Increased scalability and sequencing quality of an epigenetic age prediction assay.

Epigenetic ageing in a human context, has been used to better understand the relationship between age and factors such as lifestyle and genetics. In an ecological setting, it has been used to predict the age of individual animals for wildlife management. Despite the importance of epigenetic ageing in a range of research fields, the assays to measure epigenetic ageing are either expensive on a large scale or complex. In this study, we aimed to improve the efficiency and sequencing quality of an existing epigenetic ageing assay for the Australian Lungfish (Neoceratodus forsteri). We used an enzyme-based alternative to bisulfite conversion to reduce DNA fragmentation and evaluated its performance relative to bisulfite conversion. We found the sequencing quality to be 12% higher with the enzymatic alternative compared to bisulfite treatment (p-value < 0.01). This new enzymatic based approach, although currently double the cost of bisulfite treatment can increases the throughput and sequencing quality. We envisage this assay setup being adopted increasingly as the scope and scale of epigenetic ageing research continues to grow.

The genetic signature left by the range expansion of red foxes in Australia is detectable after more than 80 years of population stability.

Reconstructing biological invasions from historical sources can provide insights into how they occur but are difficult to do when invasions are poorly documented. Genetic signatures left by invaders can also offer insights into invasion routes, points of origin and general biology but often present conclusions that are contradictory to expectations. Here, we test the ability of continental-wide microsatellite genotype data from 29 loci and 3122 samples to reconstruct the well-documented invasion of red foxes Vulpes vulpes from the United Kingdom into Australia over 150 years ago, an invasion that has led to the extinction of many native species. Our analysis reveals several key signals of invasion evident in Australian foxes. They display lower levels of diversity than foxes sampled from the UK, exhibit clines in diversity from the point of introduction (south-east Australia) to the edge of their range, and show strong evidence of allele surfing in westerly and north-easterly directions. These characteristics are consistent with a single point of origin followed by rapid expansion in westerly and north-easterly directions as suggested by historical records. We also find little genetic structure in foxes across Australia with only the vast Nullarbor Plains and Great Victoria Desert region presenting a detectable barrier to their dispersal. As such, no mainland region within the current range of foxes can be considered genetically isolated and therefore appropriate for localised eradication efforts. Overall, our analyses demonstrate the ability of comprehensive population genetic studies to reconstruct invasion histories even after more than 80 years since colonisation was stabilised.

Calibrating epigenetic clocks with training data error.

Animal age data are valuable for management of wildlife populations. Yet, for most species, there is no practical method for determining the age of unknown individuals. However, epigenetic clocks, a molecular-based method, are capable of age prediction by sampling specific tissue types and measuring DNA methylation levels at specific loci. Developing an epigenetic clock requires a large number of samples from animals of known ages. For most species, there are no individuals whose exact ages are known, making epigenetic clock calibration inaccurate or impossible. For many epigenetic clocks, calibration samples with inaccurate age estimates introduce a degree of error to epigenetic clock calibration. In this study, we investigated how much error in the training data set of an epigenetic clock can be tolerated before it resulted in an unacceptable increase in error for age prediction. Using four publicly available data sets, we artificially increased the training data age error by iterations of 1% and then tested the model against an independent set of known ages. A small effect size increase (Cohen's d >0.2) was detected when the error in age was higher than 22%. The effect size increased linearly with age error. This threshold was independent of sample size. Downstream applications for age data may have a more important role in deciding how much error can be tolerated for age prediction. If highly precise age estimates are required, then it may be futile to embark on the development of an epigenetic clock when there is no accurately aged calibration population to work with. However, for other problems, such as determining the relative age order of pairs of individuals, a lower-quality calibration data set may be adequate.

Ocean connectivity and habitat characteristics predict population genetic structure of seagrass in an extreme tropical setting.

Understanding patterns of gene flow and processes driving genetic differentiation is important for a broad range of conservation practices. In marine organisms, genetic differentiation among populations is influenced by a range of spatial, oceanographic, and environmental factors that are attributed to the seascape. The relative influences of these factors may vary in different locations and can be measured using seascape genetic approaches. Here, we applied a seascape genetic approach to populations of the seagrass, Thalassia hemprichii, at a fine spatial scale (~80 km) in the Kimberley coast, western Australia, a complex seascape with strong, multidirectional currents greatly influenced by extreme tidal ranges (up to 11 m, the world's largest tropical tides). We incorporated genetic data from a panel of 16 microsatellite markers, overwater distance, oceanographic data derived from predicted passive dispersal on a 2 km-resolution hydrodynamic model, and habitat characteristics from each meadow sampled. We detected significant spatial genetic structure and asymmetric gene flow, in which meadows 12-14 km apart were less connected than ones 30-50 km apart. This pattern was explained by oceanographic connectivity and differences in habitat characteristics, suggesting a combined scenario of dispersal limitation and facilitation by ocean current with local adaptation. Our findings add to the growing evidence for the key role of seascape attributes in driving spatial patterns of gene flow. Despite the potential for long-distance dispersal, there was significant genetic structuring over small spatial scales implicating dispersal and recruitment bottlenecks and highlighting the importance of implementing local-scale conservation and management measures.

Fish species lifespan prediction from promoter cytosine-phosphate-guanine density.

Lifespan is a key attribute of a species' life cycle and varies extensively among major lineages of animals. In fish, lifespan varies by several orders of magnitude, with reported values ranging from less than 1 year to approximately 400 years. Lifespan information is particularly useful for species management, as it can be used to estimate invasion potential, extinction risk and sustainable harvest rates. Despite its utility, lifespan is unknown for most fish species. This is due to the difficulties associated with accurately identifying the oldest individual(s) of a given species, and/or deriving lifespan estimates that are representative for an entire species. Recently it has been shown that CpG density in gene promoter regions can be used to predict lifespan in mammals and other vertebrates, with variable accuracy across taxa. To improve accuracy of lifespan prediction in a non-mammalian vertebrate group, here we develop a fish-specific genomic lifespan predictor. Our new model includes more than eight times the number of fish species included in the previous vertebrate model (n = 442) and uses fish-specific gene promoters as reference sequences. The model predicts fish lifespan from genomic CpG density alone (measured as CpG observed/expected ratio), explaining 64% of the variance between known and predicted lifespans. The predictions are highly robust to variation in genome quality and are applicable to all classes of fish; a taxonomically diverse and speciose group. The results demonstrate the value of promoter CpG density as a universal predictor of fish lifespan that can applied where empirical data are unavailable, or impracticable to obtain.

Comparison of materials for rapid passive collection of environmental DNA.

Passive collection is an emerging sampling method for environmental DNA (eDNA) in aquatic systems. Passive eDNA collection is inexpensive and efficient, and requires minimal equipment, making it suited to high-density sampling and remote deployment. Here, we compare the effectiveness of nine membrane materials for passively collecting fish eDNA from a 3-million-litre marine mesocosm. We submerged materials (cellulose, cellulose with 1% and 3% chitosan, cellulose overlayed with electrospun nanofibres and 1% chitosan, cotton fibres, hemp fibres, and sponge with either zeolite or active carbon) for intervals between 5 and 1080 min. We show that for most materials, with as little as 5 min of submersion, mitochondrial fish eDNA measured with qPCR, and fish species richness measured with metabarcoding, was comparable to that collected by conventional filtering. Furthermore, PCR template DNA concentrations and species richness were generally not improved significantly by longer submersion. Species richness detected for all materials ranged between 11 and 37 species, with a median of 27, which was comparable to the range for filtered eDNA (19-32). Using scanning electron microscopy, we visualized biological matter adhering to the surface of materials, rather than entrapped, with images also revealing a diversity in size and structure of putative eDNA particles. eDNA can be collected rapidly from seawater with a passive approach and using a variety of materials. This will suit cost- and time-sensitive biological surveys, and where access to equipment is limited.

Age prediction of green turtles with an epigenetic clock.

Age is a fundamental life history attribute that is used to understand the dynamics of wild animal populations. Unfortunately, most animals do not have a practical or nonlethal method to determine age. This makes it difficult for wildlife managers to carry out population assessments, particularly for elusive and long-lived fauna such as marine turtles. In this study, we present an epigenetic clock that predicts the age of marine turtles from skin biopsies. The model was developed and validated using DNA from known-age green turtles (Chelonia mydas) from two captive populations, and mark-recapture wild turtles with known time intervals between captures. Our method, based on DNA methylation levels at 18 CpG sites, was highly accurate with a median absolute error of 2.1 years (4.7% of maximum age in data set). This is the first epigenetic clock developed for a reptile and illustrates their broad applicability across a broad variety of vertebrate species. It has the potential to transform marine turtle management through a nonlethal and inexpensive method to provide key life history information.

Influence of offshore oil and gas structures on seascape ecological connectivity.

Offshore platforms, subsea pipelines, wells and related fixed structures supporting the oil and gas (O&G) industry are prevalent in oceans across the globe, with many approaching the end of their operational life and requiring decommissioning. Although structures can possess high ecological diversity and productivity, information on how they interact with broader ecological processes remains unclear. Here, we review the current state of knowledge on the role of O&G infrastructure in maintaining, altering or enhancing ecological connectivity with natural marine habitats. There is a paucity of studies on the subject with only 33 papers specifically targeting connectivity and O&G structures, although other studies provide important related information. Evidence for O&G structures facilitating vertical and horizontal seascape connectivity exists for larvae and mobile adult invertebrates, fish and megafauna; including threatened and commercially important species. The degree to which these structures represent a beneficial or detrimental net impact remains unclear, is complex and ultimately needs more research to determine the extent to which natural connectivity networks are conserved, enhanced or disrupted. We discuss the potential impacts of different decommissioning approaches on seascape connectivity and identify, through expert elicitation, critical knowledge gaps that, if addressed, may further inform decision making for the life cycle of O&G infrastructure, with relevance for other industries (e.g. renewables). The most highly ranked critical knowledge gap was a need to understand how O&G structures modify and influence the movement patterns of mobile species and dispersal stages of sessile marine species. Understanding how different decommissioning options affect species survival and movement was also highly ranked, as was understanding the extent to which O&G structures contribute to extending species distributions by providing rest stops, foraging habitat, and stepping stones. These questions could be addressed with further dedicated studies of animal movement in relation to structures using telemetry, molecular techniques and movement models. Our review and these priority questions provide a roadmap for advancing research needed to support evidence-based decision making for decommissioning O&G infrastructure.

Distinct Endophytic Bacterial Communities Inhabiting Seagrass Seeds.

Seagrasses are marine angiosperms that can live completely or partially submerged in water and perform a variety of significant ecosystem services. Like terrestrial angiosperms, seagrasses can reproduce sexually and, the pollinated female flower develop into fruits and seeds, which represent a critical stage in the life of plants. Seed microbiomes include endophytic microorganisms that in terrestrial plants can affect seed germination and seedling health through phytohormone production, enhanced nutrient availability and defence against pathogens. However, the characteristics and origins of the seagrass seed microbiomes is unknown. Here, we examined the endophytic bacterial community of six microenvironments (flowers, fruits, and seeds, together with leaves, roots, and rhizospheric sediment) of the seagrass Halophila ovalis collected from the Swan Estuary, in southwestern Australia. An amplicon sequencing approach (16S rRNA) was used to characterize the diversity and composition of H. ovalis bacterial microbiomes and identify core microbiome bacteria that were conserved across microenvironments. Distinct communities of bacteria were observed within specific seagrass microenvironments, including the reproductive tissues (flowers, fruits, and seeds). In particular, bacteria previously associated with plant growth promoting characteristics were mainly found within reproductive tissues. Seagrass seed-borne bacteria that exhibit growth promoting traits, the ability to fix nitrogen and anti-pathogenic potential activity, may play a pivotal role in seed survival, as is common for terrestrial plants. We present the endophytic community of the seagrass seeds as foundation for the identification of potential beneficial bacteria and their selection in order to improve seagrass restoration.

Optimal sample size for calibrating DNA methylation age estimators.

Age is a fundamental parameter in wildlife management as it is used to determine the risk of extinction, manage invasive species, and regulate sustainable harvest. In a broad variety of vertebrates species, age can be determined by measuring DNA methylation. Animals with known ages are initially required during development, calibration, and validation of these epigenetic clocks. However, wild animals with known ages are frequently difficult to obtain. Here, we perform Monte-Carlo simulations to determine the optimal sample size required to create an accurate calibration model for age estimation by elastic net regression modelling of cytosine-phosphate-guanine methylation data. Our results suggest a minimum calibration population size of 70, but ideally 134 individuals or more for accurate and precise models. We also provide estimates to the extent a model can be extrapolated beyond a distribution of ages that was used during calibration. The findings can assist researchers to better design age estimation models and decide if their model is adequate for determining key population attributes.

Passive eDNA collection enhances aquatic biodiversity analysis.

Environmental DNA (eDNA) metabarcoding is a sensitive and widely used approach for species detection and biodiversity assessment. The most common eDNA collection method in aquatic systems is actively filtering water through a membrane, which is time consuming and requires specialized equipment. Ecological studies investigating species abundance or distribution often require more samples than can be practically collected with current filtration methods. Here we demonstrate how eDNA can be passively collected in both tropical and temperate marine systems by directly submerging filter membranes (positively charged nylon and non-charged cellulose ester) in the water column. Using a universal fish metabarcoding assay, we show that passive eDNA collection can detect fish as effectively as active eDNA filtration methods in temperate systems and can also provide similar estimates of total fish biodiversity. Furthermore, passive eDNA collection enables greater levels of biological sampling, which increases the range of ecological questions that eDNA metabarcoding can address.

A DNA methylation age predictor for zebrafish.

Changes in DNA methylation at specific CpG sites have been used to build predictive models to estimate animal age, predominantly in mammals. Little testing for this effect has been conducted in other vertebrate groups, such as bony fish, the largest vertebrate class. The development of most age-predictive models has relied on a genome-wide sequencing method to obtain a DNA methylation level, which makes it costly to deploy as an assay to estimate age in many samples. Here, we have generated a reduced representation bisulfite sequencing data set of caudal fin tissue from a model fish species, zebrafish (Danio rerio), aged from 11.9-60.1 weeks. We identified changes in methylation at specific CpG sites that correlated strongly with increasing age. Using an optimised unique set of 26 CpG sites we developed a multiplex PCR assay that predicts age with an average median absolute error rate of 3.2 weeks in zebrafish between 10.9-78.1 weeks of age. We also demonstrate the use of multiplex PCR as an efficient quantitative approach to measure DNA methylation for the use of age estimation. This study highlights the potential further use of DNA methylation as an age estimation method in non-mammalian vertebrate species.

Extreme seascape drives local recruitment and genetic divergence in brooding and spawning corals in remote north-west Australia.

Management strategies designed to conserve coral reefs threatened by climate change need to incorporate knowledge of the spatial distribution of inter- and intra-specific genetic diversity. We characterized patterns of genetic diversity and connectivity using single nucleotide polymorphisms (SNPs) in two reef-building corals to explore the eco-evolutionary processes that sustain populations in north-west Australia. Our sampling focused on the unique reefs of the Kimberley; we collected the broadcast spawning coral Acropora aspera (n = 534) and the brooding coral Isopora brueggemanni (n = 612) across inter-archipelago (tens to hundreds of kilometres), inter-reef (kilometres to tens of kilometres) and within-reef (tens of metres to a few kilometres) scales. Initial analysis of A. aspera identified four highly divergent lineages that were co-occurring but morphologically similar. Subsequent population analyses focused on the most abundant and widespread lineage, Acropora asp-c. Although the overall level of geographic subdivision was greater in the brooder than in the spawner, fundamental similarities in patterns of genetic structure were evident. Most notably, limits to gene flow were observed at scales <35 kilometres. Further, we observed four discrete clusters and a semi-permeable barrier to dispersal that were geographically consistent between species. Finally, sites experiencing bigger tides were more connected to the metapopulation and had greater gene diversity than those experiencing smaller tides. Our data indicate that the inshore reefs of the Kimberley are genetically isolated from neighbouring oceanic bioregions, but occasional dispersal between inshore archipelagos is important for the redistribution of evolutionarily important genetic diversity. Additionally, these results suggest that networks of marine reserves that effectively protect reefs from local pressures should be spaced within a few tens of kilometres to conserve the existing patterns of demographic and genetic connectivity.

Lifespan estimation in marine turtles using genomic promoter CpG density.

Maximum lifespan for most animal species is difficult to define. This is challenging for wildlife management as it is critical for estimating important aspects of population biology such as mortality rate, population viability, and period of reproductive potential. Recently, it has been shown cytosine-phosphate-guanine (CpG) density is predictive of maximum lifespan in vertebrates. This has made it possible to predict lifespan in long-lived species, which are generally the most intractable. In this study, we use gene promoter CpG density to predict the lifespan of five marine turtle species. Marine turtles are a particularly difficult group for lifespan estimation because of their migratory behaviour, longevity and high juvenile mortality rates, which all restrict individual tracking over their lifespan. Sanger sequencing was used to determine the CpG density in selected promoters. We predicted the lifespans for marine turtle species ranged from 50.4 years (flatback turtle, Natator depressus) to 90.4 years (leatherback turtle, Dermochelys coriacea). These lifespan predictions have broad applications in marine turtle research such as better understanding life cycles and determining population viability.

Setting priorities for conservation at the interface between ocean circulation, connectivity, and population dynamics.

Population persistence in the marine environment is driven by patterns of ocean circulation, larval dispersal, ecological interactions, and demographic rates. For habitat-forming organisms in particular, understanding the relationship between larval connectivity and meta-population dynamics aids in planning for marine spatial management. Here, we estimate networks of connectivity between fringing coral reefs in the northwest shelf of Australia by combining a particle tracking model based on shelf circulation with models of subpopulation dynamics of individual reefs. Coral cover data were used as a proxy for overall habitat quality, which can change as a result of natural processes, human-driven impacts, and management initiatives. We obtain three major results of conservation significance. First, the dynamics of the ecological network result from the interplay between network connectivity and ecological processes on individual reefs. The maximum coral cover a zone can sustain imposes a significant nonlinearity on the role an individual reef plays within the dynamics of the network, and thus on the impact of conservation interventions on specific reefs. Second, the role of an individual reef within these network dynamics changes considerably depending on the overall state of the system: a reef's role in sustaining the system's state can be different from the same reef's role in helping the system recover following major disturbance. Third, patterns of network connectivity change significantly as a function of yearly shelf circulation trends, and nonlinearity in network dynamics make mean connectivity a poor representation of yearly variations. From a management perspective, the priority list of reefs that are targets for management interventions depends crucially on what type of stressors (system-wide vs. localized) need addressing. This choice also depends not only on the ultimate purpose of management, but also on future oceanographic, climate change, and development scenarios that will determine the network connectivity and habitat quality.

A genomic predictor of lifespan in vertebrates.

Biological ageing and its mechanistic underpinnings are of immense biomedical and ecological significance. Ageing involves the decline of diverse biological functions and places a limit on a species' maximum lifespan. Ageing is associated with epigenetic changes involving DNA methylation. Furthermore, an analysis of mammals showed that the density of CpG sites in gene promoters, which are targets for DNA methylation, is correlated with lifespan. Using 252 whole genomes and databases of animal age and promotor sequences, we show a pattern across vertebrates. We also derive a predictive lifespan clock based on CpG density in a selected set of promoters. The lifespan clock accurately predicts maximum lifespan in vertebrates (R2 = 0.76) from the density of CpG sites within only 42 selected promoters. Our lifespan clock provides a wholly new method for accurately estimating lifespan using genome sequences alone and enables estimation of this challenging parameter for both poorly understood and extinct species.

DNA metabarcoding assays reveal a diverse prey assemblage for Mobula rays in the Bohol Sea, Philippines.

Diet studies provide base understanding of trophic structure and are a valuable initial step for many fields of marine ecology, including conservation and fisheries biology. Considerable complexity in marine trophic structure can exist due to the presence of highly mobile species with long life spans. Mobula rays are highly mobile, large, planktivorous elasmobranchs that are frequently caught either directly or as bycatch in fisheries, which, combined with their conservative life history strategy, makes their populations susceptible to decline in intensely fished regions. Effective management of these iconic and vulnerable species requires an understanding of the diets that sustain them, which can be difficult to determine using conventional sampling methods. We use three DNA metabarcode assays to identify 44 distinct taxa from the stomachs (n = 101) of four sympatric Mobula ray species (Mobula birostris, Mobula tarapacana, Mobula japanica, and Mobula thurstoni) caught over 3 years (2013-2015) in a direct fishery off Bohol in the Philippines. The diversity and incidence of bony fishes observed in ray diets were unprecedented. Nevertheless, rays showed dietary overlap, with krill (Euphausia) dominating their diet. Our results provide a more detailed assessment of sympatric ray diets than was previously described and reveal the complexity that can exist in food webs at critical foraging habitats.

Marine environmental DNA biomonitoring reveals seasonal patterns in biodiversity and identifies ecosystem responses to anomalous climatic events.

Marine ecosystems are changing rapidly as the oceans warm and become more acidic. The physical factors and the changes to ocean chemistry that they drive can all be measured with great precision. Changes in the biological composition of communities in different ocean regions are far more challenging to measure because most biological monitoring methods focus on a limited taxonomic or size range. Environmental DNA (eDNA) analysis has the potential to solve this problem in biological oceanography, as it is capable of identifying a huge phylogenetic range of organisms to species level. Here we develop and apply a novel multi-gene molecular toolkit to eDNA isolated from bulk plankton samples collected over a five-year period from a single site. This temporal scale and level of detail is unprecedented in eDNA studies. We identified consistent seasonal assemblages of zooplankton species, which demonstrates the ability of our toolkit to audit community composition. We were also able to detect clear departures from the regular seasonal patterns that occurred during an extreme marine heatwave. The integration of eDNA analyses with existing biotic and abiotic surveys delivers a powerful new long-term approach to monitoring the health of our world's oceans in the context of a rapidly changing climate.