Meta‐analysis shows that environmental DNA outperforms traditional surveys,but warrants better reporting standards

1. Decades of environmental DNA (eDNA) method application, spanning a wide variety of taxa and habitats, has advanced our understanding of eDNA and underlined its value as a tool for conservation practitioners. The general consensus is that eDNA methods are more accurate and cost‐effective than traditional survey methods. However, they are formally approved for just a few species globally (e.g., Bighead Carp, Silver Carp, Great Crested Newt). We conducted a meta‐analysis of studies that directly compare eDNA with traditional surveys to evaluate the assertion that eDNA methods are consistently “better.”
2. Environmental DNA publications for multiple species or single macro‐organism detection were identified using the Web of Science, by searching “eDNA” and “environmental DNA” across papers published between 1970 and 2020. The methods used, focal taxa, habitats surveyed, and quantitative and categorical results were collated and analyzed to determine whether and under what circumstances eDNA outperforms traditional surveys.
3. Results show that eDNA methods are cheaper, more sensitive, and detect more species than traditional methods. This is, however, taxa‐dependent, with amphibians having the highest potential for detection by eDNA survey. Perhaps most strikingly, of the 535 papers reviewed just 49 quantified the probability of detection for both eDNA and traditional survey methods and studies were three times more likely to give qualitative statements of performance.
4. Synthesis and applications: The results of this meta‐analysis demonstrate that where there is a direct comparison, eDNA surveys of macro‐organisms are more accurate and efficient than traditional surveys. This conclusion, however, is based on just a fraction of available eDNA papers as most do not offer this granularity. We recommend that conclusions are substantiated with comparable and quantitative data. Where a direct comparison has not been made, we caution against the use of qualitative statements about relative performance. This consistency and rigor will simplify how the eDNA research community tracks methods‐based advances and will also provide greater clarity for conservation practitioners. To this end suggest reporting standards for eDNA studies.

     

Effects of consumer surface sterilization on diet DNA metabarcoding data of terrestrial invertebrates in natural environments and feeding trials

1. DNA metabarcoding is an emerging tool used to quantify diet in environments and consumer groups where traditional approaches are unviable, including small‐bodied invertebrate taxa. However, metabarcoding of small taxa often requires DNA extraction from full body parts (without dissection), and it is unclear whether surface contamination from body parts alters presumed diet presence or diversity.
2. We examined four different measures of diet (presence, rarefied read abundance, richness, and species composition) for a terrestrial invertebrate consumer (the spider Heteropoda venatoria) both collected in its natural environment and fed an offered diet item in contained feeding trials using DNA metabarcoding of full body parts (opisthosomas). We compared diet from consumer individuals surface sterilized to remove contaminants in 10% commercial bleach solution followed by deionized water with a set of unsterilized individuals.
3. We found that surface sterilization did not significantly alter any measure of diet for consumers in either a natural environment or feeding trials. The best‐fitting model predicting diet detection in feeding trial consumers included surface sterilization, but this term was not statistically significant (β = −2.3, p‐value = .07).
4. Our results suggest that surface contamination does not seem to be a significant concern in this DNA diet metabarcoding study for consumers in either a natural terrestrial environment or feeding trials. As the field of diet DNA metabarcoding continues to progress into new environmental contexts with various molecular approaches, we suggest ongoing context‐specific consideration of the possibility of surface contamination.

     

Biosurveillance for invasive insect pest species using an environmental DNA metabarcoding approach and a high salt trap collection fluid

1. With the increase in global trade and warming patterns, the movement, introduction, and establishment of non‐native insect species has increased. A rapid and effective early detection biosurveillance program to identify species of concern is needed to reduce future impacts and costs associated with introduced non‐native species. One of the challenges facing insect surveillance trapping methods is the sheer volume of individual specimens in the collections. Although molecular identification methods are improving, they currently have limitations (e.g., destructive processing of specimens) and a protocol addressing these limitations can support regulatory applications that need morphological evidence to corroborate molecular data.
2. The novel protocol presented here uses a metabarcoding approach to amplify environmental DNA from a saturated salt solution trap fluid, which retains trap specimens for downstream morphological identifications. The use of a saturated salt solution to preserve specimens in traps addresses issues with the high evaporation rate of ethanol in traps, and public safety concerns with other fluid preservation options with unattended traps in public settings.
3. Using a metabarcoding approach, a 407‐nucleotide segment of the cytochrome c oxidase subunit 1 (COI) animal barcode region was successfully amplified from Lindgren funnel trap collection fluids. These traps were placed in forested areas to survey for wood‐boring beetles of regulatory concern. Our results displayed successful amplification of target taxa, including the molecular identification of the Japanese Beetle Popillia japonica, a species regulated in Canada. A second species, Anisandrus maiche, recently introduced to North America, was identified in every trap. The genus Lymantria, which contains numerous species of concern to North American woodlands, was also detected. Also, there were six other species identified of interest due to their potential impacts on native and crop flora and fauna.
4. Our results show how this protocol can be used as an efficient method for the surveillance of insects using a trap with a saturated salt solution and eDNA metabarcoding to detect species of regulatory concern.

     

Environmental DNA metabarcoding primers for freshwater fish detection and quantification: In silico and in tanks

Environmental DNA (eDNA) techniques refer to utilizing the organisms’ DNA extracted from environment samples to genetically identify target species without capturing actual organisms. eDNA metabarcoding via high‐throughput sequencing can simultaneously detect multiple fish species from a single water sample, which is a powerful tool for the qualitative detection and quantitative estimates of multiple fish species. However, sequence counts obtained from eDNA metabarcoding may be influenced by many factors, of which primer bias is one of the foremost causes of methodological error. The performance of 18 primer pairs for COI, cytb, 12S rRNA, and 16S rRNA mitochondrial genes, which are all frequently used in fish eDNA metabarcoding, were evaluated in the current study. The ribosomal gene markers performed better than the protein‐coding gene markers during in silico screening, resulting in higher taxonomic coverage and appropriate barcode lengths. Four primer pairs—AcMDB07, MiFish‐U, Ve16S1, and Ve16S3—designed for various regions of the 12S and 16S rRNA genes were screened for tank metabarcoding in a case study targeting six freshwater fish species. The four primer pairs were able to accurately detect all six species in different tanks, while only MiFish‐U, Ve16S1, and Ve16S3 revealed a significant positive relationship between species biomass and read count for the pooled tank data. The positive relationship could not be found in all species within the tanks. Additionally, primer efficiency differed depending on the species while primer preferential species varied in different fish assemblages. This case study supports the potential for eDNA metabarcoding to assess species diversity in natural ecosystems and provides an alternative strategy to evaluate the performance of candidate primers before application of eDNA metabarcoding in natural ecosystems.     

Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.     

Optimization and validation of a cost‐effective protocol for biosurveillance of invasive alien species

Environmental DNA (eDNA) metabarcoding has revolutionized biodiversity monitoring and invasive pest biosurveillance programs. The introduction of insect pests considered invasive alien species (IAS) into a non‐native range poses a threat to native plant health. The early detection of IAS can allow for prompt actions by regulating authorities, thereby mitigating their impacts. In the present study, we optimized and validated a fast and cost‐effective eDNA metabarcoding protocol for biosurveillance of IAS and characterization of insect and microorganism diversity. Forty‐eight traps were placed, following the CFIA''s annual forest insect trapping survey, at four locations in southern Ontario that are high risk for forest IAS. We collected insects and eDNA samples using Lindgren funnel traps that contained a saturated salt (NaCl) solution in the collection jar. Using cytochrome c oxidase I (COI) as a molecular marker, a modified Illumina protocol effectively identified 2,535 Barcode Index Numbers (BINs). BINs were distributed among 57 Orders and 304 Families, with the vast majority being arthropods. Two IAS (Agrilus planipennis and Lymantria dispar) are regulated by the Canadian Food Inspection Agency (CFIA) as plant health pests, are known to occur in the study area, and were identified through eDNA in collected traps. Similarly, using 16S ribosomal RNA and nuclear ribosomal internal transcribed spacer (ITS), five bacterial and three fungal genera, which contain species of regulatory concern across several Canadian jurisdictions, were recovered from all sampling locations. Our study results reaffirm the effectiveness and importance of integrating eDNA metabarcoding as part of identification protocols in biosurveillance programs.     

Integrating omics to characterize eco‐physiological adaptations: How moose diet and metabolism differ across biogeographic zones

1. With accelerated land conversion and global heating at northern latitudes, it becomes crucial to understand, how life histories of animals in extreme environments adapt to these changes. Animals may either adapt by adjusting foraging behavior or through physiological responses, including adjusting their energy metabolism or both. Until now, it has been difficult to study such adaptations in free‐ranging animals due to methodological constraints that prevent extensive spatiotemporal coverage of ecological and physiological data.
2. Through a novel approach of combining DNA‐metabarcoding and nuclear magnetic resonance (NMR)‐based metabolomics, we aim to elucidate the links between diets and metabolism in Scandinavian moose Alces alces over three biogeographic zones using a unique dataset of 265 marked individuals.
3. Based on 17 diet items, we identified four different classes of diet types that match browse species availability in respective ecoregions in northern Sweden. Individuals in the boreal zone consumed predominantly pine and had the least diverse diets, while individuals with highest diet diversity occurred in the coastal areas. Males exhibited lower average diet diversity than females.
4. We identified several molecular markers indicating metabolic constraints linked to diet constraints in terms of food availability during winter. While animals consuming pine had higher lipid, phospocholine, and glycerophosphocholine concentrations in their serum than other diet types, birch‐ and willow/aspen‐rich diets exhibit elevated concentrations of several amino acids. The individuals with highest diet diversity had increased levels of ketone bodies, indicating extensive periods of starvation for these individuals.
5. Our results show how the adaptive capacity of moose at the eco‐physiological level varies over a large eco‐geographic scale and how it responds to land use pressures. In light of extensive ongoing climate and land use changes, these findings pave the way for future scenario building for animal adaptive capacity.

     

Temperature but not ocean acidification affects energy metabolism and enzyme activities in the blue mussel,Mytilus edulis

1. In mosaic marine habitats, such as intertidal zones, ocean acidification (OA) is exacerbated by high variability of pH, temperature, and biological CO₂ production. The nonlinear interactions among these drivers can be context‐specific and their effect on organisms in these habitats remains largely unknown, warranting further investigation.
2. We were particularly interested in Mytilus edulis (the blue mussel) from intertidal zones of the Gulf of Maine (GOM), USA, for this study. GOM is a hot spot of global climate change (average sea surface temperature (SST) increasing by >0.2°C/year) with >60% decline in mussel population over the past 40 years.
3. Here, we utilize bioenergetic underpinnings to identify limits of stress tolerance in M. edulis from GOM exposed to warming and OA. We have measured whole‐organism oxygen consumption rates and metabolic biomarkers in mussels exposed to control and elevated temperatures (10 vs. 15°C, respectively) and current and moderately elevated P _CO2 levels (~400 vs. 800 µatm, respectively).
4. Our study demonstrates that adult M. edulis from GOM are metabolically resilient to the moderate OA scenario but responsive to warming as seen in changes in metabolic rate, energy reserves (total lipids), metabolite profiles (glucose and osmolyte dimethyl amine), and enzyme activities (carbonic anhydrase and calcium ATPase).
5. Our results are in agreement with recent literature that OA scenarios for the next 100–300 years do not affect this species, possibly as a consequence of maintaining its in vivo acid‐base balance.

     

Riverine distribution of mussel environmental DNA reflects a balance among density,transport, and removal processes

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Trust your guts? The effect of gut section on diet composition and impact of Mus musculus on islands using metabarcoding

1. DNA metabarcoding is widely used to characterize the diet of species, and it becomes very relevant for biodiversity conservation, allowing the understanding of trophic chains and the impact of invasive species. The need for cost‐effective biodiversity monitoring methods fostered advances in this technique. One question that arises is which sample type provides a better diet representation.
2. Therefore, with this study, we intended to evaluate if there were differences in diet estimates according to the section of the gastrointestinal tract analysed and which section(s) provided the best diet representation. Additionally, we intended to infer the ecological/economic impacts of an invader as a model of the potential effects in an originally mammal‐free ecosystem.
3. We examined the gut contents of the house mouse Mus musculus introduced to Cabo Verde, considering three sections: stomach, small intestine, and large intestine. We applied a DNA‐metabarcoding approach using two genetic markers, one specific for plants and another for invertebrates.
4. We showed that this invader consumed 131 taxa (73 plants and 58 invertebrates). We obtained significant differences in the composition of two of the three sections, with a higher incidence of invertebrates in the stomach and plants in the intestines. This may be due to stomach inhibitors acting on plants and/or to faster absorption of soft‐body invertebrates compared to the plant fibers in the intestines. We verified that the impact of this invader in the ecosystem is predominantly negative, as at least 50% of the ingested items were native, endemic, or economically important taxa, and only 19% of the diet items were exotics.
5. Overall, results showed the need to analyse only two gastrointestinal tract sections to obtain robust diet data, increasing the cost‐effectiveness of the method. Furthermore, by uncovering the native taxa most frequently preyed on by mice, this DNA‐metabarcoding approach allowed us to evaluate efficiently which are at the highest risk.

     

Using hierarchical models to compare the sensitivity of metabarcoding and qPCR for eDNA detection

Environmental DNA (eDNA) sampling—the detection of intra- or extra-cellular DNA in environmental samples—is a rapid and sensitive survey method for detecting aquatic species. Single-species detection methods (typically based on PCR or LAMP) have been shown to be more sensitive for detecting target species than multi-species detection methods, such as metabarcoding. However, previous studies have generally only compared these two eDNA detection approaches for a single target species and have used different methodological and statistical approaches. Here we present a comparison of single- and multi-species eDNA detection methods, drawing on two published case studies (one fish, one amphibian) and two new extensive datasets on a freshwater mammal (the platypus). To ensure consistent conclusions regarding the sensitivity of each eDNA method, we use the same hierarchical site occupancy-detection model for each dataset, incorporating uncertainty at the site, water sample, and technical replicate level. Overall, qPCR achieved higher detection probabilities than metabarcoding across species and datasets. However, differences in sensitivity between detection methods varied depending on methodological decisions concerning what constitutes a true positive detection (i.e., qPCR and metabarcoding thresholds). The decision as to which eDNA detection method to use should always be influenced by the study aims, but our results suggest that single-species detection methods based on qPCR may be preferable when the aim is to achieve a high detection probability for target species.     

Automatic detection of fish and tracking of movement for ecology

1. Animal movement studies are conducted to monitor ecosystem health, understand ecological dynamics, and address management and conservation questions. In marine environments, traditional sampling and monitoring methods to measure animal movement are invasive, labor intensive, costly, and limited in the number of individuals that can be feasibly tracked. Automated detection and tracking of small‐scale movements of many animals through cameras are possible but are largely untested in field conditions, hampering applications to ecological questions.
2. Here, we aimed to test the ability of an automated object detection and object tracking pipeline to track small‐scale movement of many individuals in videos. We applied the pipeline to track fish movement in the field and characterize movement behavior. We automated the detection of a common fisheries species (yellowfin bream, Acanthopagrus australis) along a known movement passageway from underwater videos. We then tracked fish movement with three types of tracking algorithms (MOSSE, Seq‐NMS, and SiamMask) and evaluated their accuracy at characterizing movement.
3. We successfully detected yellowfin bream in a multispecies assemblage (F1 score =91%). At least 120 of the 169 individual bream present in videos were correctly identified and tracked. The accuracies among the three tracking architectures varied, with MOSSE and SiamMask achieving an accuracy of 78% and Seq‐NMS 84%.
4. By employing this integrated object detection and tracking pipeline, we demonstrated a noninvasive and reliable approach to studying fish behavior by tracking their movement under field conditions. These cost‐effective technologies provide a means for future studies to scale‐up the analysis of movement across many visual monitoring systems.

     

Comparing local‐ and regional‐scale estimations of the diversity of stream fish using eDNA metabarcoding and conventional observation methods

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Detection of the elusive Dwarf sperm whale (Kogia sima) using environmental DNA at Malpelo island (Eastern Pacific,Colombia)

1. Monitoring large marine mammals is challenging due to their low abundances in general, an ability to move over large distances and wide geographical range sizes.
2. The distribution of the pygmy (Kogia breviceps) and dwarf (Kogia sima) sperm whales is informed by relatively rare sightings, which does not permit accurate estimates of their distribution ranges. Hence, their conservation status has long remained Data Deficient (DD) in the Red list of the International Union for Conservation of Nature (IUCN), which prevent appropriate conservation measures.
3. Environmental DNA (eDNA) metabarcoding uses DNA traces left by organisms in their environments to detect the presence of targeted taxon, and is here proved to be useful to increase our knowledge on the distribution of rare but emblematic megafauna.
4. Retrieving eDNA from filtered surface water provides the first detection of the Dwarf sperm whale (Kogia sima) around the remote Malpelo island (Colombia).
5. Environmental DNA collected during oceanic missions can generate better knowledge on rare but emblematic animals even in regions that are generally well sampled for other taxa.

     

How many replicates to accurately estimate fish biodiversity using environmental DNA on coral reefs?

Quantifying fish species diversity in rich tropical marine environments remains challenging. Environmental DNA (eDNA) metabarcoding is a promising tool to face this challenge through the filtering, amplification, and sequencing of DNA traces from water samples. However, because eDNA concentration is low in marine environments, the reliability of eDNA to detect species diversity can be limited. Using an eDNA metabarcoding approach to identify fish Molecular Taxonomic Units (MOTUs) with a single 12S marker, we aimed to assess how the number of sampling replicates and filtered water volume affect biodiversity estimates. We used a paired sampling design of 30 L per replicate on 68 reef transects from 8 sites in 3 tropical regions. We quantified local and regional sampling variability by comparing MOTU richness, compositional turnover, and compositional nestedness. We found strong turnover of MOTUs between replicated pairs of samples undertaken in the same location, time, and conditions. Paired samples contained non‐overlapping assemblages rather than subsets of one another. As a result, non‐saturated localized diversity accumulation curves suggest that even 6 replicates (180 L) in the same location can underestimate local diversity (for an area <1 km). However, sampling regional diversity using ~25 replicates in variable locations (often covering 10 s of km) often saturated biodiversity accumulation curves. Our results demonstrate variability of diversity estimates possibly arising from heterogeneous distribution of eDNA in seawater, highly skewed frequencies of eDNA traces per MOTU, in addition to variability in eDNA processing. This high compositional variability has consequences for using eDNA to monitor temporal and spatial biodiversity changes in local assemblages. Avoiding false‐negative detections in future biomonitoring efforts requires increasing replicates or sampled water volume to better inform management of marine biodiversity using eDNA.     

Looking for the needle in a downsized haystack: Whole‐exome sequencing unravels genomic signals of climatic adaptation in Douglas‐fir (Pseudotsuga menziesii)

1. Conifers often occur along steep gradients of diverse climates throughout their natural ranges, which is expected to result in spatially varying selection to local climate conditions. However, signals of climatic adaptation can often be confounded, because unraveled clines covary with signals caused by neutral evolutionary processes such as gene flow and genetic drift. Consequently, our understanding of how selection and gene flow have shaped phenotypic and genotypic differentiation in trees is still limited.
2. A 40‐year‐old common garden experiment comprising 16 Douglas‐fir (Pseudotsuga menziesii) provenances from a north‐to‐south gradient of approx. 1,000 km was analyzed, and genomic information was obtained from exome capture, which resulted in an initial genomic dataset of >90,000 single nucleotide polymorphisms. We used a restrictive and conservative filtering approach, which permitted us to include only SNPs and individuals in environmental association analysis (EAA) that were free of potentially confounding effects (LD, relatedness among trees, heterozygosity deficiency, and deviations from Hardy–Weinberg proportions). We used four conceptually different genome scan methods based on F_ST outlier detection and gene–environment association in order to disentangle truly adaptive SNPs from neutral SNPs.
3. We found that a relatively small proportion of the exome showed a truly adaptive signal (0.01%–0.17%) when population substructuring and multiple testing was accounted for. Nevertheless, the unraveled SNP candidates showed significant relationships with climate at provenance origins, which strongly suggests that they have featured adaptation in Douglas‐fir along a climatic gradient. Two SNPs were independently found by three of the employed algorithms, and one of them is in close proximity to an annotated gene involved in circadian clock control and photoperiodism as was similarly found in Populus balsamifera.

Synthesis. We conclude that despite neutral evolutionary processes, phenotypic and genomic signals of adaptation to climate are responsible for differentiation, which in particular explain disparity between the well‐known coastal and interior varieties of Douglas‐fir.     

Intraspecific trait variability and community assembly in hawkmoths (Lepidoptera: Sphingidae) across an elevational gradient in the eastern Himalayas,India

1. We investigated some aspects of hawkmoth community assembly at 13 elevations along a 200‐ to 2770‐m transect in the eastern Himalayas, a little studied biodiversity hot spot of global importance. We measured the morphological traits of body mass, wing loading, and wing aspect ratio of 3,301 free‐ranging individuals of 76 species without having to collect or even constrain them. We used these trait measurements and T‐statistic metrics to assess the strength of intracommunity (“internal") and extra‐community (“external”) filters which determine the composition of communities vis‐a‐vis the regional pool of species.
2. The trait distribution of constituent species turned out to be nonrandom subsets of the community‐trait distribution, providing strong evidence for internal filtering in all elevational communities. The external filter metric was more ambiguous. However, the elevational dependence of many metrics including that of the internal filter provided evidence for external (i.e., environmental) filtering. On average, a species occupied as much as 50%–75% of the total community‐trait space, yet the T‐statistic metric for internal filter was sufficiently sensitive to detect a strong nonrandom structure in the trait distribution.
3. We suggest that the change in T‐statistic metrics along the environmental gradient may provide more clues to the process of community assembly than previously envisaged. A large, smoothly varying and well‐sampled environmental span would make it easier to discern them. Developing T‐statistics for combined analysis of multiple traits will perhaps provide a more accurate picture of internal/filtering and niche complementarity. Moths are a hyperdiverse taxon and a very important component of many ecosystems. Our technique for accurately measuring body and wing dimensions of free‐ranging moths can generate trait database for a large number of individuals in a time‐ and resource‐efficient manner for a variety of community assembly studies using this important taxon.

     

Growth patterns of the pan‐European freshwater mussel,Anodonta anatina (Linnaeus, 1758) (Bivalvia: Unionidae), vary with sex and mortality in populations

1. Post‐maturation growth leading to indeterminate growth patterns is widespread in nature. However, its adaptive value is unclear. Life history theory suggests this allocation strategy may be favored by temporal pulses in the intensity of mortality and/or the capacity to produce new tissues.
2. Addressing the origin of indeterminate growth and the variability of growth patterns, we studied the growth of duck mussels, Anodonta anatina, a pan‐European unionid, in 18 Polish lakes. For each population, the sex, size, and age of collected mussels were measured to estimate Bertalanffy''s growth curve parameters. We integrated information on A. anatina mortality rates, lake trophy, biofouling by zebra mussels, Dreissena polymorpha, and the prevalence of parasitic trematode larvae to identify selective conditions in lakes.
3. We found two sources of mortality in A. anatina populations, pertaining to adverse effects of zebra mussel biofouling and trophy state on mussel survival. Additionally, populations with heavier biofouling presented a smaller abundance of parasites, indicative of a relationship between filtering intensity and contraction of water‐borne trematode larvae by filtering A. anatina.
4. Consistently for each sex, populations with a greater trophy‐related mortality were characterized in A. anatina by a smaller asymptotic size L_max, indicative of a life history response to mortality risk involving early maturation at a smaller body size. In all populations, females featured higher mortality and larger asymptotic size versus males.
5. Our findings support a theoretical view that adaptive responses to selection involve adjustments in the lifetime resource allocation patterns. These adjustments should be considered drivers of the origin of indeterminate growth strategy in species taking parental care by offspring brooding in body cavities.

     

The influence of competing root symbionts on below‐ground plant resource allocation

1. Plants typically interact with multiple above‐ and below‐ground organisms simultaneously, with their symbiotic relationships spanning a continuum ranging from mutualism, such as with arbuscular mycorrhizal fungi (AMF), to parasitism, including symbioses with plant‐parasitic nematodes (PPN).
2. Although research is revealing the patterns of plant resource allocation to mutualistic AMF partners under different host and environmental constraints, the root ecosystem, with multiple competing symbionts, is often ignored. Such competition is likely to heavily influence resource allocation to symbionts.
3. Here, we outline and discuss the competition between AMF and PPN for the finite supply of host plant resources, highlighting the need for a more holistic understanding of the influence of below‐ground interactions on plant resource allocation. Based on recent developments in our understanding of other symbiotic systems such as legume–rhizobia and AMF‐aphid‐plant, we propose hypotheses for the distribution of plant resources between contrasting below‐ground symbionts and how such competition may affect the host.
4. We identify relevant knowledge gaps at the physiological and molecular scales which, if resolved, will improve our understanding of the true ecological significance and potential future exploitation of AMF‐PPN‐plant interactions in order to optimize plant growth. To resolve these outstanding knowledge gaps, we propose the application of well‐established methods in isotope tracing and nutrient budgeting to monitor the movement of nutrients between symbionts. By combining these approaches with novel time of arrival experiments and experimental systems involving multiple plant hosts interlinked by common mycelial networks, it may be possible to reveal the impact of multiple, simultaneous colonizations by competing symbionts on carbon and nutrient flows across ecologically important scales.