Journal of Fish Biology

Freshwater ecosystems have been severely fragmented by artificial in-stream structures designed to manage water for human use. Significant efforts have been made to reconnect freshwater systems for fish movement, through the design and installation of dedicated fish passage structures (fishways) and by incorporating fish-sensitive design features into conventional infrastructure (e.g. culverts). Key to the success of these structures is making sure that the water velocities within them do not exceed the swimming capacities of the local fish species. Swimming performance data is scarce for Australian fish, which have a reduced swimming capacity when compared to many North American and European species. To help close this knowledge gap and assist fisheries management and civil engineering, we report the swimming performance capacities of twenty-one small-bodied fish and juveniles (< 10 cm) of large bodied species native to Australia as measured by critical swimming speed (Ucrit) and burst swimming speed (Usprint) in a recirculating flume. This data is complemented by endurance swim trials in a 12-meter hydraulic flume channel, and by measures of flume traverse success. Building on the utility of this dataset, we used a panel of morphological, behavioural and ecological traits to first assess their relative contributions to the observed swimming performance data, and second, to determine if they could be used to predict swimming performance capacity - a useful tool to assist in the management of species of conservation concern where access to swimming performance data may be limited. We found that body length combined with depth station (benthic, pelagic or surface) explained most of the interspecific variation in observed swimming performance data, followed by body shape and tail shape. These three traits were the most effective at predicting swimming performance in a model/unknown fish. This data will assist civil engineers and fisheries managers in Australia to mitigate the impact of in-stream structures on local fish populations.

Marine-phase salmonid aquaculture is a major component of the coastal economies of Northern Europe, North America and Chile and is under threat from numerous challenges to gill health, many of which originate from the phyto- and zooplankton. Associated losses are growing as a proportion of production year on year. A first step towards mitigating losses is to characterize the biological drivers of poor gill health. Numerous planktonic species have been implicated, including toxic and siliceous microalgae, hydrozoans and scyphozoans; however, rigorous longitudinal surveys of planktonic diversity and gill health have been lacking. In the current study, we present and assess an exhaustive identification approach combining both morphological and molecular methods (environmental DNA metabarcoding) approaches in combination with robust statistical models to identify the planktonic drivers of complex gill disease (CGD) and fish mortality. We undertook longitudinal molecular and microscopic evaluation at two marine aquaculture facilities on the west coast of Scotland using daily data collected during the 2021 growing season (March-October). Examining these two different sites, one sheltered and one exposed to the open sea, we identified new, important, and unexpected planktonic drivers (e.g. doliolids and appendicularians) of CGD and mortality and confirmed the significance of some established threats (e.g. hydrozoans and diatoms). We also explored delayed or lagged effects of planktonic abundances on gill health and undertook a comparison of environmental DNA metabarcoding and microscopy in their ability to identify and quantify planktonic species. Our data highlight the diversity of planktonic threats to salmonid aquaculture as well as the importance of using both molecular and morphological approaches to detect those. Despite our study relying on two farm sites only, our results evidence the role of the different planktonic players on salmon gill disease; there is now an urgent need to expand systematic longitudinal molecular and morphological approach across multiple sites and over multiple years. The resultant catalogue of main biological drivers will enable early warning systems, new treatments and, ultimately, a sustainable platform for future salmonid aquaculture in the marine environment.

Previous studies have estimated the size, mass, and population of hypothetical unknown animals in a large, oligotrophic freshwater loch in Scotland based on biomass and other observational considerations. The eel hypothesis proposes that the anthrozoological phenomenon at Loch Ness can be explained in part by observations of large specimens of European eel (Anguilla anguilla), as these animals are most compatible with morphological, behavioural, and environmental considerations. The present study expands upon the eel hypothesis and related literature by estimating the probability of observing eels at least as large as have been proposed, using catch data from Loch Ness and other freshwater bodies in Europe. Skew normal and generalized extreme value distributions were fitted to eel body length distributions in order to estimate cumulative distribution functions from which probabilities were obtained. The chances of finding a large eel in Loch Ness are around 1 in 50, 000 for a 1-meter specimen, which is reasonable given the lochs fish stock and suggests some sightings of smaller unknown animals may be accounted for by large eels. However, the probability of finding a specimen upwards of 6 meters is essentially zero, therefore eels probably do not account for sightings of larger animals. The existence of exceedingly large eels in the loch is not likely based on purely statistical considerations.

Calcein is a fluorescent marker commonly used to label growing calcified structures in marine organisms, but its efficacy is species- and context-specific. We evaluated calcein marking success and survival in the common periwinkle (Littorina littorea) during winter in the Gulf of Maine. Snails were immersed for 24 h in seawater containing 0, 50, or 100 mg L-1 calcein and scored for fluorescent marks 22 days later. Overall marking success was low (12.5% of exposed snails evaluated) but was strongly size-dependent: each 1 mm increase in shell length reduced the odds of acquiring a mark by 27%. Among exposed snails, higher calcein concentration (100 mg L-1) produced significantly brighter marks than the lower concentration (50 mg L-1). Survival was 100% across all treatments. The low overall marking rate likely reflects suppressed shell growth at winter temperatures. We recommend 100 mg L-1 calcein with a 24-h immersion for marking L. littorea and suggest that marking during warmer months would improve efficacy across a broader size range.

While soundscapes shape the structure and function of auditory systems over evolutionary timescales, there is limited information regarding the adaptation of wild fish populations to their natural acoustic environments. This is particularly relevant for freshwater ecosystems, which are extremely diverse and face escalating pressures from human activities and associated noise pollution. The Siamese fighting fish Betta splendens is one of the most important cultured species in the global ornamental fish market and is increasingly recognized as a model organism for genetics and behavioural studies. This air-breathing species (Anabantoidei), characterized by the presence of a suprabranchial labyrinth organ, is native to Southeast Asia and inhabits low flow freshwater ecosystems that are increasingly threatened due to habitat destruction and pollution. We characterized the underwater soundscape, along with various ecological parameters, across five marshland habitats of B. splendens, from lentic waterbodies to small canals near a lake in Chiang Rai province (Thailand). All habitats exhibited common traits of low dissolved oxygen and dense herbaceous vegetation. Soundscapes were relatively quiet with Sound Pressure Level (SPL) around 102-105 dB re 1 {micro}Pa and most spectral energy below 1000 Hz. Sound recordings captured diverse biological sounds, including potential fish vocalizations, but primarily insect sounds. Based on Auditory Evoked Potential (AEP) recordings, we identified sex-specific differences and a best hearing range within 100-400 Hz. This low-frequency tuning highlights the potential susceptibility of B. splendens to anthropogenic noise activities. This study provides first characterization of the auditory sensitivity and natural soundscape of B. splendens, establishing an important ground for future hearing research in this species. The information provided on the auditory sensory adaptation of B. splendens emphasizes the importance of preserving quiet soundscapes from lentic freshwater ecosystems.

Recent examples of rapid brain size plasticity in response to novel laboratory environments suggest that fish brain size is a flexible trait, allowing growth or shrinkage of brain tissue based on short term needs. Nevertheless, it remains to be seen if plasticity of fish brain size is relevant to natural environmental conditions. Here, using rainbow trout escaped from a farming operation as a natural experiment, we demonstrate that adult fish brain size can change rapidly in response to life in a natural lake environment. Specifically, escaped trout had on average 15% heavier brains relative to body size than captive trout after living for about 7 months in the lake. Because relative brain size of most escaped trout fell above the range of variation seen within the captive trout population, we conclude that increased brain size was achieved by plasticity after escape. Brain morphology analysis showed that the most anterior regions (olfactory bulbs and rest of telencephalon) contributed most to the increase in overall brain size in escaped trout. Relative size of the heart ventricle, another organ which can be subject to plastic changes under variable environmental conditions in fish, did not differ between escaped and captive trout. Massive and selective brain growth under the changed environmental conditions associated with escape from holding pens highlighted the plastic potential of fish brain size and suggests that a shift to increased complexity of life in the wild setting of a lake imposed greatly increased cognitive requirements on escaped trout.

Hill stream loaches (family Balitoridae and Gastromyzontidae) are thumb-sized fish that effortlessly exploit environments where flow rates are so high that potential competitors would be washed away. To cope with these extreme flow rates hill stream loaches have evolved adaptations to stick to the bottom, equivalent to the downforce generating wings and skirts of F1 racing cars, and scale architecture reminiscent of the drag-reducing riblets of Mako sharks. Hill stream loaches exhibit far more diverse flow-modifying morphological features than fast pelagic predators, suggesting as yet unknown drag reducing systems remain to be discovered. Here we describe the skeletal structure of Sewellia lineolata and Gastromyzon punctulatus and contrast that with other fish that face similar hydrodynamic challenges. We identify a major structural variation within Balitoridae pelvic sucker attachment positions which may explain fundamental constraints on the parallel development of different genera and which has not been described before. We also use high speed video capture, CT scans and Frustrated Total Internal Reflection to image and measure the sucker system in live operation and describe how it functions on a familiar activity for hill stream loaches (climbing waterfalls). We show how they can drag 3 to 4 times their own bodyweight up a vertical glass waterfall. Adaptations to high flow rates are the inspiration for this study, because there are many engineering applications where the ability to deal with high flow rates are important - either by reducing drag, or by generating the forces needed to hold an animal in place.

The pink salmon Oncorhynchus gorbuscha was introduced from its native range in the Pacific to Northwest Russia several times since the 1950s. While this species has been regularly observed in rivers in Northern Norway since that time, there has been an upsurge in the numbers of odd-year O. gorbuscha individuals observed in rivers in southern Norway in recent years, and particularly in 2017. Although the wide-scale effects of this species presence are currently uncertain, there are concerns regarding potential competition between O. gorbuscha and native species - most notably the Atlantic salmon Salmo salar. Environmental (e)DNA is becoming a widely used tool to monitor rare and invasive species in aquatic environments. In the present pilot study, primers and a probe were developed to detect O. gorbuscha from eDNA samples taken from a Norwegian river system where the species was observed. Water samples were taken at both upstream and downstream locations of the Lysakerelva river during Autumn 2017 (to coincide with spawning) and during late Spring 2018. Autumn samples were positive for O. gorbuscha at both sampling locations, whereas Spring samples showed positive detection of this species in the upstream region of the river, when smolt should have left, or be in the process of leaving the river. These findings reveal that eDNA-based methods can be used detect the presence of O. gorbuscha during their spawning season. This suggests that odd-year populations have the potential to become established in the studied river system. We recommend that eDNA sampling is repeated to determine whether individuals of this odd-year population have survived at sea and return to spawn. Our assay specificity tests indicate that the tools developed in the present study can be used for detection of O. gorbuscha in both Norwegian and other European river systems where presence/absence data is required. We also suggest some modifications to our methodology that may improve upon the detection capabilities of O. gorbuscha using eDNA.

Documenting species distributions and hybridization patterns is paramount for elucidating biogeography and understanding speciation processes. Here we combined genetic specimen analysis and environmental DNA (eDNA) to investigate the presence of American eel (Anguilla rotrata) and American x European eel (Anguilla anguilla) hybrids in Greenland freshwater. We further tested the use of eDNA to document hybridization by using European eel mtDNA as a proxy for hybrid occurrence. Overall, eDNA analysis detected mainly American eel but also European eel mtDNA. This finding was validated by DNA sequencing, which identified 3 out of 26 captured eels (14.3%) carrying European eel mtDNA. Five eels (19.2%), including all three with European mtDNA, were heterozygous for species-specific nuclear gene variants, supporting a hybrid ancestry. Further, eDNA successfully identified eels in lakes where they were caught by fyke net fishing, extending their confirmed northern range by 40 km, and indicated eel presence >200km further north. The study provides an empirical demonstration of the use of eDNA to document hybrid occurrence and extends the reported northern distribution of eels in Greenland significantly beyond previous observations. Further, the existence of hybrid eels in Greenland may be key for understanding the complex mechanisms of hybridization between American and European eels.

Male-biased adult sexual size dimorphism is often the result of intra-sex sexual selection driven by male-male competition. Capelin (Mallotus villosus) exhibit male-biased sexual size dimorphism but lack contests/fighting, and female mate choice, if present, is unrelated to male size. Consequently, it is hypothesized that adult sexual size dimorphism in capelin is due to a mating system that favours larger males with greater energy reserves, allowing them to compete for prolonged access to mating opportunities through endurance rivalry. To enable this, males need a continual supply of semen through the spawning season. However, they have unusually small testes and are predicted to deplete stored semen rapidly, and their unique sperm physiology (i.e. the only known external fertilizing vertebrate to release pre-activated semen) may constrain the ability to regenerate it. We found that the majority of capelin sampled on beaches in 2023 had adequate semen, but towards the end of the spawning season some fish had none. Capelin held in laboratory tanks could regenerate semen within two days, maintaining their ability to seize continual mating opportunities. These results support the endurance rivalry hypothesis.

The size of Astyanax mexicanus blind cavefish populations of North-East Mexico is a demographic parameter of great importance for investigating a variety of ecological, evolutionary and conservation issues. However, very few estimates have been obtained. For these mobile animals living in an environment difficult to explore as a whole, methods based on capture-mark-recapture are appropriate, but the feasibility of such approach and the interpretation of the results depend on several assumptions that must be carefully examined. Here, we provide evidence that minimally invasive genetic identification from captures at different time intervals can give insights on cavefish population size dynamics as well as other important demographic parameters of interest. We also provide tools to calibrate sampling and genotyping efforts necessary to reach a given level of precision. Our results suggest that the El Pachon cave population is currently very small, of an order of magnitude of a few hundreds of individuals, and is distributed in a relatively isolated area. The probable decline in population size in the El Pachon cave since the last census in 1971 raises serious conservation issues.

The recovery of amplifiable DNA from formaldehyde{square}fixed (FF) zooplankton samples has long been considered unfeasible. Nevertheless, advances in DNA sequencing and methods for retrieving highly degraded genetic material have demonstrated that even million{square}year{square}old samples and FF museum specimens can yield usable DNA. To access the biological information preserved in long{square}term zooplankton time series, we assessed methodologies for extracting amplifiable DNA from community samples stored for up to 28 years in formaldehyde at room temperature. On one hand, we report the failure of a method previously described as successful for FF zooplankton samples, likely due to the cold{square}storage conditions (4{square}{degrees}C) used in the original study. On the other hand, by adapting two extraction protocols designed for FF museum specimens--representing harsher and softer alternatives (HHA and HPC, respectively)--we successfully amplified and sequenced a subset of FF zooplankton samples. As expected, DNA integrity and sample pH were inversely related to preservation time, and only short DNA fragments were recovered, ruling out the use of commonly employed [&ge;]300{square}bp metabarcoding markers. While DNA integrity appeared to be a better predictor than DNA yield for amplification success, the presence of a gel band of the expected size did not always guarantee congruence with microscopy{square}based assessments. Although amplifiable DNA was recovered from most samples, including some of the oldest, community compositions concordant with microscopy were consistently recovered only from samples preserved for up to two years. Beyond this point, the HHA and HPC methods produced divergent results, reflecting a trade{square}off between the removal of formaldehyde{square}induced cross{square}linkages and the avoidance of additional DNA damage. Among the small universal markers tested ([~]120-170{square}bp), including one nuclear rRNA marker and two mitochondrial markers, only the 18S rRNA V9 region consistently amplified. We conclude by providing a set of recommendations aimed at improving the methods presented here.

Uncontrolled carbon dioxide emissions from human activities contribute to ocean warming and acidification. These alterations in ocean chemistry threaten marine organisms, such as the true giant clam, Tridacna gigas, which is already imperiled due to overharvesting and habitat destruction. To gain an understanding of the physiological and molecular responses of T. gigas and its symbiotic dinoflagellates to ocean warming and acidification, we subjected juvenile individuals to different treatments simulating predicted seawater pH (7.6 and 8.0) and temperature (28{degrees}C, 30{degrees}C, 32{degrees}C and 34{degrees}C) levels for the next century. Juvenile giant clams were able to tolerate sustained exposure to temperatures of up to 32{degrees}C and pH as low as 7.6, while exposure to higher temperature (34{degrees}C), regardless of pH level, resulted in total mortality after a week. However, symbiosis was compromised even in the sublethal treatments, as indicated by the decrease in Symbiodiniaceae density and changes in symbiont gene expression. Symbionts significantly upregulated genes involved in splicing, translation, fatty acid metabolism, and DNA repair, which may constitute an adaptive response, while downregulating genes involved in photosynthesis and transmembrane transport, suggests impaired transfer of photosynthates to the host. These findings demonstrate the vulnerability of the juvenile T. gigas holobiont to heat stress, highlighting the critical importance of continued conservation and management alongside efforts to mitigate global changes in ocean conditions to safeguard this iconic marine bivalve. Summary StatementThis study investigates physiological and molecular responses of Tridacna gigas to seawater warming and acidification, providing insights into the potential future of endangered giant clam populations in a changing ocean.

Marine anthropogenic noise pollution has risen in recent decades, driving interest in its ecological effects. While research has focused on mammals and fish, marine invertebrates remain largely understudied despite their ecological importance. This study explored sound perception in the benthic ascidian Halocynthia papillosa, assessing whether it detects acoustic signals via waterborne pressure waves or substrate vibrations. Field and laboratory experiments exposed H. papillosa to frequencies from 50-1500 Hz. Contraction responses occurred at 100 Hz, 200 Hz, and 600 Hz when played through a waterproof speaker that produced both, sound pressure and substrate vibration. In a controlled laboratory setup, only substrate vibrations triggered contractions, while pure sound pressure up to 130 dB re 1 {micro}Pa elicited no response. Repeated exposure led to habituation, reducing contraction amplitude and relaxation time. These findings indicate that ascidians rely on mechanoreception, underscoring the overlooked impact of substrate vibrations from marine noise and the necessity to reconsider study design when investigating effects of marine noise on benthic organisms.

BackgroundAnthropogenic noise is an emerging threat to marine ecosystems, yet its effects on marine invertebrates, particularly zooplanktonic species, remain poorly understood. Despite increasing evidence of behavioral and physiological impacts in invertebrates, the effects of noise on embryonic development and the molecular mechanisms underlying acoustic responses remain largely unexplored. Here, to address this gap, we investigated the impact of high-intensity underwater noise exposure on embryogenesis of the appendicularian tunicate Oikopleura dioica, a cosmopolitan zooplanktonic tunicate that plays important ecological roles in marine trophic webs and carbon cycling. Under lab-controlled conditions, we examined the effects of experimental noise exposure on early embryogenesis at both morphological and transcriptomic levels using RNA-seq in 8-cell (8c) and early tailbud (ETB) stages. ResultsNoise exposure produced no significant increase in embryo malformations compared to non-exposed controls, indicating substantial phenotypic resilience under laboratory conditions. Interestingly, transcriptomic analyses revealed a rapid molecular response of 70 differentially expressed genes (DEG) already detectable after only 30 minutes of exposure at the 8-cell stage, which became markedly amplified with 700 DEGs by the ETB stage. Together, differential expression, GO enrichment, and co-expression network analyses identified coordinated regulation of processes associated with membrane homeostasis, pyrimidine/CTP metabolism, extracellular matrix organization, cytoskeletal architecture, RNA regulation, translational control, proteostasis, mitochondrial metabolism, and developmental pathways. Importantly, both developmental stages precede the formation of differentiated mechanosensory structures, suggesting that the observed responses are unlikely to reflect conventional sound perception. ConclusionsThese findings provide the first molecular characterization of noise effects during O. dioica embryogenesis and reveal an unexpected molecular sensitivity of O. dioica embryos to underwater noise despite preserved morphological development. The transcriptional signatures support a mechanobiological framework in which acoustic exposure may directly perturb cellular mechanical homeostasis through membrane-and cytoskeleton-associated processes, triggering compensatory stress-adaptation responses involving proteostasis, RNA regulation, and metabolic reprogramming. Together, these findings establish O. dioica as a valuable emerging model for investigating the developmental and evolutionary consequences of acoustic pollution in marine ecosystems.

Among cetaceans, the Cuviers beaked whale is considered an extreme diver, very challenging to be studied with standard monitoring methods due to its elusive behaviour and a preference for deep offshore waters. These limitations seem to be mitigated by the use of new molecular methodologies capable of intercepting small traces of DNA left in the environment (eDNA) by marine organisms. Moreover, the collection of water from the superficial layer of the sea represents a perfect case study for the targeting of marine mammals, as the constraints imposed by their nature implies periodic and frequent surfacing. Therefore, we designed and tested a taxon-specific primer set to infer the Cuviers beaked whale presence, with the aims of 1) examining the effectiveness of the eDNA technique to detect the presence of deep-diving cetacean in open waters, using the Cuviers beaked whale as case study; 2) providing data on the spatiotemporal occurrence of this species within the Canyon of Caprera; and 3) assessing the species occurrence in central northern Mediterranean Sea based on molecular traces. Results from this study demonstrated that collection of superficial waters is a valid approach to monitor deep-diving cetacean species, without the need for complementary visual survey monitoring. Specifically, this study provides evidence of the regular presence of the Cuviers beaked whale in the Canyon of Caprera, with a preference for bathymetry in the range of 700-1000 m. This study also showed a potential inshore movement of this species during fall, which is possibly related to migration of its cephalopod prey or a shift in prey preferences. Overall, the data presented here are particularly relevant in the optic of proposing the Caprera Canyon as an Important Marine Mammal Area (IMMA) in the Mediterranean Sea. At a wider level, this study also showed that the stronger positive signals were recorded in sampling stations located on submarine canyon systems, demonstrating the importance of these areas as elective habitats for the Cuviers beaked whale, thus the pivotal priority to their conservation.

Using a cross-fostering experiment, we provide evidence for the contribution of both genetic differentiation and phenotypic plasticity to troglomorphic character development in the recently discovered cave form of Barbatula barbatula, an evolutionarily young lineage and first cavefish described in Europe, the northernmost record. We established reproducing populations of cave- and surface-dwelling loaches to produce cave, surface, and hybrid offspring and reared the F1 fish in a common garden setting in total darkness (DD) to simulate cave conditions as well as under the natural photoperiod (DL). We observed significant differences in the occurrence and extent of typical troglomorphic target characters among the offspring groups. Regardless of rearing conditions, cave fish exhibited smaller eyes, lighter body coloration, longer barbels, and larger olfactory epithelium than seen in surface fish. Hybrids in both rearing conditions generally showed an intermediate level of these traits. Surface and hybrid DD fish differed from the DL groups, resembling the cave fish phenotype in several traits, including eye size and body pigmentation. In contrast, cave and hybrid DL fish groups resembled surface fish phenotypes. Results confirmed that troglomorphic traits arise from heritable genetic differentiation of cave from surface forms and that phenotypic plasticity contributes to the process of adaptation to novel light conditions.

Monitoring the escapement (i.e., spawner abundance) of adult salmonids is a fundamental component of fisheries stock assessment and evaluating recovery goals. Whereas multiple methods exist for estimating escapement, they exhibit trade-offs between degree of accuracy and implementation expenses and effort. Various methods are used throughout the Central Valley to estimate escapement of the four runs of Chinook Salmon (Oncorhynchus tshawytscha), but few rivers have more than one escapement monitoring program. Fall-run Chinook Salmon escapement to the Stanislaus and Tuolumne rivers is estimated by two independent monitoring programs on each river. Since 1952, California Department of Fish and Wildlife has performed carcass surveys that provide an index of escapement. Beginning in 2003 (Stanislaus River) and 2009 (Tuolumne River), private contractors have seasonally installed and operated weirs with fish counting devices to enumerate returning adults. We used the overlapping time series to evaluate the relationship between estimates from carcass surveys and the counting weirs. With few exceptions, estimates from counting adult salmon at the weirs as they entered the spawning grounds were greater than estimates from carcass surveys on both rivers. Escapement estimates from the Stanislaus weir were on average 1.7 (SE = 0.07) times greater than estimates from carcass surveys, while estimates at the Tuolumne weir were 2.5 (SE = 0.17) times greater than estimates from carcass surveys. Estimates from both methods were highly correlated, with r2 values > 96% for both rivers. The high degree of covariation between methods indicates escapement estimates are robust and can be used in population models that incorporate additional data sources, such as estimates of juvenile production. Having multiple sources of data on fall-run Chinook Salmon will be increasingly useful for guiding and evaluating management actions in these heavily managed rivers.

There is a growing need for scalable and cost-effective genetic monitoring tools to support the management of species and populations of conservation importance, driven by the increasing biodiversity loss, need for long-term population data and limitations of invasive genetic sampling. Whale sharks (Rhincodon typus), along with other Endangered, elusive and highly migratory species, spend the majority of their lives in offshore ocean waters. This behaviour poses logistical and ethical challenges to invasive genetic sampling of the species, which is traditionally done via tissue biopsy. Here, we develop a genetic toolkit to study populations of whale sharks from seawater environmental DNA using short segments of nuclear DNA (100-300bp) containing two or more SNPs called "microhaplotypes". Amplifying these markers from seawater collected in 1L bottles behind sharks showed that we can reliably genotype sharks from water samples taken immediately behind the animal. Moreover, we observed a strong relationship between population-level allele frequencies and estimates of genetic diversity between eDNA and tissue-derived samples, demonstrating the capability of eDNA in capturing population-level genetic information with high fidelity. We also analysed a tissue dataset of 72 sharks that attended the Ningaloo Coast World Heritage Area over six-years to showcase the utility of our SNP panel to study temporal processes in these populations. Our data revealed patterns of genetic variation through time that were consistent with whole genome techniques. Contributor estimation from mock environmental samples using various combinations of tissue-derived sequence data to estimate abundance of animals in a mixed DNA sample was shown to accurately identify the number of contributors in mixtures containing [&le;] 10 individuals, beyond which biases were too large. Our findings illustrate the viability of using microhaplotype markers from seawater eDNA as a tool to study conservation genetics of whale sharks, with the potential for broader expansion of eDNA-based genetic assessments to other marine megafauna and aquatic species.

Quantifying in-situ environmental DNA (eDNA) concentrations is increasingly used to infer fish abundance and biomass in freshwater ecosystems. While various sampling strategies (i.e. the combination of sample collection, eDNA preservation and extraction protocols) have been proposed to collect eDNA, little efforts have been undertaken to evaluate how these different strategies affect taxon-specific eDNA recovery and the subsequent estimates of in-situ eDNA concentrations. In this study, we compared a point (i.e. eDNA collection from relatively small and spatially separated water samples filtered using low capacity filter units) and a spatially integrated sampling strategy (i.e. eDNA collected from spatially integrated larger water volumes filtered using high capacity filter units) in two natural lakes. Through quantitative analyses of total DNA, total fish eDNA and species-specific eDNA, we assessed the performance of both strategies to infer in-situ eDNA concentrations. Our results showed that the integrated strategy led to a reduced recovery of fish eDNA and a subsequent underestimation of in-situ eDNA concentrations compared to the point sampling strategy. While the exact mechanisms underlying this pattern require further investigation, our findings highlight the importance of carefully selecting sampling strategies according to study objectives.