A 2200-year record of Andean Condor diet and nest site usage reflects natural and anthropogenic stressors.

Understanding how animals respond to large-scale environmental changes is difficult to achieve because monitoring data are rarely available for more than the past few decades, if at all. Here, we demonstrate how a variety of palaeoecological proxies (e.g. isotopes, geochemistry and DNA) from an Andean Condor (Vultur gryphus) guano deposit from Argentina can be used to explore breeding site fidelity and the impacts of environmental changes on avian behaviour. We found that condors used the nesting site since at least approximately 2200 years ago, with an approximately 1000-year nesting frequency slowdown from ca 1650 to 650 years before the present (yr BP). We provide evidence that the nesting slowdown coincided with a period of increased volcanic activity in the nearby Southern Volcanic Zone, which resulted in decreased availability of carrion and deterred scavenging birds. After returning to the nest site ca 650 yr BP, condor diet shifted from the carrion of native species and beached marine animals to the carrion of livestock (e.g. sheep and cattle) and exotic herbivores (e.g. red deer and European hare) introduced by European settlers. Currently, Andean Condors have elevated lead concentrations in their guano compared to the past, which is associated with human persecution linked to the shift in diet.

A species-specific digital PCR assay for the endangered blue racer (Coluber constrictor foxii) in Canada.

The only population of the endangered blue racer (Coluber constrictor foxii) in Canada occurs on Pelee Island, Ontario. The species is threatened by multiple factors, including habitat degradation and loss, road mortality, persecution, and potentially predation. We designed and evaluated the performance of an environmental DNA droplet digital PCR assay that can be used for multiple facets of conservation of this species. We tested the assay in silico and in vitro using DNA of blue racers and co-occurring snake species and estimated the LOD and LOQ using synthetic DNA. As wild turkey predation has been suggested to negatively affect racers, we tested the assay on eight wild turkey faecal samples. Our assay is specific, can detect the target species at very low levels of concentration (0.002 copies/µL), and can accurately quantify copy numbers ≥ 0.26 copies/µL. We detected no racer DNA in any wild turkey faecal sample. More faecal samples collected at strategic locations during snake peak activity on Pelee Island would enable a more thorough assessment of the possibility of turkey predation. Our assay should be effective for other environmental samples and can be used for investigating other factors negatively affecting blue racers, for example, helping to quantify blue racer habitat suitability and site occupancy.

Comparative phylogeography study reveals introgression and incomplete lineage sorting during rapid diversification of Rhodiola.

BACKGROUND AND AIMS: Many plant taxa in the Qinghai-Tibetan Plateau (QTP) and the Hengduan Mountains (HM) radiated rapidly during the Quaternary but with frequent secondary contact between diverging populations. Incomplete lineage sorting and introgressive hybridization might be involved during the rapid radiation, but their effects on phylogeography have not been fully determined. METHODS: We investigated the chloroplast DNA (cpDNA)/internal transcribed spacer (ITS) sequence variations of 611 samples of Rhodiola bupleuroides, R. discolor, R. fastigiata and R. chrysanthemifolia from the QTP and HM to compare the phylogeographic patterns between the four species with different evolutionary histories, geographic ranges and reproductive modes. KEY RESULTS: The divergence times of these species were consistent with the last peak of in situ speciation in the HM. While closely related species exhibited different phylogeographic patterns, they shared several ribotypes and haplotypes in sympatric populations, suggesting introgressive hybridization. A significant phylogenetic discordance between ribotypes and haplotypes was detected in three species, implying incomplete lineage sorting. Rhodiola discolor houses an extraordinary richness of cpDNA haplotypes, and this finding may be attributed to adaptive radiation. CONCLUSION: In addition to geographic isolation and climate oscillations during the Quaternary, both introgressive hybridization and incomplete lineage sorting play important roles in species that experienced rapid diversification in the QTP and HM.

Cytonuclear discordance in the crowned-sparrows, Zonotrichia atricapilla and Zonotrichia leucophrys.

The golden-crowned (Zonotrichia atricapilla) and white-crowned (Z. leucophrys) sparrows have been presented as a compelling case for rapid speciation. They display divergence in song and plumage with overlap in their breeding ranges implying reproductive isolation, but have almost identical mitochondrial genomes. Previous research proposed hybridization and subsequent mitochondrial introgression as an alternate explanation, but lacked robust nuclear gene trees to distinguish between introgression and incomplete lineage sorting. We test for signatures of these processes between Z. atricapilla and Z. leucophrys, and investigate the relationships among Z. leucophrys subspecies, using mitochondrial sequencing and a reduced representation nuclear genomic dataset. Contrary to the paraphyly evident in mitochondrial gene trees, we confirmed the reciprocal monophyly of Z. atricapilla and Z. leucophrys using large panels of single nucleotide polymorphisms (SNPs). The pattern of cytonuclear discordance is consistent with limited, historical hybridization and mitochondrial introgression, rather than a recent origin and incomplete lineage sorting between recent sister species. We found evidence of nuclear phylogeographic structure within Z. leucophrys with two distinct clades. Altogether, our results indicate deeper divergences between Z. atricapilla and Z. leucophrys than inferred using mitochondrial markers. Our results demonstrate the limitations of relying solely on mitochondrial DNA for taxonomy, and raise questions about the possibility of selection on the mitochondrial genome during temperature oscillations (e.g. during the Pleistocene). Historical mitochondrial introgression facilitated by past environmental changes could cause erroneous dating of lineage splitting in other taxa when based on mitochondrial DNA alone.

Cryptic species and independent origins of allochronic populations within a seabird species complex (Hydrobates spp.).

Humans are inherently biased towards naming species based on morphological differences, which can lead to reproductively isolated species being mistakenly classified as one if they are morphologically similar. Recognising cryptic diversity is needed to understand drivers of speciation fully, and for accurate estimates of global biodiversity and assessments for conservation. We investigated cryptic species across the range of band-rumped storm-petrels (Hydrobates spp.): highly pelagic, nocturnal seabirds that breed on tropical and sub-tropical islands in the Atlantic and Pacific Oceans. In many breeding colonies, band-rumped storm-petrels have sympatric but temporally isolated (allochronic) populations; we sampled all breeding locations and allochronic populations. Using mitochondrial control region sequences from 754 birds, cytochrome b sequences from 69 birds, and reduced representation sequencing of the nuclear genomes of 133 birds, we uncovered high levels of genetic structuring. Population genomic analyses revealed up to seven unique clusters, and phylogenomic reconstruction showed that these represent seven monophyletic groups. We uncovered up to six independent breeding season switches across the phylogeny, spanning the continuum from genetically undifferentiated temporal populations to full allochronic species. Thus, band-rumped storm-petrels encompass multiple cryptic species, with non-geographic barriers potentially comprising strong barriers to gene flow.

Phytogeographic patterns and cryptic diversity in an aposematic toad from NW Argentina.

The Yungas Redbelly Toad, Melanophryniscus rubriventris, is patchily distributed in Argentina, confined to the upland portion (1000-2000m above sea level) of the montane forests of northern and central regions of Salta, and in central-eastern and south-eastern Jujuy. This species is known for its striking aposematic color variation across its geographic distribution, and was once treated as a complex of three subspecies based on distinctive color patterns. Here we assess the geographical genetic variation within M. rubriventris and quantify divergence in color and pattern among individuals sampled from Northwestern Argentina. We compare multi-gene phylogeography of M. rubriventris to patterns of dorsal and ventral coloration to test whether evolutionary affinities predict variation in warning color. Our results reveal two well-supported species lineages: one confined to the extreme northern portion of our sampling area, and the other extending over most of the Argentine portion of the species' range, within which there are two populations. However, these well-supported evolutionary relationships do not mirror the marked variation in warning coloration. This discordance between DNA genealogy and warning color variation may reflect selection brought about by differences in local predation pressures, potentially coupled with effects of sexual selection and thermoregulation.

Color patterns of closely related bird species are more divergent at intermediate levels of breeding-range sympatry.

Closely related species of birds often differ markedly in their color patterns. Here we examine the influence of breeding-range overlap (breeding sympatry) on the evolution of color pattern differences in a sample of closely related bird species. We used a sister-lineage method to analyze 73 phylogenetically independent comparisons among 246 species and 39 families of birds worldwide. We found that divergence of color patterns among closely related species was greater between sympatric than between allopatric lineages, but only at intermediate levels of sympatry (50%-80% breeding-range overlap). This pattern suggests that closely related species incur costs at intermediate levels of sympatry if they exhibit similar color patterns-costs that could include hybridization, interspecific aggression, competition for signaling space, or ecological interactions that secondarily influence color patterns. The decline in color pattern divergence with further increase in sympatry suggests either the relaxation of divergent selection, increased impediment of gene flow, or an increased role for counteracting selection at higher levels of sympatry. We also found that the differences in color pattern between sympatric and allopatric sister species were greatest at lower latitudes. The global scale and broad taxonomic coverage in our study suggest that the divergence of color patterns between sympatrically breeding closely related species is widespread in birds.

Phylogeography and genetic structure of two Patagonian shag species (Aves: Phalacrocoracidae).

We compared the phylogeographic and genetic structure of two sympatric shag species, Phalacrocorax magellanicus (rock shag) and Phalacrocorax atriceps (imperial shag), from Patagonia (southern South America). We used multilocus genotypes of nuclear DNA (microsatellite loci) from 324 individuals and mitochondrial DNA sequences (ATPase) from 177 individuals, to evaluate hypotheses related to the effect of physical and non-physical barriers on seabird evolution. Despite sharing many ecological traits, the focal species strongly differ in two key aspects: P. magellanicus has a strong tendency to remain at/near their breeding colonies during foraging trips and the non-breeding season, while P. atriceps exhibits the converse pattern. Both species showed similar mtDNA genetic structure, where colonies from the Atlantic Coast, Pacific Coast and Fuegian region were genetically divergent. We also found similarities in the results of Bayesian clustering analysis of microsatellites, with both species having four clusters. However population differentiation (e.g. Fst, Φst) was higher in P. magellanicus compared to P. atriceps, and average membership probabilities of individuals to specific clusters (Q-values) were also higher in the former. Phalacrocorax magellanicus has strong phylogeographic structure, consistent with the impact of Pleistocene glaciations, with diagnostic haplotypes associated with each of the three mentioned regions. The same pattern was not as evident for P. atriceps. Migration rate estimators were higher for P. atriceps than for P. magellanicus; however both species followed an n-island-like model of gene flow, this implies that dispersal occurs across the continental land mass that separates Atlantic and Pacific Oceans. Our results supported the hypothesis that non-physical barriers are important drivers of the genetic and phylogeographic structure in seabirds, and also that physical barriers constitute effective but not absolute impediments to gene flow.

Social networks reveal sex- and age-patterned social structure in Butler's gartersnakes (Thamnophis butleri).

Sex- and age-based social structures have been well documented in animals with visible aggregations. However, very little is known about the social structures of snakes. This is most likely because snakes are often considered non-social animals and are particularly difficult to observe in the wild. Here, we show that wild Butler's Gartersnakes have an age and sex assorted social structure similar to more commonly studied social animals. To demonstrate this, we use data from a 12-year capture-mark-recapture study to identify social interactions using social network analyses. We find that the social structures of Butler's Gartersnakes comprise sex- and age-assorted intra-species communities with older females often central and age segregation partially due to patterns of study site use. In addition, we find that females tended to increase in sociability as they aged while the opposite occurred in males. We also present evidence that social interaction may provide fitness benefits, where snakes that were part of a social network were more likely to have improved body condition. We demonstrate that conventional capture data can reveal valuable information on social structures in cryptic species. This is particularly valuable as research has consistently demonstrated that understanding social structure is important for conservation efforts. Additionally, research on the social patterns of animals without obvious social groups provides valuable insight into the evolution of group living.

The geographic distribution, and the biotic and abiotic predictors of select zoonotic pathogen detections in Canadian polar bears.

Increasing Arctic temperatures are facilitating the northward expansion of more southerly hosts, vectors, and pathogens, exposing naïve populations to pathogens not typical at northern latitudes. To understand such rapidly changing host-pathogen dynamics, we need sensitive and robust surveillance tools. Here, we use a novel multiplexed magnetic-capture and droplet digital PCR (ddPCR) tool to assess a sentinel Arctic species, the polar bear (Ursus maritimus; n = 68), for the presence of five zoonotic pathogens (Erysipelothrix rhusiopathiae, Francisella tularensis, Mycobacterium tuberculosis complex, Toxoplasma gondii and Trichinella spp.), and observe associations between pathogen presence and biotic and abiotic predictors. We made two novel detections: the first detection of a Mycobacterium tuberculosis complex member in Arctic wildlife and the first of E. rhusiopathiae in a polar bear. We found a prevalence of 37% for E. rhusiopathiae, 16% for F. tularensis, 29% for Mycobacterium tuberculosis complex, 18% for T. gondii, and 75% for Trichinella spp. We also identify associations with bear age (Trichinella spp.), harvest season (F. tularensis and MTBC), and human settlements (E. rhusiopathiae, F. tularensis, MTBC, and Trichinella spp.). We demonstrate that monitoring a sentinel species, the polar bear, could be a powerful tool in disease surveillance and highlight the need to better characterize pathogen distributions and diversity in the Arctic.

Non-invasive biomonitoring of polar bear feces can be used to estimate concentrations of metals of concern in traditional food.

The Arctic faces increasing exposure to environmental chemicals such as metals, posing health risks to humans and wildlife. Biomonitoring of polar bears (Ursus maritimus) can be used to quantify chemicals in the environment and in traditional foods consumed by the Inuit. However, typically, these samples are collected through invasive or terminal methods. The biomonitoring of feces could be a useful alternative to the current metal monitoring method within the Arctic. Here, we aim to 1) quantify the relationship between concentrations of metals in the feces and tissues (muscle, liver, and fat) of polar bears using predictive modeling, 2) develop an easy-to-use conversion tool for use in community-based monitoring programs to non-invasively estimate contaminant concentrations in polar bears tissues and 3) demonstrate the application of these models by examining potential exposure risk for humans from consumption of polar bear muscle. Fecal, muscle, liver, and fat samples were harvested from 49 polar bears through a community-based monitoring program. The samples were analyzed for 32 metals. Exploratory analysis indicated that mean metal concentrations generally did not vary by age or sex, and many of the metals measured in feces were positively correlated with the internal tissue concentration. We developed predictive linear regression models between internal (muscle, liver, fat) and external (feces) metal concentrations and further explored the mercury and methylmercury relationships for utility risk screening. Using the cross-validated regression coefficients, we developed a conversion tool that contributes to the One Health approach by understanding the interrelated health of humans, wildlife, and the environment in the Arctic. The findings support using feces as a biomonitoring tool for assessing contaminants in polar bears. Further research is needed to validate the developed models for other regions in the Arctic and assess the impact of environmental weathering on fecal metal concentrations.

Early Miocene origin and cryptic diversification of South American salamanders.

BACKGROUND: The currently recognized species richness of South American salamanders is surprisingly low compared to North and Central America. In part, this low richness may be due to the salamanders being a recent arrival to South America. Additionally, the number of South American salamander species may be underestimated because of cryptic diversity. The aims of our present study were to infer evolutionary relationships, lineage diversity, and timing of divergence of the South American Bolitoglossa using mitochondrial and nuclear sequence data from specimens primarily from localities in the Andes and upper Amazon Basin. We also estimated time of colonization of South America to test whether it is consistent with arrival via the Panamanian Isthmus, or land bridge connection, at its traditionally assumed age of 3 million years. RESULTS: Divergence time estimates suggest that Bolitoglossa arrived in South America from Central America by at least the Early Miocene, ca. 23.6 MYA (95% HPD 15.9-30.3 MYA), and subsequently diversified. South American salamanders of the genus Bolitoglossa show strong phylogeographic structure at fine geographic scales and deep divergences at the mitochondrial gene cytochrome b (Cytb) and high diversity at the nuclear recombination activating gene-1 (Rag1). Species often contain multiple genetically divergent lineages that are occasionally geographically overlapping. Single specimens from two southeastern localities in Ecuador are sister to the equatoriana-peruviana clade and genetically distinct from all other species investigated to date. Another single exemplar from the Andes of northwestern Ecuador is highly divergent from all other specimens and is sister to all newly studied samples. Nevertheless, all sampled species of South American Bolitoglossa are members of a single clade that is one of several constituting the subgenus Eladinea, one of seven subgenera in this large genus. CONCLUSIONS: The ancestors of South American salamanders likely arrived at least by the Early Miocene, well before the completion of the Late Pliocene Panamanian land bridge (widely accepted as ca. 3 MYA). This date is in agreement with recent, controversial, arguments that an older, perhaps short-lived, land connection may have existed between South America and present-day Panama 23-25 MYA. Since its arrival in South America, Bolitoglossa has diversified more extensively than previously presumed and currently includes several cryptic species within a relatively small geographic area. Rather than two upper Amazonian species currently recorded for this region, we propose that at least eight should be recognized, although these additional lineages remain to be formally described.

Continental phylogeography of an ecologically and morphologically diverse Neotropical songbird, Zonotrichia capensis.

BACKGROUND: The Neotropics are exceptionally diverse, containing roughly one third of all extant bird species on Earth. This remarkable species richness is thought to be a consequence of processes associated with both Andean orogenesis throughout the Tertiary, and climatic fluctuations during the Quaternary. Phylogeographic studies allow insights into how such events might have influenced evolutionary trajectories of species and ultimately contribute to a better understanding of speciation. Studies on continentally distributed species are of particular interest because different populations of such taxa may show genetic signatures of events that impacted the continent-wide biota. Here we evaluate the genealogical history of one of the world's most broadly-distributed and polytypic passerines, the rufous-collared sparrow (Zonotrichia capensis). RESULTS: We obtained control region DNA sequences from 92 Zonotrichia capensis individuals sampled across the species' range (Central and South America). Six additional molecular markers, both nuclear and mitochondrial, were sequenced for a subset of individuals with divergent control region haplotypes. Median-joining network analysis, and Bayesian and maximum parsimony phylogenetic analyses all recovered three lineages: one spanning Middle America, the Dominican Republic, and north-western South America; one encompassing the Dominican Republic, Roraima (Venezuela) and La Paz (Bolivia) south to Tierra del Fuego, Argentina; and a third, including eastern Argentina and Brazil. Phylogenetic analyses suggest that the Middle American/north-western South American clade is sister to the remaining two. Bayesian and maximum likelihood coalescent simulations used to study lineage demographic history, diversification times, migration rates and population expansion together suggested that diversification of the three lineages occurred rapidly during the Pleistocene, with negligible gene flow, leaving genetic signatures of population expansions. CONCLUSIONS: The Pleistocene history of the rufous-collared sparrow involved extensive range expansion from a probable Central American origin. Its remarkable morphological and behavioral diversity probably represents recent responses to local conditions overlying deeper patterns of lineage diversity, which are themselves produced by isolation and the history of colonization of South America.

The influence of landscape on gene flow in the eastern massasauga rattlesnake (Sistrurus c. catenatus): insight from computer simulations.

Understanding how gene flow shapes contemporary population structure requires the explicit consideration of landscape composition and configuration. New landscape genetic approaches allow us to link such heterogeneity to gene flow within and among populations. However, the attribution of cause is difficult when landscape features are spatially correlated, or when genetic patterns reflect past events. We use spatial Bayesian clustering and landscape resistance analysis to identify the landscape features that influence gene flow across two regional populations of the eastern massasauga rattlesnake, Sistrurus c. catenatus. Based on spatially explicit simulations, we inferred how habitat distribution modulates gene flow and attempted to disentangle the effects of spatially confounded landscape features. We found genetic clustering across one regional landscape but not the other, and also local differences in the effect of landscape on gene flow. Beyond the effects of isolation-by-distance, water bodies appear to underlie genetic differentiation among individuals in one regional population. Significant effects of roads were additionally detected locally, but these effects are possibly confounded with the signal of water bodies. In contrast, we found no signal of isolation-by-distance or landscape effects on genetic structure in the other regional population. Our simulations imply that these local differences have arisen as a result of differences in population density or tendencies for juvenile rather than adult dispersal. Importantly, our simulations also demonstrate that the ability to detect the consequences of contemporary anthropogenic landscape features (e.g. roads) on gene flow may be compromised when long-standing natural features (e.g. water bodies) co-exist on the landscape.

Integrating eDNA and citizen science observations to model distribution of a temperate freshwater turtle near its northern range limit.

Background: To determine species distributions and the factors underlying them, reliable occurrence data are crucial. Assembling such data can be challenging for species with cryptic life histories or that occur at low densities. Methods: We developed species-specific eDNA protocols, from sampling through data interpretation, to detect the common musk turtle (Sternotherus odoratus) and tested whether eDNA occurrences change our understanding of the species distribution and the factors that shape its northern range limit. We used Species Distribution Models (SDMs) with full parameter optimization on citizen science observations of S. odoratus in Southern Ontario alone and together with eDNA occurrences. Results: Our eDNA protocol was robust and sensitive. SDMs built from traditional observations and those supplemented with eDNA detections were comparable in prediction accuracy. However, models with eDNA detections suggested that the distribution of S. odoratus in Southern Ontario is underestimated, especially near its northern range limit, and that it is shaped by thermal conditions, hydrology, and elevation. Our study underscores the promise of eDNA for surveying cryptic aquatic organisms in undocumented areas, and how such insights can help us to improve our understanding of species distributions.

Assessing the breeding phenology of a threatened frog species using eDNA and automatic acoustic monitoring.

Background: Climate change has driven shifts in breeding phenology of many amphibians, causing phenological mismatches (e.g., predator-prey interactions), and potentially population declines. Collecting data with high spatiotemporal sensitivity on hibernation emergence and breeding times can inform conservation best practices. However, monitoring the phenology of amphibians can be challenging because of their cryptic nature over much of their life cycle. Moreover, most salamanders and caecilians do not produce conspicuous breeding calls like frogs and toads do, presenting additional monitoring challenges. Methods: In this study, we designed and evaluated the performance of an environmental DNA (eDNA) droplet digital PCR (ddPCR) assay as a non-invasive tool to assess the breeding phenology of a Western Chorus Frog population (Pseudacris maculata mitotype) in Eastern Ontario and compared eDNA detection patterns to hourly automatic acoustic monitoring. For two eDNA samples with strong PCR inhibition, we tested three methods to diminish the effect of inhibitors: diluting eDNA samples, adding bovine serum albumin to PCR reactions, and purifying eDNA using a commercial clean-up kit. Results: We recorded the first male calling when the focal marsh was still largely frozen. Chorus frog eDNA was detected on April 6th, 6 days after acoustic monitoring revealed this first calling male, but only 2 days after males attained higher chorus activity. eDNA signals were detected at more sampling locales within the marsh and eDNA concentrations increased as more males participated in the chorus, suggesting that eDNA may be a reasonable proxy for calling assemblage size. Internal positive control revealed strong inhibition in some samples, limiting detection probability and quantification accuracy in ddPCR. We found diluting samples was the most effective in reducing inhibition and improving eDNA quantification. Conclusions: Altogether, our results showed that eDNA ddPCR signals lagged behind male chorusing by a few days; thus, acoustic monitoring is preferable if the desire is to document the onset of male chorusing. However, eDNA may be an effective, non-invasive monitoring tool for amphibians that do not call and may provide a useful complement to automated acoustic recording. We found inhibition patterns were heterogeneous across time and space and we demonstrate that an internal positive control should always be included to assess inhibition for eDNA ddPCR signal interpretations.

Strong effects of food quality on host life history do not scale to impact parasitoid efficacy or life history.

Parasitoids are small insects, (e.g., small wasps or flies) that reproduce by laying eggs on or within host arthropods. Parasitoids make up a large proportion of the world's biodiversity and are popular agents of biological control. Idiobiont parasitoids paralyze their hosts upon attack and thus are expected to only target hosts large enough to support offspring development. Host resources generally impact host attributes and life histories including size, development, and life span. Some argue slow host development in response to resource quality increases parasitoid efficacy (i.e., a parasitoid's ability to successfully reproduce on or within a host) due to longer host exposure to parasitoids. However, this hypothesis is not always supported and does not consider variation in other host traits in response to resources that may be important for parasitoids (e.g., variation in host size is known to impact parasitoid efficacy). In this study we test whether trait variation within host developmental stages in response to host resources is more important for parasitoid efficacy and life histories than trait variation across host developmental stages. We exposed seed beetle hosts raised on a food quality gradient to mated female parasitoids and measured the number of hosts parasitized and parasitoid life history traits at the scale of host stage- and age-structure. Our results suggest host food quality does not cascade to impact idiobiont parasitoid life histories despite large food quality effects on host life history. Instead, variation in host life histories across host developmental stages better predicts parasitoid efficacy and life histories, suggesting finding a host in a specific instar is more important for idiobiont parasitoids than finding hosts on or within higher quality resources.

Multiomics Reveals Symbionts, Pathogens, and Tissue-Specific Microbiome of Blacklegged Ticks (Ixodes scapularis) from a Lyme Disease Hot Spot in Southeastern Ontario, Canada.

Ticks in the family Ixodidae are important vectors of zoonoses, including Lyme disease (LD), which is caused by spirochete bacteria from the Borreliella (Borrelia) burgdorferi sensu lato complex. The blacklegged tick (Ixodes scapularis) continues to expand across Canada, creating hot spots of elevated LD risk at the leading edge of its expanding range. Current efforts to understand the risk of pathogen transmission associated with I. scapularis in Canada focus primarily on targeted screens, while natural variation in the tick microbiome remains poorly understood. Using multiomics consisting of 16S metabarcoding and ribosome-depleted, whole-shotgun RNA transcriptome sequencing, we examined the microbial communities associated with adult I. scapularis (n = 32), sampled from four tissue types (whole tick, salivary glands, midgut, and viscera) and three geographical locations within a LD hot spot near Kingston, Ontario, Canada. The communities consisted of both endosymbiotic and known or potentially pathogenic microbes, including RNA viruses, bacteria, and a Babesia sp. intracellular parasite. We show that ß-diversity is significantly higher between the bacterial communities of individual tick salivary glands and midguts than that of whole ticks. Linear discriminant analysis effect size (LEfSe) determined that the three potentially pathogenic bacteria detected by V4 16S rRNA sequencing also differed among dissected tissues only, including a Borrelia strain from the B. burgdorferi sensu lato complex, Borrelia miyamotoi, and Anaplasma phagocytophilum. Importantly, we find coinfection of I. scapularis by multiple microbes, in contrast to diagnostic protocols for LD, which typically focus on infection from a single pathogen of interest (B. burgdorferi sensu stricto). IMPORTANCE As a vector of human health concern, blacklegged ticks (Ixodes scapularis) transmit pathogens that cause tick-borne diseases (TBDs), including Lyme disease (LD). Several hot spots of elevated LD risk have emerged across Canada as I. scapularis expands its range. Focusing on a hot spot in southeastern Ontario, we used high-throughput sequencing to characterize the microbiome of whole ticks and dissected salivary glands and midguts. Compared with whole ticks, salivary glands and midguts were more diverse and associated with distinct bacterial communities that are less dominated by Rickettsia endosymbiont bacteria and are enriched for pathogenic bacteria, including a B. burgdorferi sensu lato-associated Borrelia sp., Borrelia miyamotoi, and Anaplasma phagocytophilum. We also found evidence of coinfection of I. scapularis by multiple pathogens. Overall, our study highlights the challenges and opportunities associated with the surveillance of the microbiome of I. scapularis for pathogen detection using metabarcoding and metatranscriptome approaches.

A new multiplexed magnetic capture-Droplet digital PCR tool for monitoring wildlife population health and pathogen surveillance.

Anthropogenic stressors are exacerbating the emergence and spread of pathogens worldwide. In regions like the Arctic, where ecosystems are particularly susceptible, marked changes are predicted in regional diversity, intensity, and patterns of infectious diseases. To understand such rapidly changing host-pathogen dynamics and mitigate the impacts of novel pathogens, we need sensitive disease surveillance tools. We developed and validated a novel multiplexed, magnetic capture, and ddPCR tool for the surveillance of multiple pathogens in polar bears, a sentinel species that is considered susceptible to climate change and other stressors with a pan-Arctic distribution. Through sequence-specific magnetic capture, we concentrated five target template sequences from three zoonotic bacteria (Erysipelothrix rhusiopathiae, Francisella tularensis, and Mycobacterium tuberculosis complex) and two parasitic (Toxoplasma gondii and Trichinella spp.) pathogens from large quantities (<100 g) of host tissue. We then designed and validated two multiplexed probe-based ddPCR assays for the amplification and detection of the low-concentration target DNA. Validations used 48 polar bear tissues (muscle and liver). We detected 14, 1, 3, 4, and 22 tissue positives for E. rhusiopathiae, F. tularensis, M. tuberculosis complex, T. gondii, and Trichinella spp., respectively. These multiplexed assays offer a rapid, specific tool for quantifying and monitoring the changing geographical and host distributions of pathogens relevant to human and animal health.

Extensive regional variation in the phenology of insects and their response to temperature across North America.

Climate change models often assume similar responses to temperatures across the range of a species, but local adaptation or phenotypic plasticity can lead plants and animals to respond differently to temperature in different parts of their range. To date, there have been few tests of this assumption at the scale of continents, so it is unclear if this is a large-scale problem. Here, we examined the assumption that insect taxa show similar responses to temperature at 96 sites in grassy habitats across North America. We sampled insects with Malaise traps during 2019-2021 (N = 1041 samples) and examined the biomass of insects in relation to temperature and time of season. Our samples mostly contained Diptera (33%), Lepidoptera (19%), Hymenoptera (18%), and Coleoptera (10%). We found strong regional differences in the phenology of insects and their response to temperature, even within the same taxonomic group, habitat type, and time of season. For example, the biomass of nematoceran flies increased across the season in the central part of the continent, but it only showed a small increase in the Northeast and a seasonal decline in the Southeast and West. At a smaller scale, insect biomass at different traps operating on the same days was correlated up to ~75 km apart. Large-scale geographic and phenological variation in insect biomass and abundance has not been studied well, and it is a major source of controversy in previous analyses of insect declines that have aggregated studies from different locations and time periods. Our study illustrates that large-scale predictions about changes in insect populations, and their causes, will need to incorporate regional and taxonomic differences in the response to temperature.