River drainage reorganization and reticulate evolution in the Two-Lined Salamander (Eurycea bislineata) species complex.

The origin and eventual loss of biogeographic barriers can create alternating periods of allopatry and secondary contact, facilitating gene flow among distinct metapopulations and generating reticulate evolutionary histories that are not adequately described by a bifurcating evolutionary tree. One such example may exist in the two-lined salamander (Eurycea bislineata) species complex, where discordance among morphological and molecular datasets has created a "vexing taxonomic challenge". Previous phylogeographic analyses of mitochondrial DNA (mtDNA) suggested that the reorganization of Miocene paleodrainages drove vicariance and dispersal, but the inherent limitations of a single-locus dataset precluded the evaluation of subsequent gene flow. Here, we generate triple-enzyme restriction site-associated DNA sequencing (3RAD) data for >100 individuals representing all major mtDNA lineages and use a suite of complementary methods to demonstrate that discordance among earlier datasets is best explained by a reticulate evolutionary history influenced by river drainage reorganization. Systematics of such groups should acknowledge these complex histories and relationships that are not strictly hierarchical.

Phylogeography of the blacklegged tick (Ixodes scapularis) throughout the USA identifies candidate loci for differences in vectorial capacity.

The blacklegged tick (Ixodes scapularis (Journal of the Academy of Natural Sciences of Philadelphia, 1821, 2, 59)) is a vector of Borrelia burgdorferi sensu stricto (s.s.) (International Journal of Systematic Bacteriology, 1984, 34, 496), the causative bacterial agent of Lyme disease, part of a slow-moving epidemic of Lyme borreliosis spreading across the northern hemisphere. Well-known geographical differences in the vectorial capacity of these ticks are associated with genetic variation. Despite the need for detailed genetic information in this disease system, previous phylogeographical studies of these ticks have been restricted to relatively few populations or few genetic loci. Here we present the most comprehensive phylogeographical study of genome-wide markers in I. scapularis, conducted by using 3RAD (triple-enzyme restriction-site associated sequencing) and surveying 353 ticks from 33 counties throughout the species' range. We found limited genetic variation among populations from the Northeast and Upper Midwest, where Lyme disease is most common, and higher genetic variation among populations from the South. We identify five spatially associated genetic clusters of I. scapularis. In regions where Lyme disease is increasing in frequency, the I. scapularis populations genetically group with ticks from historically highly Lyme-endemic regions. Finally, we identify 10 variable DNA sites that contribute the most to population differentiation. These variable sites cluster on one of the chromosome-scale scaffolds for I. scapularis and are within identified genes. Our findings illuminate the need for additional research to identify loci causing variation in the vectorial capacity of I. scapularis and where additional tick sampling would be most valuable to further understand disease trends caused by pathogens transmitted by I. scapularis.

Population genomics indicate three different modes of divergence and speciation with gene flow in the green-winged teal duck complex.

The processes leading to divergence and speciation can differ broadly among taxa with different life histories. We examine these processes in a small clade of ducks with historically uncertain relationships and species limits. The green-winged teal (Anas crecca) complex is a Holarctic species of dabbling duck currently categorized as three subspecies (Anas crecca crecca, A. c. nimia, and A. c. carolinensis) with a close relative, the yellow-billed teal (Anas flavirostris) from South America. A. c. crecca and A. c. carolinensis are seasonal migrants, while the other taxa are sedentary. We examined divergence and speciation patterns in this group, determining their phylogenetic relationships and the presence and levels of gene flow among lineages using both mitochondrial and genome-wide nuclear DNA obtained from 1,393 ultraconserved element (UCE) loci. Phylogenetic relationships using nuclear DNA among these taxa showed A. c. crecca, A. c. nimia, and A. c. carolinensis clustering together to form one polytomous clade, with A. flavirostris sister to this clade. This relationship can be summarized as (crecca, nimia, carolinensis)(flavirostris). However, whole mitogenomes revealed a different phylogeny: (crecca, nimia)(carolinensis, flavirostris). The best demographic model for key pairwise comparisons supported divergence with gene flow as the probable speciation mechanism in all three contrasts (crecca-nimia, crecca-carolinensis, and carolinensis-flavirostris). Given prior work, gene flow was expected among the Holarctic taxa, but gene flow between North American carolinensis and South American flavirostris (M âˆ¼0.1-0.4 individuals/generation), albeit low, was not expected. Three geographically oriented modes of divergence are likely involved in the diversification of this complex: heteropatric (crecca-nimia), parapatric (crecca-carolinensis), and (mostly) allopatric (carolinensis-flavirostris). Our study shows that ultraconserved elements are a powerful tool for simultaneously studying systematics and population genomics in systems with historically uncertain relationships and species limits.

Earth history and the passerine superradiation.

Avian diversification has been influenced by global climate change, plate tectonic movements, and mass extinction events. However, the impact of these factors on the diversification of the hyperdiverse perching birds (passerines) is unclear because family level relationships are unresolved and the timing of splitting events among lineages is uncertain. We analyzed DNA data from 4,060 nuclear loci and 137 passerine families using concatenation and coalescent approaches to infer a comprehensive phylogenetic hypothesis that clarifies relationships among all passerine families. Then, we calibrated this phylogeny using 13 fossils to examine the effects of different events in Earth history on the timing and rate of passerine diversification. Our analyses reconcile passerine diversification with the fossil and geological records; suggest that passerines originated on the Australian landmass ∼47 Ma; and show that subsequent dispersal and diversification of passerines was affected by a number of climatological and geological events, such as Oligocene glaciation and inundation of the New Zealand landmass. Although passerine diversification rates fluctuated throughout the Cenozoic, we find no link between the rate of passerine diversification and Cenozoic global temperature, and our analyses show that the increases in passerine diversification rate we observe are disconnected from the colonization of new continents. Taken together, these results suggest more complex mechanisms than temperature change or ecological opportunity have controlled macroscale patterns of passerine speciation.

Tissue Distribution of Mercury in the Bodies of Wild American Alligators (Alligator mississippiensis) from a Coastal Marsh in Louisiana (USA).

Total mercury (THg) concentrations were measured in wild alligators inhabiting a coastal marsh in southern Louisiana, to determine the tissue distribution of THg among various body organs and tissue compartments. Concentrations of THg in claws and dermal tail scutes were compared to those in blood, brain, gonad, heart, kidney, liver, and skeletal muscle to determine if the former tissues, commonly available by non-lethal sampling, could be used as measures of body burdens in various internal organs. Mercury was found in all body organs and tissue compartments. However, overall, THg concentrations measured in alligators were below the FDA action level for fish consumption and were comparable to previous data reported from southwestern Louisiana. Our results suggest consumption of meat from alligators found in this region may be of little public health concern. However, the extended period of time between sampling (in this study) and the present-day highlight the need for continuous, additional, and more recent sampling to ensure consumer safety. Total mercury concentrations were highest in the kidney (3.18 ± 0.69 mg/kg dw) and liver (3.12 ± 0.76 mg/kg dw). THg levels in non-lethal samples (blood, claws, and dermal tail scutes) were positively correlated with all tissue THg concentrations (blood: R2 = 0.513-0.988; claw: R2 = 0.347-0.637, scutes: R2 = 0.333-0.649). Because THg concentrations from blood, claws, and scutes were correlated with those of the internal organs, non-lethal sampling methods may be a viable method of estimating levels of THg in other body tissues.

Escaping the fate of Sisyphus: assessing resistome hybridization baits for antimicrobial resistance gene capture.

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

An Open-Source Program (Haplo-ST) for Whole-Genome Sequence Typing Shows Extensive Diversity among Listeria monocytogenes Isolates in Outdoor Environments and Poultry Processing Plants.

A reliable and standardized classification of Listeria monocytogenes is important for accurate strain identification during outbreak investigations. Current whole-genome sequencing (WGS)-based approaches for strain characterization are either difficult to standardize, rendering them less suitable for data exchange, or are not freely available. Thus, we developed a portable and open-source tool, Haplo-ST, to improve standardization and provide maximum discriminatory potential to WGS data tied to a multilocus sequence typing (MLST) framework. Haplo-ST performs whole-genome MLST (wgMLST) for L. monocytogenes while allowing for data exchangeability worldwide. This tool takes in (i) raw WGS reads as input, (ii) cleans the raw data according to user-specified parameters, (iii) assembles genes across loci by mapping to genes from reference strains, and (iv) assigns allelic profiles to assembled genes and provides a wgMLST subtyping for each isolate. Data exchangeability relies on the tool assigning allelic profiles based on a centralized nomenclature defined by the widely used BIGSdb-Lm database. Tests of Haplo-ST's performance with simulated reads from L. monocytogenes reference strains demonstrated high sensitivity (97.5%), and coverage depths of ≥20× were found to be sufficient for wgMLST profiling. We then used Haplo-ST to characterize and differentiate between two groups of L. monocytogenes isolates derived from the natural environment and poultry processing plants. Phylogenetic reconstruction identified lineages within each group, and no lineage specificity was observed with isolate phenotypes (transient versus persistent) or origins. Genetic differentiation analyses between isolate groups identified 21 significantly differentiated loci, potentially enriched for adaptation and persistence of L. monocytogenes within poultry processing plants.IMPORTANCE We have developed an open-source tool (https://github.com/swarnalilouha/Haplo-ST) that provides allele-based subtyping of L. monocytogenes isolates at the whole-genome level. Along with allelic profiles, this tool also generates allele sequences and identifies paralogs, which is useful for phylogenetic tree reconstruction and deciphering relationships between closely related isolates. More broadly, Haplo-ST is flexible and can be adapted to characterize the genome of any haploid organism simply by installing an organism-specific gene database. Haplo-ST also allows for scalable subtyping of isolates; fewer reference genes can be used for low-resolution typing, whereas higher resolution can be achieved by increasing the number of genes used in the analysis. Our tool enabled clustering of L. monocytogenes isolates into lineages and detection of potential loci for adaptation and persistence in food processing environments. Findings from these analyses highlight the effectiveness of Haplo-ST in subtyping and evaluating relationships among isolates in studies of bacterial population genetics.

Divergence, gene flow, and speciation in eight lineages of trans-Beringian birds.

Determining how genetic diversity is structured between populations that span the divergence continuum from populations to biological species is key to understanding the generation and maintenance of biodiversity. We investigated genetic divergence and gene flow in eight lineages of birds with a trans-Beringian distribution, where Asian and North American populations have likely been split and reunited through multiple Pleistocene glacial cycles. Our study transects the speciation process, including eight pairwise comparisons in three orders (ducks, shorebirds and passerines) at population, subspecies and species levels. Using ultraconserved elements (UCEs), we found that these lineages represent conditions from slightly differentiated populations to full biological species. Although allopatric speciation is considered the predominant mode of divergence in birds, all of our best divergence models included gene flow, supporting speciation with gene flow as the predominant mode in Beringia. In our eight lineages, three were best described by a split-migration model (divergence with gene flow), three best fit a secondary contact scenario (isolation followed by gene flow), and two showed support for both models. The lineages were not evenly distributed across a divergence space defined by gene flow (M) and differentiation (FST ), instead forming two discontinuous groups: one with relatively shallow divergence, no fixed single nucleotide polymorphisms (SNPs), and high rates of gene flow between populations; and the second with relatively deeply divergent lineages, multiple fixed SNPs, and low gene flow. Our results highlight the important role that gene flow plays in avian divergence in Beringia.

Identification and characterization of microRNAs (miRNAs) and their transposable element origins in the saltwater crocodile, Crocodylus porosus.

MicroRNAs (miRNAs) are 18-24 nucleotide regulatory RNAs. They are involved in the regulation of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Data suggests a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to birds, the closest extant relatives of crocodilians. Understanding gene regulation in the saltwater crocodile in the context of relatively slow genomic change thus holds potential for the investigation of genomics, evolution, and adaptation. Utilizing eleven tissue types and sixteen small RNA libraries, we report 644 miRNAs in the saltwater crocodile with >78% of miRNAs being novel to crocodilians. We also identified potential targets for the miRNAs and analyzed the relationship of the miRNA repertoire to transposable elements (TEs). Results suggest an increased association of DNA transposons with miRNAs when compared to retrotransposons. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile. In addition, the data suggests a supporting role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation.

The drivers of tropical speciation.

Since the recognition that allopatric speciation can be induced by large-scale reconfigurations of the landscape that isolate formerly continuous populations, such as the separation of continents by plate tectonics, the uplift of mountains or the formation of large rivers, landscape change has been viewed as a primary driver of biological diversification. This process is referred to in biogeography as vicariance. In the most species-rich region of the world, the Neotropics, the sundering of populations associated with the Andean uplift is ascribed this principal role in speciation. An alternative model posits that rather than being directly linked to landscape change, allopatric speciation is initiated to a greater extent by dispersal events, with the principal drivers of speciation being organism-specific abilities to persist and disperse in the landscape. Landscape change is not a necessity for speciation in this model. Here we show that spatial and temporal patterns of genetic differentiation in Neotropical birds are highly discordant across lineages and are not reconcilable with a model linking speciation solely to landscape change. Instead, the strongest predictors of speciation are the amount of time a lineage has persisted in the landscape and the ability of birds to move through the landscape matrix. These results, augmented by the observation that most species-level diversity originated after episodes of major Andean uplift in the Neogene period, suggest that dispersal and differentiation on a matrix previously shaped by large-scale landscape events was a major driver of avian speciation in lowland Neotropical rainforests.

Microbiota of Four Tissue Types in American Alligators (Alligator mississippiensis) Following Extended Dietary Selenomethionine Exposure.

Selenium represents an essential trace nutrient that is necessary for biological functions. Deficiencies can induce disease, but excess can induce toxicity. Selenium deficiency is a major concern in underdeveloped countries, while also posing as a toxic pollutant in waterways surrounding landfills, agricultural areas, and fossil fuel production sites. We examined the microbiome of selenomethionine (SeMet) fed American alligators (Alligator mississippiensis) at the beginning and end of a 7-week exposure experiment. Alligators were randomly divided into three groups: control and 1000 or 2000 ppm SeMet. DNA from before exposure (oral and cloaca swabs) and post-exposure (oral, cloaca, small & large intestines) sampling were extracted and amplified for bacterial 16 s rRNA. While treatment did not seem to have much effect, we observed a predominance of Fusobacteriaceae and Porpyromonodaceae across all tissue types. Cetobacterium and Clostridium are the most abundant genera as potential indicators of the aquatic and carrion feeding lifestyle of alligators.

Formation of a recent hybrid zone offers insight into the geographic puzzle and maintenance of species boundaries in musk turtles.

Speciation is the result of an accumulation of reproductive barriers between populations, but pinpointing the factors involved is often difficult. However, hybrid zones can form when these barriers are not complete, especially when lineages come into contact in intermediate or modified habitats. We examine a hybrid zone between two closely related riverine turtle species, Sternotherus depressus and S. peltifer, and use dual-digest RAD sequencing to understand how this hybrid zone formed and elucidate genomic patterns of reproductive isolation. First, the geographical extent and timing of formation of the hybrid zone is established to provide context for understanding the role of extrinsic and intrinsic reproductive isolating mechanisms in this system. The strength of selection on taxon-specific contributions to maintenance of the hybrid zone is then inferred using a Bayesian genomic cline model. These analyses identify a role for selection inhibiting introgression in some genomic regions at one end of the hybrid zone and promoting introgression in many loci at the other. When selective pressures necessary to generate outliers to the genomic cline are considered with the geographical and temporal context of this hybrid zone, we conclude that habitat-specific selection probably limits introgression from S. depressus to S. peltifer in the direction of river flow. However, selection is mediating rapid, unidirectional introgression from S. peltifer to S. depressus, which is probably facilitated by anthropogenic habitat alteration. These findings indicate a potentially imminent threat of population-level genomic extinction for an already imperiled species due to ongoing human-caused habitat alteration.

Insight from an ultraconserved element bait set designed for hemipteran phylogenetics integrated with genomic resources.

Target enrichment of conserved genomic regions facilitates collecting sequences of many orthologous loci from non-model organisms to address phylogenetic, phylogeographic, population genetic, and molecular evolution questions. Bait sets for sequence capture can simultaneously target thousands of loci, which opens new avenues of research on speciose groups. Current phylogenetic hypotheses on the >103,000 species of Hemiptera have failed to unambiguously resolve major nodes, suggesting that alternative datasets and more thorough taxon sampling may be required to resolve relationships. We use a recently designed ultraconserved element (UCE) bait set for Hemiptera, with a focus on the suborder Heteroptera, or the true bugs, to test previously proposed relationships. We present newly generated UCE data for 36 samples representing three suborders, all seven heteropteran infraorders, 23 families, and 34 genera of Hemiptera and one thysanopteran outgroup. To improve taxon sampling, we also mined additional UCE loci in silico from published hemipteran genomic and transcriptomic data. We obtained 2271 UCE loci for newly sequenced hemipteran taxa, ranging from 265 to 1696 (average 904) per sample. These were similar in number to the data mined from transcriptomes and genomes, but with fewer loci overall. The amount of missing data correlates with greater phylogenetic divergence from taxa used to design the baits. This bait set hybridizes to a wide range of hemipteran taxa and specimens of varying quality, including dried specimens as old as 1973. Our estimated phylogeny yielded topologies consistent with other studies for most nodes and was strongly-supported. We also demonstrate that UCE loci are almost exclusively from the transcribed portion of the genome, thus data can be successfully integrated with existing genomic and transcriptomic resources for more comprehensive phylogenetic sampling, an important feature in the era of phylogenomics. UCE approaches can be used by other researchers for additional studies on hemipteran evolution and other research that requires well resolved phylogenies.

Conflicting Evolutionary Histories of the Mitochondrial and Nuclear Genomes in New World Myotis Bats.

The rapid diversification of Myotis bats into more than 100 species is one of the most extensive mammalian radiations available for study. Efforts to understand relationships within Myotis have primarily utilized mitochondrial markers and trees inferred from nuclear markers lacked resolution. Our current understanding of relationships within Myotis is therefore biased towards a set of phylogenetic markers that may not reflect the history of the nuclear genome. To resolve this, we sequenced the full mitochondrial genomes of 37 representative Myotis, primarily from the New World, in conjunction with targeted sequencing of 3648 ultraconserved elements (UCEs). We inferred the phylogeny and explored the effects of concatenation and summary phylogenetic methods, as well as combinations of markers based on informativeness or levels of missing data, on our results. Of the 294 phylogenies generated from the nuclear UCE data, all are significantly different from phylogenies inferred using mitochondrial genomes. Even within the nuclear data, quartet frequencies indicate that around half of all UCE loci conflict with the estimated species tree. Several factors can drive such conflict, including incomplete lineage sorting, introgressive hybridization, or even phylogenetic error. Despite the degree of discordance between nuclear UCE loci and the mitochondrial genome and among UCE loci themselves, the most common nuclear topology is recovered in one quarter of all analyses with strong nodal support. Based on these results, we re-examine the evolutionary history of Myotis to better understand the phenomena driving their unique nuclear, mitochondrial, and biogeographic histories.

Examining the Effects of Chronic Selenium Exposure on Traditionally Used Stress Parameters in Juvenile American Alligators (Alligator mississippiensis).

Environmental contaminants, such as the trace element selenium (Se), are a continuing concern to species worldwide due to their potential pathophysiological effects, including their influence on the stress response mediated through glucocorticoids (GCs; stress hormones). Environmental concentrations of Se are increasing due to anthropogenic activities, including the incomplete combustion of coal and subsequent disposal of coal combustion wastes. However, most studies examining how Se affects GCs have been focused on lower trophic organisms. The objectives of this study were to investigate the effects of long-term Se exposure on traditionally used stress parameters and to identify which of these parameters best indicate Se accumulation in liver and kidney of the American alligator (Alligator mississippiensis), a top trophic carnivore found in the southeastern United States and known to inhabit Se-containing areas. Alligators were divided into three dietary treatments and fed prey spiked with 1000 or 2000 ppm of selenomethionine (SeMet) or deionized water (control treatment) for 7 weeks. Following the 7-week treatment protocol, blood and tissue samples were obtained to measure plasma corticosterone (CORT; the main crocodilian GC), tail scute CORT, the ratio of peripheral blood heterophils (H) to lymphocytes (L) as H/L ratio, and body condition. To evaluate which parameter best indicated Se accumulation in the liver and kidney, principal component and discriminant analyses were performed. The only parameter significantly correlated with liver and kidney Se concentrations was scute CORT. Our results suggest that measurement of CORT in tail scutes compared with plasma CORT, H/L ratios, and body condition is the best indicator of Se-exposure and accumulation in crocodilians.

Resolving taxonomic turbulence and uncovering cryptic diversity in the musk turtles (Sternotherus) using robust demographic modeling.

Accurate and consistent delimitation of species and their relationships provides a necessary framework for comparative studies, understanding evolutionary relationships, and informing conservation management. Despite the ever-increasing availability of genomic data, evolutionary dynamics can still render some relationships exceedingly difficult to resolve, including underlying speciation events that are rapid, recent, or confounded by post-speciation introgression. Here we present an empirical study of musk turtles (Sternotherus), which illustrates approaches to resolve difficult nodes in the Tree of Life that robust species-tree methods fail to resolve. We sequence 4430 RAD-loci from 205 individuals. Independent coalescent-based analyses, corroborated with morphology and geography, strongly support the recognition of cryptic species within Sternotherus, but with conflicting or weak support for some intraspecific relationships. To resolve species-tree conflict, we use a likelihood-based approach to test support for alternative demographic models behind alternative speciation scenarios and argue that demographic model testing has an important role for resolving systematic relationships in recent, rapid radiations. Species-tree and demographic modeling strongly support the elevation of two nominal subspecies in Sternotherus to species and the recognition of a previously cryptic species (S. intermedius sp. nov.) described within. The evolutionary and taxonomic history of Sternotherus is discussed in the context of these new species and novel and well-supported systematic hypotheses.

Isolation and characterization of microsatellite markers for conservation management of the endangered Great-billed Seed-finch, Sporophila maximiliani (Aves, Passeriformes), and cross-amplification in other congeners.

The Great-billed Seed-finch, Sporophila maximiliani, is a threatened neotropical bird that has declined mainly due to illegal trapping, with very few records in the wild in the last two decades. Despite the existence of a considerable captive population that could be used for reintroductions into the wild, many individuals are known to be hybrids either with other species or subspecies of the genus. Forensic investigations are urgently needed to distinguish between birds born in captivity from those from illegal trade. Microsatellites can be useful tools to assess individual admixture levels and to perform parentage tests that may confirm the origin of animals, but only a few loci are available for this group of birds. Here, we provide a set of 14 microsatellite loci isolated from the S. maximiliani, many of which also amplified and were polymorphic in the Pearly-bellied Seedeater, S. pileata, and in the Copper Seedeater, S. bouvreuil. In ten loci selected for the S. maximiliani, the number of alleles per locus varied from four to nine and observed and expected heterozygosities ranged from 0.13 to 1 and 0.56 to 0.83, respectively. These loci proved to be highly informative for forensic analyses, indicating that they may be useful for conservation management plans in these endangered tropical birds.

Dietary Selenomethionine Administration and Its Effects on the American Alligator (Alligator mississippiensis): Oxidative Status and Corticosterone Levels.

Selenium (Se) is an essential nutrient which in excess causes toxicity. The disposal of incompletely combusted coal, which often is rich in Se, into aquatic settling basins is increasing the risk of Se exposure worldwide. However, very few studies have looked at the physiological effects of Se exposure on long-lived, top trophic vertebrates, such as the American alligator (Alligator mississippiensis). During a 7-week period, alligators were fed one of three dietary treatments: mice injected with deionized water or mice injected with water containing 1000 or 2000 ppm selenomethionine (SeMet). One week after the last feeding alligators were bled within 3 min of capture for plasma corticosterone (CORT). A few days later, all alligators were euthanized and whole blood and tail tissue were harvested to measure oxidative damage, an antioxidant-associated transcription factor, and antioxidant enzymes [glutathione peroxidase-1 (GPX1), superoxide dismutase-1 (SOD1), and SOD2] by Western blotting. There was a dose-dependent increase in baseline CORT levels in alligators administered SeMet. Except for blood SOD2 levels, SeMet treatment had no effect (p > 0.05 for all) on oxidative status: oxidative damage, GPX1, SOD1, and muscle SOD2 levels were similar among treatments. Our results illustrate that high levels of Se may act as a stressor to crocodilians. Future studies should investigate further the physiological effects of Se accumulation in long-lived, top-trophic vertebrates.

Avoiding Missing Data Biases in Phylogenomic Inference: An Empirical Study in the Landfowl (Aves: Galliformes).

Production of massive DNA sequence data sets is transforming phylogenetic inference, but best practices for analyzing such data sets are not well established. One uncertainty is robustness to missing data, particularly in coalescent frameworks. To understand the effects of increasing matrix size and loci at the cost of increasing missing data, we produced a 90 taxon, 2.2 megabase, 4,800 locus sequence matrix of landfowl using target capture of ultraconserved elements. We then compared phylogenies estimated with concatenated maximum likelihood, quartet-based methods executed on concatenated matrices and gene tree reconciliation methods, across five thresholds of missing data. Results of maximum likelihood and quartet analyses were similar, well resolved, and demonstrated increasing support with increasing matrix size and sparseness. Conversely, gene tree reconciliation produced unexpected relationships when we included all informative loci, with certain taxa placed toward the root compared with other approaches. Inspection of these taxa identified a prevalence of short average contigs, which potentially biased gene tree inference and caused erroneous results in gene tree reconciliation. This suggests that the more problematic missing data in gene tree-based analyses are partial sequences rather than entire missing sequences from locus alignments. Limiting gene tree reconciliation to the most informative loci solved this problem, producing well-supported topologies congruent with concatenation and quartet methods. Collectively, our analyses provide a well-resolved phylogeny of landfowl, including strong support for previously problematic relationships such as those among junglefowl (Gallus), and clarify the position of two enigmatic galliform genera (Lerwa, Melanoperdix) not sampled in previous molecular phylogenetic studies.

Genistein prevention of hyperglycemia and improvement of glucose tolerance in adult non-obese diabetic mice are associated with alterations of gut microbiome and immune homeostasis.

Although studies have linked soy phytoestrogen 4,7,4-trihydroxyisoflavone genistein (GEN) to reduced type 1 diabetes (T1D) risk, the mechanism of dietary GEN on T1D remains unknown. In our studies, adult non-obese diabetic (NOD) mouse model was employed to investigate the effects of GEN exposure on blood glucose level (BGL), glucose tolerance, gut microbiome, and immune responses. Adult male and female NOD mice were fed with either soy-based or casein-based diet, and received GEN at 20mg/kg body weight by gavage daily. The BGL and immune responses (represented by serum antibodies, cytokines and chemokines, and histopathology) were monitored, while the fecal gut microbiome was sequenced for 16S ribosomal RNA to reveal any alterations in gut microbial communities. A significantly reduced BGL was found in NOD males fed with soy-based diet on day 98 after initial dosing, and an improved glucose tolerance was observed on both diets. In addition, an anti-inflammatory response (suggested by reduced IgG2b and cytokine/chemokine levels, and alterations in the microbial taxonomy) was accompanied by an altered ß-diversity in gut microbial species. Among the NOD females exposed to GEN, a later onset of T1D was observed. However, the profiles of gut microbiome, antibodies and cytokines/chemokines were all indicative of pro-inflammation. This study demonstrated an association among GEN exposure, gut microbiome alteration, and immune homeostasis in NOD males. Although the mechanisms underlying the protective effects of GEN in NOD mice need to be explored further, the current study suggested a GEN-induced sex-specific effect in inflammatory status and gut microbiome.