False‐negative detections from environmental DNA collected in the presence of large numbers of killer whales (Orcinus orca)

While environmental DNA (eDNA) is becoming increasingly established in biodiversity monitoring of freshwater ecosystems, the use of eDNA surveys in the marine environment is still in its infancy. Here, we use two approaches: targeted quantitative PCR (qPCR) and whole-genome enrichment capture followed by shotgun sequencing in an effort to amplify killer whale DNA from seawater samples. Samples were collected in close proximity to killer whales in inshore and offshore waters, in varying sea conditions and from the surface and subsurface but none returned strongly positive detections of killer whale eDNA. We validated our laboratory methodologies by successfully amplifying a dilution series of a positive control of killer whale DNA. Furthermore, DNA of Atlantic mackerel, which was present at all sites during sampling, was successfully amplified from the same seawater samples, with positive detections found in ten of the eighteen eDNA extracts. We discuss the various eDNA collection and amplification methodologies used and the abiotic and biotic factors that influence eDNA detection. We discuss possible explanations for the lack of positive killer whale detections, potential pitfalls, and the apparent limitations of eDNA for genetic research on cetaceans, particularly in offshore regions

Framing Cutting-Edge Integrative Deep-Sea Biodiversity Monitoring via Environmental DNA and Optoacoustic Augmented Infrastructures

Deep-sea ecosystems are reservoirs of biodiversity that are largely unexplored, but their exploration and biodiscovery are becoming a reality thanks to biotechnological advances (e.g., omics technologies) and their integration in an expanding network of marine infrastructures for the exploration of the seas, such as cabled observatories. While still in its infancy, the application of environmental DNA (eDNA) metabarcoding approaches is revolutionizing marine biodiversity monitoring capability. Indeed, the analysis of eDNA in conjunction with the collection of multidisciplinary optoacoustic and environmental data, can provide a more comprehensive monitoring of deep-sea biodiversity. Here, we describe the potential for acquiring eDNA as a core component for the expanding ecological monitoring capabilities through cabled observatories and their docked Internet Operated Vehicles (IOVs), such as crawlers. Furthermore, we provide a critical overview of four areas of development: (i) Integrating eDNA with optoacoustic imaging; (ii) Development of eDNA repositories and cross-linking with other biodiversity databases; (iii) Artificial Intelligence for eDNA analyses and integration with imaging data; and (iv) Benefits of eDNA augmented observatories for the conservation and sustainable management of deep-sea biodiversity. Finally, we discuss the technical limitations and recommendations for future eDNA monitoring of the deep-sea. It is hoped that this review will frame the future direction of an exciting journey of biodiscovery in remote and yet vulnerable areas of our planet, with the overall aim to understand deep-sea biodiversity and hence manage and protect vital marine resources

Multiplexing with three-primer PCR for rapid and economical microsatellite validation

The next generation sequencing revolution has enabled rapid discovery of genetic markers, however, development of fully functioning new markers still requires a long and costly process of marker validation. This study reports a rapid and economical approach for the validation and deployment of polymorphic microsatellite markers obtained from a 454 pyrosequencing library of Atlantic cod, Gadus morhua, Linnaeus 1758. Primers were designed from raw reads to amplify specific amplicon size ranges, allowing effective PCR multiplexing. Multiplexing was combined with a three-primer PCR approach using four universal tails to label amplicons with separate fluorochromes. A total of 192 primer pairs were tested, resulting in 73 polymorphic markers. Of these, 55 loci were combined in six multiplex panels each containing between six and eleven markers. Variability of the loci was assessed on G. morhua from the Celtic Sea (n = 46) and the Scotian Shelf (n = 46), two locations that have shown genetic differentiation in previous studies. Multilocus FST between the two samples was estimated at 0.067 (P = 0.001). After three loci potentially under selection were excluded, the global FST was estimated at 0.043 (P = 0.001). Our technique combines three-primer and multiplex PCR techniques, allowing simultaneous screening and validation of relatively large numbers of microsatellite loci

Identification of naturally occurring hybrids between two overexploited sciaenid species along the South African coast

Hybridization between fish species can play a significant role in evolutionary processes and can influence management and conservation planning, however, this phenomenon has been widely understudied, especially in marine organisms. The distribution limits of two sciaenid species (silver kob, Argyrosomus inodorus, and dusky kob, A. japonicus) partly overlap along the South African coast, where both species have undergone severe depletion due to overfishing. Following the identification of a number of possible cases of species misidentification or hybridization (21 out of 422 individuals), nuclear and mitochondrial DNA data (12 microsatellite loci and 562 bp of the COI gene) were analysed to investigate the genetic composition of these individuals. Results indicated a field-based species misidentification rate of approximately 2.8% and a rate of natural hybridization of 0.7%. Interestingly, all hybrid fish resulted from first-generation (F1) hybridization events, which occurred exclusively between silver kob females and dusky kob males. Whether hybridization is the result of natural events (such as secondary contact following a shift in distribution range), or anthropogenic activities (size-selective pressure due to overfishing), these findings have important implications for critical recovery and future management of these species in the wild.DAFF (South Africa)http://www.elsevier.com/locate/ympevhb201

Evidence for distinct coastal and offshore communities of bottlenose dolphins in the north east Atlantic.

Bottlenose dolphin stock structure in the northeast Atlantic remains poorly understood. However, fine scale photo-id data have shown that populations can comprise multiple overlapping social communities. These social communities form structural elements of bottlenose dolphin (Tursiops truncatus) [corrected] populations, reflecting specific ecological and behavioural adaptations to local habitats. We investigated the social structure of bottlenose dolphins in the waters of northwest Ireland and present evidence for distinct inshore and offshore social communities. Individuals of the inshore community had a coastal distribution restricted to waters within 3 km from shore. These animals exhibited a cohesive, fission-fusion social organisation, with repeated resightings within the research area, within a larger coastal home range. The offshore community comprised one or more distinct groups, found significantly further offshore (>4 km) than the inshore animals. In addition, dorsal fin scarring patterns differed significantly between inshore and offshore communities with individuals of the offshore community having more distinctly marked dorsal fins. Specifically, almost half of the individuals in the offshore community (48%) had characteristic stereotyped damage to the tip of the dorsal fin, rarely recorded in the inshore community (7%). We propose that this characteristic is likely due to interactions with pelagic fisheries. Social segregation and scarring differences found here indicate that the distinct communities are likely to be spatially and behaviourally segregated. Together with recent genetic evidence of distinct offshore and coastal population structures, this provides evidence for bottlenose dolphin inshore/offshore community differentiation in the northeast Atlantic. We recommend that social communities should be considered as fundamental units for the management and conservation of bottlenose dolphins and their habitat specialisations

Fine-scale population structure and connectivity of bottlenose dolphins, Tursiops truncatus, in European waters and implications for conservation

Funding: Fyssen post-doctoral fellowship, Fondation Total, a bridge funding from the School of Biology of the University of St Andrews and People’s Trust for Endangered Species (ML).1. Protecting species often involves the designation of protected areas, wherein suitable management strategies are applied either at the taxon or ecosystem level. Special Areas of Conservation (SACs) have been created in European waters under the Habitats Directive to protect bottlenose dolphins, Tursiops truncatus, which forms two ecotypes, pelagic and coastal. 2. The SACs have been designated in coastal waters based on photo‐identification studies that have indicated that bottlenose dolphins have relatively high site fidelity. However, individuals can carry out long‐distance movements, which suggests potential for demographic connectivity between the SACs as well as with other areas. 3. Connectivity can be studied using genetic markers. Previous studies on the species in this area used different sets of genetic markers and therefore inference on the fine‐scale population structure and demographic connectivity has not yet been made at a large scale. A common set of microsatellite markers was used in this study to provide the first comprehensive estimate of genetic structure of bottlenose dolphins in European Atlantic waters. 4. As in previous studies, a high level of genetic differentiation was found between coastal and pelagic populations. Genetic structure was defined at an unprecedented fine‐scale level for coastal dolphins, leading to identification of five distinct coastal populations inhabiting the following areas: Shannon estuary, west coast of Ireland, English Channel, coastal Galicia, east coast of Scotland and Wales/west Scotland. Demographic connectivity was very low among most populations with <10% migration rate, suggesting no demographic coupling among them. Each local population should therefore be monitored separately. 5. The results of this study have the potential to be used to identify management units for bottlenose dolphins in this region and thus offer a significant contribution to the conservation of the species in European Atlantic waters. Future studies should prioritize obtaining biopsies from free‐living dolphins from areas where only samples from stranded animals were available, i.e. Wales, west Scotland and Galicia, in order to reduce uncertainty caused by sample origin doubt, as well as from areas not included in this study (e.g. Iroise Sea, France). Furthermore, future management strategies should include monitoring local population dynamics and could also consider other options, such as population viability analysis or the incorporation of genetic data with ecological data (e.g. stable isotope analysis) in the designation of management units.PostprintPeer reviewe

Emaciated enigma: decline in body conditions of common dolphins in the Celtic Seas ecoregion

Monitoring the nutritional health of cetaceans has become increasingly important in a changing environment, where multiple stressors impact animals. Particularly for those species that require consumption of energy‐dense prey, such as the common dolphin. Thus far, no uniform measure for monitoring body condition has been recommended across cetaceans, and species‐specific measures may need to be developed if employed as a population condition indicator under Descriptor 1 of the Marine Strategy Framework Directive. Here, nine morphometric body condition indices were applied to long‐term common dolphin stranding data sets originating from Ireland and the UK. We report a recent decline in the nutritional health of common dolphins in the Celtic Seas ecoregion comparing animals from 2017 to 2019 to animals from 1990 to 2006, with an increase in cases of animals dying due to starvation. Using ordinal regression trees, ventral blubber thickness (VBT) was identified as the most important index to predict nutritional status, defined at necropsy, followed by the scaled mass index (SMI). Using generalised linear models, both the VBT and SMI indices differentiated individuals that died from chronic and acute causes of death (i.e., bycatch), where animals in chronic conditions had significantly thinner VBT and lower SMI. Both significant temporal and seasonal patterns in VBT were identified, with poorer body conditions observed during the autumn and better body conditions observed during the spring, as well as an overall decline detected in VBT during the study period. While VBT was positively correlated with total body length, SMI showed the opposite trend. The VBT index is recommended for monitoring nutritional health within the species when total body length and season are considered. Further research is needed to understand the underlying causes for the observed decline, including shifts in prey availability and/or quality, to inform targeted conservation management strategies

Advancing fishery-independent stock assessments for the Norway lobster (Nephrops norvegicus) with new monitoring techn

The Norway lobster, Nephrops norvegicus, supports a key European fishery. Stock assessments for this species are mostly based on trawling and UnderWater TeleVision (UWTV) surveys. However, N. norvegicus are burrowing organisms and these survey methods are unable to sample or observe individuals in their burrows. To account for this, UWTV surveys generally assume that “1 burrow system = 1 animal”, due to the territorial behavior of N. norvegicus. Nevertheless, this assumption still requires in-situ validation. Here, we outline how to improve the accuracy of current stock assessments for N. norvegicus with novel ecological monitoring technologies, including: robotic fixed and mobile camera-platforms, telemetry, environmental DNA (eDNA), and Artificial Intelligence (AI). First, we outline the present status and threat for overexploitation in N. norvegicus stocks. Then, we discuss how the burrowing behavior of N. norvegicus biases current stock assessment methods. We propose that state-of-the-art stationary and mobile robotic platforms endowed with innovative sensors and complemented with AI tools could be used to count both animals and burrows systems in-situ, as well as to provide key insights into burrowing behavior. Next, we illustrate how multiparametric monitoring can be incorporated into assessments of physiology and burrowing behavior. Finally, we develop a flowchart for the appropriate treatment of multiparametric biological and environmental data required to improve current stock assessment methods

Advancing fishery-independent stock assessments for the Norway lobster (Nephrops norvegicus) with new monitoring technologies

The Norway lobster, Nephrops norvegicus, supports a key European fishery. Stock assessments for this species are mostly based on trawling and UnderWater TeleVision (UWTV) surveys. However, N. norvegicus are burrowing organisms and these survey methods are unable to sample or observe individuals in their burrows. To account for this, UWTV surveys generally assume that "1 burrow system = 1 animal", due to the territorial behavior of N. norvegicus. Nevertheless, this assumption still requires in-situ validation. Here, we outline how to improve the accuracy of current stock assessments for N. norvegicus with novel ecological monitoring technologies, including: robotic fixed and mobile camera-platforms, telemetry, environmental DNA (eDNA), and Artificial Intelligence (AI). First, we outline the present status and threat for overexploitation in N. norvegicus stocks. Then, we discuss how the burrowing behavior of N. norvegicus biases current stock assessment methods. We propose that state-of-the-art stationary and mobile robotic platforms endowed with innovative sensors and complemented with AI tools could be used to count both animals and burrows systems in-situ, as well as to provide key insights into burrowing behavior. Next, we illustrate how multiparametric monitoring can be incorporated into assessments of physiology and burrowing behavior. Finally, we develop a flowchart for the appropriate treatment of multiparametric biological and environmental data required to improve current stock assessment methods