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.

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.

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.

Evaluating the correlation between genome-wide diversity and the release of plastic phenotypic variation in experimental translocations to novel natural environments.

Phenotypic reaction norms are often shaped and constrained by selection and are important for allowing organisms to respond to environmental change. However, selection cannot constrain reaction norms for environmental conditions that populations have not experienced. Consequently, cryptic neutral genetic variation for the reaction norm can accumulate such that a release of phenotypic variation occurs upon exposure to novel14 conditions. Most genomic diversity behaves as if functionally neutral. Therefore, genome-wide diversity metrics may correlate with levels of cryptic genetic variation and, as a result, exhibit a positive relationship with a release of phenotypic variation in novel environments. To test this hypothesis, we conducted translocations of juvenile brook trout (Salvelinus fontinalis) from 12 populations to novel uninhabited ponds that represented a gradient of environmental conditions. We assessed reaction norms for morphological traits (body size and four morphometric relative warps) across pond environmental gradients and evaluated the effect of genome-wide heterozygosity on phenotypic variability. All traits displayed plastic reaction norms. Overall, we found some evidence that a release of phenotypic variation consistent with cryptic genetic variation can occur in novel environmental conditions. However, the extent to which this release correlated with average genome-wide diversity was limited to only one of five traits examined. Our results suggest a limited link between genomic diversity26 and the accumulation of cryptic genetic variation in reaction norms. Similarly, reaction norms were constrained for many of the morphological traits examined. Past conditions may have constrained reaction norms in the putatively novel environments despite significant deviations from contemporary source population habitat. Additionally, as a generalist colonizing species brook trout may exhibit plastic phenotypes across a wide range of environmental conditions.

Population correlates of rapid captive-induced maladaptation in a wild fish.

Understanding the extent to which captivity generates maladaptation in wild species can inform species recovery programs and elucidate wild population responses to novel environmental change. Although rarely quantified, effective population size (N e ) and genetic diversity should influence the magnitude of plastic and genetic changes manifested in captivity that reduce wild fitness. Sexually dimorphic traits might also mediate consequences of captivity. To evaluate these relationships, we generated >600 full- and half-sibling families from nine wild brook trout populations, reared them for one generation under common, captive environmental conditions and contrasted several fitness-related traits in wild versus captive lines. We found substantial variation in lifetime success (lifetime survival and reproductive success) and life history traits among wild populations after just one captive generation (fourteen- and threefold ranges across populations, respectively). Populations with lower heterozygosity showed lower captive lifetime success, suggesting that captivity generates maladaptation within one generation. Greater male-biased mortality in captivity occurred in populations having disproportionately higher growth rates in males than females. Wild population N e and allelic diversity had little or no influence on captive trait expression and lifetime success. Our results have four conservation implications: (i) Trait values and lifetime success were highly variable across populations following one generation of captivity. (ii) Maladaptation induced by captive breeding might be particularly intense for the very populations practitioners are most interested in conserving, such as those with low heterozygosity. (iii) Maladaptive sex differences in captivity might be associated with population-dependent growth costs of reproduction. (iv) Heterozygosity can be a good indicator of short-term, intraspecific responses to novel environmental change.

Are heritability and selection related to population size in nature? Meta-analysis and conservation implications.

It is widely thought that small populations should have less additive genetic variance and respond less efficiently to natural selection than large populations. Across taxa, we meta-analytically quantified the relationship between adult census population size (N) and additive genetic variance (proxy: h (2)) and found no reduction in h (2) with decreasing N; surveyed populations ranged from four to one million individuals (1735 h (2) estimates, 146 populations, 83 species). In terms of adaptation, ecological conditions may systematically differ between populations of varying N; the magnitude of selection these populations experience may therefore also differ. We thus also meta-analytically tested whether selection changes with N and found little evidence for systematic differences in the strength, direction or form of selection with N across different trait types and taxa (7344 selection estimates, 172 populations, 80 species). Collectively, our results (i) indirectly suggest that genetic drift neither overwhelms selection more in small than in large natural populations, nor weakens adaptive potential/h (2) in small populations, and (ii) imply that natural populations of varying sizes experience a variety of environmental conditions, without consistently differing habitat quality at small N. However, we caution that the data are currently insufficient to determine whether some small populations may retain adaptive potential definitively. Further study is required into (i) selection and genetic variation in completely isolated populations of known N, under-represented taxonomic groups, and nongeneralist species, (ii) adaptive potential using multidimensional approaches and (iii) the nature of selective pressures for specific traits.

Does source population size affect performance in new environments?

Small populations are predicted to perform poorly relative to large populations when experiencing environmental change. To explore this prediction in nature, data from reciprocal transplant, common garden, and translocation studies were compared meta-analytically. We contrasted changes in performance resulting from transplantation to new environments among individuals originating from different sized source populations from plants and salmonids. We then evaluated the effect of source population size on performance in natural common garden environments and the relationship between population size and habitat quality. In 'home-away' contrasts, large populations exhibited reduced performance in new environments. In common gardens, the effect of source population size on performance was inconsistent across life-history stages (LHS) and environments. When transplanted to the same set of new environments, small populations either performed equally well or better than large populations, depending on life stage. Conversely, large populations outperformed small populations within native environments, but only at later life stages. Population size was not associated with habitat quality. Several factors might explain the negative association between source population size and performance in new environments: (i) stronger local adaptation in large populations and antagonistic pleiotropy, (ii) the maintenance of genetic variation in small populations, and (iii) potential environmental differences between large and small populations.