Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate ...occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In many North American rivers, populations of multiple species of non-native cyprinid fishes are present, including black carp (Mylpharyngodon piceus), grass carp (Ctenopharyngodon idella), bighead ...carp (Hypophthalmichthys nobilis), silver carp (Hypophthalmichthys molitrix), common carp (Cyprinus carpio), and goldfish (Carassius auratus). All six of these species are found in the Mississippi River basin and tracking their invasion has proven difficult, particularly where abundance is low. Knowledge of the location of the invasion front is valuable to natural resource managers because future ecological and economic damages can be most effectively prevented when populations are low. To test the accuracy of environmental DNA (eDNA) as an early indicator of species occurrence and relative abundance, we applied eDNA technology to the six non-native cyprinid species putatively present in a 2.6 river mile stretch of the Chicago (IL, USA) canal system that was subsequently treated with piscicide. The proportion of water samples yielding positive detections increased with relative abundance of the six species, as indicated by the number of carcasses recovered after poisoning. New markers for black carp, grass carp, and a common carp/goldfish are reported and details of the marker testing to ensure specificity are provided.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Non-invasively collected faecal samples are an alternative source of DNA to tissue samples, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although ...several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new 'High Volume- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol' (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of single nucleotide polymorphism (SNP) markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To inform management and conservation decisions, environmental DNA (eDNA) methods are used to detect genetic material shed into the water by imperiled and invasive species. Methodological ...enhancements are needed to reduce filter clogging, PCR inhibition, and false-negative detections when eDNA is at low concentrations. In the first of three simple experiments, we sought to ameliorate filter clogging from particulates and organic material through a scaled-up, multi-filter protocol. We combined four filters in a 5 mL Phenol-Chloroform-Isoamyl (PCI) procedure to allow for larger volumes of water (~1 L) to be filtered rapidly. Increasing the filtered water volume by four times resulted in 4.4X the yield of target DNA. Next, inhibition from organic material can reduce or block eDNA detections in PCR-based assays. To remove inhibitory compounds retained during eDNA isolation, we tested three methods to chemically strip inhibitors from eDNA molecules. The use of CTAB as a short-term (5-8 day) storage buffer, followed by a PCI isolation, resulted in the highest eDNA yields. Finally, as opposed to a linear relationship among increasing concentrations of filtered genomic eDNA, we observed a sharp change between the lower (70-280 ng) and higher (420-560 ng) amounts. This may be important for effectively precipitating eDNA during protocol testing.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The northern shrimp (Pandalus borealis Krøyer) population in the Gulf of Maine collapsed during an extreme heatwave that occurred across the Northwest Atlantic Ocean in 2012. Northern shrimp is a ...boreal species, and reaches its southern limit in the Gulf of Maine. Here we investigate proximate causes for the population collapse using data from fishery-independent surveys, environmental monitoring, and the commercial fishery. We first examined spatial data to confirm that the decline in population estimates was not due to a major displacement of the population, and then tested hypotheses related to fishing mortality and shifts in predation pressure. Fishing mortality may have contributed but could not explain the magnitude of the decline or the disappearance of pre-exploitable size individuals. Stomach contents analysis and biomass trends revealed no new fish predators of shrimp. However, longfin squid (Doryteuthis pealeii Lesueur) was unique among all species in showing time-series biomass peaks during spring, summer and fall of 2012, and spatial overlap with northern shrimp was unusually high in 2012. Longfin squid is a voracious and opportunistic predator that consumes crustaceans as well as fish. We hypothesize that the warmer temperatures of 2012 not only led to expansion of longfin squid distribution in Gulf of Maine, but had differential effects on migration phenology that further increased spatial overlap with northern shrimp. The weight of our evidence suggests that longfin squid predation was likely a significant factor in the collapse of northern shrimp in the Gulf of Maine.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Global conservation policy and action have largely neglected protecting and monitoring genetic diversity—one of the three main pillars of biodiversity. Genetic diversity (diversity within ...species) underlies species’ adaptation and survival, ecosystem resilience, and societal innovation. The low priority given to genetic diversity has largely been due to knowledge gaps in key areas, including the importance of genetic diversity and the trends in genetic diversity change; the perceived high expense and low availability and the scattered nature of genetic data; and complicated concepts and information that are inaccessible to policymakers. However, numerous recent advances in knowledge, technology, databases, practice, and capacity have now set the stage for better integration of genetic diversity in policy instruments and conservation efforts. We review these developments and explore how they can support improved consideration of genetic diversity in global conservation policy commitments and enable countries to monitor, report on, and take action to maintain or restore genetic diversity.
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The rapidly emerging field of macrogenetics focuses on analysing publicly accessible genetic datasets from thousands of species to explore large-scale patterns and predictors of intraspecific genetic ...variation. Facilitated by advances in evolutionary biology, technology, data infrastructure, statistics and open science, macrogenetics addresses core evolutionary hypotheses (such as disentangling environmental and life-history effects on genetic variation) with a global focus. Yet, there are important, often overlooked, limitations to this approach and best practices need to be considered and adopted if macrogenetics is to continue its exciting trajectory and reach its full potential in fields such as biodiversity monitoring and conservation. Here, we review the history of this rapidly growing field, highlight knowledge gaps and future directions, and provide guidelines for further research.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Background
Environmental DNA (eDNA) analysis is increasingly being used to detect the presence and relative abundance of rare species, especially invasive or imperiled aquatic species. The rapid ...progress in the eDNA field has resulted in numerous studies impacting conservation and management actions. However, standardization of eDNA methods and reporting across the field is yet to be fully established, with one area being the calculation and interpretation of assay limit of detection (LOD) and limit of quantification (LOQ).
Aims
Here, we propose establishing consistent methods for determining and reporting of LOD and LOQ for single‐species quantitative PCR (qPCR) eDNA studies.
Materials & Methods/ Results
We utilize datasets from multiple cooperating laboratories to demonstrate both a discrete threshold approach and a curve‐fitting modeling approach for determining LODs and LOQs for eDNA qPCR assays. We also provide details of an R script developed and applied for the modeling method.
Discussion/Conclusions
Ultimately, standardization of how LOD and LOQ are determined, interpreted, and reported for eDNA assays will allow for more informed interpretation of assay results, more meaningful interlaboratory comparisons of experiments, and enhanced capacity for assessing the relative technical quality and performance of different eDNA qPCR assays.
We propose establishing consistent methods for determining and reporting of LOD and LOQ for single‐species quantitative PCR (qPCR) eDNA studies. We demonstrate the use of both a discrete threshold approach and a curve‐fitting modeling approach for determining LODs and LOQs for these assays. Ultimately, standardization of how LOD and LOQ are determined, interpreted, and reported for eDNA assays will allow for more informed interpretation of assay results, more meaningful interlaboratory comparisons of experiments, and enhanced capacity for assessing the relative technical quality and performance of different eDNA qPCR assays.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The International Union for Conservation of Nature (IUCN) Red List is an important and widely used tool for conservation assessment. The IUCN uses information about a species’ range, population size, ...habitat quality and fragmentation levels, and trends in abundance to assess extinction risk. Genetic diversity is not considered, although it affects extinction risk. Declining populations are more strongly affected by genetic drift and higher rates of inbreeding, which can reduce the efficiency of selection, lead to fitness declines, and hinder species’ capacities to adapt to environmental change. Given the importance of conserving genetic diversity, attempts have been made to find relationships between red‐list status and genetic diversity. Yet, there is still no consensus on whether genetic diversity is captured by the current IUCN Red List categories in a way that is informative for conservation. To assess the predictive power of correlations between genetic diversity and IUCN Red List status in vertebrates, we synthesized previous work and reanalyzed data sets based on 3 types of genetic data: mitochondrial DNA, microsatellites, and whole genomes. Consistent with previous work, species with higher extinction risk status tended to have lower genetic diversity for all marker types, but these relationships were weak and varied across taxa. Regardless of marker type, genetic diversity did not accurately identify threatened species for any taxonomic group. Our results indicate that red‐list status is not a useful metric for informing species‐specific decisions about the protection of genetic diversity and that genetic data cannot be used to identify threat status in the absence of demographic data. Thus, there is a need to develop and assess metrics specifically designed to assess genetic diversity and inform conservation policy, including policies recently adopted by the UN's Convention on Biological Diversity Kunming‐Montreal Global Biodiversity Framework.
La diversidad genética y los estados de la Lista Roja de la UICN
Resumen
La Lista Roja de la Unión Internacional para la Conservación de la Naturaleza (UICN) es una importante herramienta de uso extendido para evaluar la conservación. La UICN utiliza datos sobre la distribución y tamaño poblacional de una especie, la calidad y niveles de fragmentación de su hábitat y sus tendencias de abundancia para valorar su riesgo de extinción, A pesar de que la diversidad genética afecta al riesgo de extinción, la UICN no la considera. La deriva génica y las tasas altas de endogamia afectan con mayor fuerza a las poblaciones en declinación, lo que puede reducir la eficiencia de la selección, derivar en la disminución de la aptitud y dificultar la capacidad de una especie de adaptarse ante el cambio ambiental. Se ha intentado encontrar la relación entre la diversidad genética y el estado en las listas rojas ya que su conservación es muy importante. Aun con lo anterior, no hay un consenso actual sobre si la diversidad genética está capturada en las categorías vigentes de la Lista Roja de la UICN de manera que sea informativa para la conservación. Para poder evaluar el poder predictivo de la correlación entre la diversidad genética y el estado en la Lista Roja de los vertebrados, sintetizamos trabajos previos y analizamos de nuevo los conjuntos de datos con base en tres tipos de información genética: ADN mitocondrial, microsatélites y genomas completos. Las especies con un estado de riesgo de extinción más alto fueron propensas a una diversidad genética más baja para todos los tipos de marcadores, aunque estas relaciones fueron débiles y variaron entre los taxones, lo cual es coherente con trabajos anteriores. Sin importar el tipo de marcador, la diversidad genética no fue un identificador certero de las especies amenazadas en ninguno de los grupos taxonómicos. Nuestros resultados indican que el estado de lista roja no es una medida útil para guiar las decisiones específicas por especie en relación con la protección de la diversidad genética. También indican que los datos genéticos no pueden usarse para identificar el estado de amenaza si no se tienen los datos demográficos. Por lo tanto, es necesario desarrollar y evaluar las medidas diseñadas específicamente para valorar la diversidad genética e informar las políticas de conservación, incluidas las que adoptó recientemente la ONU en el Convenio del Marco Mundial Kunming‐Montreal de la Diversidad Biológica.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The federally endangered Cape Sable seaside sparrow (
Ammospiza maritima mirabilis
) is endemic to the Greater Everglades ecosystem in southern Florida, inhabiting fragmented marl prairies in six ...individual subpopulations. The subspecies is threatened by loss of breeding habitat from fire and water management. Genetic information is severely limited for the subspecies but could help inform decisions regarding subpopulation protections and potential translocations for genetic rescue. To provide genetic data and inform management efforts, feather samples were collected across five subpopulations (designated A–E) and protocols were tested to optimize DNA extraction yields. We assessed four mitochondrial DNA markers (N = 36–69) and 12 nuclear microsatellite loci (N = 55) in 108 sparrows. Mitochondrial DNA sequences revealed low haplotype diversity, with NADH dehydrogenase-2 haplotypes matching to most other extant subspecies and to the Atlantic coast subspecies. Nuclear diversity was low compared to other subspecies, but similar across subpopulations. Samples grouped as one population when analyzed by Principal Component Analysis, Bayesian modelling and genetic distance metrics. Limited genetic emigration was detected from one putative migrant. Relatedness was significantly different for sparrows in the most geographically distant subpopulation (A), likely reflecting high self-recruitment and natal site fidelity (
P
= 0.003). The low to moderate effective population size (N
E
= 202.4; N
E
:N
C
= 0.06) and generation time estimates indicated that unique genetic variation could be lost quickly during stochastic events. The sample sizes were limited, which reduced the power to comprehensively address recent population size reductions and any subsequent loss of genetic diversity.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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