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.     

Use of genetic markers to identify the illegal trade of billfish in the second largest fishing warehouse of Latin America

1. Billfish are oceanic pelagic species that are often caught by tuna fleets and are of great interest for sport fishing. Two species of billfish have specific legislation prohibiting their marketing and export in Brazil.
2. DNA barcoding is a universal system of molecular identification based on a sequence of mitochondrial DNA cytochrome oxidase subunit I (COI), which serves as a diagnostic genomic marker in each species.
3. The barcode DNA technique was used to identify billfish marketed in the second largest fishing warehouse in Latin America, the CEAGESP (Companhia de Entrepostos e Armazéns Gerais de São Paulo), located in São Paulo, Brazil. Seventy‐nine samples of billfish were collected during three inspection visits carried out by Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis.
4. After DNA sequencing, 70 samples (88.60%) were identified to the species level; 21 (30.00%) were identified as Xiphias gladius, 43 (61.42%) as Istiophorus platypterus and six (8.57%) as Kajikia albida. The sale of this latter species is prohibited in Brazil and it is considered Vulnerable on the list of endangered species of the IUCN and in the official list of species of endangered Fauna – Fish and Aquatic Invertebrates.
5. Molecular analyses proved to be very efficient at uncovering irregularities in the identification of the white marlin (K. albida), which was traded illegally in CEAGESP, demonstrating the ineffectiveness of the current monitoring techniques used and emphasizing the need for the adoption of better public policies for the conservation of the species.

     

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

1. Documenting the occurrence and habitat occupancy of rare aquatic species is an ongoing challenge for conservation. Characterization of environmental DNA (eDNA) from bulk water samples has emerged as a powerful tool to infer species presence or absence without the need to observe or handle organisms.
2. Previous eDNA studies have yet to develop species‐specific markers that target taxa with many potentially sympatric confamilials. Forty‐one freshwater pearly mussel species (Unionidae) are found in southern Ontario, Canada, with many of these listed as threatened, endangered, or of conservation concern; however, locating populations for protection can be challenging owing to morphological crypsis and species scarcity.
3. Species‐specific eDNA markers were developed to target four unionid species. Following in silico and in vitro validation, markers were validated in the field by comparing eDNA results from water samples to detections based on quadrat sampling.
4. Target species were detected by eDNA sampling at all sites where they had previously been located by quadrat sampling.
5. The paired sampling design showed that species‐specific markers can be designed even within speciose families, and that eDNA detection of mussels is at least as sensitive as quadrat sampling. Furthermore, detection probabilities were not affected by sampling depth, and eDNA concentrations were positively correlated with mussel densities.
6. These findings confirm that eDNA assays are a valuable complement to traditional methods for locating and managing imperilled unionid populations.

     

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.

     

A simple,rapid method for detecting seven common invasive fish species in Europe from environmental DNA

1. Biological invasions are a global threat to biodiversity, and many arise from deliberate introductions.
2. The American freshwater fish Micropterus salmoides and Ameiurus spp. (Ameiurus melas and Ameiurus nebulosus) were introduced to Europe for recreational fishing, Gambusia holbrooki and Gambusia affinis were introduced for mosquito population control, and Lepomis gibbosus was introduced as an ornamental species. The Asiatic Pseudorasbora parva was acquired inadvertently as an accompanying species in fish consignments.
3. This article presents a novel approach for detecting these species directly from water samples based on a panel of five taxon‐specific primers within 16S rDNA.
4. The primers were validated from tissue, in aquarium experiments, and from Ebro River water samples (Spain). With a simple polymerase chain reaction (PCR) protocol, followed by visualization in agarose gel or capillary electrophoresis, it was possible to detect these species from environmental DNA concentrations as low as 0.89–100 pg mL^–1.
5. This sensitive and economical tool can be used to control European invasions of these species and to preserve native biodiversity.

     

Identification of microsatellite loci and examination of genetic structure for the endangered springsnails Juturnia kosteri and Pyrgulopsis roswellensis in the Chihuahuan Desert

1. Desert spring ecosystems harbour many endemic species that are threatened with extinction owing to human activities. Springsnails (superfamily Truncatelloidea) are among the most diverse freshwater snail groups in desert springs of western North America. They occupy geographically narrow ranges, often consisting of a single spring or spring complex.
2. Microsatellite markers were developed via high‐throughput de novo sequencing for two endangered springsnail species, Juturnia kosteri and Pyrgulopsis roswellensis, endemic to springs on Bitter Lake National Wildlife Refuge (BLNWR) near Roswell, New Mexico, USA. Genetic diversity, population structure, effective population size (N_e), and historic demography were assessed in two populations for each species.
3. A large number of putative markers were identified, of which 14 loci for J. kosteri and 18 loci for P. roswellensis showed polymorphism. Genetic diversity and N_e were greater in P. roswellensis than J. kosteri. Significant genetic differentiation (F_ST = 0.187–0.288) was found among populations for both species, indicating currently limited gene flow. Neither species showed signs of recent population bottlenecks.
4. Analyses with microsatellite markers revealed fine‐scale population genetic structure in both species, unlike a previous study using mitochondrial DNA sequences. Although these species co‐occur on BLNWR, slightly different genetic characteristics between them suggest that they may have different evolutionary and demographic histories. Small N_e relative to census size indicates strong genetic drift in these populations. Future efforts should be directed to identifying causes of such strong drift.
5. Because there are limited numbers of microsatellite markers available for springsnails, the markers identified in this study should be useful for congeners via cross‐species amplification. Understanding current genetic variation and structure of these species is not only required for species recovery, but will also be useful for comparisons among many aquatic organisms endemic to springs of western North America.

Copyright © 2016 John Wiley & Sons, Ltd.     

Niche‐based species distribution models and conservation planning for endangered freshwater crayfish in south‐western Germany

1. Niche‐based species distribution models (SDMs) can help conservation planning by forecasting environmental suitability for an endangered species. Here, SDMs were constructed for stone crayfish (Austropotamobius torrentium) and white‐clawed crayfish (Austropotamobius pallipes s. str.) to identify catchments in south‐western Germany where environmental conditions are favourable for reintroduction.
2. Maximum‐entropy modelling (Maxent) was used with presence‐only data to forecast environmental suitability for the two crayfish species based on five climate variables, slope, land cover, and a human impact index.
3. SDMs showed good to excellent performance and were able to capture the range of both Austropotamobius species. Presence probabilities were mostly determined by climate variables, and climate niches partly overlap, with white‐clawed crayfish occurring at conditions with less extreme winter temperatures and lower temperature seasonality than stone crayfish. Human impact contributed between 10 and 27% to the models and was negatively related to presence probabilities. Contribution of land cover was low (5%) but showed a positive relationship with deciduous broadleaf forest in both species.
4. Both SDMs indicated several catchments with high predicted environmental suitability but no present occurrence records. Subsequent crayfish and habitat surveys in these catchments revealed four streams considered suitable for reintroduction and led to discovery of five previously unknown white‐clawed crayfish populations. Overall, SDMs proved to be a powerful tool for conservation planning of freshwater crayfish species.

     

Illegal trade of the guitarfish Rhinobatos horkelii on the coasts of central and southern Brazil: genetic identification to aid conservation

1. Among the diverse species of guitarfish, Rhinobatos horkelii is endemic to the south‐west Atlantic and is primarily found off the Brazilian coast. The IUCN has classified this species as being in critical danger of extinction owing to widespread exploitation.
2. Currently, this species is protected under Brazilian conservation laws. However, the morphological similarity of R. horkelii to other species precludes effective protection from fishing.
3. Guitarfish samples were obtained from fishermen in different regions along the Brazilian coast and were identified using a genetic forensic method (multiplex‐PCR). The analysis showed that 56% of the samples analysed were from R. horkelii, 25% from Rhinobatos percellens and 19% from Zapteryx brevirostris confirming that R. horkelii continues to be caught, despite conservation legislation.
4. These results stress the need for effective conservation measures and may help to alert others to the occurrence of R. horkelii poaching. In addition, this work aims to establish an effective method of species identification to help prevent poaching of protected species such as R. horkelii.

Copyright © 2012 John Wiley & Sons, Ltd.     

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.

     

Development and multiplexing of microsatellite loci for the near threatened freshwater mussel Potomida littoralis (Cuvier, 1798) using 454 sequencing

1. The Unionidae are among the most endangered fauna in the world and globally in decline. They are particularly vulnerable to habitat loss and fragmentation, susceptible to flow, pollution and climatic disturbances and introduction of invasive species. Despite their well‐recognized ecological and conservation importance, there is a surprising lack of genomic resources currently available for European species.
2. The aim of this study was to develop and characterize microsatellites for the near threatened freshwater mussel, Potomida littoralis using 454 sequencing.
3. In order to improve genotyping throughput as well as cost‐effectiveness, two multiplex‐PCR reactions were designed to amplify 16 new loci. All the new 16 microsatellites were successfully combined in two multiplexed PCR with the number of alleles ranging from 2 to 25 per locus (with a mean of 10), confirming the utility of the new markers.
4. The new genetic markers can therefore be used for studying the population genetic structure and evolution of this species, e.g. to examine current levels of genetic variability within and between populations and thus to contribute to conservation and management. Copyright © 2013 John Wiley & Sons, Ltd.