DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.
Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine et al.: Biomonitoring macroinvertebrates with molecular methods 50 application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
The emergence of a novel infectious disease, chytridiomycosis, is now widely recognized as a major cause of amphibian declines and biodiversity loss across local and global scales. Amphibian mortalities caused by the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd) were first recorded in Iberia, Europe over a decade ago. In August 2009, hundreds of post‐metamorphic common midwife toads (Alytes obstetricans) were found dead in the water and margins of a pond in the Serra da Estrela Natural Park, north‐central Portugal. Histological and genetic analyses confirmed their infection with Bd. Given the likelihood of a new outbreak of chytridiomycosis, we evaluated the possible impacts of this disease on populations of A. obstetricans within the Park by conducting field surveys during 2010 and 2011. We compared the present distribution and abundance of A. obstetricans with historical records, and quantified the present prevalence and intensity of infection by Bd. Results showed that (1) A. obstetricans disappeared from 67% of the 1 × 1 km squares where it was recorded in the past, (2) breeding is currently limited to 16% of the confirmed breeding sites in the past, and that (3) larvae are now less abundant, as well as are highly infected by Bd in the remaining sites. These effects were most pronounced at altitudes above 1200 m. Our findings suggest that an outbreak of chytridiomycosis is responsible for the rapid decline of A. obstetricans in Serra da Estrela, and we believe that urgent conservation measures are needed to prevent local extinction of the species.
Freshwater ecosystems are among the most threatened in the world, and downstream reaches of stream networks are often the most affected by multiple anthropogenic stressors. In these circumstances, many species currently restricted to stream headwaters may represent remnants of once larger populations, though this is generally difficult to ascertain due to limited historical data. Here, we document a case of range contraction into headwaters of the globally threatened Pyrenean desman Galemys pyrenaicus, a species endemic to South‐western Europe, from surveys carried out in 1993–96 and 2014–15. Geostatistical logistic mixed models were used to relate the probabilities of desman occurrence and extinction to environmental variables in 74 sites within two watersheds in NE Portugal, while controlling for linear (Euclidean) and hydrologic spatial dependencies. In 1993–96, desmans were recorded at 85.1% of sites, and their presence was positively related to stream order and slope. In 2014–15, desmans were only found at 31.1% of sites, and their presence was positively related to slope steepness and negatively to stream order and maximum temperature of the warmest month. The extinction rate across periods was 63.5%, and the probability of extinction increased with stream order and declined with slope steepness. Maps of predicted distribution and extinction indicated that while the species was widely distributed in the first period, it largely disappeared thereafter from the main rivers and the largest tributaries, persisting mostly in stream headwaters in more mountainous areas. The severe decline found in ours and other studies suggests that the conservation status of the Pyrenean desman may need to be upgraded, and highlights the importance of headwater streams for its conservation. Furthermore, this study supports the view that headwater streams may be increasingly important for biodiversity conservation in modified river systems, favouring the permanence of remnant, albeit fragmented populations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.