TICI: a taxon-independent community index for eDNA-based ecological health assessment.

Global biodiversity is declining at an ever-increasing rate. Yet effective policies to mitigate or reverse these declines require ecosystem condition data that are rarely available. Morphology-based bioassessment methods are difficult to scale, limited in scope, suffer prohibitive costs, require skilled taxonomists, and can be applied inconsistently between practitioners. Environmental DNA (eDNA) metabarcoding offers a powerful, reproducible and scalable solution that can survey across the tree-of-life with relatively low cost and minimal expertise for sample collection. However, there remains a need to condense the complex, multidimensional community information into simple, interpretable metrics of ecological health for environmental management purposes. We developed a riverine taxon-independent community index (TICI) that objectively assigns indicator values to amplicon sequence variants (ASVs), and significantly improves the statistical power and utility of eDNA-based bioassessments. The TICI model training step uses the Chessman iterative learning algorithm to assign health indicator scores to a large number of ASVs that are commonly encountered across a wide geographic range. New sites can then be evaluated for ecological health by averaging the indicator value of the ASVs present at the site. We trained a TICI model on an eDNA dataset from 53 well-studied riverine monitoring sites across New Zealand, each sampled with a high level of biological replication (n = 16). Eight short-amplicon metabarcoding assays were used to generate data from a broad taxonomic range, including bacteria, microeukaryotes, fungi, plants, and animals. Site-specific TICI scores were strongly correlated with historical stream condition scores from macroinvertebrate assessments (macroinvertebrate community index or MCI; R2 = 0.82), and TICI variation between sample replicates was minimal (CV = 0.013). Taken together, this demonstrates the potential for taxon-independent eDNA analysis to provide a reliable, robust and low-cost assessment of ecological health that is accessible to environmental managers, decision makers, and the wider community.

Non-invasive real-time genomic monitoring of the critically endangered kakapo.

We used non-invasive real-time genomic approaches to monitor one of the last surviving populations of the critically endangered kakapo (Strigops habroptilus). We first established an environmental DNA metabarcoding protocol to identify the distribution of kakapo and other vertebrate species in a highly localized manner using soil samples. Harnessing real-time nanopore sequencing and the high-quality kakapo reference genome, we then extracted species-specific DNA from soil. We combined long read-based haplotype phasing with known individual genomic variation in the kakapo population to identify the presence of individuals, and confirmed these genomically informed predictions through detailed metadata on kakapo distributions. This study shows that individual identification is feasible through nanopore sequencing of environmental DNA, with important implications for future efforts in the application of genomics to the conservation of rare species, potentially expanding the application of real-time environmental DNA research from monitoring species distribution to inferring fitness parameters such as genomic diversity and inbreeding.

Gut content metabarcoding of specialized feeders is not a replacement for environmental DNA assays of seawater in reef environments.

In tropical marine ecosystems, the coral-based diet of benthic-feeding reef fishes provides a window into the composition and health of coral reefs. In this study, for the first time, we compare multi-assay metabarcoding sequences of environmental DNA (eDNA) isolated from seawater and partially digested gut items from an obligate corallivore butterflyfish (Chaetodon lunulatus) resident to coral reef sites in the South China Sea. We specifically tested the proportional and statistical overlap of the different approaches (seawater vs gut content metabarcoding) in characterizing eukaryotic community composition on coral reefs. Based on 18S and ITS2 sequence data, which differed in their taxonomic sensitivity, we found that gut content detections were only partially representative of the eukaryotic communities detected in the seawater based on low levels of taxonomic overlap (3 to 21%) and significant differences between the sampling approaches. Overall, our results indicate that dietary metabarcoding of specialized feeders can be complimentary to, but is no replacement for, more comprehensive environmental DNA assays of reef environments that might include the processing of different substrates (seawater, sediment, plankton) or traditional observational surveys. These molecular assays, in tandem, might be best suited to highly productive but cryptic oceanic environments (kelp forests, seagrass meadows) that contain an abundance of organisms that are often small, epiphytic, symbiotic, or cryptic.

Symbiont Identity Impacts the Microbiome and Volatilome of a Model Cnidarian-Dinoflagellate Symbiosis.

The symbiosis between cnidarians and dinoflagellates underpins the success of reef-building corals in otherwise nutrient-poor habitats. Alterations to symbiotic state can perturb metabolic homeostasis and thus alter the release of biogenic volatile organic compounds (BVOCs). While BVOCs can play important roles in metabolic regulation and signalling, how the symbiotic state affects BVOC output remains unexplored. We therefore characterised the suite of BVOCs that comprise the volatilome of the sea anemone Exaiptasia diaphana ('Aiptasia') when aposymbiotic and in symbiosis with either its native dinoflagellate symbiont Breviolum minutum or the non-native symbiont Durusdinium trenchii. In parallel, the bacterial community structure in these different symbiotic states was fully characterised to resolve the holobiont microbiome. Based on rRNA analyses, 147 unique amplicon sequence variants (ASVs) were observed across symbiotic states. Furthermore, the microbiomes were distinct across the different symbiotic states: bacteria in the family Vibrionaceae were the most abundant in aposymbiotic anemones; those in the family Crocinitomicaceae were the most abundant in anemones symbiotic with D. trenchii; and anemones symbiotic with B. minutum had the highest proportion of low-abundance ASVs. Across these different holobionts, 142 BVOCs were detected and classified into 17 groups based on their chemical structure, with BVOCs containing multiple functional groups being the most abundant. Isoprene was detected in higher abundance when anemones hosted their native symbiont, and dimethyl sulphide was detected in higher abundance in the volatilome of both Aiptasia-Symbiodiniaceae combinations relative to aposymbiotic anemones. The volatilomes of aposymbiotic anemones and anemones symbiotic with B. minutum were distinct, while the volatilome of anemones symbiotic with D. trenchii overlapped both of the others. Collectively, our results are consistent with previous reports that D. trenchii produces a metabolically sub-optimal symbiosis with Aiptasia, and add to our understanding of how symbiotic cnidarians, including corals, may respond to climate change should they acquire novel dinoflagellate partners.

Optimal sample type and number vary in small shallow lakes when targeting non-native fish environmental DNA.

Non-native fish have been shown to have deleterious impacts on freshwater ecosystems in New Zealand. Early detection is critical for their effective management. Traditional capture-based techniques may not detect newly introduced fish, especially if they are present in low abundance. Molecular techniques that target environmental DNA (eDNA) have been shown, in many instances, to be more sensitive, cost-effective and require lower sampling effort. However, appropriate sampling strategies are needed to ensure robust and interpretable data are obtained. In this study we used droplet digital PCR assays to investigate the presence of two non-native fish in New Zealand, the European perch (Perca fluviatilis) and rudd (Scardinius erythrophthalmus) in three small lakes. Samples were collected from water and surface sediment at near-shore and mid-lake sites. Probabilistic modelling was used to assess the occupancy of fish eDNA and develop guidance on sampling strategies. Based on the detection probability measures from the present study, at least six sites and five replicates per site are needed to reliably detect fish eDNA in sediment samples, and twelve sites with eight replicates per site for water samples. The results highlight the potential of developing monitoring and surveillance programs adapted to lakes, that include the use of assays targeting eDNA. This study focused on small shallow lakes, and it is likely that these recommendations may vary in larger, deeper, and more geomorphologically complex lakes, and this requires further research.

PeskAAS: A near-real-time, open-source monitoring and analytics system for small-scale fisheries.

Small-scale fisheries are responsible for landing half of the world's fish catch, yet there are very sparse data on these fishing activities and associated fisheries production in time and space. Fisheries-dependent data underpin scientific guidance of management and conservation of fisheries systems, but it is inherently difficult to generate robust and comprehensive data for small-scale fisheries, particularly given their dispersed and diverse nature. In tackling this challenge, we use open source software components including the Shiny R package to build PeskAAS; an adaptable and scalable digital application that enables the collation, classification, analysis and visualisation of small-scale fisheries catch and effort data. We piloted and refined this system in Timor-Leste; a small island developing nation. The features that make PeskAAS fit for purpose are that it is: (i) fully open-source and free to use (ii) component-based, flexible and able to integrate vessel tracking data with catch records; (iii) able to perform spatial and temporal filtering of fishing productivity by fishing method and habitat; (iv) integrated with species-specific length-weight parameters from FishBase; (v) controlled through a click-button dashboard, that was co-designed with fisheries scientists and government managers, that enables easy to read data summaries and interpretation of context-specific fisheries data. With limited training and code adaptation, the PeskAAS workflow has been used as a framework on which to build and adapt systematic, standardised data collection for small-scale fisheries in other contexts. Automated analytics of these data can provide fishers, managers and researchers with insights into a fisher's experience of fishing efforts, fisheries status, catch rates, economic efficiency and geographic preferences and limits that can potentially guide management and livelihood investments.

Phylogenetic analysis of cell-cycle regulatory proteins within the Symbiodiniaceae.

In oligotrophic waters, cnidarian hosts rely on symbiosis with their photosynthetic dinoflagellate partners (family Symbiodiniaceae) to obtain the nutrients they need to grow, reproduce and survive. For this symbiosis to persist, the host must regulate the growth and proliferation of its symbionts. One of the proposed regulatory mechanisms is arrest of the symbiont cell cycle in the G1 phase, though the cellular mechanisms involved remain unknown. Cell-cycle progression in eukaryotes is controlled by the conserved family of cyclin-dependent kinases (CDKs) and their partner cyclins. We identified CDKs and cyclins in different Symbiodiniaceae species and examined their relationship to homologs in other eukaryotes. Cyclin proteins related to eumetazoan cell-cycle-related cyclins A, B, D, G/I and Y, and transcriptional cyclin L, were identified in the Symbiodiniaceae, alongside several alveolate-specific cyclin A/B proteins, and proteins related to protist P/U-type cyclins and apicomplexan cyclins. The largest expansion of Symbiodiniaceae cyclins was in the P/U-type cyclin groups. Proteins related to eumetazoan cell-cycle-related CDKs (CDK1) were identified as well as transcription-related CDKs. The largest expansion of CDK groups was, however, in alveolate-specific groups which comprised 11 distinct CDK groups (CDKA-J) with CDKB being the most widely distributed CDK protein. As a result of its phylogenetic position, conservation across Symbiodiniaceae species, and the presence of the canonical CDK motif, CDKB emerged as a likely candidate for a Saccharomyces cerevisiae Cdc28/Pho85-like homolog in Symbiodiniaceae. Similar to cyclins, two CDK-groups found in Symbiodiniaceae species were solely associated with apicomplexan taxa. A comparison of Breviolum minutum CDK and cyclin gene expression between free-living and symbiotic states showed that several alveolate-specific CDKs and two P/U-type cyclins exhibited altered expression in hospite, suggesting that symbiosis influences the cell cycle of symbionts on a molecular level. These results highlight the divergence of Symbiodiniaceae cell-cycle proteins across species. These results have important implications for host control of the symbiont cell cycle in novel cnidarian-dinoflagellate symbioses.

Environmental DNA can act as a biodiversity barometer of anthropogenic pressures in coastal ecosystems.

Loss of biodiversity from lower to upper trophic levels reduces overall productivity and stability of coastal ecosystems in our oceans, but rarely are these changes documented across both time and space. The characterisation of environmental DNA (eDNA) from sediment and seawater using metabarcoding offers a powerful molecular lens to observe marine biota and provides a series of 'snapshots' across a broad spectrum of eukaryotic organisms. Using these next-generation tools and downstream analytical innovations including machine learning sequence assignment algorithms and co-occurrence network analyses, we examined how anthropogenic pressures may have impacted marine biodiversity on subtropical coral reefs in Okinawa, Japan. Based on 18 S ribosomal RNA, but not ITS2 sequence data due to inconsistent amplification for this marker, as well as proxies for anthropogenic disturbance, we show that eukaryotic richness at the family level significantly increases with medium and high levels of disturbance. This change in richness coincides with compositional changes, a decrease in connectedness among taxa, an increase in fragmentation of taxon co-occurrence networks, and a shift in indicator taxa. Taken together, these findings demonstrate the ability of eDNA to act as a barometer of disturbance and provide an exemplar of how biotic networks and coral reefs may be impacted by anthropogenic activities.

Multi-gene incongruence consistent with hybridisation in Cladocopium (Symbiodiniaceae), an ecologically important genus of coral reef symbionts.

Coral reefs rely on their intracellular dinoflagellate symbionts (family Symbiodiniaceae) for nutritional provision in nutrient-poor waters, yet this association is threatened by thermally stressful conditions. Despite this, the evolutionary potential of these symbionts remains poorly characterised. In this study, we tested the potential for divergent Symbiodiniaceae types to sexually reproduce (i.e. hybridise) within Cladocopium, the most ecologically prevalent genus in this family. With sequence data from three organelles (cob gene, mitochondrion; psbAncr region, chloroplast; and ITS2 region, nucleus), we utilised the Incongruence Length Difference test, Approximately Unbiased test, tree hybridisation analyses and visual inspection of raw data in stepwise fashion to highlight incongruences between organelles, and thus provide evidence of reticulate evolution. Using this approach, we identified three putative hybrid Cladocopium samples among the 158 analysed, at two of the seven sites sampled. These samples were identified as the common Cladocopium types C40 or C1 with respect to the mitochondria and chloroplasts, but the rarer types C3z, C3u and C1# with respect to their nuclear identity. These five Cladocopium types have previously been confirmed as evolutionarily distinct and were also recovered in non-incongruent samples multiple times, which is strongly suggestive that they sexually reproduced to produce the incongruent samples. A concomitant inspection of next generation sequencing data for these samples suggests that other plausible explanations, such as incomplete lineage sorting or the presence of co-dominance, are much less likely. The approach taken in this study allows incongruences between gene regions to be identified with confidence, and brings new light to the evolutionary potential within Symbiodiniaceae.

Inter-partner specificity limits the acquisition of thermotolerant symbionts in a model cnidarian-dinoflagellate symbiosis.

The ability of corals and other cnidarians to survive climate change depends partly on the composition of their endosymbiont communities. The dinoflagellate family Symbiodiniaceae is genetically and physiologically diverse, and one proposed mechanism for cnidarians to acclimate to rising temperatures is to acquire more thermally tolerant symbionts. However, cnidarian-dinoflagellate associations vary in their degree of specificity, which may limit their capacity to alter symbiont communities. Here, we inoculated symbiont-free polyps of the sea anemone Exaiptasia pallida (commonly referred to as 'Aiptasia'), a model system for the cnidarian-dinoflagellate symbiosis, with simultaneous or sequential mixtures of thermally tolerant and thermally sensitive species of Symbiodiniaceae. We then monitored symbiont success (relative proportional abundance) at normal and elevated temperatures across two to four weeks. All anemones showed signs of bleaching at high temperature. During simultaneous inoculations, the native, thermally sensitive Breviolum minutum colonized polyps most successfully regardless of temperature when paired against the non-native but more thermally tolerant Symbiodinium microadriaticum or Durusdinium trenchii. Furthermore, anemones initially colonized with B. minutum and subsequently exposed to S. microadriaticum failed to acquire the new symbiont. These results highlight how partner specificity may place strong limitations on the ability of certain cnidarians to acquire more thermally tolerant symbionts, and hence their adaptive potential under climate change.

aphid: an R package for analysis with profile hidden Markov models.

SUMMARY: Hidden Markov models (HMMs) and profile HMMs form an integral part of biological sequence analysis, supporting an ever-growing list of applications. The aphid R package can be used to derive, train, plot, import and export HMMs and profile HMMs in the R environment. Computationally-intensive dynamic programing recursions, such as the Viterbi, forward and backward algorithms are implemented in C++ and parallelized for increased speed and efficiency. AVAILABILITY AND IMPLEMENTATION: The aphid package is released under the GPL-3 license, and is freely available for download from CRAN and GitHub (https://github.com/shaunpwilkinson/aphid). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Thermal Shock Induces Host Proteostasis Disruption and Endoplasmic Reticulum Stress in the Model Symbiotic Cnidarian Aiptasia.

Coral bleaching has devastating effects on coral survival and reef ecosystem function, but many of the fundamental cellular effects of thermal stress on cnidarian physiology are unclear. We used label-free liquid chromatography-tandem mass spectrometry to compare the effects of rapidly (33.5 °C, 24 h) and gradually (30 and 33.5 °C, 12 days) elevated temperatures on the proteome of the model symbiotic anemone Aiptasia. We identified 2133 proteins in Aiptasia, 136 of which were differentially abundant between treatments. Thermal shock, but not acclimation, resulted in significant abundance changes in 104 proteins, including those involved in protein folding and synthesis, redox homeostasis, and central metabolism. Nineteen abundant structural proteins showed particularly reduced abundance, demonstrating proteostasis disruption and potential protein synthesis inhibition. Heat shock induced antioxidant mechanisms and proteins involved in stabilizing nascent proteins, preventing protein aggregation and degrading damaged proteins, which is indicative of endoplasmic reticulum stress. Host proteostasis disruption occurred before either bleaching or symbiont photoinhibition was detected, suggesting host-derived reactive oxygen species production as the proximate cause of thermal damage. The pronounced abundance changes in endoplasmic reticulum proteins associated with proteostasis and protein turnover indicate that these processes are essential in the cellular response of symbiotic cnidarians to severe thermal stress.

Isolation by resistance across a complex coral reef seascape.

A detailed understanding of the genetic structure of populations and an accurate interpretation of processes driving contemporary patterns of gene flow are fundamental to successful spatial conservation management. The field of seascape genetics seeks to incorporate environmental variables and processes into analyses of population genetic data to improve our understanding of forces driving genetic divergence in the marine environment. Information about barriers to gene flow (such as ocean currents) is used to define a resistance surface to predict the spatial genetic structure of populations and explain deviations from the widely applied isolation-by-distance model. The majority of seascape approaches to date have been applied to linear coastal systems or at large spatial scales (more than 250 km), with very few applied to complex systems at regional spatial scales (less than 100 km). Here, we apply a seascape genetics approach to a peripheral population of the broadcast-spawning coral Acropora spicifera across the Houtman Abrolhos Islands, a high-latitude complex coral reef system off the central coast of Western Australia. We coupled population genetic data from a panel of microsatellite DNA markers with a biophysical dispersal model to test whether oceanographic processes could explain patterns of genetic divergence. We identified significant variation in allele frequencies over distances of less than 10 km, with significant differentiation occurring between adjacent sites but not between the most geographically distant ones. Recruitment probabilities between sites based on simulated larval dispersal were projected into a measure of resistance to connectivity that was significantly correlated with patterns of genetic divergence, demonstrating that patterns of spatial genetic structure are a function of restrictions to gene flow imposed by oceanographic currents. This study advances our understanding of the role of larval dispersal on the fine-scale genetic structure of coral populations across a complex island system and applies a methodological framework that can be tailored to suit a variety of marine organisms with a range of life-history characteristics.

Intra-genomic variation in symbiotic dinoflagellates: recent divergence or recombination between lineages?

BACKGROUND: The symbiosis between corals and the dinoflagellate alga Symbiodinium is essential for the development and survival of coral reefs. Yet this fragile association is highly vulnerable to environmental disturbance. A coral's ability to tolerate temperature stress depends on the fitness of its resident symbionts, whose thermal optima vary extensively between lineages. However, the in hospite population genetic structure of Symbiodinium is poorly understood and mostly based on analysis of bulk DNA extracted from thousands to millions of cells. Using quantitative single-cell PCR, we enumerated DNA polymorphisms in the symbionts of the reef-building coral Pocillopora damicornis, and applied a model selection approach to explore the potential for recombination between coexisting Symbiodinium populations. RESULTS: Two distinct Symbiodinium ITS2 sequences (denoted C100 and C109) were retrieved from all P. damicornis colonies analysed. However, the symbiont assemblage consisted of three distinct Symbiodinium populations: cells featuring pure arrays of ITS2 type C109, near-homogeneous cells of type C100 (with trace ITS2 copies of type C109), and those with co-dominant C100 and C109 ITS2 repeats. The symbiont consortia of some colonies consisted almost entirely of these putative C100 × C109 recombinants. CONCLUSIONS: Our results are consistent with the occurrence of sexual recombination between Symbiodinium types C100 and C109. While the multiple-copy nature of the ITS2 dictates that the observed pattern of intra-genomic co-dominance may be a result of incomplete concerted evolution of intra-genomic polymorphisms, this is a less likely explanation given the occurrence of homogeneous cells of the C109 type. Conclusive evidence for inter-lineage recombination and introgression in this genus will require either direct observational evidence or a single-cell genotyping approach targeting multiple, single-copy loci.