Drying as an effective method to store soil samples for DNA-based microbial community analyses: a comparative study.

Soil sampling for environmental DNA in remote and semi-remote locations is often limited due to logistical constraints surrounding sample preservation, including no or limited access to a freezer. Freezing at - 20 °C is a common DNA preservation strategy, however, other methods such as desiccation, ethanol or commercial preservatives are available as potential alternative DNA preservation methods for room temperature storage. In this study, we assessed five preservation methods (CD1 solution, 95% Ethanol, Dry & Dry silica gel packs, RNAlater, LifeGuard) along with freezing at - 20 °C, against immediate extraction on organic and mineral soils for up to three weeks of preservation. We assessed direct effects on DNA concentration and quality, and used DNA metabarcoding to assess effects on bacterial and fungal communities. Drying with Dry & Dry led to no significant differences from immediate extraction. RNAlater led to lower DNA concentrations, but effects on community structures were comparable to freezing. CD1, LifeGuard and Ethanol either caused immediate significant shifts in community structure, degradation of DNA quality or changes in diversity metrics. Overall, our study supports the use of drying with silica gel packs as a cost-effective, and easily applied method for the short-term storage at room temperature for DNA-based microbial community analyses.

All boreal forest successional stages needed to maintain the full suite of soil biodiversity, community composition, and function following wildfire.

Wildfire is a natural disturbance in boreal forest systems that has been predicted to increase in frequency, intensity, and extent due to climate change. Most studies tend to assess the recovery of one component of the community at a time but here we use DNA metabarcoding to simultaneously monitor soil bacteria, fungi, and arthropods along an 85-year chronosequence following wildfire in jack pine-dominated ecosites. We describe soil successional and community assembly processes to better inform sustainable forest management practices. Soil taxa showed different recovery trajectories following wildfire. Bacteria shared a large core community across stand development stages (~ 95-97% of their unique sequences) and appeared to recover relatively quickly by crown closure. By comparison fungi and arthropods shared smaller core communities (64-77% and 68-69%, respectively) and each stage appeared to support unique biodiversity. We show the importance of maintaining a mosaic ecosystem that represents each stand development stage to maintain the full suite of biodiversity in soils following wildfire, especially for fungi and arthropods. These results will provide a useful baseline for comparison when assessing the effects of human disturbance such as harvest or for assessing the effects of more frequent wildfire events due to climate change.

Forest soil biotic communities show few responses to wood ash applications at multiple sites across Canada.

There is interest in utilizing wood ash as an amendment in forestry operations as a mechanism to return nutrients to soils that are removed during harvesting, with the added benefit of diverting this bioenergy waste material from landfill sites. Existing studies have not arrived at a consensus on what the effects of wood ash amendments are on soil biota. We collected forest soil samples from studies in managed forests across Canada that were amended with wood ash to evaluate the effects on arthropod, bacterial and fungal communities using metabarcoding of F230, 16S, 18S and ITS2 sequences as well as enzyme analyses to assess its effects on soil biotic function. Ash amendment did not result in consistent effects across sites, and those effects that were detected were small. Overall, this study suggests that ash amendment applied to managed forest systems in amounts (up to 20 Mg ha-1) applied across the 8 study sties had little to no detectable effects on soil biotic community structure or function. When effects were detected, they were small, and site-specific. These non-results support the application of wood ash to harvested forest sites to replace macronutrients (e.g., calcium) removed by logging operations, thereby diverting it from landfill sites, and potentially increasing stand productivity.

Avian Predation in a Declining Outbreak Population of the Spruce Budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae).

The impact of avian predation on a declining population of the spruce budworm, Choristoneura fumifereana (Clem.), was measured using single-tree exclosure cages in a mature stand of balsam fir, Abies balsamea (L.), and white spruce, Picea glauca (Moench.) Voss. Bird population censuses and observations of foraging and nest-feeding activity were also made to determine the response of budworm-linked warblers to decreasing food availability. Seasonal patterns of foraging. as well as foraging success in the declining prey population was compared to similar information from birds observed in another stand where the spruce budworm population was rising. Avian predation was an important source of mortality between the 4th instar and moth emergence in the declining outbreak population. Mortality by predation increased from negligible to over 98% as budworm density dropped from 100 to <1 larva/kg of host foliage, over 3 years. Calculations based on nest-feeding activity and basic metabolic demands support these observed rates. Seasonal and yearly differences in predation rates observed between the two host-tree species correspond to equivalent shifts in bird foraging behavior in response to dropping insect density. In particular, a preference for searching on white spruce disappeared, although budworm-linked birds remained more efficient at finding food on this plant. The ability to change foraging behavior as prey density dropped differed between bird species.

The Ecobiomics project: Advancing metagenomics assessment of soil health and freshwater quality in Canada.

Transformative advances in metagenomics are providing an unprecedented ability to characterize the enormous diversity of microorganisms and invertebrates sustaining soil health and water quality. These advances are enabling a better recognition of the ecological linkages between soil and water, and the biodiversity exchanges between these two reservoirs. They are also providing new perspectives for understanding microorganisms and invertebrates as part of interacting communities (i.e. microbiomes and zoobiomes), and considering plants, animals, and humans as holobionts comprised of their own cells as well as diverse microorganisms and invertebrates often acquired from soil and water. The Government of Canada's Genomics Research and Development Initiative (GRDI) launched the Ecobiomics Project to coordinate metagenomics capacity building across federal departments, and to apply metagenomics to better characterize microbial and invertebrate biodiversity for advancing environmental assessment, monitoring, and remediation activities. The Project has adopted standard methods for soil, water, and invertebrate sampling, collection and provenance of metadata, and nucleic acid extraction. High-throughput sequencing is located at a centralized sequencing facility. A centralized Bioinformatics Platform was established to enable a novel government-wide approach to harmonize metagenomics data collection, storage and bioinformatics analyses. Sixteen research projects were initiated under Soil Microbiome, Aquatic Microbiome, and Invertebrate Zoobiome Themes. Genomic observatories were established at long-term environmental monitoring sites for providing more comprehensive biodiversity reference points to assess environmental change.

Limited initial impacts of biomass harvesting on composition of wood-inhabiting fungi within residual stumps.

Growing pressures linked to global warming are prompting governments to put policies in place to find alternatives to fossil fuels. In this study, we compared the impact of tree-length harvesting to more intensive full-tree harvesting on the composition of fungi residing in residual stumps 5 years after harvest. In the tree-length treatment, a larger amount of residual material was left around the residual stumps in contrast to the full-tree treatment where a large amount of woody debris was removed. We collected sawdust from five randomly selected residual stumps in five blocks in each of the tree-length and full-tree treatments, yielding a total of 50 samples (25 in each treatment). We characterized the fungal operational taxonomic units (OTUs) present in each stump using high-throughput DNA sequencing of the fungal ITS region. We observed no differences in Shannon diversity between tree-length and full-tree harvesting. Likewise, we observed few differences in the composition of fungal OTUs among tree-length and full-tree samples using non-metric multidimensional scaling. Using the differential abundance analysis implemented with DESeq2, we did, however, detect several associations between specific fungal taxa and the intensity of residual biomass harvest. For example, Peniophorella pallida (Bres.) KH Larss. and Tephromela sp. were found mainly in the full-tree treatment, while Phlebia livida (Pers.) Bres. and Cladophialophora chaetospira (Grove) Crous & Arzanlou were found mainly in the tree-length treatment. While none of the 20 most abundant species in our study were identified as pathogens we did identify one conifer pathogen species Serpula himantioides (Fr.) P. Karst found mainly in the full-tree treatment.

Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators.

Terrestrial arthropod fauna have been suggested as a key indicator of ecological integrity in forest systems. Because phenotypic identification is expert-limited, a shift towards DNA metabarcoding could improve scalability and democratize the use of forest floor arthropods for biomonitoring applications. The objective of this study was to establish the level of field sampling and DNA extraction replication needed for arthropod biodiversity assessments from soil. Processing 15 individually collected soil samples recovered significantly higher median richness (488-614 sequence variants) than pooling the same number of samples (165-191 sequence variants) prior to DNA extraction, and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Beta diversity was robust to changes in methodological regimes. Though our ability to identify taxa to species rank was limited, we were able to use arthropod COI metabarcodes from forest soil to assess richness, distinguish among sites, and recover site indicators based on unnamed exact sequence variants. Our results highlight the need to continue DNA barcoding local taxa during COI metabarcoding studies to help build reference databases. All together, these sampling considerations support the use of soil arthropod COI metabarcoding as a scalable method for biomonitoring.

Modelling vegetation understory cover using LiDAR metrics.

Forest understory vegetation is an important characteristic of the forest. Predicting and mapping understory is a critical need for forest management and conservation planning, but it has proved difficult with available methods to date. LiDAR has the potential to generate remotely sensed forest understory structure data, but this potential has yet to be fully validated. Our objective was to examine the capacity of LiDAR point cloud data to predict forest understory cover. We modeled ground-based observations of understory structure in three vertical strata (0.5 m to < 1.5 m, 1.5 m to < 2.5 m, 2.5 m to < 3.5 m) as a function of a variety of LiDAR metrics using both mixed-effects and Random Forest models. We compared four understory LiDAR metrics designed to control for the spatial heterogeneity of sampling density. The four metrics were highly correlated and they all produced high values of variance explained in mixed-effects models. The top-ranked model used a voxel-based understory metric along with vertical stratum (Akaike weight = 1, explained variance = 87%, cross-validation error = 15.6%). We found evidence of occlusion of LiDAR pulses in the lowest stratum but no evidence that the occlusion influenced the predictability of understory structure. The Random Forest model results were consistent with those of the mixed-effects models, in that all four understory LiDAR metrics were identified as important, along with vertical stratum. The Random Forest model explained 74.4% of the variance, but had a lower cross-validation error of 12.9%. We conclude that the best approach to predict understory structure is using the mixed-effects model with the voxel-based understory LiDAR metric along with vertical stratum, because it yielded the highest explained variance with the fewest number of variables. However, results show that other understory LiDAR metrics (fractional cover, normalized cover and leaf area density) would still be effective in mixed-effects and Random Forest modelling approaches.

Cross-scale effects of spruce budworm outbreaks on boreal warblers in eastern Canada.

Insect outbreaks are major natural disturbance events that affect communities of forest birds, either directly by affecting the food supply or indirectly by changing the vegetation composition of forest canopies. An examination of correlations between measures of bird and insect abundance across different spatial scales and over varying time lag effects may provide insight into underlying mechanisms. We developed a hierarchical Bayesian model to assess correlations between counts of eight warbler species from the Breeding Bird Survey in eastern Canada, 1966 to 2009, with the presence of spruce budworm (Choristoneura fumiferana Clem.) at immediate local scales and time-lagged regional scales, as measured by extent of defoliation on host tree species. Budworm-associated species Cape May warbler (Setophaga tigrina), bay-breasted warbler (Setophaga castanea), and Tennessee warbler (Oreothlypis peregrina) responded strongly and positively to both local and regional effects. In contrast, non-budworm-associated species, Blackburnian warbler (Setophaga fusca), magnolia warbler (Setophaga magnolia), Canada warbler (Cardellina canadensis), black-throated blue warbler (Setophaga caerulescens), and black-throated green warbler (Setophaga virens), only responded to regional effects in a manner that varied across eastern Canada. The complex responses by forest birds to insect outbreaks involve both increased numerical responses to food supply and to longer term responses to changes in forest structure and composition. These effects can vary across spatial scales and be captured in hierarchical population models, which can serve to disentangle common trends from data when examining drivers of population dynamics like forest management or climate change.

Author Correction: DNA metabarcoding and morphological macroinvertebrate metrics reveal the same changes in boreal watersheds across an environmental gradient.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

DNA metabarcoding and morphological macroinvertebrate metrics reveal the same changes in boreal watersheds across an environmental gradient.

Cost-effective, ecologically relevant, sensitive, and standardized indicators are requisites of biomonitoring. DNA metabarcoding of macroinvertebrate communities is a potentially transformative biomonitoring technique that can reduce cost and time constraints while providing information-rich, high resolution taxonomic data for the assessment of watershed condition. Here, we assess the utility of DNA metabarcoding to provide aquatic indicator data for evaluation of forested watershed condition across Canadian eastern boreal watersheds, subject to natural variation and low-intensity harvest management. We do this by comparing the similarity of DNA metabarcoding and morphologically derived macroinvertebrate metrics (i.e. richness, % Ephemeroptera, Plecoptera and Trichoptera, % chironomid), and the ability of DNA metabarcoding and morphological metrics to detect key gradients in stream condition linked to forested watershed features. Our results show consistency between methods, where common DNA metabarcoding and morphological macroinvertebrate metrics are positively correlated and indicate the same key gradients in stream condition (i.e. dissolved oxygen, and dissolved organic carbon, total nitrogen and conductivity) linked to watershed size and shifts in forest composition across watersheds. Our study demonstrates the potential usefulness of macroinvertebrate DNA metabarcoding to future application in broad-scale biomonitoring of watershed condition across environmental gradients.

Bioenergy and Biodiversity: Key Lessons from the Pan American Region.

Understanding how large-scale bioenergy production can affect biodiversity and ecosystems is important if society is to meet current and future sustainable development goals. A variety of bioenergy production systems have been established within different contexts throughout the Pan American region, with wide-ranging results in terms of documented and projected effects on biodiversity and ecosystems. The Pan American region is home to the majority of commercial bioenergy production and therefore the region offers a broad set of experiences and insights on both conflicts and opportunities for biodiversity and bioenergy. This paper synthesizes lessons learned focusing on experiences in Canada, the United States, and Brazil regarding the conflicts that can arise between bioenergy production and ecological conservation, and benefits that can be derived when bioenergy policies promote planning and more sustainable land-management systems. We propose a research agenda to address priority information gaps that are relevant to biodiversity concerns and related policy challenges in the Pan American region.

Peyerimhoffiajaschhoforum (Diptera, Sciaridae), a new deadwood inhabiting species from Canada.

A new species of black fungus gnat from Canada, Peyerimhoffiajaschhoforum sp. n., is presented with a description, illustrations, biotope information and a brief discussion of the placement and concept of the genus Peyerimhoffia. P.jaschhoforum is characterized by a unique gonostylar structure where the apex is hollowed but not enclosed and contains a mass of mega setae housed within. P.jaschhoforum was reared from decomposing jack pine (Pinusbanksiana Lamb.) deadwood using both in-situ and ex-situ photoeclectors. We documented three additional specimens originating from Fennoscandia that resemble P.jaschhoforum but differ based on a broader tegmen, placement of setigerous papillae behind the tegmen and the fused intercoxal area. Based on this, these specimens are assigned to a new subspecies, Peyerimhoffiajaschhoforumfennoscandica ssp. n.

Initial responses of rove and ground beetles (Coleoptera, Staphylinidae, Carabidae) to removal of logging residues following clearcut harvesting in the boreal forest of Quebec, Canada.

Increased interest in biomass harvesting for bioenergetic applications has raised questions regarding the potential ecological consequences on forest biodiversity. Here we evaluate the initial changes in the abundance, species richness and community composition of rove (Staphylinidae) and ground beetles (Carabidae), immediately following 1) stem-only harvesting (SOH), in which logging debris (i.e., tree tops and branches) are retained on site, and 2) whole-tree harvesting (WTH), in which stems, tops and branches are removed in mature balsam fir stands in Quebec, Canada. Beetles were collected throughout the summer of 2011, one year following harvesting, using pitfall traps. Overall catch rates were greater in uncut forest (Control) than either stem-only or whole-tree harvested sites. Catch rates in WTH were greater than SOH sites. Uncut stands were characterized primarily by five species: Atheta capsularis, Atheta klagesi, Atheta strigosula, Tachinus fumipennis/frigidus complex (Staphylinidae) and to a lesser extent to Pterostichus punctatissimus(Carabidae). Increased catch rates in WTH sites, where post-harvest biomass was less, were attributable to increased catches of rove beetles Pseudopsis subulata, Quedius labradorensis and to a lesser extent Gabrius brevipennis. We were able to characterize differences in beetle assemblages between harvested and non-harvested plots as well as differences between whole tree (WTH) and stem only (SOH) harvested sites where logging residues had been removed or left following harvest. However, the overall assemblage response was largely a recapitulation of the responses of several abundant species.