Molecular ecology of microbiomes in the wild: Common pitfalls, methodological advances and future directions.

The study of microbiomes across organisms and environments has become a prominent focus in molecular ecology. This perspective article explores common challenges, methodological advancements, and future directions in the field. Key research areas include understanding the drivers of microbiome community assembly, linking microbiome composition to host genetics, exploring microbial functions, transience and spatial partitioning, and disentangling non-bacterial components of the microbiome. Methodological advancements, such as quantifying absolute abundances, sequencing complete genomes, and utilizing novel statistical approaches, are also useful tools for understanding complex microbial diversity patterns. Our aims are to encourage robust practices in microbiome studies and inspire researchers to explore the next frontier of this rapidly changing field.

Temporal change in floral availability leads to periods of resource limitation and affects diet specificity in a generalist pollinator.

Generalist species are core components of ecological networks and crucial for the maintenance of biodiversity. Generalist species and networks are expected to be more resilient, and therefore understanding the dynamics of specialization and generalization in ecological networks is a key focus in a time of rapid global change. Whilst diet generalization is frequently studied, our understanding of how it changes over time is limited. Here we explore temporal variation in diet specificity in the honeybee (Apis mellifera), using pollen DNA metabarcoding of honey samples, through the foraging season, over two years. We find that, overall, honeybees are generalists that visit a wide range of plants, but there is temporal variation in the degree of specialization. Temporal specialization of honeybee colonies corresponds to periods of resource limitation, identified as a lack of honey stores. Honeybees experience a lack of preferred resources in June when switching from flowering trees in spring to shrubs and herbs in summer. Investigating temporal patterns in specialization can identify periods of resource limitation that may lead to species and network vulnerability. Diet specificity must therefore be explored at different temporal scales in order to fully understand species and network stability in the face of ecological change.

Comparative population genomics of manta rays has global implications for management.

Understanding population connectivity and genetic diversity is of fundamental importance to conservation. However, in globally threatened marine megafauna, challenges remain due to their elusive nature and wide-ranging distributions. As overexploitation continues to threaten biodiversity across the globe, such knowledge gaps compromise both the suitability and effectiveness of management actions. Here, we use a comparative framework to investigate genetic differentiation and diversity of manta rays, one of the most iconic yet vulnerable groups of elasmobranchs on the planet. Despite their recent divergence, we show how oceanic manta rays (Mobula birostris) display significantly higher heterozygosity than reef manta rays (Mobula alfredi) and that M. birostris populations display higher connectivity worldwide. Through inferring modes of colonization, we reveal how both contemporary and historical forces have likely influenced these patterns, with important implications for population management. Our findings highlight the potential for fisheries to disrupt population dynamics at both local and global scales and therefore have direct relevance for international conservation of marine species.

High throughput shotgun sequencing of eRNA reveals taxonomic and derived functional shifts across a benthic productivity gradient.

Benthic macrofauna is regularly used in monitoring programmes, however the vast majority of benthic eukaryotic biodiversity lies mostly in microscopic organisms, such as meiofauna (invertebrates < 1 mm) and protists, that rapidly responds to environmental change. These communities have traditionally been hard to sample and handle in the laboratory, but DNA sequencing has made such work less time consuming. While DNA sequencing captures both alive and dead organisms, environmental RNA (eRNA) better targets living organisms or organisms of recent origin in the environment. Here, we assessed the biodiversity of three known bioindicator microeukaryote groups (nematodes, foraminifera, and ciliates) in sediment samples collected at seven coastal sites along an organic carbon (OC) gradient. We aimed to investigate if eRNA shotgun sequencing can be used to simultaneously detect differences in (i) biodiversity of multiple microeukaryotic communities; and (ii) functional feeding traits of nematodes. Results showed that biodiversity was lower for nematodes and foraminifera in high OC (6.2%-6.9%), when compared to low OC sediments (1.2%-2.8%). Dissimilarity in community composition increased for all three groups between Low OC and High OC, as well as the classified feeding type of nematode genera (with more nonselective deposit feeders in high OC sediment). High relative abundant genera included nematode Sabatieria and foraminifera Elphidium in high OC, and Cryptocaryon-like ciliates in low OC sediments. Considering that future sequencing technologies are likely to decrease in cost, the use of eRNA shotgun sequencing to assess biodiversity of benthic microeukaryotes could be a powerful tool in recurring monitoring programmes.

Ecosystems monitoring powered by environmental genomics: A review of current strategies with an implementation roadmap.

A decade after environmental scientists integrated high-throughput sequencing technologies in their toolbox, the genomics-based monitoring of anthropogenic impacts on the biodiversity and functioning of ecosystems is yet to be implemented by regulatory frameworks. Despite the broadly acknowledged potential of environmental genomics to this end, technical limitations and conceptual issues still stand in the way of its broad application by end-users. In addition, the multiplicity of potential implementation strategies may contribute to a perception that the routine application of this methodology is premature or "in development", hence restraining regulators from binding these tools into legal frameworks. Here, we review recent implementations of environmental genomics-based methods, applied to the biomonitoring of ecosystems. By taking a general overview, without narrowing our perspective to particular habitats or groups of organisms, this paper aims to compare, review and discuss the strengths and limitations of four general implementation strategies of environmental genomics for monitoring: (a) Taxonomy-based analyses focused on identification of known bioindicators or described taxa; (b) De novo bioindicator analyses; (c) Structural community metrics including inferred ecological networks; and (d) Functional community metrics (metagenomics or metatranscriptomics). We emphasise the utility of the three latter strategies to integrate meiofauna and microorganisms that are not traditionally utilised in biomonitoring because of difficult taxonomic identification. Finally, we propose a roadmap for the implementation of environmental genomics into routine monitoring programmes that leverage recent analytical advancements, while pointing out current limitations and future research needs.

Connecting high-throughput biodiversity inventories: Opportunities for a site-based genomic framework for global integration and synthesis.

High-throughput sequencing (HTS) is increasingly being used for the characterization and monitoring of biodiversity. If applied in a structured way, across broad geographical scales, it offers the potential for a much deeper understanding of global biodiversity through the integration of massive quantities of molecular inventory data generated independently at local, regional and global scales. The universality, reliability and efficiency of HTS data can potentially facilitate the seamless linking of data among species assemblages from different sites, at different hierarchical levels of diversity, for any taxonomic group and regardless of prior taxonomic knowledge. However, collective international efforts are required to optimally exploit the potential of site-based HTS data for global integration and synthesis, efforts that at present are limited to the microbial domain. To contribute to the development of an analogous strategy for the nonmicrobial terrestrial domain, an international symposium entitled "Next Generation Biodiversity Monitoring" was held in November 2019 in Nicosia (Cyprus). The symposium brought together evolutionary geneticists, ecologists and biodiversity scientists involved in diverse regional and global initiatives using HTS as a core tool for biodiversity assessment. In this review, we summarize the consensus that emerged from the 3-day symposium. We converged on the opinion that an effective terrestrial Genomic Observatories network for global biodiversity integration and synthesis should be spatially led and strategically united under the umbrella of the metabarcoding approach. Subsequently, we outline an HTS-based strategy to collectively build an integrative framework for site-based biodiversity data generation.

Phylogenomics and species delimitation for effective conservation of manta and devil rays.

Practical biodiversity conservation relies on delineation of biologically meaningful units. Manta and devil rays (Mobulidae) are threatened worldwide, yet morphological similarities and a succession of recent taxonomic changes impede the development of an effective conservation strategy. Here, we generate genome-wide single nucleotide polymorphism (SNP) data from a geographically and taxonomically representative set of manta and devil ray samples to reconstruct phylogenetic relationships and evaluate species boundaries under the general lineage concept. We show that nominal species units supported by alternative data sources constitute independently evolving lineages, and find robust evidence for a putative new species of manta ray in the Gulf of Mexico. Additionally, we uncover substantial incomplete lineage sorting indicating that rapid speciation together with standing variation in ancestral populations has driven phylogenetic uncertainty within Mobulidae. Finally, we detect cryptic diversity in geographically distinct populations, demonstrating that management below the species level may be warranted in certain species. Overall, our study provides a framework for molecular genetic species delimitation that is relevant to wide-ranging taxa of conservation concern, and highlights the potential for genomic data to support effective management, conservation and law enforcement strategies.

Epistatic regulation of growth in Atlantic salmon revealed: a QTL study performed on the domesticated-wild interface.

BACKGROUND: Quantitative traits are typically considered to be under additive genetic control. Although there are indications that non-additive factors have the potential to contribute to trait variation, experimental demonstration remains scarce. Here, we investigated the genetic basis of growth in Atlantic salmon by exploiting the high level of genetic diversity and trait expression among domesticated, hybrid and wild populations. RESULTS: After rearing fish in common-garden experiments under aquaculture conditions, we performed a variance component analysis in four mapping populations totaling ~ 7000 individuals from six wild, two domesticated and three F1 wild/domesticated hybrid strains. Across the four independent datasets, genome-wide significant quantitative trait loci (QTLs) associated with weight and length were detected on a total of 18 chromosomes, reflecting the polygenic nature of growth. Significant QTLs correlated with both length and weight were detected on chromosomes 2, 6 and 9 in multiple datasets. Significantly, epistatic QTLs were detected in all datasets. DISCUSSION: The observed interactions demonstrated that the phenotypic effect of inheriting an allele deviated between half-sib families. Gene-by-gene interactions were also suggested, where the combined effect of two loci resulted in a genetic effect upon phenotypic variance, while no genetic effect was detected when the two loci were considered separately. To our knowledge, this is the first documentation of epistasis in a quantitative trait in Atlantic salmon. These novel results are of relevance for breeding programs, and for predicting the evolutionary consequences of domestication-introgression in wild populations.

Salinity drives meiofaunal community structure dynamics across the Baltic ecosystem.

Coastal benthic biodiversity is under increased pressure from climate change, eutrophication, hypoxia, and changes in salinity due to increase in river runoff. The Baltic Sea is a large brackish system characterized by steep environmental gradients that experiences all of the mentioned stressors. As such it provides an ideal model system for studying the impact of on-going and future climate change on biodiversity and function of benthic ecosystems. Meiofauna (animals < 1 mm) are abundant in sediment and are still largely unexplored even though they are known to regulate organic matter degradation and nutrient cycling. In this study, benthic meiofaunal community structure was analysed along a salinity gradient in the Baltic Sea proper using high-throughput sequencing. Our results demonstrate that areas with higher salinity have a higher biodiversity, and salinity is probably the main driver influencing meiofauna diversity and community composition. Furthermore, in the more diverse and saline environments a larger amount of nematode genera classified as predators prevailed, and meiofauna-macrofauna associations were more prominent. These findings show that in the Baltic Sea, a decrease in salinity resulting from accelerated climate change will probably lead to decreased benthic biodiversity, and cause profound changes in benthic communities, with potential consequences for ecosystem stability, functions and services.

How quantitative is metabarcoding: A meta-analytical approach.

Metabarcoding has been used in a range of ecological applications such as taxonomic assignment, dietary analysis and the analysis of environmental DNA. However, after a decade of use in these applications there is little consensus on the extent to which proportions of reads generated corresponds to the original proportions of species in a community. To quantify our current understanding, we conducted a structured review and meta-analysis. The analysis suggests that a weak quantitative relationship may exist between the biomass and sequences produced (slope = 0.52 ± 0.34, p < 0.01), albeit with a large degree of uncertainty. None of the tested moderators, sequencing platform type, the number of species used in a trial or the source of DNA, were able to explain the variance. Our current understanding of the factors affecting the quantitative performance of metabarcoding is still limited: additional research is required before metabarcoding can be confidently utilized for quantitative applications. Until then, we advocate the inclusion of mock communities when metabarcoding as this facilitates direct assessment of the quantitative ability of any given study.

Deep segregation in the open ocean: Macaronesia as an evolutionary hotspot for low dispersal marine invertebrates.

Diversification and speciation of terrestrial organisms are anticipated in oceanic islands such as Macaronesia, a group of Atlantic islands that have remained unconnected to continental landmasses. Hitherto, the diversification of marine organisms in oceanic islands, especially those with low vagility, has received little direct empirical analysis using molecular markers. Here, we focus on such a case study, through applying a multilocus molecular approach to investigate the diversity and evolution of a group that lacks a planktonic larval stage, the isopod genus Dynamene, in Macaronesia and Northeast Atlantic. Sequences of two mitochondrial (cytochrome c oxidase subunit I and 16S rRNA) and two nuclear (18S rRNA and 28S rRNA) loci were obtained from specimens of Dynamene edwardsi (Lucas, 1849), Dynamene magnitorata Holdich, 1968 and Dynamene bidentata (Adams, 1800) collected along the Northeast Atlantic and Macaronesia. Although no major phylogeographic structure was detected in D. bidentata and D. magnitorata, from five to nine deeply divergent lineages were evident within D. edwardsi. The divergent lineages displayed genetic distances comparable to those found among established species of peracarids. D. edwardsi exhibits a long, rich and complex phylogeographic history in Macaronesia, where the geodynamics of the islands possibly associated with founder effects and subsequent lack of gene flow among populations confounds patterns based on geographic proximity of targeted populations. Our findings collectively suggest a much larger role of oceanic islands in the diversification of marine invertebrates than previously anticipated. The work provides insights into the origins and dynamics of ongoing geographic segregation and associated deep divergence among sister evolutionary lineages in Macaronesia.

Cryptic diets of forage fish: jellyfish consumption observed in the Celtic Sea and western English Channel.

To establish if fishes' consumption of jellyfish changes through the year, we conducted a molecular gut-content assessment on opportunistically sampled species from the Celtic Sea in October and compared these with samples previously collected in February and March from the Irish Sea. Mackerel Scomber scombrus were found to feed on hydrozoan jellyfish relatively frequently in autumn, with rare consumption also detected in sardine Sardina pilchardus and sprat Sprattus sprattus. By October, moon jellyfish Aurelia aurita appeared to have escaped predation, potentially through somatic growth and the development of stinging tentacles. This is in contrast with sampling in February and March where A. aurita ephyrae were heavily preyed upon. No significant change in predation rate was observed in S. sprattus, but jellyfish predation by S. scombrus feeding in autumn was significantly higher than that seen during winter. This increase in consumption appears to be driven by the consumption of different, smaller jellyfish species than were targeted during the winter.

Whole genome duplication and transposable element proliferation drive genome expansion in Corydoradinae catfishes.

Genome size varies significantly across eukaryotic taxa and the largest changes are typically driven by macro-mutations such as whole genome duplications (WGDs) and proliferation of repetitive elements. These two processes may affect the evolutionary potential of lineages by increasing genetic variation and changing gene expression. Here, we elucidate the evolutionary history and mechanisms underpinning genome size variation in a species-rich group of Neotropical catfishes (Corydoradinae) with extreme variation in genome size-0.6 to 4.4 pg per haploid cell. First, genome size was quantified in 65 species and mapped onto a novel fossil-calibrated phylogeny. Two evolutionary shifts in genome size were identified across the tree-the first between 43 and 49 Ma (95% highest posterior density (HPD) 36.2-68.1 Ma) and the second at approximately 19 Ma (95% HPD 15.3-30.14 Ma). Second, restriction-site-associated DNA (RAD) sequencing was used to identify potential WGD events and quantify transposable element (TE) abundance in different lineages. Evidence of two lineage-scale WGDs was identified across the phylogeny, the first event occurring between 54 and 66 Ma (95% HPD 42.56-99.5 Ma) and the second at 20-30 Ma (95% HPD 15.3-45 Ma) based on haplotype numbers per contig and between 35 and 44 Ma (95% HPD 30.29-64.51 Ma) and 20-30 Ma (95% HPD 15.3-45 Ma) based on SNP read ratios. TE abundance increased considerably in parallel with genome size, with a single TE-family (TC1-IS630-Pogo) showing several increases across the Corydoradinae, with the most recent at 20-30 Ma (95% HPD 15.3-45 Ma) and an older event at 35-44 Ma (95% HPD 30.29-64.51 Ma). We identified signals congruent with two WGD duplication events, as well as an increase in TE abundance across different lineages, making the Corydoradinae an excellent model system to study the effects of WGD and TEs on genome and organismal evolution.

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities.

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing ("HTS") platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed "environmental DNA" or "eDNA"). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called "eDNA metabarcoding" and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.

Competition and phylogeny determine community structure in Müllerian co-mimics.

Until recently, the study of negative and antagonistic interactions (for example, competition and predation) has dominated our understanding of community structure, maintenance and assembly. Nevertheless, a recent theoretical model suggests that positive interactions (for example, mutualisms) may counterbalance competition, facilitating long-term coexistence even among ecologically undifferentiated species. Müllerian mimics are mutualists that share the costs of predator education and are therefore ideally suited for the investigation of positive and negative interactions in community dynamics. The sole empirical test of this model in a Müllerian mimetic community supports the prediction that positive interactions outweigh the negative effects of spatial overlap (without quantifying resource acquisition). Understanding the role of trophic niche partitioning in facilitating the evolution and stability of Müllerian mimetic communities is now of critical importance, but has yet to be formally investigated. Here we show that resource partitioning and phylogeny determine community structure and outweigh the positive effects of Müllerian mimicry in a species-rich group of neotropical catfishes. From multiple, independent reproductively isolated allopatric communities displaying convergently evolved colour patterns, 92% consist of species that do not compete for resources. Significant differences in phylogenetically conserved traits (snout morphology and body size) were consistently linked to trait-specific resource acquisition. Thus, we report the first evidence, to our knowledge, that competition for trophic resources and phylogeny are pivotal factors in the stable evolution of Müllerian mimicry rings. More generally, our work demonstrates that competition for resources is likely to have a dominant role in the structuring of communities that are simultaneously subject to the effects of both positive and negative interactions.

Plasticity in growth of farmed and wild Atlantic salmon: is the increased growth rate of farmed salmon caused by evolutionary adaptations to the commercial diet?

BACKGROUND: Domestication of Atlantic salmon for commercial aquaculture has resulted in farmed salmon displaying substantially higher growth rates than wild salmon under farming conditions. In contrast, growth differences between farmed and wild salmon are much smaller when compared in the wild. The mechanisms underlying this contrast between environments remain largely unknown. It is possible that farmed salmon have adapted to the high-energy pellets developed specifically for aquaculture, contributing to inflated growth differences when fed on this diet. We studied growth and survival of 15 families of farmed, wild and F1 hybrid salmon fed three contrasting diets under hatchery conditions; a commercial salmon pellet diet, a commercial carp pellet diet, and a mixed natural diet consisting of preserved invertebrates commonly found in Norwegian rivers. RESULTS: For all groups, despite equal numbers of calories presented by all diets, overall growth reductions as high 68 and 83%, relative to the salmon diet was observed in the carp and natural diet treatments, respectively. Farmed salmon outgrew hybrid (intermediate) and wild salmon in all treatments. The relative growth difference between wild and farmed fish was highest in the carp diet (1: 2.1), intermediate in the salmon diet (1:1.9) and lowest in the natural diet (1:1.6). However, this trend was non-significant, and all groups displayed similar growth reaction norms and plasticity towards differing diets across the treatments. CONCLUSIONS: No indication of genetic-based adaptation to the form or nutritional content of commercial salmon diets was detected in the farmed salmon. Therefore, we conclude that diet alone, at least in the absence of other environmental stressors, is not the primary cause for the large contrast in growth differences between farmed and wild salmon in the hatchery and wild. Additionally, we conclude that genetically-increased appetite is likely to be the primary reason why farmed salmon display higher growth rates than wild salmon when fed ad lib rations under hatchery conditions. Our results contribute towards an understanding of the potential genetic changes that have occurred in farmed salmon in response to domestication, and the potential mechanisms underpinning genetic and ecological interactions between farmed escapees and wild salmonids.

Convergence of multiple markers and analysis methods defines the genetic distinctiveness of cryptic pitvipers.

Using multiple markers and multiple analytical approaches is critical for establishing species boundaries reliably, especially so in the case of cryptic species. Despite development of new and powerful analytical methods, most studies continue to adopt a few, with the choice often being subjective. One such example is routine analysis of Amplified Fragment Length Polymorphism (AFLP) data using population genetic models despite disparity between method assumptions and data properties. The application of newly developed methods for analyzing this dominant marker may not be entirely clear in the context of species delimitation. In this study, we use AFLPs and mtDNA to investigate cryptic speciation in the Trimeresurus macrops complex that belongs to a taxonomically difficult lineage of Asian pitvipers. We analyze AFLPs using population genetic, phylogenetic, multivariate statistical, and Bayes Factor Delimitation methods. A gene tree from three mtDNA markers provided additional evidence. Our results show that the inferences about species boundaries that can be derived from population genetic analysis of AFLPs have certain limitations. In contrast, four multivariate statistical analyses produced clear clusters that are consistent with each other, as well as with Bayes Factor Delimitation results, and with mtDNA and total evidence phylogenies. Furthermore, our results concur with allopatric distributions and patterns of variation in individual morphological characters previously identified in the three proposed species: T. macrops sensu stricto, T. cardamomensis, and T. rubeus. Our study provides evidence for reproductive isolation and genetic distinctiveness that define these taxa as full species. In addition, we re-emphasize the importance of examining congruence of results from multiple methods of AFLP analysis for inferring species diversity.

An integrated spatio-temporal view of riverine biodiversity using environmental DNA metabarcoding.

Anthropogenically forced changes in global freshwater biodiversity demand more efficient monitoring approaches. Consequently, environmental DNA (eDNA) analysis is enabling ecosystem-scale biodiversity assessment, yet the appropriate spatio-temporal resolution of robust biodiversity assessment remains ambiguous. Here, using intensive, spatio-temporal eDNA sampling across space (five rivers in Europe and North America, with an upper range of 20-35 km between samples), time (19 timepoints between 2017 and 2018) and environmental conditions (river flow, pH, conductivity, temperature and rainfall), we characterise the resolution at which information on diversity across the animal kingdom can be gathered from rivers using eDNA. In space, beta diversity was mainly dictated by turnover, on a scale of tens of kilometres, highlighting that diversity measures are not confounded by eDNA from upstream. Fish communities showed nested assemblages along some rivers, coinciding with habitat use. Across time, seasonal life history events, including salmon and eel migration, were detected. Finally, effects of environmental conditions were taxon-specific, reflecting habitat filtering of communities rather than effects on DNA molecules. We conclude that riverine eDNA metabarcoding can measure biodiversity at spatio-temporal scales relevant to species and community ecology, demonstrating its utility in delivering insights into river community ecology during a time of environmental change.