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

1. Knowledge of species distribution is of utmost importance for conservation and management of endangered freshwater mussels. Conventional monitoring approaches are often time consuming and costly. The use of environmental DNA (eDNA) is a relatively new approach and considered as an effective tool to detect the presence of target species in aquatic environments. The aim of this study was to establish a nested PCR system to detect eDNA of Margaritifera margaritifera and to discuss the advantages and disadvantages of eDNA in mussel surveys compared with classical monitoring.
2. DNA of M. margaritifera was detected in 2 L water samples collected directly downstream (25 m) from pearl mussel populations (population size from 800–20 000), with an internal‐nested PCR approach greatly increasing the detection sensitivity (down to 10 fg target DNA). eDNA detection at greater distances downstream (500 and 1 000 m) of these populations failed, possibly due to DNA degradation or dilution processes. eDNA was also detected downstream of an extinct population, most likely resulting from overlooked mussels or the release of DNA from dead shells.
3. The eDNA approach proposed herein may be helpful in initial screening of streams that are otherwise difficult to monitor, or in the detection of buried juvenile mussels without disturbing their habitat. However, it cannot replace monitoring of population demography, and of other important information for conservation, and should thus only be seen as a supplementary tool.

Copyright © 2015 John Wiley & Sons, Ltd.     

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

1. Determining the occurrence and site occupancy of rare and endangered species can be challenging, particularly without causing harm or stress to the species of concern.
2. Environmental DNA (eDNA) detection was used to assess habitat occupancy by spotted gar (Lepisosteus oculatus), which is federally listed as Threatened in Canada, with known occurrences limited to a small number of locations in southern Ontario.
3. Quantitative polymerase chain reaction (qPCR) assays were developed to detect spotted gar eDNA, which was detected in all but one previously recorded location. The eDNA method was shown to be more effective than traditional netting for detecting spotted gar habitat use.
4. The use of qPCR allowed for quantification of substantial variation in detection strength (copy number) among replicate eDNA samples, with implications for establishing sampling designs for detection and surveillance.
5. The use of eDNA for detection and monitoring of aquatic species of conservation concern shows great potential as a non‐invasive method for assessing species occurrences and habitat occupancy, as well as for informing targeted sampling by conventional capture methods.

Copyright © 2016 John Wiley & Sons, Ltd.     

Monitoring a loss: Detection of the semi‐aquatic crocodile lizard (Shinisaurus crocodilurus) in inaccessible habitats via environmental DNA

1. Assessing the conservation status of a species is strongly dependent upon data on species distribution and abundance. With the emergence of novel methods for species monitoring – such as the use of environmental DNA (eDNA) – monitoring success can be improved at reduced expenditure in the field, particularly in remote regions and terrains where access is difficult or dangerous.
2. The highly endangered crocodile lizard (Shinisaurus crocodilurus Ahl, 1930) inhabits fragmented sites of the remaining evergreen forest with running water systems in a narrow distribution range in southern China and north‐east Vietnam. Crocodile lizards spend most of the day within or above water bodies, which are commonly remote and inaccessible.
3. To monitor recent spatial occurrences, and to confirm the persistence or extinction of previously reported populations (especially in heavily altered habitats), the suitability of using eDNA and quantitative polymerase chain reaction (qPCR) was tested as an alternative method for monitoring this semiaquatic lizard.
4. To assess the accuracy and limitations of this method, eDNA results from the field were compared with eDNA data from mesocosms and census data on the actual abundance of this species in the field.
5. Environmental DNA of the crocodile lizard was detected in all of the positive controls, and in four of six natural sites; thus, all data collected using traditional field surveys were confirmed with eDNA results.
6. eDNA monitoring was found to be a reliable method for assessing the viability of populations; we suggest that it should be developed as a tool for efficient wildlife management, particularly under difficult field and funding conditions.

     

Developing a standard approach for monitoring freshwater pearl mussel (Margaritifera margaritifera) populations in European rivers

1. The freshwater pearl mussel (Margaritifera margaritifera) is an endangered species in Europe, protected nationally and internationally, but with a steadily declining range and abundance owing to pressures such as pollution, river engineering, and illegal exploitation. Despite this, no consistent approaches have been developed around Europe for monitoring pearl mussel populations and their habitats.
2. To address this need, experts on pearl mussel ecology from 11 countries met at a series of workshops in order to develop a protocol for monitoring, published under the auspices of the European Committee for Standardization (CEN). This standard is unique, as it is the first CEN standard dedicated to a single species of conservation concern.
3. The standard is aimed at scientists, conservation bodies, and environmental regulators, and can be used for designing national monitoring programmes as well as reporting on the conservation status of pearl mussel populations under the European Habitats Directive. It contains guidance at the individual site level to determine why populations are failing to recruit, but also addresses the need for a wider‐scale approach to ensure that catchment developments do not have adverse impacts on rivers containing pearl mussels.
4. A pearl mussel monitoring programme needs to investigate the size and viability of populations, as well as the fish hosts (Atlantic salmon, Salmo salar, or brown trout, Salmo trutta) on which pearl mussel larvae depend. Water quality, including variables such as dissolved oxygen, acid–base chemistry, and nutrient levels, is also an essential monitoring component, together with the physical features of the river bed, river flow regimes, and sediment dynamics.
5. It is hoped that this pan‐European approach will improve the ability to compare data across many countries, and will ultimately ensure that the results of monitoring are translated into measures for improving the conservation status of the freshwater pearl mussel throughout its range.

     

Reproductive ecology of the threatened and endemic freshwater mussel Lampsilis bracteata

1. The Texas fatmucket, Lampsilis bracteata, is a unionid mussel endemic to the Colorado and upper Guadalupe River basins of Central Texas and a candidate for federal listing under the Endangered Species Act. There is increased interest in propagation and population restoration of threatened mussels in Texas as a potential conservation method, but still little is known about their life histories and how local populations may differ in aspects of their reproductive ecology, e.g. timing of brooding and potential local adaptations to host fish.
2. The purpose of this study was to compare host fish compatibilities and seasonality of reproduction between two populations: (a) by cross‐infesting fish from the San Saba and Llano Rivers in the Colorado River basin with sympatric and allopatric mussel larvae in the laboratory (hatchery‐produced Guadalupe bass and largemouth bass were also infested); and (b) by assessing gamete concentration, brooding period, viability of larvae and sex ratios using monthly sampling events between February 2017 and February 2018.
3. Reproduction varied with season and between populations. The proportion of females brooding tended to be lower in the summer and the autumn, and higher during winter and spring months before peak water temperatures were reached. The sex ratio in both populations did not significantly differ from 1:1. Fecundity and larvae viability were higher in the Llano River population compared with the San Saba population. Trematode flatworms were found in several female gonad samples from the San Saba population and in a few samples from the Llano population.
4. The highest metamorphosis success occurred on wild green sunfish and largemouth bass, and hatchery‐produced largemouth and Guadalupe bass. The average metamorphosis success tended to be higher for some mussel–fish pairings originating from the same tributary, suggesting that mussels may be locally adapted to host fish, which should be considered in conservation efforts.

     

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.

     

Insights into population genetics,connectivity and demographic history of the longnosed skate Dipturus oxyrinchus (Linnaeus, 1758) in the western Mediterranean Sea

1. Genetic information is crucial for the conservation of Dipturus oxyrinchus (Linnaeus, 1758), a threatened large skate with declining populations over most of its geographical range. The main aim of the present study was to investigate the genetic structure, connectivity and demographic history of the longnosed skate in Sardinia (western Mediterranean Sea).
2. Patterns of population structure were assessed in 175 specimens from six sampling sites. Variation in two mitochondrial genes (cytochrome c oxidase subunit I (COI) and control region) highlighted high genetic diversity and low but significant genetic differentiation among sites, which clustered into three groups corresponding to the north‐west, north‐east and south Sardinian coasts.
3. The observed genetic structuring could presumably depend on a combination of past geological events, contemporary restrictions to dispersal and biological characteristics of the species (e.g. site‐fidelity, no pelagic larval stage, limited dispersal of juveniles and/or adults).
4. Demographic analyses showed signs of past population expansion, but substantial current stability of Sardinian populations. From a conservation perspective, these results are encouraging, and indicate that Sardinian populations are still large and stable, and seem not to have suffered negative side‐effects from the ever‐growing fishing pressure in the region.
5. The occurrence of genetic structuring strongly supported the close monitoring of populations to identify any erosion of their gene pool, and high genetic variability of the Sardinian D. oxyrinchus populations could thus represent priority populations for conservation purposes, providing potential sources for recolonization in cases of local extinctions in other areas of the distribution range of the species.
6. When the sequences from Sardinia were compared with those available from other areas, the data seem to exclude the possibility that the Atlantic and Mediterranean host totally isolated populations or even different species, as recently suggested. However, additional markers and a larger sampling sites are needed to confirm these findings.

     

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

1. European freshwater pearl mussels are among the most critically threatened bivalve molluscs. Margaritifera margaritifera and Margaritifera durrovensis are endangered, and both occur in Ireland and are currently listed separately in Annex II of the European Habitats Directive. This study had the objective of guiding the conservation of freshwater pearl mussels in Ireland based on a genetic characterization of the most important Irish populations of both species using microsatellite and mitochondrial DNA (mtDNA) analyses.
2. Margaritifera durrovensis from the Nore was found to have the highest genetic divergence from all the populations studied; however, there was substantial relatedness between the genetic constitution of the Nore and of central–eastern M. margaritifera populations, placing the Nore pearl mussel within the M. margaritifera taxon (Habitats Directive species code 1029). Two main conservation units of pearl mussel were identified: a mostly salmon‐dependent western cluster and a trout‐dependent central–eastern cluster.
3. The genetic diversity of western Irish freshwater pearl mussels, as expressed by allelic richness and observed heterozygosity, was greater by a factor of two than in central European and central–eastern Irish populations, suggesting a genetic diversity hot spot and low effects of genetic drift and selection. The trout‐dependent central and eastern populations had much lower genetic variability, but had the greatest differentiation and uniqueness.
4. Conservation of freshwater pearl mussels in Ireland should recognize the existence of a minimum of two conservation units (western and central–eastern) that differ in their use of host fish and in geographic isolation. Low levels of genetic drift and inbreeding in western populations should be secured by sustaining optimal habitat conditions favourable for the recruitment of mussels and their migratory salmonid hosts. The small population sizes of central–eastern populations and problems with recruitment require urgent action, e.g. by captive breeding and augmentation, to prevent any further erosion of their genetic variability.

     

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.

     

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

1. Environmental DNA (eDNA) assays are valuable tools for monitoring the presence and distribution of cryptic species.
2. Like many freshwater mussels, the numbers of dwarf wedgemussel, Alasmidonta heterodon, have dwindled and its range has diminished. As of its listing in 1993, only 10–20 locations were known to persist out of the 70 Atlantic slope locations known historically.
3. A quantitative polymerase chain reaction (qPCR) assay to detect the presence of A. heterodon was developed that uses two probes to accommodate a single‐nucleotide polymorphism (SNP) in the probe‐binding site within the cytochrome c oxidase subunit I (COI) gene. This SNP defines northern and southern major phylogenetic lineages.
4. The primers match exactly the previously determined COI sequences of 20 dwarf wedgemussel individuals representing Atlantic slope populations from North Carolina, Virginia, Maryland, New York, and New Hampshire. Other than for the qPCR assay described here, these primers can be used for sequencing or metabarcoding to further delineate dwarf wedgemussel populations phylogenetically.
5. A simple eDNA preparation method is introduced using flocculation to concentrate free DNA in solution as well as cellular material (including shed animal cells, bacteria, viruses, and dissolved DNA).
6. In addition to the specific application described here, the methodological approaches used in this study are widely applicable to conservation, including, but not limited to, general aquatic biodiversity, phylogenetic studies, and the detection of pathogenic microbes.