent bat lineages. Bioinformatic analysis for unknown PAX9 regulatory motifs indicated a novel post-transcriptional regulatory mechanism involving a Musashi protein. This regulation was assessed through fluorescent reporter assays and gene knockdowns. Results are compatible with the hypothesis that the number of Musashi binding-elements in PAX9 mRNA proportionally regulates protein translation rate. Although a connection between morphology and binding element frequency was not apparent, results indicate this regulation would vary among craniofacially divergent bat species, but be static among conserved species. Under this model, Musashi’s regulatory control of alternative human PAX9 isoforms would also vary. The presence of Musashi-binding elements within PAX9 of all mammals examined, chicken, zebrafish, and the fly homolog of PAX9, indicates this regulatory mechanism is ancient, originating basal to much of the animal phylogeny.

Prevention of antibiotic-associated metabolic syndrome in mice by intestinal alkaline phosphatase.

To examine whether co‐administration of intestinal alkaline phosphatase (IAP) with antibiotics early in life may have a preventive role against metabolic syndrome (MetS) in mice.

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.

Elevated mitochondrial genome variation after 50 generations of radiation exposure in a wild rodent

Currently, the effects of chronic, continuous low dose environmental irradiation on the mitochondrial genome of resident small mammals are unknown. Using the bank vole (Myodes glareolus) as a model system, we tested the hypothesis that approximately 50 generations of exposure to the Chernobyl environment has significantly altered genetic diversity of the mitochondrial genome. Using deep sequencing, we compared mitochondrial genomes from 131 individuals from reference sites with radioactive contamination comparable to that present in northern Ukraine before the 26 April 1986 meltdown, to populations where substantial fallout was deposited following the nuclear accident. Population genetic variables revealed significant differences among populations from contaminated and uncontaminated localities. Therefore, we rejected the null hypothesis of no significant genetic effect from 50 generations of exposure to the environment created by the Chernobyl meltdown. Samples from contaminated localities exhibited significantly higher numbers of haplotypes and polymorphic loci, elevated genetic diversity, and a significantly higher average number of substitutions per site across mitochondrial gene regions. Observed genetic variation was dominated by synonymous mutations, which may indicate a history of purify selection against nonsynonymous or insertion/deletion mutations. These significant differences were not attributable to sample size artifacts. The observed increase in mitochondrial genomic diversity in voles from radioactive sites is consistent with the possibility that chronic, continuous irradiation resulting from the Chernobyl disaster has produced an accelerated mutation rate in this species over the last 25 years. Our results, being the first to demonstrate this phenomenon in a wild mammalian species, are important for understanding genetic consequences of exposure to low‐dose radiation sources.

TEMPORAL DYNAMICS OF RELATIVE ABUNDANCES AND BACTERIAL SUCCESSION IN CHRONIC WOUND COMMUNITIES

Polymicrobial bacterial infection is an important factor contributing to wound chronicity. Consequently, clinicians frequently adopt a biofilm‐based wound care approach, in which wounds are treated utilizing DNA sequencing information about microbial communities. While more successful than treatment not using community information, there is little information about temporal dynamics of wound communities and optimal approaches over the course of treatment. To characterize these dynamics, temporal analysis over three sampling points was conducted for 167 chronic wounds. Across sampling intervals, wound communities from the same patients changed in composition, and most commonly shared less than 50% of observed species. There was a significant relationship between community similarity and time between sampling. Classifying wounds into state types, we found that communities frequently transitioned from Pseudomonas or Staphylococcus dominated, into a highly variable state type. Although low abundance microbial species are typically disregarded due to uncertainty of biological importance, we found that 80% of wound microbiomes included common or dominant species at subsequent time points that were in low abundance in earlier samples. Moreover, these species were often those known to frequently infect wounds. Results document compositional shifts through the course of treatment and suggest that routine consideration of low abundance species may improve biofilm‐based wound care. Moreover, findings indicate that integrating ecological modeling to understand wound microbiome succession may lead to more informed therapy.