The primers pairs used in this study, which were utilized to amplify a 12S fragment, are able to distinguish all native Acipenseridae species known to inhabit the Danube. In addition, analysis of samples collected ex situ and the integrative sampling strategy we adopted in situ confirmed that, based on teleo markers and an eDNA metabarcoding approach, are sufficiently sensitive for detecting the occurrence of the endangered Danube sturgeons. Moreover, in this study, we present the first comprehensive large-scale biogeographic snapshot for the sterlet.
Reports regarding the detection of sturgeon species are generally very limited in terms temporal and spatial coverage. This is perhaps not surprising, given that sturgeons are typically bottom-dweller that are nowadays exceptionally rare species in many large river systems, and are exceedingly difficult to detect using traditional monitoring methods. Consequently, most of the currently available data are derived from small-scale scientific studies (sometimes conducted over distances of only several hundred meters). Although fisheries data can provide a broader pictures on larger scales, they are of limited value in certain respects (Guy and Brown 2007). To the best of our knowledge, the only systematic basin-wide traditional fish assessments of the Danube Basin have been the Joint Danube surveys (conducted in 2007, 2013, and 2019) (Bammer et al. 2021; Bammer et al. 2015; Wiesner et al. 2008). However, despite the high sampling effort of more than 2500 electro-fished stretches covering close to 3 Mio m2, only 3 sturgeon individuals at 2 sites have been caught. The use of electrified benthic frame trawls and trammel nets did, nevertheless, enable the capture of sturgeons from 3 additional sampling sites. These findings accordingly serve to highlight that traditional sampling methods have hitherto failed to provide a comprehensive picture regarding the distribution of sturgeon species across a large geographical scale along a longitudinal profile of the Danube. eDNA-based surveys have the potential to overcome these limitations in aquatic systems that are generally unamenable to monitoring using traditional methods (Jerde 2021), and can serve as an initial first step in identifying key habitats and sampling sites for the targeted monitoring of population sizes and recruitment.
However, although there is a growing body of literature highlighting the various merits of eDNA sampling compared with traditional sampling (e.g. Czeglédi et al. 2021; Fediajevaite et al. 2021; Hänfling et al. 2016; Pont et al. 2018; Valentini et al. 2016), particularly with respect to large water bodies and rare species, the technique does, nevertheless, have certain drawbacks and restrictions. Capture-based methods have a distinct advantage in that they can provide information relating to population structure and the condition of individual fish, as well as the certainty of having physical specimens (Jerde 2021). A further limitation is that identification of the presence of a species does not necessarily pinpoint the exact location of fish, particularly in the case of flowing water bodies. Under conditions of hydrological transport, eDNA essentially functions as a carrier of material containing genetic information on the biodiversity of upstream catchments (Deiner et al. 2016). Moreover, detection distances can vary significantly from a few kilometers in small streams to more than 100 km in large rivers (Pont et al. 2018 and references therein). In this regard, this limitation could also be viewed as an advantage, as longer river sections can be integrated in a single sample. A further issue of particular importance in the context of surveys conducted for rare, low-density, or possibly extinct animals is the occurrence of false positive (see further Darling et al. 2021) and false negative readings. For example, false positives can arise in instances in which although target DNA is present in a sample, no living organisms are present in the sampled system. In this case, eDNA could plausibly be derived via contamination from external sources, such as sewage and wastewater discharge or effluents from aquaculture farms upstream of the sampling site (Rees et al. 2014). This is potential applicable in the case of sturgeon species, particularly in the Lower Danube catchment, in which sturgeon are traded in fish markets, served in restaurants, and cultured in fish farms, and hence it is conceivable that DNA originating from these sources might enter river systems. Conversely, false negatives (e.g., organisms in the system but no detectable DNA) could, for example, arise if the amount of target DNA falls below the limit of detection, DNA of non-target species interfere with the reaction, inhibitors are present in the sample, or in the case of insufficient sampling effort (Rees et al. 2014). The effect of eDNA concentration dilution is an issue of particular concern with respect to the detection of rare species in large rivers. However, when using eDNA metabarcoding in such environments, there are several strategies that can be adopted to potentially enhance the probability of detection, notably, increasing sample replication at either one or both of the following 2 levels: sample collection and molecular replicates (e.g., number of PCR assays in the laboratory) (Ficetola et al. 2015). In the case of detecting rare eDNA in samples with very poor molecular detection probability, an increase in molecular replicates is particularly advisable (Erickson et al. 2019). Accordingly, in this study we performed 2 replicate samplings and 12 PCR replicates (for each replicate sample), which is considered a more than sufficient replication effort for species with low detection probability (Ficetola et al. 2015). Collecting a larger number of samples would also be beneficial and is particularly important in the context of metabarcoding studies that set minimum acceptance thresholds for the number of reads considered indicative of a true positive signal. To overcome this issue, we performed an integrative sampling strategy in space (an entire section of the river) and time (approx. half an hour) and by collecting relatively large volumes of water (28.73 L per sample on average). Systematically collecting from a large number of sampling sites in this way would certainly contribute to maximizing the likelihood of detection (Cantera et al. 2019). Furthermore, when practical, we would recommend sampling at sites with low stream flows and at different times throughout the year. Enhancing the association between eDNA concentration and absolute species abundance would clearly contribute to incorporating the eDNA approach into conservation programs (Thomsen and Willerslev 2015), and indeed, a combination of total eDNA qPCR and eDNA metabarcoding has already been proven to be a useful approach in biomonitoring and bioassessment surveys in which a rough estimate of the absolute abundance of fish species is sufficient (Pont et al. submitted).
Among the sturgeon species known to inhabit the Danube, the sterlet (A. ruthenus) is the only species that is encountered occasionally throughout the entire length of the river (Friedrich et al. 2019). However, despite being of a certain economic value in the Middle and Lower Danube catchments (Guti 2008; Vassilev and Pehlivanov 2003), the current status of the population remains almost completely unknown, although the consensus among most authors is that stocks are declining (e.g. Bacalbasa-Dobrovici 1991; Paraschiv et al. 2006) and aging (Kubala et al. 2021). In the present study, approximately half of the samples collected from the Danube yielded a positive signal for the sterlet, and notably, we identified 3 areas with higher densities of this species, as indicated by a high frequency of detection and a high number of reads (namely, the Danube Delta, Iron gate to Belgrade, and Budapest to Gabčíkovo sections of the river). The highest rates of sterlet detection were obtained for the Delta region of the Lower Danube (rkm 0–862) with a subsequent reduction in detection until rkm 235. An absence of positive signals further upstream between rkm 375 and 700 would tend to be indicative of a discontinuous longitudinal distribution of populations within River Danube. However, genetic analysis has indicated the occurrence of gene flow and a low level of sub-structuring within the Danube (Cvijanović et al. 2017; Reinartz et al. 2011). The aforementioned stretch of the Danube has, nonetheless, experienced a marked reduction in sterlet stocks over the past few decades (Vassilev and Pehlivanov 2003), and thus further research is needed to explain these results. In contrast, with the exception of a single site at rkm 1560, sterlet were successfully detected at all sampling points in the Middle Danube (rkm 943–1790), with the highest relative species abundances being recorded from the stretches between Iron gate and Belgrade and from Budapest to Gabčíkovo. Even so, recent massive declines have been reported for this section of the Danube, which have mainly been attributed to the destruction of important spawning habitats during construction of the Gabčikovo Dam (Guti 2008). In the Upper Danube (rkm > 1790) sterlets were detected at only 2 sampling sites, namely, downstream of Vienna (rkm 1920) in the tailwater of the Freudenau hydropower plant and in Deggendorf (rkm 2282). Since the 20th century, it appear that sterlets have been present in the Upper Danube in only low numbers within a few fragmented populations (Friedrich et al. 2019), most of which are actively supplemented by periodic re-stocking (Friedrich 2018). A similar practice is in place at Vienna, where a remnant wild population of an estimated few dozen individuals is actively maintained by re-stocking (Friedrich et al. 2016). The sample at Jochenstein with a known small reproductive population (Zauner 1997) did not detect the species in this study. Although there are historical reports of the presence of sterlet at the most upstream site at Deggendorf (Reinartz 2008 references therein; e.g. Streibl 1920), their occasional detection by commercial fisheries in this area can probably be ascribed to stocking activities.
With respect to the other sturgeon species, we successfully detected the targeted species in water samples collected in an ex situ environment (see Table 2). Notably, in these analyses, we also detected the presence of A. transmontanus, even though this species had been removed from the sampled tank more than a week prior to collecting water samples. In this regard, it has been established that eDNA can persist and remain detectable by PCR for between a day and approximately one month after the removal of animals, depending on environmental conditions (Barnes et al. 2014; Dejean et al. 2011). However, our analysis of field samples revealed only single occurrences of A. stellatus (Danube Delta) and A. gueldenstaedtii (River Inn), the latter of which we suspect originated from rearing ponds upstream of the sampling site. The fact that none of the other target species were detected, would thus tend to indicate that these are either practically absent from the sampling sites or that the amounts of DNA in samples were below the limit of detection of our current methodology, both of which are plausible. A. nudiventris is considered to be functionally extinct in the Danube (Reinartz and Slavcheva 2016; Simonovic et al. 2005) and all other anadromous species (H. huso, A. stellatus, and A. gueldenstaedtii) are currently listed as critically endangered, occurring in the Lower Danube in only small numbers up to the Iron Gate site (Friedrich et al. 2018). A further factor that might account for our inability to detect these rare anadromous species is the timing of sampling. For Ponto-Caspian sturgeons, there are 4 known patterns of migration with differing peaks that occur either in spring or during fall (Berg 1934; Holčík 1989). However, whereas sampling in July (as conducted in the present study) may not be conducive to detecting the adults of early-spawning species such as the beluga sturgeon, this timing should be ideal for detecting the presence of either the young‐of‐the‐year and/or mature adults of all the targeted species.
One consistent request by several organizations, action plans, and projects that have targeted sturgeon conservation is the establishment of a permanent and standardized monitoring program (e.g. Friedrich 2018; Friedrich et al. 2018; ICPDR 2018; Sandu et al. 2013), which would contribute to documenting changes in population dynamics for adaptive management. Such a permanent monitoring network could be readily implemented if based on repeated eDNA sampling campaigns. In addition, this would also facilitate further studies on seasonal migration patterns from the marine environment into rivers (see Stoeckle et al. 2017), as well as assessments of the efficacy of population support actions and the characterization of key habitats, such as spawning, overwintering, or nursery areas, that could subsequently be investigated and, if necessary, protected. Devising effective sampling methods for monitoring contributes to meeting one of the urgent priority needs of supporting the informed management of freshwater biodiversity, which is a vital step in enhancing coordinated action for its sustainable management and conservation (Maasri et al. 2021).
In conclusion, in this study, we demonstrate the practical utility of the eDNA metabarcoding approach as a tool for monitoring sturgeon species in large rivers, as illustrated by our survey of the entire Danube. Moreover, we present the first comprehensive whole-river snapshot study of Acipenser ruthenus conducted on a large geographical scale. Given certain limitations of our current methodology, the sampling strategy will need to be modified for the assessment of other endangered sturgeon species to counteract the dilution effect of very low eDNA concentrations. An in-depth understanding of species distribution and population dynamics is essential for developing adaptive conservation management plans, and in this regard, the benefits of an eDNA approach for conservation efforts, fisheries management, and scientific studies are numerous, particularly for rare bottom-dwelling species inhabiting large rivers. Techniques based on short species-specific eDNA fragments are potentially more sensitive than traditional survey methods, as well as being more cost-effective, non-invasive, and facilitating time-limited coverage of large geographical areas, thereby enabling the implementation of conservation measures within an ecologically and politically actionable time scale.