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.

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.

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.

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.

Comparative evaluation for the performance of environmental DNA and RNA analyses targeting mitochondrial and nuclear genes from ayu (Plecoglossus altivelis).

Environmental DNA and RNA (eDNA and eRNA; collectively eNA) analyses have the potential for non-invasive and cost-efficient biomonitoring compared with traditional capture-based surveys. Although various types of eNA particles, including not only mitochondrial eDNA but also nuclear eDNA and their transcripts, are present in the water, performances of eNA detection and quantification have not yet been evaluated sufficiently across multiple mitochondrial and nuclear genes. We conducted a tank experiment with ayu (Plecoglossus altivelis) to compare the detection sensitivity, yields per water sample, and quantification variability between replicates of each type of eNAs. The assay targeting the multi-copy nuclear gene exhibited a higher sensitivity than the assay targeting the mitochondrial gene, and both the target eDNA and eRNA concentrations per water sample were higher for the nuclear gene. On the contrary, variation in eRNA quantifications per sample does not necessarily correspond to that in eDNA, and the intra-sample quantification variability (represented as the CVs between PCR replicates) tended to be larger for eRNA than eDNA. Our results suggested that, even if suitable to the sensitive detection of species occurrence, the use of eRNA particularly derived from multi-copy nuclear gene may not be necessarily appropriate for the reliable assessment of species abundance. The findings in this study would help optimize eNA analyses for making biomonitoring and stock assessment in aquatic environments more efficient and reliable.

Exploring uncharted territory: new frontiers in environmental DNA for tropical fisheries management.

Tropical ecosystems host a significant share of global fish diversity contributing substantially to the global fisheries sector. Yet their sustainable management is challenging due to their complexity, diverse life history traits of tropical fishes, and varied fishing techniques involved. Traditional monitoring techniques are often costly, labour-intensive, and/or difficult to apply in inaccessible sites. These limitations call for the adoption of innovative, sensitive, and cost-effective monitoring solutions, especially in a scenario of climate change. Environmental DNA (eDNA) emerges as a potential game changer for biodiversity monitoring and conservation, especially in aquatic ecosystems. However, its utility in tropical settings remains underexplored, primarily due to a series of challenges, including the need for a comprehensive barcode reference library, an understanding of eDNA behaviour in tropical aquatic environments, standardized procedures, and supportive biomonitoring policies. Despite these challenges, the potential of eDNA for sensitive species detection across varied habitats is evident, and its global use is accelerating in biodiversity conservation efforts. This review takes an in-depth look at the current state and prospects of eDNA-based monitoring in tropical fisheries management research. Additionally, a SWOT analysis is used to underscore the opportunities and threats, with the aim of bridging the knowledge gaps and guiding the more extensive and effective use of eDNA-based monitoring in tropical fisheries management. Although the discussion applies worldwide, some specific experiences and insights from Indian tropical fisheries are shared to illustrate the practical application and challenges of employing eDNA in a tropical context.

Utilizing the state of environmental DNA (eDNA) to incorporate time-scale information into eDNA analysis.

Environmental DNA (eDNA) analysis allows cost-effective and non-destructive biomonitoring with a high detection sensitivity in terrestrial and aquatic environments. However, the eDNA results can sometimes include false-positive inferences of target organisms owing to the detection of aged eDNA that has long since been released from the individual and is more likely to be detected at a site further away from its source. In order to address the issue, this manuscript focuses on the state of eDNA, proposing new methodologies to estimate the age of eDNA: (1) DNA damage rate, (2) eDNA particle size distribution, and (3) viable cell-derived eDNA. In addition, the manuscript also focuses on the shorter persistence of environmental RNA (eRNA) compared with eDNA, highlighting the application of eRNA and environmental nucleic acid ratio for assessing the age of the genetic materials in water. Although substantial further research is essential to support the feasibility of these methodologies, incorporating time-scale information into eDNA analysis would update current eDNA analysis, improve the accuracy and reliability of eDNA-based monitoring, and further refine eDNA analysis as a useful monitoring tool in ecology, fisheries and various environmental sciences.

Environmental transcriptomics under heat stress: Can environmental RNA reveal changes in gene expression of aquatic organisms?

To safeguard biodiversity in a changing climate, taxonomic information about species turnover and insights into the health of organisms are required. Environmental DNA approaches are increasingly used for species identification, but cannot provide functional insights. Transcriptomic methods reveal the physiological states of macroorganisms, but are currently species-specific and require tissue sampling or animal sacrifice, making community-wide assessments challenging. Here, we test whether broad functional information (expression level of the transcribed genes) can be harnessed from environmental RNA (eRNA), which includes extra-organismal RNA from macroorganisms along with whole microorganisms. We exposed Daphnia pulex as well as phytoplankton prey and microorganism colonizers to control (20°C) and heat stress (28°C) conditions for 7 days. We sequenced eRNA from tank water (after complete removal of Daphnia) as well as RNA from Daphnia tissue, enabling comparisons of extra-organismal and organismal RNA-based gene expression profiles. Both RNA types detected similar heat stress responses of Daphnia. Using eRNA, we identified 32 Daphnia genes to be differentially expressed following heat stress. Of these, 17 were also differentially expressed and exhibited similar levels of relative expression in organismal RNA. In addition to the extra-organismal Daphnia response, eRNA detected community-wide heat stress responses consisting of distinct functional profiles and 121 differentially expressed genes across eight taxa. Our study demonstrates that environmental transcriptomics based on extra-organismal eRNA can noninvasively reveal gene expression responses of macroorganisms following environmental changes, with broad potential implications for the biomonitoring of health across the trophic chain.

Application of an eDNA assay for the detection of Pseudoloma neurophilia (Microsporidia) in zebrafish (Danio rerio) facilities.

Environmental DNA (eDNA) water assays are beginning to be implemented for many important pathogens in confined aquaculture systems. Recirculating systems are rapidly being developed for fin fish aquaculture. Zebrafish (Danio rerio) are reared in these systems, and Pseudoloma neurophilia (Microsporidia) represents a serious challenge for zebrafish research facilities. Diagnosis of the pathogen has traditionally used histology or PCR of tissues with lethal sampling. However, with the development of a nonlethal assay to detect P. neurophilia in tank water, facilities will be able to integrate the assay into routine surveillance efforts to couple with their established protocols. Here, we first describe a modified protocol to extract and quantify parasite DNA from the environment for nonlethal detection of P. neurophilia in adult zebrafish populations. Using this modified assay, we then evaluated water samples from a longitudinal experimental infection study, targeting timepoints during initial infection. The parasite was detectable in the water immediately after initial exposure until week 4 post exposure (pe), when the parasite was undetectable until 7 weeks pe. After that time, the parasite was sporadically detected in the water for the 10-month study, likely correlating with the lifecycle of the parasite. Using water samples from the Zebrafish International Resource Center, we also validated the clinical relevance of the assay in a large zebrafish facility. The integration of this assay at ZIRC will significantly compliment surveillance and control efforts for the microsporidian parasite.

Pooling of intra-site measurements inflates variability of the correlation between environmental DNA concentration and organism abundance.

Environmental DNA (eDNA) analysis can promote efficient ecosystem monitoring and resource management. However, limited knowledge of the factors affecting the relationship between eDNA concentration and organism abundance causes uncertainty in relative abundance estimates based on eDNA concentration. Pooling of data points obtained from multiple locations within a site has been used to mitigate intra-site variation in eDNA and abundance estimates, but decreases the sample size used for estimating the relationship. I here assessed how the pooling of intra-site measurements of eDNA concentration and organism abundance impacted the reliability of the correlative relationship between eDNA concentration and organism abundance. Mathematical models were developed to simulate measurements of eDNA concentrations and organism abundances from multiple locations in a given survey site, and the CVs (coefficient of variability) of the correlations were compared depending on whether data points from different locations were individually treated or pooled. Although the mean and median values of the correlation coefficients were similar between the scenarios, the CVs of the simulated correlations were substantially higher under the pooled scenario than the individual scenario. Additionally, I re-analyzed two empirical studies conducted in lakes, both showing higher CVs of the correlations by pooling intra-site measurements. This study suggests that it would make eDNA-based abundance estimation more reliable and reproducible to individually analyze target eDNA concentrations and organism abundance estimates.

Can nuclear aquatic environmental DNA be a genetic marker for the accurate estimation of species abundance?

Environmental DNA (eDNA) analysis is a promising tool for the sensitive and effective monitoring of species distribution and abundance. Traditional eDNA analysis has targeted mitochondrial DNA (mtDNA) fragments due to their abundance in cells; however, the quantification may vary depending on cell type and physiology. Conversely, some recent eDNA studies have targeted multi-copy nuclear DNA (nuDNA) fragments, such as ribosomal RNA genes, in water, and reported a higher detectability and more rapid degradation than mitochondrial eDNA (mt-eDNA). These properties suggest that nuclear eDNA (nu-eDNA) may be useful for the accurate estimation of species abundance relative to mt-eDNA, but which remains unclear. In this study, we compiled previous studies and re-analyzed the relationships between mt- and nu-eDNA concentration and species abundance by comparing the R2 values of the linear regression. We then performed an aquarium experiment using zebrafish (Danio rerio) to compare the relationships across genetic regions, including single-copy nuDNA. We found more accurate relationships between multi-copy nu-eDNA and species abundance than mt-eDNA in these datasets, although the difference was not significant upon weighted-averaging the R2 values. Moreover, we compared the decay rate constants of zebrafish eDNA across genetic regions and found that multi-copy nu-eDNA degraded faster than mt-eDNA under pH 7, implying a quick turnover of multi-copy nu-eDNA in the field. Although further empirical studies of nu-eDNA applications are necessary to support our findings, this study provides the groundwork for improving the estimation accuracy of species abundance via eDNA analysis.

Dual colorimetric strategy for specific DNA detection by nicking endonuclease-assisted gold nanoparticle signal amplification.

The continuous spread of invasive alien species, as zebra mussel (Dreissena polymorpha), is a major global concern and it is urgent to stop it. Early stages of an invasion are crucial and challenging; however, detection tools based on environmental DNA analysis are promising alternatives. We present an alternative DNA target amplification strategy for signal enhancement followed by dual-mode colorimetric naked eye and optical smartphone analysis for the early detection of zebra mussel environmental DNA. Target amplification was designed based on the nicking endonuclease probe cleavage upon probe and complementary target hybridization. The cleaved/intact probe interacts with DNA-modified nanoparticles for colorimetric detection. We have demonstrated that enzyme amplification strategy enhanced 12-fold the sensitivity by naked eye detection, achieving a detection limit of ~8 nM (4.48×1010 copies) in controlled conditions, whereas target in complex environmental samples allowed the detection of 22.5 nM (1.26×1011 copies). Competitive assays also showed that the system can discriminate specific zebra mussel DNA sequences from other DNA sequences. Additionally, smartphone analysis for DNA quantification further improved the sensitivity of its detection by 130-fold, more than 2 orders of magnitude, when applied to environmental samples. The limit of detection to 0.17 nM (9.52×108 copies) is based on RGB coordinates, which is especially relevant to monitor early aggregation stages, being more accurate and reducing naked eye detection subjectivity. DNA extracted from zebra mussel meat, zebra mussel contaminated river water, and non-contaminated river water samples were successfully tested. Dual-mode colorimetric detection is useful in field analysis without the need for expensive laboratory equipment.

Environmental DNA study on aquatic ecosystem monitoring and management: Recent advances and prospects.

Environmental DNA (eDNA) is organismal DNA that can be detected in the environment and is derived from cellular material of organisms shed into aquatic or terrestrial environments. It can be sampled and monitored using molecular methods, which is important for the early detection of invasive and native species as well as the discovery of rare and cryptic species. While few reviews have summarized the latest findings on eDNA for most aquatic animal categories in the aquatic ecosystem, especially for aquatic eDNA processing and application. In the present review, we first performed a bibliometric network analysis of eDNA studies on aquatic animals. Subsequently, we summarized the abiotic and biotic factors affecting aquatic eDNA occurrence. We also systematically discussed the relevant experiments and analyses of aquatic eDNA from various aquatic organisms, including fish, molluscans, crustaceans, amphibians, and reptiles. Subsequently, we discussed the major achievements of eDNA application in studies on the aquatic ecosystem and environment. The application of eDNA will provide an entirely new paradigm for biodiversity conservation, environment monitoring, and aquatic species management at a global scale.

DNA metabarcoding-based study on bacteria and fungi associated with house dust mites (Dermatophagoides spp.) in settled house dust.

House dust mites (HDMs) including Dermatophagoides spp. are an important cause of respiratory allergies. However, their relationship with microorganisms in house dust has not been fully elucidated. Here, we characterized bacteria and fungi associated with HDMs in house dust samples collected in 107 homes in Korea by using DNA barcode sequencing of bacterial 16S rRNA gene, fungal internal transcribed spacer 2 (ITS2) region, and arthropod cytochrome c oxidase I (COI) gene. Our inter-kingdom co-occurrence network analysis and/or indicator species analysis identified that HDMs were positively related with a xerophilic fungus Wallemia, mycoparasitic fungi such as Cystobasidium, and some human skin-related bacterial and fungal genera, and they were negatively related with the hygrophilous fungus Cephalotrichum. Overall, our study has succeeded in adding novel insights into HDM-related bacteria and fungi in the house dust ecosystem, and in confirming the historically recognized fact that HDMs are associated with xerophilic fungi such as Wallemia. Understanding the microbial ecology in house dust is thought to be important for elucidating the etiology of human diseases including allergies, and our study revealed baseline information of house dust ecology in relation to HDMs. The findings could be useful from a perspective of human health.

Assessing the impact of the threatened crucian carp (Carassius carassius) on pond invertebrate diversity: A comparison of conventional and molecular tools.

Fishes stocked for recreation and angling can damage freshwater habitats and negatively impact biodiversity. The pond-associated crucian carp (Carassius carassius) is rare across Europe and is stocked for conservation management in England, but its impacts on pond biota are understudied. Freshwater invertebrates contribute substantially to aquatic biodiversity, encompassing many rare and endemic species, but their small size and high abundance complicate their assessment. Practitioners have employed sweep-netting and kick-sampling with microscopy (morphotaxonomy), but specimen size/quality and experience can bias identification. DNA and environmental DNA (eDNA) metabarcoding offer alternative means of invertebrate assessment. We compared invertebrate diversity in ponds (N = 18) with and without crucian carp using morphotaxonomic identification, DNA metabarcoding and eDNA metabarcoding. Five 2 L water samples and 3 min sweep-net samples were collected at each pond. Inventories produced by morphotaxonomic identification of netted samples, DNA metabarcoding of bulk tissue samples and eDNA metabarcoding of water samples were compared. Alpha diversity was greatest with DNA or eDNA metabarcoding, depending on whether standard or unbiased methods were considered. DNA metabarcoding reflected morphotaxonomic identification, whereas eDNA metabarcoding produced markedly different communities. These complementary tools should be combined for comprehensive invertebrate assessment. Crucian carp presence minimally reduced alpha diversity in ponds, but positively influenced beta diversity through taxon turnover (i.e., ponds with crucian carp contained different invertebrates to fishless ponds). Crucian carp presence contributes to landscape-scale invertebrate diversity, supporting continued conservation management in England. Our results show that molecular tools can enhance freshwater invertebrate assessment and facilitate development of more accurate and ecologically effective pond management strategies.

Study on the correlation between soil microbial diversity and ambient environmental factors influencing the safflower distribution in Xinjiang.

The effects of soil microbial properties and physiographical factors on safflower distributions in the main safflower plantations of Xinjiang province in China were studied. This study may help determine the basis of the environmental factors for evaluating the geoherbalism of this medicinal plant. The soil microbial biodiversity in the bulk soil and rhizosphere of safflower at different growth stages and from different sampling plots were characterized by analyzing the environmental DNAs in the samples. With general primers targeting the 16S ribosomal DNA for bacteria and the internal transcribed spacer 1 gene for fungi, the study was performed using marker gene amplification coupled with Illumina HiSeq high-throughput sequencing technologies. Correlation analysis and a distance-based redundancy analysis were performed to determine the dominant factors affecting the distribution of the microorganism in safflower soils. A total of 16517 bacterial operational taxonomic units (OTUs) were obtained from all the 108 soil samples of nine safflower sampling plots. At the phylum level, 48 phyla have been identified with Actinobacteria (32.9%) and proteobacteria (28.7%) being predominant. For fungi, 8746 OTUs were obtained, which belonged to seven phyla with Ascomycota overwhelmingly superior in relative abundance. A significant positive correlation was found between soil microbe quantity and ASL (above sea level). Safflower was sensitive to changes in elevation, growing more abundantly in the mountainous regions at heights of around 1,200 m above sea level. It is concluded that the dominant factors affecting the distribution of microorganisms in safflower soils were soil moisture, available N, and ASL.

Lake sedimentary DNA accurately records 20th Century introductions of exotic conifers in Scotland.

Sedimentary DNA (sedDNA) has recently emerged as a new proxy for reconstructing past vegetation, but its taphonomy, source area and representation biases need better assessment. We investigated how sedDNA in recent sediments of two small Scottish lakes reflects a major vegetation change, using well-documented 20th Century plantations of exotic conifers as an experimental system. We used next-generation sequencing to barcode sedDNA retrieved from subrecent lake sediments. For comparison, pollen was analysed from the same samples. The sedDNA record contains 73 taxa (mainly genus or species), all but one of which are present in the study area. Pollen and sedDNA shared 35% of taxa, which partly reflects a difference in source area. More aquatic taxa were recorded in sedDNA, whereas taxa assumed to be of regional rather than local origin were recorded only as pollen. The chronology of the sediments and planting records are well aligned, and sedDNA of exotic conifers appears in high quantities with the establishment of plantations around the lakes. SedDNA recorded other changes in local vegetation that accompanied afforestation. There were no signs of DNA leaching in the sediments or DNA originating from pollen.

Ancient plant DNA in lake sediments.

Contents 924 I. 925 II. 925 III. 927 IV. 929 V. 930 VI. 930 VII. 931 VIII. 933 IX. 935 X. 936 XI. 938 938 References 938 SUMMARY: Recent advances in sequencing technologies now permit the analyses of plant DNA from fossil samples (ancient plant DNA, plant aDNA), and thus enable the molecular reconstruction of palaeofloras. Hitherto, ancient frozen soils have proved excellent in preserving DNA molecules, and have thus been the most commonly used source of plant aDNA. However, DNA from soil mainly represents taxa growing a few metres from the sampling point. Lakes have larger catchment areas and recent studies have suggested that plant aDNA from lake sediments is a more powerful tool for palaeofloristic reconstruction. Furthermore, lakes can be found globally in nearly all environments, and are therefore not limited to perennially frozen areas. Here, we review the latest approaches and methods for the study of plant aDNA from lake sediments and discuss the progress made up to the present. We argue that aDNA analyses add new and additional perspectives for the study of ancient plant populations and, in time, will provide higher taxonomic resolution and more precise estimation of abundance. Despite this, key questions and challenges remain for such plant aDNA studies. Finally, we provide guidelines on technical issues, including lake selection, and we suggest directions for future research on plant aDNA studies in lake sediments.

Precision wildlife medicine: applications of the human-centred precision medicine revolution to species conservation.

The current species extinction crisis is being exacerbated by an increased rate of emergence of epizootic disease. Human-induced factors including habitat degradation, loss of biodiversity and wildlife population reductions resulting in reduced genetic variation are accelerating disease emergence. Novel, efficient and effective approaches are required to combat these epizootic events. Here, we present the case for the application of human precision medicine approaches to wildlife medicine in order to enhance species conservation efforts. We consider how the precision medicine revolution, coupled with the advances made in genomics, may provide a powerful and feasible approach to identifying and treating wildlife diseases in a targeted, effective and streamlined manner. A number of case studies of threatened species are presented which demonstrate the applicability of precision medicine to wildlife conservation, including sea turtles, amphibians and Tasmanian devils. These examples show how species conservation could be improved by using precision medicine techniques to determine novel treatments and management strategies for the specific medical conditions hampering efforts to restore population levels. Additionally, a precision medicine approach to wildlife health has in turn the potential to provide deeper insights into human health and the possibility of stemming and alleviating the impacts of zoonotic diseases. The integration of the currently emerging Precision Medicine Initiative with the concepts of EcoHealth (aiming for sustainable health of people, animals and ecosystems through transdisciplinary action research) and One Health (recognizing the intimate connection of humans, animal and ecosystem health and addressing a wide range of risks at the animal-human-ecosystem interface through a coordinated, collaborative, interdisciplinary approach) has great potential to deliver a deeper and broader interdisciplinary-based understanding of both wildlife and human diseases.

Identifying a breeding habitat of a critically endangered fish, Acheilognathus typus, in a natural river in Japan.

Freshwater biodiversity has been severely threatened in recent years, and to conserve endangered species, their distribution and breeding habitats need to be clarified. However, identifying breeding sites in a large area is generally difficult. Here, by combining the emerging environmental DNA (eDNA) analysis with subsequent traditional collection surveys, we successfully identified a breeding habitat for the critically endangered freshwater fish Acheilognathus typus in the mainstream of Omono River in Akita Prefecture, Japan, which is one of the original habitats of this species. Based on DNA cytochrome B sequences of A. typus and closely related species, we developed species-specific primers and a probe that were used in real-time PCR for detecting A. typus eDNA. After verifying the specificity and applicability of the primers and probe on water samples from known artificial habitats, eDNA analysis was applied to water samples collected at 99 sites along Omono River. Two of the samples were positive for A. typus eDNA, and thus, small fixed nets and bottle traps were set out to capture adult fish and verify egg deposition in bivalves (the preferred breeding substrate for A. typus) in the corresponding regions. Mature female and male individuals and bivalves containing laid eggs were collected at one of the eDNA-positive sites. This was the first record of adult A. typus in Omono River in 11 years. This study highlights the value of eDNA analysis to guide conventional monitoring surveys and shows that combining both methods can provide important information on breeding sites that is essential for species' conservation.