Deep Ocean Environments Research Articles

Influencing Factors and Model of Shallow Gas Enrichment in the Quaternary Sediments of the Qiongdongnan Basin, South China Sea

Investigating the primary influencing factors that regulate the enrichment of shallow gas not only deepens our understanding of the rules governing shallow gas enrichment in deep-ocean environments but also has the potential to enhance the success rate of locating shallow gas reservoirs. Recent drilling activities in the LS36 gas field located in the central Qiongdongnan Basin have revealed a substantial shallow gas reserve within the sediments of the Quaternary Ledong Formation, marking it as the first shallow gas reservoir discovered in the offshore region of China with confirmed natural gas geological reserves surpassing 100 billion cubic meters. However, the formation mechanism and influencing factors of shallow gas enrichment remain elusive due to the limited availability of 3D seismic and well data. This study employs seismic interpretation and digital simulation to decipher the dynamics of shallow gas accumulation and utilizes the carbon isotope composition of methane to ascertain the origin of the shallow gas. Our results show that the shallow gas is primarily concentrated within a large-scale submarine fan, covering a distribution region of up to 2800 km2, situated in the deep-sea plain. The δ13 C1 methane carbon isotope data ranges from −69.7‰ to −45.2‰ and all δ13 C2 values are above −28‰, suggesting that the shallow gas within the Ledong Formation is derived from a mix of biogenic gas produced in shallow strata and thermogenic gas generated in deeper source rocks. The results of gas sources, seismic profiles, and digital simulations suggest that thermogenic gas originating from the Lingshui and Beijiao sags was transported to the Quaternary submarine fan via a complex system that includes faults, gas chimneys, and channel sands. The mass-transported deposits (MTDs) in the upper reaches of the submarine fan have effectively acted as a seal, preventing the escape of shallow gas from the fan. Therefore, the factors contributing to the enrichment of shallow gas in the Qiongdongnan Basin include the presence of favorable submarine fan reservoirs, the availability of two distinct gas sources, the effective sealing of MTDs, and the presence of two efficient transport pathways. A conceptual model for the accumulation of shallow gas is developed, illuminating the complex formation–migration–accumulation process. This study underscores the importance of aligning multiple influencing factors in the process of shallow gas accumulation, and the suggested accumulation model may be pertinent to shallow gas exploration in other marginal sea basins.

Segmented propagation model for the paths and travel times of surface duct leaky signals.

In the deep ocean environment with a surface duct, sound propagating within the duct leaks into the geometric shadow zone below it. However, the propagation paths and time of these leaky parts have not been fully characterized. This paper investigates the mechanism of surface duct leaky (SDL) signals based on normal mode theory. It reveals that SDL signals are caused by specific modes with grazing angles close to zero at the bottom of the surface duct. Combining the theory of diffracted sound rays, the study proposes a Segmented Propagation model (SPM) for SDL signals. The propagation paths of SDL signals are divided into three segments: S1, which extends from the source to the surface duct; S2, the segment propagating within the surface duct; and S3, the segment leading from the surface duct to the receiver. The proposed SPM describes the propagation mechanism of SDL signals and allows for precise calculation of their propagation time. Experimental data from the western Pacific are used to verify the SPM.

Deep-Learning-Based Source Localization Using a Local 3-D Acoustic Intensity Field in a Sound-Speed Mismatched Deep-Ocean Environment

Deep-Learning-Based Source Localization Using a Local 3-D Acoustic Intensity Field in a Sound-Speed Mismatched Deep-Ocean Environment

Impacts of Hydrostatic Pressure on Distributed Temperature-Sensing Optical Fibers for Extreme Ocean and Ice Environments

Optical fiber is increasingly used for both communication and distributed sensing of temperature and strain in environmental studies. In this work, we demonstrate the viability of unreinforced fiber tethers (bare fiber) for Raman-based distributed temperature sensing in deep ocean and deep ice environments. High-pressure testing of single-mode and multimode optical fiber showed little to no changes in light attenuation over pressures from atmospheric to 600 bars. Most importantly, the differential attenuation between Stokes and anti-Stokes frequencies, critical for the evaluation of distributed temperature sensing, was shown to be insignificantly affected by fluid pressures over the range of pressures tested for single-mode fiber, and only very slightly affected in multimode fiber. For multimode fiber deployments to ocean depths as great as 6000 m, the effect of pressure-dependent differential attenuation was shown to impact the estimated temperatures by only 0.15 °K. These new results indicate that bare fiber tethers, in addition to use for communication, can be used for distributed temperature or strain in fibers subjected to large depth (pressure) in varying environments such as deep oceans, glaciers and potentially the icy moons of Saturn and Jupiter.

Composite repair method for internally damaged cylinders subjected to external pressure

In deep ocean environment, it is feasible to effectively repair damaged cylinders by externally wrapping them with lightweight composite materials. An analytical method was developed to determine the optimal thickness and length for composite repairs of internally damaged cylinders under uniform external pressure. The repair layer thickness was evaluated based on the assumption of full thickness reduction, and the repair length was determined by axial shearing theory. Repairs were designed for three groups of damaged cylinders, and the repaired and damaged cylinders were compared with pristine cylinders. The finite-element method was used to validate the analytical method and investigate the failure modes of the cylinders. Furthermore, the analytical and numerical results were validated through experiments. The results indicate that the load-bearing capacity of damaged cylinders can be completely restored using the designed repair method. Moreover, at collapse, the instability locations of the repaired cylinders exist near an end instead of the middle region. The analytical, numerical, and experimental results are in favorable agreement. The research contributes to offering a robust solution to repairing internally damaged cylindrical structures, promising substantial advantages in terms of maintenance and extending the lifespan of these structures.

Foraging behaviour and ecology of transient killer whales within a deep submarine canyon system.

Transient killer whales have been documented hunting marine mammals across a variety of habitats. However, relatively little has been reported about their predatory behaviours near deep submarine canyons and oceanic environments. We used a long-term database of sightings and encounters with these predators in and around the Monterey Submarine Canyon, California to describe foraging behaviour, diet, seasonal occurrence, and habitat use patterns. Transient killer whales belonging to the outer coast subpopulation were observed within the study area 261 times from 2006-2021. Occurrences, behaviours, and group sizes all varied seasonally, with more encounters occurring in the spring as grey whales migrated northward from their breeding and calving lagoons in Mexico (March-May). Groups of killer whales foraged exclusively in open water, with individuals within the groups following the contours of the submarine canyon as they searched for prey. Focal follows revealed that killer whales spent 51% of their time searching for prey (26% of their time along the shelf-break and upper slope of the canyon, and 25% in open water). The remainder of their time was spent pursuing prey (10%), feeding (23%), travelling (9%), socializing (6%), and resting (1%). Prey species during 87 observed predation events included California sea lions, grey whale calves, northern elephant seals, minke whales, common dolphins, Pacific white-sided dolphins, Dall's porpoise, harbour porpoise, harbour seals, and sea birds. The calculated kill rates (based on 270 hours of observing 50 predation events) were 0.26 California sea lions per killer whale over 24 hours, 0.11 grey whale calves, and 0.15 for all remaining prey species combined. These behavioural observations provide insights into predator-prey interactions among apex predators over submarine canyons and deep pelagic environments.

The BBNJ Agreement: Through the Prism of Deep-Sea Vulnerable Marine Ecosystems

On 19 June 2023, member States of the United Nations adopted, by consensus, the Agreement under the United Nations Convention on the Law of the Sea on the Conservation and Sustainable Use of Marine Biological Diversity of Areas Beyond National Jurisdiction (BBNJ Agreement). The BBNJ Agreement seeks to address one of the greatest challenges of the 21st century—managing our deep ocean environment, including its ecosystems and resources, in a way that does not lead to its long-term decline and maintains its potential to meet the needs of present and future generations. This article assesses the BBNJ Agreement through an analysis of its application to deep-sea vulnerable marine ecosystems (VMEs). As VMEs represent a key component of marine biodiversity and are particularly susceptible to anthropogenic disturbance, they provide a useful medium for undertaking a legal analysis of the provisions, rules, and procedures of the recently adopted instrument.

Comparing eDNA metabarcoding and conventional pelagic netting to inform biodiversity monitoring in deep ocean environments

Abstract The performance of environmental DNA (eDNA) metabarcoding has rarely been evaluated against conventional sampling methods in deep ocean mesopelagic environments. We assessed the biodiversity patterns generated with eDNA and two co-located conventional methods, oblique midwater trawls and vertical multinets, to compare regional and sample-level diversity. We then assessed the concordance of ecological patterns across water column habitats and evaluated how DNA markers and the level of sampling effort influenced the inferred community. We found eDNA metabarcoding characterized regional diversity well, detecting more taxa while identifying similar ecological patterns as conventional samples. Within sampling locations, eDNA metabarcoding rarely detected taxa across more than one replicate. While more taxa were found in eDNA than oblique midwater trawls within sample stations, fewer were found compared to vertical multinets. Our simulations show greater eDNA sampling effort would improve concordance with conventional methods. We also observed that using taxonomic data from multiple markers generated ecological patterns most similar to those observed with conventional methods. Patterns observed with Exact Sequence Variants were more stable across markers suggesting they are more powerful for detecting change. eDNA metabarcoding is a valuable tool for identifying and monitoring biological hotspots but some methodological adjustments are recommended for deep ocean environments.

Serpentinization as the source of energy, electrons, organics, catalysts, nutrients and pH gradients for the origin of LUCA and life.

Serpentinization in hydrothermal vents is central to some autotrophic theories for the origin of life because it generates compartments, reductants, catalysts and gradients. During the process of serpentinization, water circulates through hydrothermal systems in the crust where it oxidizes Fe (II) in ultramafic minerals to generate Fe (III) minerals and H2. Molecular hydrogen can, in turn, serve as a freely diffusible source of electrons for the reduction of CO2 to organic compounds, provided that suitable catalysts are present. Using catalysts that are naturally synthesized in hydrothermal vents during serpentinization H2 reduces CO2 to formate, acetate, pyruvate, and methane. These compounds represent the backbone of microbial carbon and energy metabolism in acetogens and methanogens, strictly anaerobic chemolithoautotrophs that use the acetyl-CoA pathway of CO2 fixation and that inhabit serpentinizing environments today. Serpentinization generates reduced carbon, nitrogen and - as newer findings suggest - reduced phosphorous compounds that were likely conducive to the origins process. In addition, it gives rise to inorganic microcompartments and proton gradients of the right polarity and of sufficient magnitude to support chemiosmotic ATP synthesis by the rotor-stator ATP synthase. This would help to explain why the principle of chemiosmotic energy harnessing is more conserved (older) than the machinery to generate ion gradients via pumping coupled to exergonic chemical reactions, which in the case of acetogens and methanogens involve H2-dependent CO2 reduction. Serpentinizing systems exist in terrestrial and deep ocean environments. On the early Earth they were probably more abundant than today. There is evidence that serpentinization once occurred on Mars and is likely still occurring on Saturn's icy moon Enceladus, providing a perspective on serpentinization as a source of reductants, catalysts and chemical disequilibrium for life on other worlds.

Investigations of the acoustic-field autoproducts in three dimensions

Acoustic remote sensing tasks, such as remote unknown source localization, are commonly completed via signal processing schemes that are implemented in the bandwidth of the acoustic recordings. Interestingly, many such schemes can also be implemented at frequencies outside of the bandwidth of the recorded field—despite their absence from the recordings—by using the frequency-difference and frequency-sum autoproducts [Worthmann and Dowling, J. Acoust. Soc. Am., 141 (2017) 4579–4590]. Recent acoustic-field autoproduct studies have involved beamforming and source localization in shallow- and deep-ocean environments, and have addressed the impacts of refraction, shadow zones, rough surface scattering, and noise. These previous efforts primarily explored the properties of the autoproducts when the sound field primarily varies in two spatial dimensions. This presentation provides the results of a study of the autoproducts formed from the simple but fully three-dimensional acoustic fields arising from an isolated source placed in a uniform-sound-speed environment bounded by reflecting surfaces that are not parallel. This study indicates the extent to which the autoproduct properties associated with two dimensional acoustic-field variations extend to three dimensional acoustic-field variations. The results from theory and simulations are presented, and from laboratory water-tank measurements if time allows.

Combining uncertain machine learning predictions and numerical simulation results for the extreme value analysis of cyclone-induced wave heights – Application in Guadeloupe

Assessing return level RL (with return period typically ranging from 100 to 500 years) for extreme waves in cyclone-prone regions is often made problematic by the lack of sufficient representative samples to properly fit the extreme value probability distributions. Motivated by the Guadeloupe context (French West Indies), we address this problem when the wave numerical model is too expensive to be run a large number of times. A possible option is to use a machine learning (ML) model in place of the long-running numerical mode. By construction, ML predictions are however uncertain, because they are learned from a limited number of pre-calculated wave simulation results. We propose to account for this type of error along the inference of the parameters of the extreme value probability distribution within an Approximate Bayesian Computation (ABC) scheme. Using a subset of the database of numerically computed HS for Guadeloupe (for approximately 700 synthetic cyclones representative of 1000 years of cyclonic activity), we train a random forest (RF) regression model to relate the cyclone characteristics (radius, atmospheric pressure, distance to cyclone eye) to HS. The quantile RF model is then used to model the prediction error within the ABC scheme. By comparison to the 100-year and 500-year RL reference solutions (calculated using the entire database), we show that the ML-based approach achieves low bias and high reliability of the RL estimates at locations both along Guadeloupe coasts and in deep ocean environments for low sample size (down to ∼70). Provided that the prediction error is low-to-moderate, integrating this type of uncertainty improves the performance of the ML-based approach, and decreases the computational burden of the return level estimation.

Hydrodynamic experiment of submerged floating tunnel under regular wave and current actions during construction period

Submerged floating tunnel (SFT) is an innovative cable-supported structural system for crossing deep and long-distance ocean environments. In the complex ocean environment, the construction of SFT needs to consider wave and current forces. Specific construction measures and control also require in-depth study and understanding of the dynamic response of SFT under such environmental loads. In this study, the dynamic response of SFT and cable forces under the action of waves alone and wave-current interactions are investigated by using a large wave-current basin. A total of 138 regular wave and wave-current cases were conducted during the experiments, and the influence of waves and wave-current interactions on the dynamic response of SFT and cable forces are discussed in detail by combining experimental data with corresponding analysis. Results show that the wave height, current velocity, and ratio of wavelength to structure size are important factors affecting the dynamic response of SFT and cable forces. The multi-anchor cable arrangement used in the present experimental tests distribute cable force more effectively and reduce the potential safety hazard caused by cable breakage. This study can provide a useful reference for the construction and control of the single SFT segment under construction in a complex ocean environment, especially under the interaction of waves and currents.

The role of hydrodynamics for the spatial distribution of high-temperature hydrothermal vent-endemic fauna in the deep ocean environment

Active hydrothermal vents provide the surrounding submarine environment with substantial amounts of matter and energy, thus serving as important habitats for diverse megabenthic communities in the deep ocean and constituting a unique, highly productive chemosynthetic ecosystem on Earth. Vent-endemic biological communities gather near the venting site and are usually not found beyond a distance of the order of 100 m from the vent. This is surprising because one would actually expect matter ejected from high-temperature vents, which generate highly turbulent buoyancy plumes, to be suspended and carried far away by the plume flows and deep-sea currents. Here, we study this problem from a fluid dynamics perspective by simulating the vent hydrodynamics using a numerical model that couples the plume flow with induced matter and energy transport. We find that both low- and high-temperature vents deposit most vent matter relatively close to the plume. In particular, the tendency of turbulent buoyancy plumes to carry matter far away is strongly counteracted by generated entrainment flows back into the plume stem. The deposition ranges of organic and inorganic hydrothermal particles obtained from the simulations for various natural high-temperature vents are consistent with the observed maximum spatial extent of biological communities, evidencing that plume hydrodynamics exercises strong control over the spatial distribution of vent-endemic fauna. While other factors affecting the spatial distribution of vent-endemic fauna, such as geology and geochemistry, are site-specific, the main physical features of plume hydrodynamics unraveled in this study are largely site-unspecific and therefore universal across vent sites on Earth.

Micro-Raman mapping of critical metals (Li, Co, Ni) in a rhythmically laminated deep-ocean ferromanganese deposit

Deep-ocean ferromanganese deposits represent one of the most important strategic reservoirs for rare and critical metals. In particular, Mn-oxyhydroxides, such as asbolane and lithiophorite, concentrate large amounts of Li, Ni, and Co into polymetallic nodules and crusts. However, because of their poor crystallinity and the presence of finely intermixed additional phases, these minerals cannot be unambiguously identified by standard X-ray powder diffraction methods. In addition, Li cannot be routinely detected by standard X-ray spectroscopy techniques.In this work we show how the spatial distribution of asbolane (the Ni-Co-rich Mn-oxide) and lithiophorite (the Li-rich Mn-oxide) across strongly inhomogeneous ferromanganese mineralizations can be investigated at high-resolution (∼ 1 μm) via fast and easily accessible Raman scattering measurements. Because of the strong selectivity of these minerals to the incorporation of critical metals, the obtained micro-Raman maps provide also an indirect map of the Co and Ni vs. Li distribution in the crusts. The described results thus show that our spectroscopic approach could represent an efficient and valuable in situ tool for mineral chemistry and resource evaluation of these elements in ferromanganese deposits from deep-ocean environments. This research opens a new frontier for the application of Raman spectroscopy in ore prospecting for critical minerals and metals.

A rotating piezoelectric-electromagnetic hybrid harvester for water flow energy

Water contains a great amount of potential energy, out of this, an energy harvesting and converting solution from water is very promising for green renewable energy. In this paper, a hybrid energy harvester (HEH) is proposed, which has a combination of piezoelectric and electromagnetic mechanisms for energy conversion. Two piezoelectric energy harvesters (PEH) and two electromagnetic energy harvesters (EMH) can work synchronously to achieve high-performance output. The structural variables of the prototype are studied and analyzed through the self-designed experimental test system. When the prototype is assembled with optimal structural parameters, the maximum output voltages of a single PEH and EMH are 56.24 V and 1.016 V. The maximum output power of HEH is 22.78mW, which is 21 % higher than that of PEH and 253011 % higher than that of EMH. Subsequent underwater tests and a series of application tests were performed to verify the performance of the HEH, showing that it can easily power some commercially available sensors and small electronic devices. Experimental results show that HEH is promising in wireless self-powering and self-sensing. It provides a new power supply scheme for laying power supply without grid for monitoring equipment in deep sea and ocean environment.

Complete genome sequence of Bacillus cereus 2-6A, a marine exopolysaccharide-producing bacterium isolated from deep-sea hydrothermal sediment of the Pacific Ocean

Bacillus cereus 2-6A, was isolated from the sediments in the hydrothermal area of the Pacific Ocean with a water depth of 2628m. In this study, we report the whole genome sequence of strain 2-6A and analyze that to understand its metabolic capacities and biosynthesis potential of natural products. The genome of strain 2-6A consists of a circular chromosome of 5,191,018bp with a GC content of 35.3mol% and two plasmids of 234,719bp and 411,441bp, respectively. Genomic data mining reveals that strain 2-6A has several gene clusters involved in exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs) production and complex polysaccharides degradation. It also possesses a variety of genes for allowing strain 2-6A to cope with osmotic stress, oxidative stress, heat shock, cold shock and heavy metal stress, which could play a vital role in the adaptability of the strain to hydrothermal environments. Gene clusters for secondary metabolite production, such as lasso peptide and siderophore, are also predicted. Therefore, genome sequencing and data mining provide insights into the molecular mechanisms of Bacillus in adapting to hydrothermal deep ocean environments and can facilitate further experimental exploration.

Spatial Changes in Gas Transport and Sediment Stiffness Influenced by Regional Stress: Observations From Piezometer Data Along Vestnesa Ridge, Eastern Fram Strait

AbstractGas transport through sediments to the seabed and seepage occurs via advection through pores, faults, and fractures, and as solubility driven gas diffusion. The pore pressure gradient is a key factor in these processes. Yet, in situ measurements for quantitative studies of fluid dynamics and sediment deformation in deep ocean environments remain scarce. In this study, we integrate piezometer data, geotechnical tests, and sediment core analyses to study the pressure regime that controls gas transport along the Vestnesa Ridge in the eastern Fram Strait. The data show a progressive westward decrease in induced pore pressure (i.e., from c. 180 to c. 50 kPa) upon piezometer penetration and undrained shear strength of the sediments, interpreted as a decrease in sediment stiffness. In addition, the data suggest that the upper c. 6 m of sediments may be mechanically damaged due to variations in gas diffusion rates and exsolution. Background pore pressures are mostly at hydrostatic conditions, but localized excess pore pressures (i.e., up to 10 kPa) exist and point toward external controls. When analyzed in conjunction with observations from geophysical data and sediment core analyses, the pore pressure data suggest a spatial change from an advection dominated to a diffusion dominated fluid flow system, influenced by the behavior of sedimentary faults. Understanding gas transport mechanisms and their effect on fine‐grained sediments of deep ocean settings is critical for constraining gas hydrate inventories, seepage phenomena and sub‐seabed sediment deformations and instabilities.

Inline small-angle X-ray scattering-coupled chromatography under extreme hydrostatic pressure.

As continuing discoveries highlight the surprising abundance and resilience of deep ocean and subsurface microbial life, the effects of extreme hydrostatic pressure on biological structure and function have attracted renewed interest. Biological small-angle X-ray scattering (BioSAXS) is a widely used method of obtaining structural information from biomolecules in solution under a wide range of solution conditions. Due to its ability to reduce radiation damage, remove aggregates, and separate monodisperse components from complex mixtures, size-exclusion chromatography-coupled SAXS (SEC-SAXS) is now the dominant form of BioSAXS at many synchrotron beamlines. While BioSAXS can currently be performed with some difficulty under pressure with non-flowing samples, it has not been clear how, or even if, continuously flowing SEC-SAXS, with its fragile media-packed columns, might work in an extreme high-pressure environment. Here we show, for the first time, that reproducible chromatographic separations coupled directly to high-pressure BioSAXS can be achieved at pressures up to at least 100 MPa and that pressure-induced changes in folding and oligomeric state and other properties can be observed. The apparatus described here functions at a range of temperatures (0°C-50°C), expanding opportunities for understanding biomolecular rules of life in deep ocean and subsurface environments.

Experimental Investigation of the Dynamic Behavior of Submerged Floating Tunnels under Regular Wave Conditions

Submerged floating tunnels (SFTs) are an innovative traffic structure for transportation in deep and long-distance ocean environments. SFTs have the risk of being subjected to wave action in the complex ocean environment. Therefore, in order to ensure the safety and stability of SFTs during their service, the dynamic behavior of an SFT subjected to the action of waves is experimentally investigated in this study. Based on the wave–current flume, a physical scale-model experiment of an SFT for studying the dynamic behavior of an SFT subjected to regular waves interactions is deeply and thoroughly explored. The results show that the wave outputs from the wave-making system are verified to be reliable. The influence of major load parameters on the dynamic behavior of the SFT are deeply and thoroughly explored. The effects of wave height and wave period on acceleration, wave pressure, anchor cable force and displacement in an SFT are discussed. This experimental study has theoretical and practical significant for SFT design and safety.

Improved biodiversity detection using a large-volume environmental DNA sampler with in situ filtration and implications for marine eDNA sampling strategies

Metabarcoding analysis of environmental DNA samples is a promising new tool for marine biodiversity and conservation. Typically, seawater samples are obtained using Niskin bottles and filtered to collect eDNA. However, standard sample volumes are small relative to the scale of the environment, conventional collection strategies are limited, and the filtration process is time consuming. To overcome these limitations, we developed a new large – volume eDNA sampler with in situ filtration, capable of taking up to 12 samples per deployment. We conducted three deployments of our sampler on the robotic vehicle Mesobot in the Flower Garden Banks National Marine Sanctuary in the northwestern Gulf of Mexico and collected samples from 20 to 400 m depth. We compared the large volume (∼40–60 L) samples collected by Mesobot with small volume (∼2 L) samples collected using the conventional CTD rosette – mounted Niskin bottle approach. We sequenced the V9 region of 18S rRNA, which detects a broad range of invertebrate taxa, and found that while both methods detected biodiversity changes associated with depth, our large volume samples detected approximately 66% more taxa than the CTD small volume samples. We found that the fraction of the eDNA signal originating from metazoans relative to the total eDNA signal decreased with sampling depth, indicating that larger volume samples may be especially important for detecting metazoans in mesopelagic and deep ocean environments. We also noted substantial variability in biological replicates from both the large volume Mesobot and small volume CTD sample sets. Both of the sample sets also identified taxa that the other did not – although the number of unique taxa associated with the Mesobot samples was almost four times larger than those from the CTD samples. Large volume eDNA sampling with in situ filtration, particularly when coupled with robotic platforms, has great potential for marine biodiversity surveys, and we discuss practical methodological and sampling considerations for future applications.