Validation of environmental DNA (eDNA) as a detection tool for at‐risk freshwater pearly mussel species (Bivalvia: Unionidae)

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Environmental DNA as a monitoring tool for the endangered freshwater pearl mussel (Margaritifera margaritifera L.): a substitute for classical monitoring approaches?

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Comparison of traditional and environmental DNA survey methods for detecting rare and abundant freshwater fish

1. Detecting rare species is often a necessity for conservation and management, yet challenging for many field survey methods. Environmental DNA (eDNA) is a highly promising solution that has been shown to outperform many established survey methods.
2. Macquarie perch (Macquaria australasica) is an endangered native species that has declined significantly in range and abundance. Detection of M. australasica was compared with an abundant alien fish species (Oncorhynchus mykiss) using eDNA and three conventional survey methods: gill nets, electrofishing and fyke nets.
3. eDNA occupancy estimates for both fish species were compared using four different models to investigate what effect these differences have on false positives and false negatives for the rare and common fish species. These models used unadjusted eDNA detections in water samples, eDNA detections that have been screened using a limit of detection method to remove potential false positives, eDNA data supplemented with a second survey method, or eDNA data augmented with sequencing of positive polymerase chain reaction replicates.
4. eDNA surveying as a single detection method was found to be more efficient and sensitive compared with each capture method separately and combined. Occupancy estimates for the common and rare species did not vary significantly between the four site occupancy-detection models, suggesting that supplementary data may not have as much effect on occupancy estimates compared with other approaches such as temporal or spatial sampling.
5. We conclude that eDNA outperforms the three established survey methods for both a rare and common freshwater fish species. Although there was no significant effect of augmenting eDNA survey methods with other survey data, additional data may improve confidence in detection, and provide confirmatory evidence for unexpected or new detections of a species.

     

Dead or alive — Old empty shells do not prompt false-positive results in environmental DNA surveys targeting the freshwater pearl mussel (Margaritifera margaritifera L.)

1. Environmental DNA (eDNA) from water samples is increasingly used to detect the presence and distribution of species in aquatic ecosystems. However, before implementing eDNA in monitoring programmes, various species-specific sampling or analytical issues remain to be resolved in order to minimize frequencies of false-positive and -negative results. For example, empty shells from freshwater pearl mussels (Margaritifera margaritifera) contain extractable DNA (chemical extraction from ground-up shells) suggesting a risk of false-positive samples at stream sites with extinct populations but with empty shell material remaining.
2. The aim of this study was to investigate whether empty and naturally degrading shells from M. margaritifera can cause false-positive eDNA signals in water samples.
3. Water samples were collected from outdoor stream channels (in Lemming, Denmark) with living freshwater pearl mussels or empty shell material (density ~10 individuals m⁻²) during a 3-week experimental period. Living freshwater pearl mussels were collected from Hemgravs stream in Sweden and transported to Denmark according to permissions granted by the Swedish and Danish authorities.
4. All water samples from stream channels containing empty shells were negative for eDNA indicating that eDNA traces in stream water are most likely to originate from living individuals located upstream of the sampling site. Water samples collected from stream channels containing living individuals of M. margaritifera were consistently positive for eDNA except for one sample (interpreted as a false negative).
5. The study shows that positive eDNA signals for freshwater pearl mussels most likely reflect the presence of living individuals. Consequently, we suggest that eDNA should be used to locate remaining population fragments of M. margaritifera in deep and turbulent streams, providing a platform for faster and more efficient decision making when launching investigative and mitigation initiatives.

     

Detecting the undetectable: Characterization,optimization, and validation of an eDNA detection assay for the federally endangered dwarf wedgemussel,Alasmidonta heterodon (Bivalvia: Unionoida)

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Environmental DNA (eDNA) detection and habitat occupancy of threatened spotted gar (Lepisosteus oculatus)

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Improving the detection of rare native fish species in environmental DNA metabarcoding surveys

1. The presence of threatened or endangered species often strongly influences management and conservation decisions. Within the Murray–Darling Basin (MDB), Australia, the presence of threatened native fish affects the management and allocation of water resources. In New South Wales, these decisions are currently based on traditional fisheries data and a predictive MaxEnt model. However, it is important to verify the model's predictive power given the implication it may have, but this requires methods with a high detection sensitivity for rare species.
2. Although the use of environmental DNA (eDNA) monitoring, in particular eDNA metabarcoding, achieves a higher detection sensitivity compared with traditional methods, earlier surveys in the MDB have shown that the highly abundant and invasive common carp (Cyprinus carpio) can reduce detection probabilities for rare species. Consequently, a polymerase chain reaction (PCR) blocking primer designed to block the amplification of carp eDNA could increase the detection probabilities for rare native species while simultaneously reducing the required sampling effort and survey costs. Although PCR blocking primers are often used in ancient DNA and dietary studies, no aquatic eDNA metabarcoding study to date has evaluated the potential benefits of using PCR blocking primers.
3. A laboratory and field-based pilot study was used to address this knowledge gap and assess the impact of a blocking primer, targeting cyprinid fishes (including carp), on the detection probabilities of native species and the minimum sampling effort required.
4. Results showed that the inclusion of the blocking primer increased the detection probabilities for native species by 10–20% and reduced the minimum required sampling effort by 25–50%. These findings provide important insights into possible methods for optimizing eDNA metabarcoding surveys for the detection of rare aquatic species.

     

Improved detection sensitivity using an optimal eDNA preservation and extraction workflow and its application to threatened sawfishes

1. Pressures on coastal ecosystems are increasing and aquatic species that are restricted to these habitats are facing the threat of extinction. However, the true extent of many threatened and rare aquatic species, especially elasmobranchs, remains unclear due to high levels of data deficiency and poor efficacy of traditional survey methods. Sawfishes (Pristidae), a family of shark-like rays, are among the most threatened and rare elasmobranch species and are difficult to detect in turbid, coastal habitats. Reliable cost-effective tools to detect these species are urgently needed to increase their conservation potential.
2. Characterization of environmental DNA (eDNA) extracted from water samples has garnered significant appeal for detection of rare and threatened species. To assist conservation and monitoring efforts for sawfishes using eDNA, species-specific TaqMan quantitative polymerase chain reaction assays were developed and validated to detect 1.25–5 copies of a 12S rRNA gene fragment. Filter samples were collected in Northern Territory, Australia to assess the utility of the developed eDNA assays and compare the efficacy of preservation and extraction workflows for detecting rare species.
3. Dwarf sawfish (Pristis clavata) were detected in three of 20 sites, and there was a significant effect of preservation and extraction workflow on total eDNA yield and subsequent detection success. Longmire's preserved samples extracted using glycogen-aided precipitation yielded a significantly higher concentration of total eDNA (n = 60; β = 1.27, t(95) = 8.172, P < 0.0001) and yielded positive P. clavata eDNA detections compared to ethanol preserved samples extracted using QIAGEN DNeasy kit, which did not yield any positive detections.
4. The optimized eDNA assays were developed to support monitoring efforts for endangered sawfishes. Importantly, this study demonstrates that choice of preservation and extraction workflow requires careful consideration, especially when detection of rare or threatened species can have important management and conservation outcomes.

     

Averting co-extinction: Successful mussel translocation rescues an endangered population of the European bitterling,Rhodeus amarus

1. Local extinctions break species interactions and have cascading effects throughout ecosystems; parasites are often severely affected. The European bitterling, Rhodeus amarus, is a cyprinid fish that parasitizes unionid mussels by laying eggs into the mussel gill cavity, where embryos develop and emerge as active juveniles; this relationship is obligatory for the bitterling.
2. This article describes a field experiment aimed at averting the secondary extinction of the European bitterling after a complete die-off of a freshwater mussel community, as a result of habitat destruction.
3. Approximately 5,000 unionid mussels were reintroduced within the short time frame in which the remnant bitterling population was still present at the site. Mussel survival was high, and bitterling resumed reproduction, with vigorous courtship observed within 24 hours of mussel release. Recruitment was successful, as evidenced by increased occupancy, densities, and relative frequencies in the fish assemblage. The frequency of sub-adults and young-of-the-year changed from 0% before mussel reintroduction to 80% a year later, and 50% 2 years later, when young-of-the-year contributed to about half of the young fish. No bitterling were observed at two control sites where mussels were not reintroduced.
4. This study exemplifies how the timely restitution of affiliate species can avert co-extinction. It also shows how the conservation of the bitterling within its historical distribution range can serve mussel conservation, including species that although not legally protected, are important keystone species and ecosystem engineers, shaping the structure and function of a broad range of freshwater habitats.

     

An alien fish threatens an endangered parasitic bivalve: the relationship between brook trout (Salvelinus fontinalis) and freshwater pearl mussel (Margaritifera margaritifera) in northern Europe

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Prediction of potential freshwater pearl mussel habitat aids in discovery of a large population of Margaritifera laevis in Iturup Island,Russia

1. Freshwater pearl mussels (family Margaritiferidae) are sensitive to human impact and environmental changes. Large declines in their abundance have prompted studies of distribution and estimations of population densities.
2. Iturup Island, a part of Kuril Islands, Russia, is considered to be within the distribution area of freshwater pearl mussels, but this information is based on only two specimens collected several decades ago. Detailed survey of the island is challenging as most of its territory is nearly impassable.
3. A preliminary analysis of island rivers was carried out prior to the surveys to discover potential freshwater pearl mussel habitats. Based on previous experience from European Russia, freshwater pearl mussel habitat was expected to occur in rivers flowing out of lowland lakes.
4. Live individuals of Margaritifera laevis were found during surveys in one river together with the unionid Beringiana beringiana, which were also found in two other rivers where the predicted habitat occurred. Based on prediction and surveys, the occurrence of freshwater pearl mussels in other rivers of Iturup Island is unlikely.
5. Pearl mussels were concentrated in a small section of the Zmeika River with a population of about 100,000 mussels. Present threats to the population include overharvest of host fish masou salmon (Oncorhynchus masou).
6. Surveys using similar methods can help to reveal unknown freshwater mussel populations on other Kuril Islands and on the mainland.

     

Genetic structure of Irish freshwater pearl mussels (Margaritifera margaritifera and Margaritifera durrovensis): Validity of subspecies,roles of host fish,and conservation implications

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Freshwater mussels in Mediterranean-climate regions: Species richness,conservation status,threats, and Conservation Actions Needed

1. The five global Mediterranean-climate regions are experiencing alarming rates of freshwater biodiversity loss. Although freshwater mussels are recognized as important functional components in aquatic ecosystems, and are among the most threatened faunal groups globally, there has been no synthesis of the plight of this group within these regions.
2. Data from the International Union for Conservation of Nature (IUCN) Red List were reviewed to compare the conservation status, threats, and conservation actions needed for freshwater mussel species occurring in Mediterranean-climate regions (med-mussels) with those of other freshwater mussel species globally. The first comprehensive catalogue of med-mussel species was compiled using existing taxonomic literature.
3. There are 41 med-mussel species, 30 of which occur in the Mediterranean basin. Many regions have just a single species, and regions where multiple species occur generally only have between one and four species per river basin. Med-mussel species are almost twice as likely to be ‘Imperilled’, are affected by 2.4 times more threats, and require 3.5 times more conservation actions than non-med mussels. In many cases, the exact threats have not been identified.
4. In combination with low species richness, this level of imperilment means that Mediterranean-climate regions are at risk of losing the benefits that mussels provide to broader ecosystem functioning. The conservation of med-mussels can be improved by increasing our knowledge of species distributions, including the identification of cryptic species and significant management units, through population genetic work. In addition, recognizing the potential of ‘novel’ habitats and refuge areas could augment the management of this important functional group.

     

Taxonomic and geographical representation of freshwater environmental DNA research in aquatic conservation

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Conservation status assessment and a new method for establishing conservation priorities for freshwater mussels (Bivalvia: Unionida) in the middle and lower reaches of the Yangtze River drainage

1. The freshwater mussel (Unionida) fauna of the Yangtze River is among the most diverse on Earth. In recent decades, human activities have caused habitat degradation in the river, and previous studies estimated that up to 80% of the mussel species in the Yangtze River are Threatened or Near Threatened with extinction. However, a comprehensive and systematic evaluation of the conservation status of this fauna has yet to be completed.
2. This study evaluated the conservation status of the 69 recognized freshwater mussel species in the middle and lower reaches of the Yangtze River, using the criteria published by the International Union for Conservation of Nature (IUCN). A new method for prioritizing species for conservation was then developed and applied termed Quantitative Assessment of Species for Conservation Prioritization (QASCP), which prioritizes species according to quantifiable data on their distribution and population status, life history, and recovery importance and potential.
3. IUCN assessments showed that 35 (51%) species in the study region are Threatened or Near Threatened (11 Endangered, 20 Vulnerable, 4 Near Threatened). In addition, 16 species (23%) could not be assessed owing to data deficiency. Key threats to the freshwater mussel biodiversity of the Yangtze River include pollution, habitat loss and fragmentation, loss of access to host fish, and overharvesting of mussels and their host fish. The genera Aculamprotula, Gibbosula, Lamprotula, Pseudodon, Ptychorhynchus, and Solenaia were identified as particularly threatened.
4. Data availability allowed QASCP assessment of 44 species. Only Solenaia carinata, regionally Endangered under IUCN criteria, achieved the highest QASCP rank, i.e. First Priority. The five species assessed as Second Priority were considered either regionally Endangered (one), Vulnerable (three), or Data Deficient (one) under IUCN criteria. The 23 Third Priority species were assessed as regionally Endangered (two), Vulnerable (15), Near Threatened (two), or Least Concern (four).

     

A new genus and two new,rare freshwater mussel (Bivalvia: Unionidae) species endemic to Borneo are threatened by ongoing habitat destruction

1. Most of the Bornean endemic freshwater mussel (Unionida) species known to date have not been recorded by science for the past 50 years or more, owing to a lack of research effort and presumed population losses.
2. This study assessed current patterns and recent changes in the diversity and distribution of freshwater mussels in north-eastern Borneo. Physical surveys and interviews were conducted at 24 sites, and anecdotal evidence for current or historical presence of mussels was collected for a further 13 sites.
3. Native species, i.e. Schepmania sp. and Khairuloconcha sahanae gen. & sp. nov., were only found in one small stream of the Kinabatangan River basin within the Gomantong Forest Reserve, whereas the non-native Sinanodonta cf. woodiana was common across the study area.
4. Molecular phylogenetics (five genes) of the native taxa, including comparative material from West Kalimantan and Sarawak, revealed: (i) the presence of a new genus with two new, rare species: Khairuloconcha lunbawangorum sp. nov. in the Limbang River basin and K. sahanae in the Kinabatangan River basin; (ii) that Khairuloconcha and Ctenodesma form the Bornean endemic tribe Ctenodesmini trib. nov.; and (iii) that Schepmania represents another Bornean endemic tribe Schepmaniini trib. nov.
5. Both Khairuloconcha gen. nov. species are known from a single stream each and are apparently restricted to forest stream habitats where they occur at very low densities. Schepmania appears to have a severely contracted range in the Kinabatangan and adjacent basins. We urgently call for full protection of the currently known sites of K. lunbawangorum and K. sahanae, and development of an action plan to save the Bornean freshwater mussel fauna.

     

Predation on native fish eggs by invasive round goby revealed by species‐specific gut content DNA analyses

1. Conservation of riverine fish often aims to improve access to spawning grounds and restore longitudinal connectivity by removing migration barriers, and involves substantial investments. However, these investments also enable non‐native predators to invade upstream into spawning areas and potentially adversely affect the recruitment of threatened freshwater fish through egg or fry predation.
2. Detecting egg predation is often challenging. Visual inspections of fish gut contents may underestimate predation of soft materials such as eggs and fry, which limits the discovery of predators preying upon these life‐stages. DNA‐based detection assays may offer a more sensitive tool to assess predation of soft materials.
3. A conservation issue was confirmed by developing and applying a species‐specific DNA‐based detection assay: invasive round goby (Neogobius melanostomus) prey on the eggs or fry of the threatened common nase (Chondrostoma nasus) in Switzerland.
4. DNA‐based detection assays were also developed for five other valuable native fish species, including endangered salmonid and cyprinid river spawners. The applicability of the assays was confirmed in a series of laboratory and field feeding experiments involving eggs and fish tissue. In addition, this work provides a guiding framework for conservation managers regarding the use and applicability of different DNA‐based detection approaches for gut content analysis.
5. The results of this study could inform local conservation measures – such as temporary reductions in the density of round goby at spawning sites prior to spawning – and demonstrate how targeted application of species‐specific molecular markers may advance freshwater fish management.

     

Demonstrating the practical impact of studies on biotic interactions and adaptation of a threatened unionoid mussel (Margaritifera margaritifera) to its host fish (Salmo trutta)

1. To understand the ecological factors behind the decline of functionally important threatened species with complex life cycles, many different life‐cycle stages need to be investigated. The highly threatened unionoid freshwater mussels, with their complex life cycle, including a parasitic stage on host fish, often have a large influence on biodiversity and ecosystem functioning.
2. The overall aim of the present article is to summarize and discuss the impact of two articles published in Aquatic Conservation: Marine and Freshwater Ecosystems (AQC) on biotic interactions and adaptation of a threatened unionoid mussel (Margaritifera margaritifera) to its host fish (Salmo trutta).
3. The two AQC publications described research on the influence of population size and density of mussels and host fish, and host–parasite interactions between mussels and their host fish, on the recruitment of juvenile mussels.
4. The results from these publications filled gaps in knowledge and resulted in recommendations and incentives for conservation. The results and method development have been used in practical conservation work with threatened mussel species and have been implemented and cited in management handbooks. The outcome of the publications has been implemented in large conservation and restoration projects, and in several recent scientific publications.
5. Specifically, the results from one publication showed that ecological parameters such as mussel and host fish density and population size influenced recruitment of the threatened freshwater pearl mussel. The results from the second publication showed that understanding host–parasite interactions is important for comparing the suitability of host fish strains, and that host fish strains differ in their suitability for mussel infestations. In combination, the articles show that integrating ecological parameters of threatened mussels and their host fish with host–parasite interaction experiments can be an important influence on conservation recommendations, adaptive management and national management programmes for threatened species.

     

Trophic niche overlap between native freshwater mussels (Order: Unionida) and the invasive Corbicula fluminea

1. Freshwater mussels (Order Unionida) are highly threatened. Interspecific competition for food sources with invasive alien species is considered to be one of the factors responsible for their decline because successful invaders are expected to have wider trophic niches and more flexible feeding strategies than their native counterparts.
2. In this study, carbon (δ¹³C: ¹³C/¹²C) and nitrogen (δ¹⁵N: ¹⁵N/¹⁴N) stable isotopes were used to investigate the trophic niche overlap between the native freshwater mussel species, Anodonta anatina, Potomida littoralis, and Unio delphinus, and the invasive bivalve Corbicula fluminea living in sympatry in the Tua basin (south-west Europe).
3. The species presenting the widest trophic niches were C. fluminea and A. anatina, which indicate that they have broader diets than U. delphinus and P. littoralis. Nonetheless, all the species assimilated microphytobenthos, sediment organic matter, and detritus derived from vascular plants, although with interspecific variability in the assimilated proportions of each source. The trophic niche of the invasive species overlapped with the trophic niche of all the native species, with the extent varying between sites and according to the species.
4. From the three native species analysed, Potomida littoralis may be at a higher risk for competition for food with C. fluminea in the Tua basin, if food sources become limited, because this native mussel presented the narrowest trophic niche across sites and the highest probability of overlapping with the trophic niche of C. fluminea.
5. Given the global widespread distribution of C. fluminea, the implementation of management measures devoted to the control or even eradication of this invasive alien species should be a conservation priority given its potential for competition with highly threatened native freshwater mussels.

     

Conservation status of two endangered freshwater mussel species in Bavaria,Germany: Habitat quality,threats, and implications for conservation management

1. The freshwater pearl mussel (Margaritifera margaritifera) and the thick‐shelled river mussel (Unio crassus) are relatively widespread across Europe, but are strongly declining and are now protected by the European Habitats Directive. In the course of this study, 20 pearl mussel and 14 thick‐shelled river mussel streams in Bavaria, Germany, were investigated.
2. The mussel populations were mapped to determine population size and age structure. For the assessment of habitat quality, host fish abundance and physicochemical parameters were investigated, e.g. substratum quality, water chemistry, redox potential, and turbidity. Furthermore, potential risks for the populations such as predation or river maintenance were also recorded and assessed.
3. The average population size and recruitment rates of M. margaritifera populations were lower than in U. crassus populations, with 3517 (2.2% juveniles) compared with 5566 (41.4% juveniles) individuals, respectively. On average, 22.3% of particles were smaller than 0.85 mm in diameter at M. margaritifera sites, whereas the mean proportion of fine particles at U. crassus sites was twice as high, at 41.3%. Other parameters such as redox potential or electric conductivity also indicated more favourable habitat conditions in M. margaritifera streams. Unio crassus seems to be less vulnerable to adverse substratum texture and increased nutrient levels than M. margaritifera.
4. The main threats for U. crassus were physical habitat destruction, predation by muskrat, or a lack of host fish, whereas M. margaritifera mainly suffered from siltation leading to a lack of oxygen supply to the interstitial zone, affecting recruitment. Consequently, conservation strategies need to be species‐specific and address stream‐specific reasons for decline. As a basis, accurate and comparable monitoring data are necessary, which implies the standardization of monitoring protocols.