ABSTRACT
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.
Subject(s)
DNA, Environmental , Ecosystem , Animals , Biodiversity , Lakes , Earth, PlanetABSTRACT
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.
ABSTRACT
Mutation rate variation has been under intense investigation for decades. Despite these efforts, little is known about the extent to which environmental stressors accelerate mutation rates and influence the genetic load of populations. Moreover, most studies on stressors have focused on unicellular organisms and point mutations rather than large-scale deletions and duplications (copy number variations [CNVs]). We estimated mutation rates in Daphnia pulex exposed to low levels of environmental stressors as well as the effect of selection on de novo mutations. We conducted a mutation accumulation (MA) experiment in which selection was minimized, coupled with an experiment in which a population was propagated under competitive conditions in a benign environment. After an average of 103 generations of MA propagation, we sequenced 60 genomes and found significantly accelerated rates of deletions and duplications in MA lines exposed to ecologically relevant concentrations of metals. Whereas control lines had gene deletion and duplication rates comparable to other multicellular eukaryotes (1.8 × 10-6 per gene per generation), the presence of nickel and copper increased these rates fourfold. The realized mutation rate under selection was reduced to 0.4× that of control MA lines, providing evidence that CNVs contribute to mutational load. Our CNV breakpoint analysis revealed that nonhomologous recombination associated with regions of DNA fragility is the primary source of CNVs, plausibly linking metal-induced DNA strand breaks with higher CNV rates. Our findings suggest that environmental stress, in particular multiple stressors, can have profound effects on large-scale mutation rates and mutational load of multicellular organisms.
Subject(s)
Base Sequence , Copper/toxicity , DNA Breaks , Daphnia/genetics , Nickel/therapeutic use , Sequence Deletion , Animals , Daphnia/metabolism , Dose-Response Relationship, Drug , Environmental Exposure/adverse effectsABSTRACT
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.
Subject(s)
Copper , Daphnia , Animals , Biological Evolution , Copper/toxicity , Daphnia/genetics , Genetic Variation , GenotypeABSTRACT
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.
Subject(s)
Daphnia , Gene Flow , Animals , Biological Evolution , Daphnia/genetics , Genetic Speciation , Geography , PhylogenyABSTRACT
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.
Subject(s)
DNA, Environmental , Animals , Aquatic Organisms , Biodiversity , Ecosystem , Environmental Monitoring , Fishes/genetics , Fresh WaterABSTRACT
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.
Subject(s)
DNA, Environmental , Ecosystem , Animals , Lakes , Seasons , TroutABSTRACT
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.
Subject(s)
Alternative Splicing/drug effects , Arthropod Proteins/genetics , Copper/adverse effects , Daphnia/physiology , Animals , Daphnia/classification , Daphnia/drug effects , Evolution, Molecular , Gene Expression Profiling , Gene Expression Regulation/drug effects , Gene Ontology , High-Throughput Nucleotide Sequencing , Sequence Analysis, RNA , Species Specificity , Stress, PhysiologicalABSTRACT
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.
Subject(s)
Daphnia/classification , Daphnia/genetics , Genetic Speciation , Animals , Crosses, Genetic , Ecology , Ecosystem , Female , Gene Flow , Male , Reproductive IsolationABSTRACT
Daphnia pulex is a keystone species for aquatic habitats and an ecological/evolution model organism. Although significant progress has been made on characterizing its genome, the D. pulex proteome remains largely uncharacterized partially due to abnormally high protein degradation during homogenization and emphasis on genomic analysis. In this study, various sample preparation and mass spectrometry acquisition methods are performed for the purpose of improving D. pulex proteome exploration. Benefits for employing both in-gel and in-solution methods of trypsin digestion are observed. Furthermore, acquisition methods employing ion mobility separation greatly increase peptide identification and more than doubled the proteome coverage. Bioinformatic analysis suggests that mitochondrial and hydrolytic activities are enriched in D. pulex compared to closely related invertebrates or Homo sapiens. Also, novel D. pulex proteins possessing putative genome modifying functional domains are identified. Data are available via ProteomeXchange with identifier PXD008455.
Subject(s)
Daphnia/metabolism , Mass Spectrometry/methods , Proteins/metabolism , Proteome/analysis , Animals , Computational BiologyABSTRACT
Understanding the rates, spectra, and fitness effects of spontaneous mutations is fundamental to answering key questions in evolution, molecular biology, disease genetics, and conservation biology. To estimate mutation rates and evaluate the effect of selection on new mutations, we propagated mutation accumulation (MA) lines of Daphnia pulex for more than 82 generations and maintained a non-MA population under conditions where selection could act. Both experiments were started with the same obligate asexual progenitor clone. By sequencing 30 genomes and implementing a series of validation steps that informed the bioinformatic analyses, we identified a total of 477 single nucleotide mutations (SNMs) in the MA lines, corresponding to a mutation rate of 2.30 × 10-9 (95% CI 1.90-2.70 × 10-9) per nucleotide per generation. The high overall rate of loss of heterozygosity (LOH) of 4.82 × 10-5 per site per generation was due to a large ameiotic recombination event spanning an entire arm of a chromosome (â¼6 Mb) and several hemizygous deletion events spanning â¼2 kb each. In the non-MA population, we found significantly fewer mutations than expected based on the rate derived from the MA experiment, indicating purifying selection was likely acting to remove new deleterious mutations. We observed a surprisingly high level of genetic variability in the non-MA population, which we propose to be driven by balancing selection. Our findings suggest that both positive and negative selection on new mutations is powerful and effective in a strictly clonal population.
Subject(s)
Daphnia/genetics , Genetic Fitness , Mutation Accumulation , Animals , Base Sequence , Biological Evolution , Computational Biology/methods , Daphnia/metabolism , Environment , Evolution, Molecular , Female , Male , Mutation , Mutation Rate , Selection, GeneticABSTRACT
The combination of DNA barcoding and high-throughput (next-generation) sequencing (metabarcoding) provides many promises but also serious challenges. Generating a reliable comparable estimate of biodiversity remains a central challenge to the application of the technology. Many approaches have been used to turn millions of sequences into distinct taxonomic units. However, the extent to which these methods impact the outcome of simple ecological analyses is not well understood. Here we performed a simple analysis of dietary overlap by skinks and shrews on Ile Aux Aigrettes, Mauritius. We used a combination of filtering thresholds and clustering algorithms on a COI metabarcoding dataset and demonstrate that all bioinformatics parameters will have interacting effects on molecular operational taxonomic unit (MOTU) recovery rates. These effects generated estimates covering two orders of magnitude. However, the effect on a simple ecological analysis was not large and, despite the wide variation in estimates of niche overlap, the same ecological conclusion was drawn in most cases. We advise that a conservative clustering programme coupled with larger sequence divergences to define a cluster, the removal of singletons, rigorous length filtering, and stringent match criteria for Molecular Identifier tags are preferable to avoid MOTU inflation and that the same parameters be used in all comparative analyses.
Subject(s)
Biodiversity , DNA Barcoding, Taxonomic , Ecosystem , Computational Biology/methods , DNA Barcoding, Taxonomic/methods , Ecology , Electron Transport Complex IV/genetics , High-Throughput Nucleotide SequencingABSTRACT
A diverse array of molecular markers and constantly evolving analytical approaches have been employed to reconstruct the invasion histories of the most notorious invasions. Detailed information on the source(s) of introduction, invasion route, type of vectors, number of independent introductions and pathways of secondary spread has been corroborated for a large number of biological invasions. In this review, I present the promises and limitations of current techniques while discussing future directions. Broad phylogeographic surveys of native and introduced populations have traced back invasion routes with surprising precision. These approaches often further clarify species boundaries and reveal complex patterns of genetic relationships with noninvasive relatives. Moreover, fine-scale analyses of population genetics or genomics allow deep inferences on the colonization dynamics across invaded ranges and can reveal the extent of gene flow among populations across various geographical scales, major demographic events such as genetic bottlenecks as well as other important evolutionary events such as hybridization with native taxa, inbreeding and selective sweeps. Genetic data have been often corroborated successfully with historical, geographical and ecological data to enable a comprehensive reconstruction of the invasion process. The advent of next-generation sequencing, along with the availability of extensive databases of repository sequences generated by barcoding projects opens the opportunity to broadly monitor biodiversity, to identify early invasions and to quantify failed invasions that would otherwise remain inconspicuous to the human eye.
Subject(s)
Biological Evolution , Genetics, Population/methods , Introduced Species , Animals , Gene Flow , Genetic Markers , Genetics, Population/trends , Hybridization, Genetic , Inbreeding , Phylogeny , Phylogeography , Zooplankton/geneticsABSTRACT
Understanding the response of organisms to multiple stressors is critical for predicting if populations can adapt to rapid environmental change. Natural and anthropogenic stressors often interact, complicating general predictions. In this study, we examined the interactive and cumulative effects of two common environmental stressors, lowered calcium concentration, an anthropogenic stressor, and predator presence, a natural stressor, on the water flea Daphnia pulex. We analyzed expression changes of five genes involved in calcium homeostasis - cuticle proteins (Cutie, Icp2), calbindin (Calb), and calcium pump and channel (Serca and Ip3R) - using real-time quantitative PCR (RT-qPCR) in a full factorial experiment. We observed strong synergistic interactions between low calcium concentration and predator presence. While the Ip3R gene was not affected by the stressors, the other four genes were affected in their transcriptional levels by the combination of the stressors. Transcriptional patterns of genes that code for cuticle proteins (Cutie and Icp2) and a sarcoplasmic calcium pump (Serca) only responded to the combination of stressors, changing their relative expression levels in a synergistic response, while a calcium-binding protein (Calb) responded to low calcium stress and the combination of both stressors. The expression pattern of these genes (Cutie, Icp2, and Serca) were nonlinear, yet they were dose dependent across the calcium gradient. Multiple stressors can have complex, often unexpected effects on ecosystems. This study demonstrates that the dominant interaction for the set of tested genes appears to be synergism. We argue that gene expression patterns can be used to understand and predict the type of interaction expected when organisms are exposed simultaneously to natural and anthropogenic stressors.
Subject(s)
Calcium Signaling , Daphnia/metabolism , Stress, Physiological , Animals , Calbindins/genetics , Calcium/metabolism , Daphnia/genetics , Gene Expression Regulation , HomeostasisSubject(s)
Genetics , Periodicals as Topic/history , Genome , History, 20th Century , History, 21st CenturyABSTRACT
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.
Subject(s)
DNA, Environmental , RNA , Animals , RNA/genetics , Environmental Monitoring , Biodiversity , Ecology , Demography , DNA Barcoding, Taxonomic , EcosystemABSTRACT
Despite the great utility of mitochondrial DNA (mtDNA) sequence data in population genetics and phylogenetics, key parameters describing the process of mitochondrial mutation (e.g., the rate and spectrum of mutational change) are based on few direct estimates. Furthermore, the variation in the mtDNA mutation process within species or between lineages with contrasting reproductive strategies remains poorly understood. In this study, we directly estimate the mtDNA mutation rate and spectrum using Daphnia pulex mutation-accumulation (MA) lines derived from sexual (cyclically parthenogenetic) and asexual (obligately parthenogenetic) lineages. The nearly complete mitochondrial genome sequences of 82 sexual and 47 asexual MA lines reveal high mtDNA mutation rate of 1.37 × 10(-7) and 1.73 × 10(-7) per nucleotide per generation, respectively. The Daphnia mtDNA mutation rate is among the highest in eukaryotes, and its spectrum is dominated by insertions and deletions (70%), largely due to the presence of mutational hotspots at homopolymeric nucleotide stretches. Maximum likelihood estimates of the Daphnia mitochondrial effective population size reveal that between five and ten copies of mitochondrial genomes are transmitted per female per generation. Comparison between sexual and asexual lineages reveals no statistically different mutation rates and highly similar mutation spectra.
Subject(s)
DNA, Mitochondrial/genetics , Daphnia/genetics , Genome, Mitochondrial , Mutation Rate , Animals , Biological Evolution , Evolution, Molecular , Mitochondria/genetics , Mutation , Phylogeny , Recombination, GeneticABSTRACT
The molecular mechanisms leading to asexuality remain little understood despite their substantial bearing on why sexual reproduction is dominant in nature. Here, we examine the role of hybridization in the origin and spread of obligate asexuality in Daphnia pulex, arguably the best-documented case of contagious asexuality. Obligately parthenogenetic (OP) clones of D. pulex have traditionally been separated into 'hybrid' (Ldh SF) and 'nonhybrid' (Ldh SS) forms because the lactase dehydrogenase (Ldh) locus distinguishes the cyclically parthenogenetic (CP) lake dwelling Daphnia pulicaria (Ldh FF) from its ephemeral pond dwelling sister species D. pulex (Ldh SS). The results of our population genetic analyses based on microsatellite loci suggest that both Ldh SS and SF OP individuals can originate from the crossing of CP female F1 (D. pulex × D. pulicaria) and backcross with males from OP lineages carrying genes that suppress meiosis specifically in female offspring. In previous studies, a suite of diagnostic markers was found to be associated with OP in Ldh SS D. pulex lineages. Our association mapping supports a similar genetic mechanism for the spread of obligate parthenogenesis in Ldh SF OP individuals. Interestingly, our study shows that CP D. pulicaria carry many of the diagnostic microsatellite alleles associated with obligate parthenogenesis. We argue that the assemblage of mutations that suppress meiosis and underlie obligate parthenogenesis in D. pulex originated due to a unique historical hybridization and introgression event between D. pulex and D. pulicaria.