Researchers will attempt to answer critical questions regarding the optimal amount of sampling effort, timing, sample location, as well as field and laboratory methods for detecting AIS across a range of lake types and AIS abundances. Christopher M Merkes. Prior to his current position, he was a conservation scientist at the Orianne Society from November 2009 through August 2016. After an in vitro validation step, following the protocol outlined in Appendix S7 (Tréguier et al., 2014), all assays were shown to be species‐specific and gave high reliability in both in situ and ex situ trials. We thank the Editor and the three anonymous reviewers for their constructive comments.

We targeted two threatened killifish and one non‐native invasive species, known to be spreading across Europe at a rapid rate (Freyhof et al., 2014; Grapputo, Bisazza, & Pilastro, 2006). The updates, modifications, and improvements have also been validated in several laboratories, and have been peer-reviewed, as part of the on-going GLRI funded interagency eDNA Calibration Study. However, we opted for the larger pore size of the two as, in the field, filters can get clogged by sediment, affecting the amount of water that can be filtered (Goldberg et al., 2016). Such high densities may therefore be playing a yet undetermined role on the reliability or sensitivity of this assay. The most prevalent use in current research is using eDNA to study the location… Using our novel assays, we were able to highlight six sites where V. letourneuxi were found and six sites for V. robertae. 1122 0 obj <>stream When electrofishing was undertaken at depths < 1.5 m (wadable sites), a single 100 m electrofishing pass was conducted from downstream to upstream. DNA was extracted from tissue samples of these species using the Qiagen DNeasy® Blood and Tissue Kit following the manufacturer's instructions. Any queries (other than missing content) should be directed to the corresponding author for the article. The manuscript was written by Q.M and M.S and reviewed by all authors. As two filter types were compared for assessing their efficiency for eDNA collection, five of these samples were filtrated through a sterile 0.45 µm Sterivex ™ HV filters (Sterivex™ filter unit, HV with luer lock outlet, Merck®, Millipore®), and the remaining five were filtrated through a sterile 0.22 µm Sterivex ™ GP filters (Sterivex™ filter unit, GP with luer lock outlet, Merck®, Millipore®). Such a result which is also common with physical monitoring methods can and often does mean management decisions designed on such data is not 100% perfect, notably, as failure to detect the organism in question is not proof of absence. The range of both Valencia species is thought to now be restricted to only 12 systems, and these populations are thought to be vulnerable.

At the four brackish sites (“7B,” “8B,” “11B,” and “13B,” Appendix S7, Table S2), sampling was conducted with a D‐shaped net (minimum 8 sweeps and maximum 22 sweeps). Institute of Marine Biological Resources and Inland Waters, Hellenic Centre for Marine Research, Anavissos, Greece, Research Institute for Nature and Forest, Geraardsbergen, Belgium. Once widely distributed in Western Greece (Barbieri, Daoulas, Psarras, Stoumboudi, & Economou, 2000), the distribution ranges of the two native species have now been drastically reduced over the last 40 years. The species was much more adaptable than originally thought and exhibited high behavioral plasticity further ensuring its success even in degraded habitats (Vargas & de Sostoa, 1996).

These requirements lead to constraints on the number and extent of surveys that can be undertaken. Environmental DNA (eDNA) detection is now widely utilized as an alternative tool for monitoring a number of species (Sepulveda et al., 2019; Thomas et al., 2019; Vilaça et al., 2020; Wacker et al., 2019).

Although the target fragments of our assays ranged between 113 and 167 bp, we saw no such variation in the reliability of the assays, all performing equally well and accurately. Working off-campus? Regardless of the cause, such a result highlights an important issue which needs to be dealt with. Long‐term and recent trends in global wetland area, Effects of large river restoration on currently used bioindicators and alternative metrics, ednaoccupancy : An r package for multiscale occupancy modelling of environmental DNA data, The freshwater ichthyofauna of Greece ‐ an update based on a hydrographic basin survey, Environmental DNA (eDNA) detects the pool frog (, Sampling designs for landscape‐level eDNA monitoring programs, Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding, Mitochondrial genome sequencing and development of genetic markers for the detection of DNA of invasive bighead and silver carp (, Introduction pathways and establishment rates of invasive aquatic species in Europe, Critical considerations for the application of environmental DNA methods to detect aquatic species, Invasion success despite reduction of genetic diversity in the European populations of eastern mosquitofish (Gambusia holbrooki), Environmental DNA assays for invasive populations of the Black Carp, Mylopharyngodon piceus, in North America (eDNA) Assays for Invasive Populations of Black Carp in North America, Climate change, keystone predation, and biodiversity loss, Needle in a haystack? Declining biodiversity, driven primarily by increasing anthropogenic activities such as habitat degradation and/or overexploitation, coupled with the effects of climate change, is significantly altering nearly every ecosystem on our planet (Cardinale et al., 2012; Harley, 2011; Hooper et al., 2012). In this study, we assess the use of environmental DNA detection as a rapid and effective tool for monitoring the occurrence of two threatened freshwater killifish species: Valencia letourneuxi (Sauvage, 1880), Valencia robertae (Freyhof, Kärst, & Geiger, 2014), and of the alien invasive Gambusia holbrooki (Girard, 1859) in Greek aquatic systems. Statistical analyses were performed with R, version 3.4.1 (R Core Team, 2018). Q.M performed DNA extraction and qPCR. All eDNA samples collected during this trial were tested with both killifish assays as a complimentary step for assessing the specificity and reliability of our method. E-mail address: rerickson@usgs.gov. Feedback from French restoration projects, Standards for ecologically successful river restoration: Ecological success in river restoration, Alien freshwater fish species in the Balkans‐Vectors and pathways of introduction, R: A language and environment for statistical computing, Emerging threats and persistent conservation challenges for freshwater biodiversity, Non‐native fish impacts on Mediterranean freshwater ecosystems: Current knowledge and research needs: NON‐NATIVE FISHES IN MEDITERRANEAN FRESHWATERS, Ecosystem approaches to fishery management through essential fish habitat, Using occupancy modelling to compare environmental DNA to traditional field methods for regional‐scale monitoring of an endangered aquatic species, Site occupancy models in the analysis of environmental DNA presence/absence surveys: A case study of an emerging amphibian pathogen (N Yoccoz, Ed), Improved detection of rare, endangered and invasive trout in using a new large‐volume sampling method for eDNA capture, Monitoring the near‐extinct European weather loach in Denmark based on environmental DNA from water samples, Invited overview: Conclusions from a review of electrofishing and its harmful effects on fish, Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter, Rangewide tidewater goby occupancy survey using environmental DNA, A system for rapid eDNA detection of aquatic invasive species, Monitoring endangered freshwater biodiversity using environmental DNA: species monitoring by environmental DNA, Environmental DNA – An emerging tool in conservation for monitoring past and present biodiversity, Environmental DNA surveillance for invertebrate species: advantages and technical limitations to detect invasive crayfishProcambarus clarkiiin freshwater ponds, A pound of prevention, plus a pound of cure: Early detection and eradication of invasive species in the Laurentian Great Lakes, Life history of Gambusia holbrooki (Pisces, Poeciliidae) in the Ebro delta (NE Iberian peninsula), Detection of spatiotemporal variation in ranavirus distribution using eDNA, Reduced genetic variation and strong genetic population structure in the freshwater killifish Valencia letourneuxi (Valenciidae) based on nuclear and mitochondrial markers, Downstream transport and seasonal variation in freshwater pearl mussel (, Early detection of a highly invasive bivalve based on environmental DNA (eDNA), Environmental DNA monitoring for short‐term reproductive migration of endemic anadromous species, Shishamo smelt (, https://doi.org/10.1373/clinchem.2008.112797, https://doi.org/10.1038/s41598‐018‐23740‐5, https://doi.org/10.1371/journal.pone.0117803, https://doi.org/10.1080/11250000500502111, https://doi.org/10.1038/s41598‐019‐40977‐w, https://doi.org/10.1016/j.ecolind.2019.02.058, https://doi.org/10.1371/journal.pone.0121655, https://doi.org/10.2478/s11756‐009‐0231‐3, https://doi.org/10.1093/bioinformatics/bts199, https://doi.org/10.1016/j.biocon.2014.11.020, https://doi.org/10.1890/0012‐9658(2002)083[2248:ESORWD]2.0.CO;2, https://doi.org/10.1111/j.1365‐2664.2005.01098.x, http://doi.org/10.1038/s41598‐018‐37001‐y, http://doi.org/10.1038/s41598‐019‐50571‐9, https://doi.org/10.1016/j.jenvman.2014.02.010, https://doi.org/10.1111/j.1365‐2664.2005.01004.x, https://doi.org/10.1111/j.1365‐2400.2011.00842.x, https://doi.org/10.1016/j.biocon.2014.11.023, https://doi.org/10.1007/s11160‐004‐1095‐9, https://doi.org/10.1007/s10592‐019‐01161‐9, https://doi.org/10.1111/j.1365‐294X.2011.05418.x, https://doi.org/10.1016/j.biocon.2014.11.019, http://doi.org/10.1016/j.jglr.2009.11.002, https://doi.org/10.1007/s10530‐017‐1545‐7.

We identified the LOD as the last dilution of the standard curve in which the targeted DNA is amplified with a cycle threshold (Ct) below 45 (Mauvisseau, Burian, et al., 2019; Mauvisseau, Davy‐Bowker, et al., 2019). For the three species, the dilution series ranged from 10–1 to 10–8 using 10 “technical replicates” (i.e., qPCR replicates) for each dilution step, allowing for the assessment of the limit of detection (LOD) and limit of quantification (LOQ) (Figure 1) (Bustin et al., 2009; Mauvisseau, Burian, et al., 2019; Tréguier et al., 2014). In conclusion, our field efforts confirmed the usefulness of eDNA monitoring for the detection of both threatened killifishes and a non‐native and highly invasive species, regarded as responsible for the decline of many native, and often threatened freshwater species across Europe (Freyhof et al., 2014; Grapputo et al., 2006). eDNA Further, as of yet we do not understand the temporal effect of eDNA sampling on either the killifish or G. holbrooki and further studies should assess whether there is an optimal time for sampling or more importantly when results are less reliable (a time not to survey). However, in our case (due to the limited number of habitats hosting our target species and thus the relatively low number of locations surveyed), occupancy modeling may not have helped in this regard. This suggests that the eggs may have increased the amount of eDNA in the system; alternatively, the result may also be explained by an increase in the sloughing rates of the fishes tissue and/or mucus directly (Klymus, Richter, Chapman, & Paukert, 2015). However, that said, the noninvasive nature of eDNA sampling and the comparable results in the field between our assays and more traditional survey methods suggest eDNA is still a sufficient tool for the detection of these species. Sites “1B” to “12B” are within the geographical range or.

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