Environmental DNA for freshwater fish monitoring: Insights for conservation within a protected area

Background Many fish species have been introduced in wild ecosystems around the world to provide food or leisure, deliberately or from farm escapes. Some of those introductions have had large ecological effects. The north American native rainbow trout (Oncorhynchus mykiss Walbaum, 1792) is one of the most widely farmed fish species in the world. It was first introduced in Spain in the late 19th century for sport fishing (Elvira 1995) and nowadays is used there for both fishing and aquaculture. On the other hand, the European native brown trout (Salmo trutta L.) is catalogued as vulnerable in Spain. Detecting native and invasive fish populations in ecosystem monitoring is crucial, but it may be difficult from conventional sampling methods such as electrofishing. These techniques encompass some mortality, thus are not adequate for some ecosystems as the case of protected areas. Environmental DNA (eDNA) analysis is a sensitive and non-invasive method that can be especially useful for rare and low-density species detection and inventory in water bodies. Methods In this study we employed two eDNA based methods (qPCR and nested PCR-RFLP) to detect salmonid species from mountain streams within a protected area, The Biosphere Reserve and Natural Park of Redes (Upper Nalón Basin, Asturias, Northern Spain), where brown trout is the only native salmonid. We also measured some habitat variables to see how appropriate for salmonids the area is. The sampling area is located upstream impassable dams and contains one rainbow trout fish farm. Results Employing qPCR methodology, brown trout eDNA was detected in all the nine sampling sites surveyed, while nested PCR-RFLP method failed to detect it in two sampling points. Rainbow trout eDNA was detected with both techniques at all sites in the Nalón River’ (n1, n2 and n3). Salmonid habitat units and water quality were high from the area studied. Discussion In this study, a high quantity of rainbow trout eDNA was found upstream and downstream of a fish farm located inside a Biosphere Reserve. Unreported escapes from the fish farm are a likely explanation of these results. Since salmonid habitat is abundant and the water quality high, the establishment of rainbow trout populations would be favored should escapes occur. Environmental DNA has here proved to be a valuable tool for species detection in freshwater environments, and the probe-based qPCR highly sensitive technique for detection of scarce species. We would recommend this method for routine monitoring and early detection of introduced species within natural reserves

Geographic patterns of genetic variation in a broadly distributed marine vertebrate: new insights into loggerhead turtle stock structure from expanded mitochondrial DNA sequences

Previous genetic studies have demonstrated that natal homing shapes the stock structure of marine turtle nesting populations. However, widespread sharing of common haplotypes based on short segments of the mitochondrial control region often limits resolution of the demographic connectivity of populations. Recent studies employing longer control region sequences to resolve haplotype sharing have focused on regional assessments of genetic structure and phylogeography. Here we synthesize available control region sequences for loggerhead turtles from the Mediterranean Sea, Atlantic, and western Indian Ocean basins. These data represent six of the nine globally significant regional management units (RMUs) for the species and include novel sequence data from Brazil, Cape Verde, South Africa and Oman. Genetic tests of differentiation among 42 rookeries represented by short sequences (380 bp haplotypes from 3,486 samples) and 40 rookeries represented by long sequences (~800 bp haplotypes from 3,434 samples) supported the distinction of the six RMUs analyzed as well as recognition of at least 18 demographically independent management units (MUs) with respect to female natal homing. A total of 59 haplotypes were resolved. These haplotypes belonged to two highly divergent global lineages, with haplogroup I represented primarily by CC-A1, CC-A4, and CC-A11 variants and haplogroup II represented by CC-A2 and derived variants. Geographic distribution patterns of haplogroup II haplotypes and the nested position of CC-A11.6 from Oman among the Atlantic haplotypes invoke recent colonization of the Indian Ocean from the Atlantic for both global lineages. The haplotypes we confirmed for western Indian Ocean RMUs allow reinterpretation of previous mixed stock analysis and further suggest that contemporary migratory connectivity between the Indian and Atlantic Oceans occurs on a broader scale than previously hypothesized. This study represents a valuable model for conducting comprehensive international cooperative data management and research in marine ecology

Barcodes of marine invertebrates from north Iberian ports: Native diversity and resistance to biological invasions

Ports are gateways for many marine organisms transported by ships worldwide, especially non-indigenous species (NIS). In this study carried out in North Iberian ports (Cantabrian Sea, Bay of Biscay) we have observed 38% of exotic macroinvertebrates. Four species, namely the barnacle Austrominius modestus, the tubeworm Ficopomatus enigmaticus, the Pacific oyster Crassostrea gigas and the pygmy mussel Xenostrobus securis, exhibited clear signs of invasiveness. A total of 671 barcode (cytochrome oxidase subunit I or 18S rRNA) genes were obtained and confirmed the species status of some cryptic NIS. Negative and significant correlation between diversity estimators of native biota and proportion of NIS suggests biotic resistance in ports. This could be applied to management of port biota for contributing to prevent the settlement of biopollutants in these areas which are very sensitive to biological invasions.Versión del editor2,359

Mitochondrial DNA reveals Pleistocenic colonisation of the Mediterranean by loggerhead turtles (Caretta caretta)

As the loggerhead turtle (Caretta caretta) is a philopatric species with a strong genetic structure, the analysis of mtDNA can be used to track evolutionary and colonisation events. In this study we use a genetic approach to understand the population structure of C. caretta in the Mediterranean Sea and to test whether loggerheads could have colonised the Mediterranean during the Pleistocene and survived the cold phases in warm refugia. We amplified a long mtDNA D-loop fragment (815. bp) from 168 dead hatchlings sampled from a selection of rookeries in the Eastern Mediterranean: Libya, Israel, Lebanon, Cyprus and Greece. Previously published data from Turkey and Calabria (Southern Italy) were also included in the analyses. The population nesting in Libya emerged as the oldest population in the Mediterranean, dating from the Pleistocene ca. 65,000. years ago (20,000-200,000). This reveals that the Libyan population might have settled in the Mediterranean basin before the end of the last glacial period. The remaining nesting sites, except Calabria, were subsequently colonised as the population expanded. The populations nesting in Eastern Turkey and Western Greece settled ca. 30,000. years ago (10,000-100,000), whereas the remaining populations originated as a result of a more recent Holocenic expansion. As Calabria presented a unique Atlantic haplotype, found nowhere else in the Mediterranean, we consider this nesting site as the result of an independent colonisation event from the Atlantic and not the recent spread of Mediterranean populations. This reveals that the current genetic structure of C. caretta rookeries in the Mediterranean would be the result of at least two colonisation events from the Atlantic, the oldest one in Libya and a most recent in Calabria, combined with local extinctions during Pleistocenic glaciations and re-colonisations from glacial refugia in Libya, Eastern Turkey and Western Greece. © 2012 Elsevier B.V.Peer Reviewe

Fine ‑ scale distribution of juvenile Atlantic and Mediterranean loggerhead turtles (Caretta caretta) in the Mediterranean Sea

Loggerhead turtles nesting in the Mediterranean Sea exhibit remarkable genetic structuring. This paper tests the hypothesis that young loggerhead turtles from different rookeries do not distribute homogeneously among the major Mediterranean foraging grounds, due to a complex pattern of surface currents. We extracted long fragments of mitochondrial DNA from 275 stranded or bycaught juvenile turtles from six foraging grounds (Catalano-Balearic Sea, Algerian basin, Tyrrhenian Sea, Adriatic Sea, northern Ionian Sea and southern Levantine Sea). We used a Bayesian mixed-stock analysis to estimate the contributions from rookeries in the Mediterranean, the North-west Atlantic and Cape Verde to the studied foraging grounds. Differences were found in the relative contribution of juvenile turtles of Atlantic and Mediterranean origin to each foraging ground. A decreasing proportion of Atlantic juveniles was detected along the main surface current entering the Mediterranean, with a high prevalence of turtles from eastern Florida in the Algerian basin and lower numbers elsewhere. In regard to the turtles of Mediterranean origin, juveniles from Libya prevailed in central and western Mediterranean foraging grounds other than the Algerian basin. Conversely, the Adriatic Sea was characterised by a large presence of individuals from western Greece, while the southern Levantine Sea was inhabited by a heterogeneous mix of turtles from the eastern Mediterranean rookeries (Turkey, Lebanon and Israel). Overall, the distribution of juveniles may be related to surface circulation patterns in the Mediterranean and suggests that fisheries might have differential effects on each population depending on the overlap degree between foraging and fishing grounds