Revealing population demographics with environmental RNA.

The analyses of environmental DNA (eDNA) and environmental RNA (eRNA) released by organisms into their surrounding environment (water, soil and air) have emerged as powerful tools for monitoring biodiversity. While eDNA has been widely adopted for the non-invasive detection of species and characterization of community composition, the utilization of eRNA is still in its infancy. Due to its functional nature, eRNA holds intriguing potential for biodiversity monitoring offering new avenues of research beyond species detection. For example, conspecifics that are almost genetically identical can exhibit distinct transcriptomic differences depending on their life stage. In this issue of Molecular Ecology Resources, Parsley and Goldberg (2024) demonstrate, through a lab-validated field study, that eRNA can be used to detect distinct life stages of amphibians. This study elegantly demonstrates that eRNA can be used not only to detect invasive or endangered species but also to reveal population demographic information important for guiding effective conservation strategies.

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.

Freshwater connectivity transforms spatially integrated signals of biodiversity.

Aquatic ecosystems offer a continuum of water flow from headwater streams to inland lakes and coastal marine systems. This spatial connectivity influences the structure, function and dynamics of aquatic communities, which are among the most threatened and degraded on the Earth. Here, we determine the spatial resolution of environmental DNA (eDNA) in dendritic freshwater networks, which we use as a model for connected metacommunities. Our intensive sampling campaign comprised over 420 eDNA samples across 21 connected lakes, allowing us to analyse detections at a variety of scales, from different habitats within a lake to entire lake networks. We found strong signals of within-lake variation in eDNA distribution reflective of typical habitat use by both fish and zooplankton. Most importantly, we also found that connecting channels between lakes resulted in an accumulation of downstream eDNA detections in lakes with a higher number of inflows, and as networks increased in length. Environmental DNA achieves biodiversity surveys in these habitats in a high-throughput, spatially integrated way. These findings have profound implications for the interpretation of eDNA detections in aquatic ecosystems in global-scale biodiversity monitoring observations.

Environmental nucleic acids: A field-based comparison for monitoring freshwater habitats using eDNA and eRNA.

Nucleic acids released by organisms and isolated from environmental substrates are increasingly being used for molecular biomonitoring. While environmental DNA (eDNA) has received much attention, the potential of environmental RNA as a biomonitoring tool remains under-explored. Several recent studies using paired DNA and RNA metabarcoding of bulk samples suggest that RNA might better reflect "metabolically active" parts of the community. However, such studies mainly capture organismal eDNA and eRNA. For larger eukaryotes, isolation of extra-organismal RNA will be important, but viability needs to be examined in a field-based setting. In this study we evaluate (a) whether extra-organismal eRNA release from macroeukaryotes can be detected given its supposedly rapid degradation, and (b) if the same field collection methods for eDNA can be applied to eRNA. We collected eDNA and eRNA from water in lakes where fish community composition is well documented, enabling a comparison between the two nucleic acids in two different seasons with monitoring using conventional methods. We found that eRNA is released from macroeukaryotes and can be filtered from water and metabarcoded in a similar manner as eDNA to reliably provide species composition information. eRNA had a small but significantly greater true positive rate than eDNA, indicating that it correctly detects more species known to exist in the lakes. Given relatively small differences between the two molecules in describing fish community composition, we conclude that if eRNA provides significant advantages in terms of lability, it is a strong candidate to add to the suite of molecular monitoring tools.

Environmental RNA degrades more rapidly than environmental DNA across a broad range of pH conditions.

Although the use and development of molecular biomonitoring tools based on environmental nucleic acids (eDNA and eRNA; collectively known as eNAs) have gained broad interest for the quantification of biodiversity in natural ecosystems, studies investigating the impact of site-specific physicochemical parameters on eNA-based detection methods (particularly eRNA) remain scarce. Here, we used a controlled laboratory microcosm experiment to comparatively assess the environmental degradation of eDNA and eRNA across an acid-base gradient following complete removal of the progenitor organism (Daphnia pulex). Using water samples collected over a 30-day period, eDNA and eRNA copy numbers were quantified using a droplet digital PCR (ddPCR) assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene of D. pulex. We found that eRNA decayed more rapidly than eDNA at all pH conditions tested, with detectability-predicted by an exponential decay model-for up to 57 h (eRNA; neutral pH) and 143 days (eDNA; acidic pH) post organismal removal. Decay rates for eDNA were significantly higher in neutral and alkaline conditions than in acidic conditions, while decay rates for eRNA did not differ significantly among pH levels. Collectively, our findings provide the basis for a predictive framework assessing the persistence and degradation dynamics of eRNA and eDNA across a range of ecologically relevant pH conditions, establish the potential for eRNA to be used in spatially and temporally sensitive biomonitoring studies (as it is detectable across a range of pH levels), and may be used to inform future sampling strategies in aquatic habitats.

Genotype diversity promotes the persistence of Daphnia populations exposed to severe copper stress.

When environmental stressors of high intensity are sustained for long periods of time, populations face high probabilities of being extirpated. However, depending on the intensity of the stressor, large populations with sufficient genetic diversity may persist. We report the results of an experiment that tracked the persistence of Daphnia populations exposed to copper contamination. We assessed whether genotypic diversity reduced the risk of extinction. We created monoclonal and multiclonal populations and monitored their population sizes during a 32-week experiment. Cu was applied at a sub-lethal concentration and then increased every week until the population sizes dropped to about 10% of the carrying capacity (Cu at 180 µg/L). The concentration was then increased up to 186 µg/L and held stable until the end of the experiment. A survival analysis showed that clonal diversity extended the persistence of Daphnia populations, but copper contamination caused a substantial genetic erosion followed by population extirpation. However, some Cu-treated populations, mostly multiclonal, showed U-shaped patterns of growth consistent with evolutionary rescue but these did not lead to lasting population recovery. These results highlight the importance of genetic variation for population persistence, but they also show how quickly it can be lost in contaminated environments.

Towards reproducible metabarcoding data: Lessons from an international cross-laboratory experiment.

Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (Australia, Canada, New Zealand and the USA) were distributed to 12 independent laboratories. Participants were asked to follow one of two HTS library preparation workflows. While DNA extraction, primers and bioinformatic analyses were purposefully standardized to allow comparison, many other technical variables were allowed to vary among laboratories (amplification protocols, type of instrument used, etc.). Despite substantial variation observed in raw results, the primary signal in the data was consistent, with the samples grouping strongly by geographical origin for all data sets. Simple post hoc data clean-up by removing low-quality samples gave the best improvement in sample classification for nuclear 18S rRNA gene data, with an overall 92.81% correct group attribution. For mitochondrial COI gene data, the best classification result (95.58%) was achieved after correction for contamination errors. The identified critical methodological factors that introduced the greatest variability (preservation buffer, sample defrosting, template concentration, DNA polymerase, PCR enhancer) should be of great assistance in standardizing future biodiversity studies using metabarcoding.

Integrating physiology and environmental dynamics to operationalize environmental DNA (eDNA) as a means to monitor freshwater macro-organism abundance.

Research has demonstrated consistent positive correlations between organism abundance and absolute environmental DNA (eDNA) concentrations. Robust correlations in laboratory experiments indicate strong functional links, suggesting the potential for eDNA to monitor organism abundance in nature. However, correlations between absolute eDNA concentrations and organism abundance in nature tend to be weaker because myriad biotic and abiotic factors influence steady-state eDNA concentrations, decoupling its direct functional link with abundance. Additional technical challenges can also weaken correlations between relative organism abundance and relative eDNA data derived from metabarcoding. Future research must account for these factors to improve the inference of organism abundance from eDNA, including integrating the effects of organism physiology on eDNA production, eDNA dynamics in lentic/lotic systems, and key environmental parameters that impact estimated steady-state concentrations. Additionally, it is critical to manage expectations surrounding the accuracy and precision that eDNA can provide - eDNA, for example, cannot provide abundance estimates comparable to intensively managed freshwater fisheries that enumerate every individual fish. Recent developments, however, are encouraging. Current methods could provide meaningful information regarding qualitative conservation thresholds and emergent research has demonstrated that eDNA concentrations in natural ecosystems can provide rough quantitative estimates of abundance, particularly when models integrate physiology and/or eDNA dynamics. Operationalizing eDNA to infer abundance will probably require more than simple correlations with organism biomass/density. Nevertheless, the future is promising - models that integrate eDNA dynamics in nature could represent an effective means to infer abundance, particularly when traditional methods are considered too "costly" or difficult to obtain.

Assessment of current taxonomic assignment strategies for metabarcoding eukaryotes.

The effective use of metabarcoding in biodiversity science has brought important analytical challenges due to the need to generate accurate taxonomic assignments. The assignment of sequences to genus or species level is critical for biodiversity surveys and biomonitoring, but it is particularly challenging as researchers must select the approach that best recovers information on species composition. This study evaluates the performance and accuracy of seven methods in recovering the species composition of mock communities by using COI barcode fragments. The mock communities varied in species number and specimen abundance, while upstream molecular and bioinformatic variables were held constant, and using a set of COI fragments. We evaluated the impact of parameter optimization on the quality of the predictions. Our results indicate that BLAST top hit competes well with more complex approaches if optimized for the mock community under study. For example, the two machine learning methods that were benchmarked proved more sensitive to reference database heterogeneity and completeness than methods based on sequence similarity. The accuracy of assignments was impacted by both species and specimen counts (query compositional heterogeneity) which ultimately influence the selection of appropriate software. We urge researchers to: (i) use realistic mock communities to allow optimization of parameters, regardless of the taxonomic assignment method employed; (ii) carefully choose and curate the reference databases including completeness; and (iii) use QIIME, BLAST or LCA methods, in conjunction with parameter tuning to better assign taxonomy to diverse communities, especially when information on species diversity is lacking for the area under study.

Environmental RNA: A Revolution in Ecological Resolution?

Current advancements in environmental RNA (eRNA) exploit its relatively fast turnover rate relative to environmental DNA (eDNA) to assess 'metabolically active' or temporally/spatially recent community diversity. However, this focus significantly underutilizes the trove of potential ecological information encrypted in eRNA. Here, we argue for pushing beyond current species-level eDNA detection capabilities by using eRNA to detect any organisms with unique eRNA profiles, potentially including different life-history stages, sexes, or even specific phenotypes within a species. We also discuss the future of eRNA as a means of assessing the physiological status of organisms and the ecological health of populations and communities, reflecting ecosystem-level conditions. We posit that eRNA has the potential to significantly improve the resolution of organism detection, biological monitoring, and biomonitoring applications in ecology.

Speciation in Daphnia.

The microcrustacean Daphnia is arguably one of the most studied zooplankton species, having a well understood ecology, life history, and a relatively well studied evolutionary history. Despite this wealth of knowledge, species boundaries within closely related species in this genus often remain elusive and the major evolutionary forces driving the diversity of daphniids remain controversial. This genus contains more than 80 species with multiple cryptic species complexes, with many closely related species able to hybridize. Here, we review speciation research in Daphnia within the framework of current speciation theory. We evaluate the role of geography, ecology, and biology in restricting gene flow and promoting diversification. Of the 253 speciation studies on Daphnia, the majority of studies examine geographic barriers (55%). While evidence shows that geographic barriers play a role in species divergence, ecological barriers are also probably prominent in Daphnia speciation. We assess the contribution of ecological and nonecological reproductive isolating barriers between closely related species of Daphnia and found that none of the reproductive isolating barriers are restricting gene flow completely. Research on reproductive isolating barriers has disproportionally focused on two species complexes, the Daphnia pulex and Daphnia longispina species complexes. Finally, we identify areas of research that remain relatively unexplored and discuss future research directions that build our understanding of speciation in daphniids.

Thermal stratification and fish thermal preference explain vertical eDNA distributions in lakes.

Significant advances have been made towards surveying animal and plant communities using DNA isolated from environmental samples. Despite rapid progress, we lack a comprehensive understanding of the "ecology" of environmental DNA (eDNA), particularly its temporal and spatial distribution and how this is shaped by abiotic and biotic processes. Here, we tested how seasonal variation in thermal stratification and animal habitat preferences influences the distribution of eDNA in lakes. We sampled eDNA depth profiles of five dimictic lakes during both summer stratification and autumn turnover, each containing warm- and cool-water fishes as well as the cold-water stenotherm, lake trout (Salvelinus namaycush). Habitat use by S. namaycush was validated by acoustic telemetry and was significantly related to eDNA distribution during stratification. Fish eDNA became "stratified" into layers during summer months, reflecting lake stratification and the thermal niches of the species. During summer months, S. namaycush, which rarely ventured into shallow waters, could only be detected at the deepest layers of the lakes, whereas the eDNA of warm-water fishes was much more abundant above the thermocline. By contrast, during autumn lake turnover, the fish species assemblage as detected by eDNA was homogenous throughout the water column. These findings contribute to our overall understanding of the "ecology" of eDNA within lake ecosystems, illustrating how the strong interaction between seasonal thermal structure in lakes and thermal niches of species on very localized spatial scales influences our ability to detect species.

Freshwater zooplankton metapopulations and metacommunities respond differently to environmental and spatial variation.

Theory predicts that population genetic structure and metacommunity structure are linked by the common processes of drift and migration, but how population genetic structure and metacommunity structure are related in nature is still unknown. Deeper understanding of the processes influencing both genetic and community diversity is vital for better predicting how environmental change will impact biodiversity patterns. We examined how crustacean zooplankton and rotifer species' metapopulation genetic structure and metacommunities respond to environmental and spatial variation both within and across four regions of boreal Canada. Metapopulation and metacommunity variation partitioning results were compared within and across the four regions. Metapopulations and metacommunities responded differently to environmental variation and spatial structure both within and across regions, as metapopulations were influenced by different environmental variables compared to metacommunities. At larger spatial scales both metapopulations and metacommunities exhibited greater spatial and environmental structuring, again responding to a different subset of environmental variables. Our findings suggest that even though both genetic and species diversity are linked by the same processes, regional variation in environmental characteristics and spatial structure influence resulting biodiversity patterns differently. To date, no other empirical research has explored relationships between entire metapopulation and metacommunity assemblages at large regional spatial scales.

Metal exposure causes rDNA copy number to fluctuate in mutation accumulation lines of Daphnia pulex.

Ribosomal (r)DNA is a highly dynamic, conserved, multigene family whose sequence homogeneity is thought to be maintained by intra- and interchromosomal recombination, which are capable of changing rDNA copy number. It is generally not known how environmental stress such as sublethal exposure to environmentally relevant concentrations of metals impacts rDNA copy number. To determine how chronic metal exposure affects rDNA, we measured copy number of the 18S rRNA gene in 355 copper and nickel-exposed samples and 132 metal-free samples derived from 325 mutation accumulation (MA) lines of two genetically distinct Daphnia pulex lineages. The MA lines were sampled at four time points over 100+ generations of clonal propagation. The copy number of rDNA was also measured in 15 individuals sampled from a metal-free non-MA control population established from the same progenitor as one of the MA lineages. We found that mean rDNA copy number fluctuated across lines exposed to metals with a tendency to decrease over time. In contrast, mean rDNA copy number in the metal-free control lines and the non-MA population remained stable over time. It is generally accepted that extreme rDNA loss results in the loss of organism fitness. Thus, fluctuations in rDNA copy number, including losses, could affect the long-term viability of natural populations of Daphnia in metal-contaminated habitats.

Breaking ecological barriers: Anthropogenic disturbance leads to habitat transitions, hybridization, and high genetic diversity.

Genetic diversity is expected to erode in disturbed habitats through strong selection, local extinctions, and recolonization associated with genetic bottlenecks and restricted gene flow. Despite this general prediction and over three decades of population genetics studies, our understanding of the long-term effect of environmental disturbance on local and regional genetic diversity remains limited. We conducted a population genetic survey of the microcrustacean Daphnia across a landscape subject to anthropogenic stressors from a century of industrial mining. At the local scale we found moderate genetic diversity (i.e., low clonal diversity), characteristic of habitat-specific selective sweeps and local extinctions, but high diversity and strong genetic structure at the regional scale despite the shared watershed of many lakes and exceptional dispersal ability of daphniids. Many habitats experienced changes in species assemblages, with the obligate asexual Daphnia pulex lineages-known only to inhabit ponds-dominating disrupted urban lakes. This habitat transition (pond to lake) was likely facilitated by the disruption of ecological barriers maintaining the genomic separation of these young species. Thus, disrupted habitats can exhibit complex and unexpected genetic patterns of local extinctions and recolonizations, followed by habitat transitions, hybridization and potential speciation events that are difficult to predict and should not be underestimated.

Alternative splicing is highly variable among Daphnia pulex lineages in response to acute copper exposure.

BACKGROUND: Despite being one of the primary mechanisms of gene expression regulation in eukaryotes, alternative splicing is often overlooked in ecotoxicogenomic studies. The process of alternative splicing facilitates the production of multiple mRNA isoforms from a single gene thereby greatly increasing the diversity of the transcriptome and proteome. This process can be important in enabling the organism to cope with stressful conditions. Accurate identification of splice sites using RNA sequencing requires alignment to independent exonic positions within the genome, presenting bioinformatic challenges, particularly when using short read data. Although technological advances allow for the detection of splicing patterns on a genome-wide scale, very little is known about the extent of intraspecies variation in splicing patterns, particularly in response to environmental stressors. In this study, we used RNA-sequencing to study the molecular responses to acute copper exposure in three lineages of Daphnia pulex by focusing on the contribution of alternative splicing in addition to gene expression responses. RESULTS: By comparing the overall gene expression and splicing patterns among all 15 copper-exposed samples and 6 controls, we identified 588 differentially expressed (DE) genes and 16 differentially spliced (DS) genes. Most of the DS genes (13) were not found to be DE, suggesting unique transcriptional regulation in response to copper that went unnoticed with conventional DE analysis. To understand the influence of genetic background on gene expression and alternative splicing responses to Cu, each of the three lineages was analyzed separately. In contrast to the overall analysis, each lineage had a higher proportion of unique DS genes than DE genes suggesting that genetic background has a larger influence on DS than on DE. Gene Ontology analysis revealed that some pathways involved in stress response were jointly regulated by DS and DE genes while others were regulated by only transcription or only splicing. CONCLUSIONS: Our findings suggest an important role for alternative splicing in shaping transcriptome diversity in response to metal exposure in Daphnia, highlighting the importance of integrating splicing analyses with gene expression surveys to characterize molecular pathways in evolutionary and environmental studies.

Release and degradation of environmental DNA and RNA in a marine system.

Over the last decade, there has been growing interest in the analysis of environmental DNA (eDNA) to infer the presence of organisms in aquatic environments. The efficacy of eDNA/eRNA based tools are highly depend on the turnover rate of the molecule (their release and degradation). Environmental DNA has been shown to persist for days, weeks or years in environmental samples. Environmental RNA (eRNA) is thought to degrade faster than eDNA, however to our knowledge, no experimental studies have explored this. Here we present an aquarium study to investigate eDNA and eRNA shedding rates and degradation for two sessile marine invertebrates. The copy numbers for eDNA and eRNA were assessed using droplet digital PCR targeting the mitochondrial Cytochrome c Oxidase subunit 1 (COI) gene. Environmental RNA persisted after organism removal for much longer than expected with detections for up to 13 h. In contrast, eDNA was detected is samples collected up to 94 h after organism removal. There was no evidence that the decay rates constants for eDNA and eRNA were different (p = 0.6, Kruskal-Wallis tests). Both eDNA and eRNA was detected in biofilms collected at the end of the experiment (day 21). This suggests binding with organic or inorganic compounds or stabilization of these molecules in the biofilm matrix. The finding of the prolonged persistence of eRNA may provide new opportunities for improved biodiversity surveys through reducing false positives caused by legacy DNA and could also facilitate new research on environmental transcriptomics.

The evolution of reproductive isolation in Daphnia.

BACKGROUND: The process by which populations evolve to become new species involves the emergence of various reproductive isolating barriers (RIB). Despite major advancements in understanding this complex process, very little is known about the order in which RIBs evolve or their relative contribution to the total restriction of gene flow during various stages of speciation. This is mainly due to the difficulties of studying reproductive isolation during the early stages of species formation. This study examines ecological and non-ecological RIB within and between Daphnia pulex and Daphnia pulicaria, two recently diverged species that inhabit distinct habitats and exhibit an unusual level of intraspecific genetic subdivision. RESULTS: We find that while ecological prezygotic barriers are close to completion, none of the non-ecological barriers can restrict gene flow between D. pulex and D. pulicaria completely when acting alone. Surprisingly, we also identified high levels of postzygotic reproductive isolation in 'conspecific' interpopulation crosses of D. pulex. CONCLUSIONS: While the ecological prezygotic barriers are prevalent during the mature stages of speciation, non-ecological barriers likely dominated the early stages of speciation. This finding indicates the importance of studying the very early stages of speciation and suggests the contribution of postzygotic isolation in initiating the process of speciation.