Soil Microbial Community Composition and Tolerance to Contaminants in an Urban Brownfield Site.

Brownfields are unused sites that contain hazardous substances due to previous commercial or industrial use. The sites are inhospitable for many organisms, but some fungi and microbes can tolerate and thrive in the nutrient-depleted and contaminated soils. However, few studies have characterized the impacts of long-term contamination on soil microbiome composition and diversity at brownfields. This study focuses on an urban brownfield-a former rail yard in Los Angeles that is contaminated with heavy metals, volatile organic compounds, and petroleum-derived pollutants. We anticipate that heavy metals and organic pollutants will shape soil microbiome diversity and that several candidate fungi and bacteria will be tolerant to the contaminants. We sequence three gene markers (16S ribosomal RNA, 18S ribosomal RNA, and the fungal internal transcribed spacer (FITS)) in 55 soil samples collected at five depths to (1) profile the composition of the soil microbiome across depths; (2) determine the extent to which hazardous chemicals predict microbiome variation; and (3) identify microbial taxonomic groups that may metabolize these contaminants. Detected contaminants in the samples included heavy metals, petroleum hydrocarbons, polycyclic aromatic hydrocarbons, and volatile organic compounds. Bacterial, eukaryotic, and fungal communities all varied with depth and with concentrations of arsenic, chromium, cobalt, and lead. 18S rRNA microbiome richness and fungal richness were positively correlated with lead and cobalt levels, respectively. Furthermore, bacterial Paenibacillus and Iamia, eukaryotic Actinochloris, and fungal Alternaria were enriched in contaminated soils compared to uncontaminated soils and represent taxa of interest for future bioremediation research. Based on our results, we recommend incorporating DNA-based multi-marker microbial community profiling at multiple sites and depths in brownfield site assessment standard methods and restoration.

Author Correction: Effects of sampling effort on biodiversity patterns estimated from environmental DNA metabarcoding surveys.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Global invasion history of the agricultural pest butterfly Pieris rapae revealed with genomics and citizen science.

The small cabbage white butterfly, Pieris rapae, is a major agricultural pest of cruciferous crops and has been introduced to every continent except South America and Antarctica as a result of human activities. In an effort to reconstruct the near-global invasion history of P. rapae, we developed a citizen science project, the "Pieris Project," and successfully amassed thousands of specimens from 32 countries worldwide. We then generated and analyzed nuclear (double-digest restriction site-associated DNA fragment procedure [ddRAD]) and mitochondrial DNA sequence data for these samples to reconstruct and compare different global invasion history scenarios. Our results bolster historical accounts of the global spread and timing of P. rapae introductions. We provide molecular evidence supporting the hypothesis that the ongoing divergence of the European and Asian subspecies of P. rapae (∼1,200 y B.P.) coincides with the diversification of brassicaceous crops and the development of human trade routes such as the Silk Route (Silk Road). The further spread of P. rapae over the last ∼160 y was facilitated by human movement and trade, resulting in an almost linear series of at least 4 founding events, with each introduced population going through a severe bottleneck and serving as the source for the next introduction. Management efforts of this agricultural pest may need to consider the current existence of multiple genetically distinct populations. Finally, the international success of the Pieris Project demonstrates the power of the public to aid scientists in collections-based research addressing important questions in invasion biology, and in ecology and evolutionary biology more broadly.

Water Flow and Biofilm Cover Influence Environmental DNA Detection in Recirculating Streams.

The increasing use of environmental DNA (eDNA) for determination of species presence in aquatic ecosystems is an invaluable technique for both ecology as a field and for the management of aquatic ecosystems. We examined the degradation dynamics of fish eDNA using an experimental array of recirculating streams, also using a "nested" primer assay to estimate degradation among eDNA fragment sizes. We introduced eDNA into streams with a range of water velocities (0.1-0.8 m s-1) and substrate biofilm coverage (0-100%) and monitored eDNA concentrations over time (∼10 d) to assess how biophysical conditions influence eDNA persistence. We found that the presence of biofilm significantly increased initial decay rates relative to previous studies conducted in nonflowing microcosms, suggesting important differences in detection and persistence in lentic vs lotic systems. Lastly, by using a nested primer assay that targeted different size eDNA fragments, we found that fragment size altered both the estimated rate constant coefficients, as well as eDNA detectability over time. Larger fragments (>600 bp) were quickly degraded, while shorter fragments (<100 bp) remained detectable for the entirety of the experiment. When using eDNA as a stream monitoring tool, understanding environmental factors controlling eDNA degradation will be critical for optimizing eDNA sampling strategies.

Effects of sampling effort on biodiversity patterns estimated from environmental DNA metabarcoding surveys.

Environmental DNA (eDNA) metabarcoding can greatly enhance our understanding of global biodiversity and our ability to detect rare or cryptic species. However, sampling effort must be considered when interpreting results from these surveys. We explored how sampling effort influenced biodiversity patterns and nonindigenous species (NIS) detection in an eDNA metabarcoding survey of four commercial ports. Overall, we captured sequences from 18 metazoan phyla with minimal differences in taxonomic coverage between 18 S and COI primer sets. While community dissimilarity patterns were consistent across primers and sampling effort, richness patterns were not, suggesting that richness estimates are extremely sensitive to primer choice and sampling effort. The survey detected 64 potential NIS, with COI identifying more known NIS from port checklists but 18 S identifying more operational taxonomic units shared between three or more ports that represent un-recorded potential NIS. Overall, we conclude that eDNA metabarcoding surveys can reveal global similarity patterns among ports across a broad array of taxa and can also detect potential NIS in these key habitats. However, richness estimates and species assignments require caution. Based on results of this study, we make several recommendations for port eDNA sampling design and suggest several areas for future research.

Environmental DNA (eDNA) detects the invasive rusty crayfish Orconectes rusticus at low abundances.

Early detection is invaluable for the cost-effective control and eradication of invasive species, yet many traditional sampling techniques are ineffective at the low population abundances found at the onset of the invasion process. Environmental DNA (eDNA) is a promising and sensitive tool for early detection of some invasive species, but its efficacy has not yet been evaluated for many taxonomic groups and habitat types.We evaluated the ability of eDNA to detect the invasive rusty crayfish Orconectes rusticus and to reflect patterns of its relative abundance, in upper Midwest, USA, inland lakes. We paired conventional baited trapping as a measure of crayfish relative abundance with water samples for eDNA, which were analysed in the laboratory with a qPCR assay. We modelled detection probability for O. rusticus eDNA using relative abundance and site characteristics as covariates and also tested the relationship between eDNA copy number and O. rusticus relative abundance.We detected O. rusticus eDNA in all lakes where this species was collected by trapping, down to low relative abundances, as well as in two lakes where trap catch was zero. Detection probability of O. rusticus eDNA was well predicted by relative abundance of this species and lake water clarity. However, there was poor correspondence between eDNA copy number and O. rusticus relative abundance estimated by trap catches. Synthesis and applications. Our study demonstrates a field and laboratory protocol for eDNA monitoring of crayfish invasions, with results of statistical models that provide guidance of sampling effort and detection probabilities for researchers in other regions and systems. We propose eDNA be included as a tool in surveillance for invasive or imperilled crayfishes and other benthic arthropods.

Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms.

Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs.

Estimating species richness using environmental DNA.

The foundation for any ecological study and for the effective management of biodiversity in natural systems requires knowing what species are present in an ecosystem. We assessed fish communities in a stream using two methods, depletion-based electrofishing and environmental DNA metabarcoding (eDNA) from water samples, to test the hypothesis that eDNA provides an alternative means of determining species richness and species identities for a natural ecosystem. In a northern Indiana stream, electrofishing yielded a direct estimate of 12 species and a mean estimated richness (Chao II estimator) of 16.6 species with a 95% confidence interval from 12.8 to 42.2. eDNA sampling detected an additional four species, congruent with the mean Chao II estimate from electrofishing. This increased detection rate for fish species between methods suggests that eDNA sampling can enhance estimation of fish fauna in flowing waters while having minimal sampling impacts on fish and their habitat. Modern genetic approaches therefore have the potential to transform our ability to build a more complete list of species for ecological investigations and inform management of aquatic ecosystems.

Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding.

Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206-L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina-sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction.

Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol-chloroform-isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.

Environmental conditions influence eDNA persistence in aquatic systems.

Environmental DNA (eDNA) surveillance holds great promise for improving species conservation and management. However, few studies have investigated eDNA dynamics under natural conditions, and interpretations of eDNA surveillance results are clouded by uncertainties about eDNA degradation. We conducted a literature review to assess current understanding of eDNA degradation in aquatic systems and an experiment exploring how environmental conditions can influence eDNA degradation. Previous studies have reported macrobial eDNA persistence ranging from less than 1 day to over 2 weeks, with no attempts to quantify factors affecting degradation. Using a SYBR Green quantitative PCR assay to observe Common Carp ( Cyprinus carpio ) eDNA degradation in laboratory mesocosms, our rate of Common Carp eDNA detection decreased over time. Common Carp eDNA concentration followed a pattern of exponential decay, and observed decay rates exceeded previously published values for aquatic macrobial eDNA. Contrary to our expectations, eDNA degradation rate declined as biochemical oxygen demand, chlorophyll, and total eDNA (i.e., from any organism) concentration increased. Our results help explain the widely divergent, previously published estimates for eDNA degradation. Measurements of local environmental conditions, consideration of environmental influence on eDNA detection, and quantification of local eDNA degradation rates will help interpret future eDNA surveillance results.

Microsatellite fragment analysis using the ABI PRISM ® 377 DNA sequencer.

The ABI PRISM (®) 377 DNA Sequencer is used for a variety of microsatellite-based research. The platform provides researchers with a cost-effective means for high-throughput genotyping, which can be further optimized by multiplexing microsatellite loci or by using a tail-labeling approach to screen large sets of markers. The goals of this chapter are to present a protocol for performing microsatellite-based analyses on the ABI 377 and to provide researchers with information on how to troubleshoot common issues associated with running the ABI 377 sequencers.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2011-31 January 2012.

This article documents the addition of 473 microsatellite marker loci and 71 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Barteria fistulosa, Bombus morio, Galaxias platei, Hematodinium perezi, Macrocentrus cingulum Brischke (a.k.a. M. abdominalis Fab., M. grandii Goidanich or M. gifuensis Ashmead), Micropogonias furnieri, Nerita melanotragus, Nilaparvata lugens Stål, Sciaenops ocellatus, Scomber scombrus, Spodoptera frugiperda and Turdus lherminieri. These loci were cross-tested on the following species: Barteria dewevrei, Barteria nigritana, Barteria solida, Cynoscion acoupa, Cynoscion jamaicensis, Cynoscion leiarchus, Cynoscion nebulosus, Cynoscion striatus, Cynoscion virescens, Macrodon ancylodon, Menticirrhus americanus, Nilaparvata muiri and Umbrina canosai. This article also documents the addition of 116 sequencing primer pairs for Dicentrarchus labrax.

Isolation and characterization of microsatellite markers in the Serra Spanish mackerel, Scomberomorus brasiliensis.

Thirteen nuclear-encoded microsatellites from a genomic DNA library of Serra Spanish mackerel, Scomberomorus brasiliensis, were isolated and characterized. The microsatellites include 10 perfect repeats (eight tetranucleotide and two dinucleotide) and three imperfect repeats (two tetranucleotide and one dinucleotide). An additional five microsatellites, isolated originally from two congeneric species (S. cavalla and S. niphonius), were characterized in S. brasiliensis. Serra Spanish mackerel support artisanal fisheries along the Caribbean and Atlantic coasts of Central and South America, from Belize to Brazil.

PERMANENT GENETIC RESOURCES: PCR primers for 100 microsatellites in red drum (Sciaenops ocellatus).

One hundred nuclear-encoded microsatellites from a genomic library of red drum (Sciaenops ocellatus) were isolated and characterized. Eight microsatellites had tetranucleotide motifs; 92 had dinucleotide motifs. The average number of alleles per microsatellite (sample of 22-24 fish) was 17.7 (range = 2-30); gene diversity averaged 0.796 (range = 0.227-1.000). Following Bonferroni correction, genotype frequencies at 90 microsatellites did not deviate significantly from Hardy-Weinberg equilibrium expectations. Occurrence of null alleles was inferred at 15 microsatellites; alleles differing by only a single base were observed at 11 microsatellites. The microsatellites developed should prove useful for population-genetic studies of 'wild' red drum and in construction of a genetic map.