Future of DNA-based insect monitoring.

Insects are crucial for ecosystem health but climate change and pesticide use are driving massive insect decline. To mitigate this loss, we need new and effective monitoring techniques. Over the past decade there has been a shift to DNA-based techniques. We describe key emerging techniques for sample collection. We suggest that the selection of tools should be broadened, and that DNA-based insect monitoring data need to be integrated more rapidly into policymaking. We argue that there are four key areas for advancement, including the generation of more complete DNA barcode databases to interpret molecular data, standardisation of molecular methods, scaling up of monitoring efforts, and integrating molecular tools with other technologies that allow continuous, passive monitoring based on images and/or laser imaging, detection, and ranging (LIDAR).

MitoFish, MitoAnnotator, and MiFish Pipeline: Updates in 10 Years.

MitoFish, MitoAnnotator, and MiFish Pipeline are comprehensive databases of fish mitochondrial genomes (mitogenomes), accurate annotation software of fish mitogenomes, and a web platform for metabarcoding analysis of fish mitochondrial environmental DNA (eDNA), respectively. The MitoFish Suite currently receives over 48,000 visits worldwide every year; however, the performance and usefulness of the online platforms can still be improved. Here, we present essential updates on these platforms, including an enrichment of the reference data sets, an enhanced searching function, substantially faster genome annotation and eDNA analysis with the denoising of sequencing errors, and a multisample comparative analysis function. These updates have made our platform more intuitive, effective, and reliable. These updated platforms are freely available at http://mitofish.aori.u-tokyo.ac.jp/.

Cyanobacterial Genomes from a Brackish Coastal Lagoon Reveal Potential for Novel Biogeochemical Functions and Their Evolution.

Cyanobacteria are recognised for their pivotal roles in aquatic ecosystems, serving as primary producers and major agents in diazotrophic processes. Currently, the primary focus of cyanobacterial research lies in gaining a more detailed understanding of these well-established ecosystem functions. However, their involvement and impact on other crucial biogeochemical cycles remain understudied. This knowledge gap is partially attributed to the challenges associated with culturing cyanobacteria in controlled laboratory conditions and the limited understanding of their specific growth requirements. This can be circumvented partially by the culture-independent methods which can shed light on the genomic potential of cyanobacterial species and answer more profound questions about the evolution of other key biogeochemical functions. In this study, we assembled 83 cyanobacterial genomes from metagenomic data generated from environmental DNA extracted from a brackish water lagoon (Chilika Lake, India). We taxonomically classified these metagenome-assembled genomes (MAGs) and found that about 92.77% of them are novel genomes at the species level. We then annotated these cyanobacterial MAGs for all the encoded functions using KEGG Orthology. Interestingly, we found two previously unreported functions in Cyanobacteria, namely, DNRA (Dissimilatory Nitrate Reduction to Ammonium) and DMSP (Dimethylsulfoniopropionate) synthesis in multiple MAGs using nirBD and dsyB genes as markers. We validated their presence in several publicly available cyanobacterial isolate genomes. Further, we identified incongruities between the evolutionary patterns of species and the marker genes and elucidated the underlying reasons for these discrepancies. This study expands our overall comprehension of the contribution of cyanobacteria to the biogeochemical cycling in coastal brackish ecosystems.

Environmental drivers of Arctic communities based on metabarcoding of marine sediment eDNA.

Our ability to assess biodiversity at relevant spatial and temporal scales for informing management is of increasing importance given this is foundational to identify and mitigate the impacts of global change. Collecting baseline information and tracking ecological changes are particularly important for areas experiencing rapid changes and representing data gaps such as Arctic marine ecosystems. Environmental DNA has the potential to provide such data. We extracted environmental DNA from 90 surface sediment samples to assess eukaryote diversity around Greenland and Svalbard using two separate primer pairs amplifying different sections of the 18S rRNA gene. We detected 27 different phyla and 99 different orders and found that temperature and the change in temperature explained the most variation in the community in a single linear model, while latitude, sea ice cover and change in temperature explained the most variation in the community when assessed by individual non-linear models. We identified potential indicator taxa for Arctic climate change, including a terebellid annelid worm. In conclusion, our study demonstrates that environmental DNA offers a feasible method to assess biodiversity and identifies warming as a key driver of differences in biodiversity across these remote ecosystems.

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA-operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing.

Environmental DNA unveils deep phylogeographic structure of a freshwater fish.

Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the B. toni eDNA-positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of B. toni, newly developed eDNA metabarcoding primers (Barba-cytb-F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome b. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue-derived DNA from B. toni. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.

The Tree of Life eDNA metabarcoding reveals a similar taxonomic richness but dissimilar evolutionary lineages between seaports and marine reserves.

Coastal areas host a major part of marine biodiversity but are seriously threatened by ever-increasing human pressures. Transforming natural coastlines into urban seascapes through habitat artificialization may result in loss of biodiversity and key ecosystem functions. Yet, the extent to which seaports differ from nearby natural habitats and marine reserves across the whole Tree of Life is still unknown. This study aimed to assess the level of α and ß-diversity between seaports and reserves, and whether these biodiversity patterns are conserved across taxa and evolutionary lineages. For that, we used environmental DNA (eDNA) metabarcoding to survey six seaports on the French Mediterranean coast and four strictly no-take marine reserves nearby. By targeting four different groups-prokaryotes, eukaryotes, metazoans and fish-with appropriate markers, we provide a holistic view of biodiversity on contrasted habitats. In the absence of comprehensive reference databases, we used bioinformatic pipelines to gather similar sequences into molecular operational taxonomic units (MOTUs). In contrast to our expectations, we obtained no difference in MOTU richness (α-diversity) between habitats except for prokaryotes and threatened fishes with higher diversity in reserves than in seaports. However, we observed a marked dissimilarity (ß-diversity) between seaports and reserves for all taxa. Surprisingly, this biodiversity signature of seaports was preserved across the Tree of Life, up to the order. This result reveals that seaports and nearby marine reserves share few taxa and evolutionary lineages along urbanized coasts and suggests major differences in terms of ecosystem functioning between both habitats.

Determinants of microbiome composition: Insights from free-ranging hybrid zebras (Equus quagga × grevyi).

The composition of mammalian gut microbiomes is highly conserved within species, yet the mechanisms by which microbiome composition is transmitted and maintained within lineages of wild animals remain unclear. Mutually compatible hypotheses exist, including that microbiome fidelity results from inherited dietary habits, shared environmental exposure, morphophysiological filtering and/or maternal effects. Interspecific hybrids are a promising system in which to interrogate the determinants of microbiome composition because hybrids can decouple traits and processes that are otherwise co-inherited in their parent species. We used a population of free-living hybrid zebras (Equus quagga × grevyi) in Kenya to evaluate the roles of these four mechanisms in regulating microbiome composition. We analysed faecal DNA for both the trnL-P6 and the 16S rRNA V4 region to characterize the diets and microbiomes of the hybrid zebra and of their parent species, plains zebra (E. quagga) and Grevy's zebra (E. grevyi). We found that both diet and microbiome composition clustered by species, and that hybrid diets and microbiomes were largely nested within those of the maternal species, plains zebra. Hybrid microbiomes were less variable than those of either parent species where they co-occurred. Diet and microbiome composition were strongly correlated, although the strength of this correlation varied between species. These patterns are most consistent with the maternal-effects hypothesis, somewhat consistent with the diet hypothesis, and largely inconsistent with the environmental-sourcing and morphophysiological-filtering hypotheses. Maternal transmittance likely operates in conjunction with inherited feeding habits to conserve microbiome composition within species.

Emerging use of air eDNA and its application to forensic investigations - A review.

Biological material is routinely collected at crime scenes and from exhibits and is a key type of evidence during criminal investigations. Improvements in DNA technologies allow collection and profiling of trace samples, comprised of few cells, significantly expanding the types of exhibits targeted for DNA analysis to include touched surfaces. However, success rates from trace and touch DNA samples tend to be poorer compared to other biological materials such as blood. Simultaneously, there have been recent advances in the utility of environmental DNA collection (eDNA) in identification and tracking of different biological organisms and species from bacteria to naked mole rats in different environments, including, soil, ice, snow, air and aquatic. This paper examines the emerging methods and research into eDNA collection, with a special emphasis on the potential forensic applications of human DNA collection from air including challenges and further studies required to progress implementation.

DNA at the whim of the water: environmental DNA as a course-based undergraduate research experience.

Course-based undergraduate research experiences (CUREs) increase student access to high impact research experiences. CUREs engage students in the scientific process by learning how to pose scientific questions, develop hypotheses, and generate data to test them. Environmental DNA (eDNA) is a growing field of research that is gaining accessibility through decreasing laboratory costs, which can make a foundation for multiple, engaging CUREs. This manuscript describes three case studies that used eDNA in an upper year undergraduate course. The first focusses on a systematic literature review of eDNA metadata reporting. The second describes the biomonitoring of brook trout in southern Ontario using eDNA. The third involves eDNA metabarcoding for freshwater fish detection in southern Ontario. Undergraduates were involved in the development and execution of experiments, scientific communication, the peer review process, and fundraising. Through this manuscript, we show the novel application of eDNA CUREs and provide a roadmap for other instructors interested in implementing similar projects. Interviews with seven students from these courses indicate the benefits experienced from taking these courses. We argue that the use of eDNA in CUREs should be expanded in undergraduate biology programs due to the benefit to students and the increasing accessibility of this technology.

Fungal diversity present in snow sampled in summer in the north-west Antarctic Peninsula and the South Shetland Islands, Maritime Antarctica, assessed using metabarcoding.

We assessed the fungal diversity present in snow sampled during summer in the north-west Antarctic Peninsula and the South Shetland Islands, maritime Antarctica using a metabarcoding approach. A total of 586,693 fungal DNA reads were obtained and assigned to 203 amplicon sequence variants (ASVs). The dominant phylum was Ascomycota, followed by Basidiomycota, Mortierellomycota, Chytridiomycota and Mucoromycota. Penicillium sp., Pseudogymnoascus pannorum, Coniochaeta sp., Aspergillus sp., Antarctomyces sp., Phenoliferia sp., Cryolevonia sp., Camptobasidiaceae sp., Rhodotorula mucilaginosa and Bannozyma yamatoana were assessed as abundant taxa. The snow fungal diversity indices were high but varied across the different locations sampled. Of the fungal ASVs detected, only 28 were present all sampling locations. The 116 fungal genera detected in the snow were dominated by saprotrophic taxa, followed by symbiotrophic and pathotrophic. Our data indicate that, despite the low temperature and oligotrophic conditions, snow can host a richer mycobiome than previously reported through traditional culturing studies. The snow mycobiome includes a complex diversity dominated by cosmopolitan, cold-adapted, psychrophilic and endemic taxa. While saprophytes dominate this community, a range of other functional groups are present.

Phenological mismatches mitigate the ecological impact of a biological invader on amphibian communities.

Horizon scans have emerged as a valuable tool to anticipate the incoming invasive alien species (IAS) by judging species on their potential impacts. However, little research has been conducted on quantifying actual impacts and assessing causes of species-specific vulnerabilities to particular IAS due to persistent methodological challenges. The underlying interspecific mechanisms driving species-specific vulnerabilities therefore remain poorly understood, even though they can substantially improve the accuracy of risk assessments. Given that interspecific interactions underlying ecological impacts of IAS are often shaped by phenological synchrony, we tested the hypothesis that temporal mismatches in breeding phenology between native species and IAS can mitigate their ecological impacts. Focusing on the invasive American bullfrog (Lithobates catesbeianus), we combined an environmental DNA (eDNA) quantitative barcoding and metabarcoding survey in Belgium with a global meta-analysis, and integrated citizen-science data on breeding phenology. We examined whether the presence of native amphibian species was negatively related to the presence or abundance of invasive bullfrogs and whether this relationship was affected by their phenological mismatches. The field study revealed a significant negative effect of increasing bullfrog eDNA concentrations on native amphibian species richness and community structure. These observations were shaped by species-specific vulnerabilities to invasive bullfrogs, with late spring- and summer-breeding species being strongly affected, while winter-breeding species remained unaffected. This trend was confirmed by the global meta-analysis. A significant negative relationship was observed between phenological mismatch and the impact of bullfrogs. Specifically, native amphibian species with breeding phenology differing by 6 weeks or less from invasive bullfrogs were more likely to be absent in the presence of bullfrogs than species whose phenology differed by more than 6 weeks with that of bullfrogs. Taken together, we present a novel method based on the combination of aqueous eDNA quantitative barcoding and metabarcoding to quantify the ecological impacts of biological invaders at the community level. We show that phenological mismatches between native and invasive species can be a strong predictor of invasion impact regardless of ecological or methodological context. Therefore, we advocate for the integration of temporal alignment between native and IAS's phenologies into invasion impact frameworks.

Fungal Spore Richness and Abundance of Allergenic Taxa: Comparing a Portable Impactor and Passive Trap Indoors and Outdoors in an Urban Setting.

Fungal spores are common airborne allergens, and fungal richness has been implicated in allergic disease. Amplicon sequencing of environmental DNA from air samples is a promising method to estimate fungal spore richness with semi-quantification of hundreds of taxa and can be combined with quantitative PCR to derive abundance estimates. However, it remains unclear how the choice of air sampling method influences these estimates. This study compared active sampling with a portable impactor and passive sampling with a passive trap over different durations to estimate fungal spore richness and the abundance of allergenic taxa. Air sampling was conducted indoors and outdoors at 12 residences, including repeated measurements with a portable impactor and passive traps with 1-day and 7-day durations. ITS2 amplicon sequence data were transformed to spore equivalents estimated by quantitative PCR, repeated active samples were combined, and abundance-based rarefaction was performed to standardize sample coverage for estimation of genus-level richness and spore abundance. Rarefied fungal richness was similar between methods indoors but higher for passive traps with a 7-day duration outdoors. Rarefied abundance of allergenic genera was similar between methods but some genera had lower abundance for passive traps with a 1-day duration, which differed indoors and outdoors indicating stochasticity in the collection of spores on collocated samplers. This study found that similar estimates of fungal spore richness and abundance of allergenic taxa can be obtained using a portable impactor or a passive trap within one day and that increased passive sample duration provides limited additional information.

Larger particle size distribution of environmental RNA compared to environmental DNA: a case study targeting the mitochondrial cytochrome b gene in zebrafish (Danio rerio) using experimental aquariums.

Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (ß) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future.

Barcoding Brazilian mammals to monitor biological diversity and threats: Trends, perspectives, and knowledge gaps.

DNA barcoding and environmental DNA (eDNA) represent significant advances for biomonitoring the world's biodiversity and its threats. However, these methods are highly dependent on the presence of species sequences on molecular databases. Brazil is one of the world's largest and most biologically diverse countries. However, many knowledge gaps still exist for describing, identifying, and monitoring of mammalian biodiversity using molecular methods. We aimed to unravel the patterns of the presence of Brazilian mammal species on molecular databases to improve our understanding of how effectively it would be to monitor them using DNA barcoding and environmental DNA, and contribute to mammalian conservation. We foundt many gaps in molecular databases, with many taxa being poorly represented, particularly from Amazonia, the order Lagomorpha, and arboreal, gomivorous, near extinct, and illegally traded species. Moreover, our analyses revealed that species description year was the most important factor determining the probability of a species to being sequenced. Primates are the group with the highest number of species considered a priority for sequencing due to their high level of combined threats. We highlight where investments are needed to fill knowledge gaps and increase the representativity of species on molecular databases to enable a better monitoring ability of Brazilian mammals encompassing different traits using DNA barcoding and environmental DNA.

Assessing the impact of river connectivity on fish biodiversity in the Yangtze River Basin using a multi-index evaluation framework.

The Yangtze River Basin, the world's third-largest river basin and a hot spot for global biodiversity conservation, is facing biodiversity crisis caused by reduced river connectivity. The deterioration arises from four dimensions: longitudinal, lateral, vertical and temporal. However, limited research has quantified the spatiotemporal connectivity of the Yangtze River Basin and further evaluated the consequent impact on fish biodiversity. In our study, a multi-index evaluation framework was developed to assess the variations in the four-dimensional connectivity of the Yangtze River Basin from 1980 to 2020, and fish biodiversity affected by reduced connectivity was detected by environmental DNA metabarcoding. Our results showed that the Yangtze River Basin suffers from a pronounced connectivity reduction, with 67% of assessed rivers experiencing deteriorated connectivity in recent years. The lost fish biodiversity along the river reaches with the worst connectivity was likely attributed to the construction of hydropower plants. The headwaters and the downstreams of most hydropower plants had a higher fish biodiversity compared with reservoirs. The free-flowing reaches in the downstream of the lowest hydropower station, had higher lotic fish abundance compared with that in the upstream. As for the entire Yangtze River Basin, 67% of threatened fish species, with 70% endemic species, were threatened by reduced river connectivity. Our result indicates that the massive loss of river connectivity changes the spatiotemporal patterns of fish community and threatens protected fish. More effective measures to restore the populations of affected fish in rivers with reduced river connectivity are required.

Environmental DNA analysis of airborne poaceae (grass) pollen reveals taxonomic diversity across seasons and climate zones.

BACKGROUND: Grasses populate most biogeographical zones, and their diversity influences allergic sensitisation to pollen. Previously, the contribution of different Poaceae subfamilies to airborne pollen has mostly been inferred from historical herbarium records. We recently applied environmental (e)DNA metabarcoding at one subtropical site revealing that successive airborne grass pollen peaks were derived from repeated flowering of Chloridoid and Panicoid grasses over a season. This study aimed to compare spatiotemporal patterns in grass pollen exposure across seasons and climate zones. METHODS: Airborne pollen concentrations across two austral pollen seasons spanning 2017-2019 at subtropical (Mutdapilly and Rocklea, Queensland) and temperate (Macquarie Park and Richmond, New South Wales) sites, were determined with a routine volumetric impaction sampler and counting by light microscopy. Poaceae rbcL metabarcode sequences amplified from daily pollen samples collected once per week were assigned to subfamily and genus using a ribosomal classifier and compared with Atlas of Living Australia sighting records. RESULTS: eDNA analysis revealed distinct dominance patterns of grass pollen at various sites: Panicoid grasses prevailed in both subtropical Mutdapilly and temperate Macquarie Park, whilst Chloridoid grasses dominated the subtropical Rocklea site. Overall, subtropical sites showed significantly higher proportion of pollen from Chloridoid grasses than temperate sites, whereas the temperate sites showed a significantly higher proportion of pollen from Pooideae grasses than subtropical sites. Timing of airborne Pooid (spring), Panicoid and Chloridoid (late spring to autumn), and Arundinoid (autumn) pollen were significantly related to number of days from mid-winter. Proportions of eDNA for subfamilies correlated with distributions grass sighting records between climate zones. CONCLUSIONS: eDNA analysis enabled finer taxonomic discernment of Poaceae pollen records across seasons and climate zones with implications for understanding adaptation of grasslands to climate change, and the complexity of pollen exposure for patients with allergic respiratory diseases.

Tree of life metabarcoding can serve as a biotic benchmark for shifting baselines in urbanized estuaries.

Urbanization of estuaries drastically changed existing shorelines and bathymetric contours, in turn modifying habitat for marine foundational species that host critical biodiversity. And yet we lack approaches to characterize a significant fraction of the biota that inhabit these ecosystems on time scales that align with rates of urbanization. Environmental DNA (or eDNA) metabarcoding that combines multiple assays targeting a broad range of taxonomic groups can provide a solution, but we need to determine whether the biological communities it detects ally with different habitats in these changing aquatic environments. In this study, we tested whether tree of life metabarcoding (ToL-metabarcoding) data extracted from filtered seawater samples correlated with four known geomorphic habitat zones across a heavily urbanized estuary (Sydney Harbour, Australia). Using this method, we substantially expanded our knowledge on the composition and spatial distribution of marine biodiversity across the tree of life in Sydney Harbour, particularly for organisms where existing records are sparse. Excluding terrestrial DNA inputs, we identified significant effects of both distance from the mouth of Sydney Harbour and geomorphic zone on biological community structure in the ToL-metabarcoding dataset (entire community), as well as in each of the taxonomic subgroups that we considered (fish, macroinvertebrates, algae and aquatic plants, bacteria). This effect appeared to be driven by taxa as a collective versus a few individual taxa, with each taxon explaining no more than 0.62% of the variation between geomorphic zones. Similarly, taxonomic richness was significantly higher within geomorphic zones with large sample sizes, but also decreased by 1% with each additional kilometer from the estuary mouth, a result consistent with a reduction in tidal inputs and available habitat in upper catchments. Based on these results, we suggest that ToL-metabarcoding can be used to benchmark biological monitoring in other urbanized estuaries globally, and in Sydney Harbour at future time points based on detection of bioindicators across the tree of life. We also suggest that robust biotic snapshots can be archived following extensive curation of taxonomic assignments that incorporates ecological affinities, supported by records from relevant and regional biodiversity repositories.

Isolation and habitat analysis of Balamuthia mandrillaris from soil.

Balamuthia mandrillaris is a free-living amoeba that causes meningoencephalitis in mammals. Over 200 cases of infection were reported worldwide, with a fatality rate of over 95%. A clear route of infection was unknown for a long time until a girl died of granulomatous amoebic encephalitis (GAE) in California, USA, in 2003 due to infection with B. mandrillaris detected in a potted plant. Since then, epidemiological studies were conducted worldwide to detect B. mandrillaris in soil and other environmental samples. We previously reported the isolation of B. mandrillaris from the soil in Japan; however, the existing B. mandrillaris culture method with BM3 medium and COS-7 cells was unsuccessful. Therefore, in this study, we aimed to conduct soil analysis to determine the growth conditions of B. mandrillaris. B. mandrillaris-positive soils were defined as soils from which B. mandrillaris was isolated and environmental DNA was PCR-positive. Soils inhabited by B. mandrillaris were alkaline, with high electrical conductivity and characteristics of nutrient-rich soils of loam and clay loam. The results of this study suggest a possible reason for the high prevalence of GAE caused by B. mandrillaris among individuals employed in agriculture-related occupations.

An updated LSU database and pipeline for environmental DNA identification of arbuscular mycorrhizal fungi.

Recent work established a backbone reference tree and phylogenetic placement pipeline for identification of arbuscular mycorrhizal fungal (AMF) large subunit (LSU) rDNA environmental sequences. Our previously published pipeline allowed any environmental sequence to be identified as putative AMF or within one of the major families. Despite this contribution, difficulties in implementation of the pipeline remain. Here, we present an updated database and pipeline with (1) an expanded backbone tree to include four newly described genera and (2) several changes to improve ease and consistency of implementation. In particular, packages required for the pipeline are now installed as a single folder (conda environment) and the pipeline has been tested across three university computing clusters. This updated backbone tree and pipeline will enable broadened adoption by the community, advancing our understanding of these ubiquitous and ecologically important fungi.