Article

Discovering hidden biodiversity: The use of complementary monitoring of fish diet based on DNA barcoding in freshwater ecosystems

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Ecological monitoring contributes to the understanding of complex ecosystem functions. The diets of fish reflect the surrounding environment and habitats and may, therefore, act as useful integrating indicators of environmental status. It is, however, often difficult to visually identify items in gut contents to species level due to digestion of soft-bodied prey beyond visual recognition, but new tools rendering this possible are now becoming available. We used a molecular approach to determine the species identities of consumed diet items of an introduced generalist feeder, brown trout (Salmo trutta), in 10 Tasmanian lakes and compared the results with those obtained from visual quantification of stomach contents. We obtained 44 unique taxa (OTUs) belonging to five phyla, including seven classes, using the barcode of life approach from cytochrome oxidase I (COI). Compared with visual quantification, DNA analysis showed greater accuracy, yielding a 1.4-fold higher number of OTUs. Rarefaction curve analysis showed saturation of visually inspected taxa, while the curves from the DNA barcode did not saturate. The OTUs with the highest proportions of haplotypes were the families of terrestrial insects Formicidae, Chrysomelidae, and Torbidae and the freshwater Chironomidae. Haplotype occurrence per lake was negatively correlated with lake depth and transparency. Nearly all haplotypes were only found in one fish gut from a single lake. Our results indicate that DNA barcoding of fish diets is a useful and complementary method for discovering hidden biodiversity.
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... introduction of exotic species) also affect ecological functions (Almeida et al., 2012;Fritts and Rodda, 1998;Jo et al., 2014Jo et al., , 2016aPimentel et al., 2000) and act as a major threat to biodiversity (Lee, 2002). Specifically, exotic fish species exert a great pressure on resident fish populations and may cause extinction of endemic and native species in lake ecosystems (Jang et al., 2006;Jo et al., 2016b;O'Grady and Cowx, 2000). ...
... 240), we randomly selected 102 guts for identification of ingested prey items by DNA barcoding (see Table A.1). To obtain the sequences from gut contents, we applied the methods of Jo et al. (2016b). The obtained sequences were aligned using Clustal W 2.0 (Larkin et al., 2007). ...
... To relate OTUs (operational taxonomic units: OTUs) to a previously sequenced species, we adopted two criteria that according to Jo et al. (2016b) ensure accurate species identification. The first criterion was acceptance of a species name if the given OTUs had ≥98% compliance with a known species. ...
Article
Worldwide, increasing amounts of dams and weirs have been established in rivers in recent decades, often with drastic effects on their ecosystems. Between late 2009 and 2011, 16 large-scale dams were built in the main channels of the four largest rivers in South Korea, eight of these along the main channel of Nakdong River (300 km, 520 km in total). We studied the effect of these constructions on the fish community in the riparian zone based on intensive fish field surveys conducted in the littoral zone during 2007–2017, analysis of fishermen's catch data and molecular analysis of the diet of the keystone species, largemouth bass (Micropterus salmoides). Our results, based on RDA and GLM analyses, showed that environmental characteristics and fish species composition changed significantly with dam construction. Total fish abundance and biomass decreased after the start of the weir construction and fish richness decreased with time. The abundance and biomass of exotic fish decreased during construction but recovered afterwards, leading to higher proportions of exotics. Dominance and Shannon indices did not change significantly from before to after construction, while the Evenness index showed a significant decreasing trend. A comparison of the diet composition of largemouth bass showed decreasing genetic variation after construction. The changes in Functional Feeding Group (FFG) of the benthic communities at the study sites did not coincide with FFG changes in the prey items of largemouth bass, indicating a transition in feeding mode from before to after construction. In conclusion, the endemic and native fish species were most sensitive and showed lower resilience to disturbance by the large-scale dam construction than the translocated and exotic species, and the lake-like ecosystems after construction markedly improved the competitive capacity of these exotic fish over the native and endemic species in the riparian zone of the river.
... This method, known as DNA metabarcoding , has shown its value in environmental DNA surveys (Shokralla et al., 2012), and has already been widely applied to understand the diets of insectivorous birds (Jedlicka et al., 2016;Trevelline et al., 2016) and bats (Vesterinen et al., 2013), although such techniques have seldom been applied to freshwater fishes. To date, the bulk of dietary DNA barcoding studies involving fish have required either direct sequencing of individual prey tissues (Aguilar et al., 2017) or molecular cloning of prey DNA from a taxonomically mixed sample followed by bacterial transformation and sequencing of individual clones (Jo et al., 2014(Jo et al., , 2016. By improving upon these DNA-based techniques with high-throughput DNA sequencing, researchers have since expanded their ability to estimate dietary composition, even in the most degraded or taxonomically rich dietary samples , and can better evaluate how dietary alteration impacts the population dynamics of affected species (Quéméré et al., 2013). ...
... Previous studies have used similar methodologies, such as molecular cloning (Carreon-Martinez et al., 2011;Jo et al., 2014Jo et al., , 2016 or Sanger sequencing of individual prey tissues (Barnett et al., 2010;Côté et al., 2013;Dunn et al., 2010). However, these methods are laborious and time consuming when attempting to classify hundreds of unique prey haplotypes in taxonomically mixed samples (Shokralla et al., 2012). ...
... While the primer set used within this study will probably not detect all prey present (Clarke et al., 2014), the average of over 50,000 high-quality prey DNA sequences per sample increases confidence that our dietary description is as complete as possible with the applied methodology. One concern with using high-throughput sequencing lies in the seemingly complex downstream bioinformatic analyses (Jo et al., 2016). Fortunately, opensource programs such as QIIME2 or Galaxy (Goecks et al., 2010) provide tools and pipelines for metabarcoding studies that can be easily adjusted for many different prey taxa and study types. ...
Article
• Providing taxonomically precise dietary characterisations for freshwater fish species is critical for gaining a deeper understanding of the trophic dynamics present in freshwater ecosystems. However, our current understanding of freshwater trophic ecology has relied almost entirely upon direct observation of foraging attempts or morphological identification of partially digested prey. Due to the limitations of morphological dietary characterisations of soft‐bodied arthropod prey, these techniques offer dietary descriptions that can lack satisfactory taxonomic resolution and may bias our interpretations of freshwater food webs. • Recent advancements in DNA‐based prey identification have allowed for species‐level prey characterisations for many terrestrial insectivores, although these techniques have seldom been applied to understand the diets of freshwater fish. This study used DNA metabarcoding with high‐throughput, next‐generation sequencing to provide species‐level descriptions of prey composition for three naturally reproducing, syntopic freshwater trout species. • Our study supports previous findings that suggested that brook trout (Salvelinus fontinalis), brown trout (Salmo trutta), and rainbow trout (Oncorhynchus mykiss) are generalist predators that display a high degree of seasonal dietary flexibility. Prey composition varied significantly across sampling periods, with detection frequency of terrestrial prey greater in the spring/summer period compared to the autumn period. • Pollution‐sensitive aquatic macroinvertebrates were detected frequently across both sampling periods, highlighting the importance of high‐quality streams that support such arthropod prey. DNA metabarcoding also detected a high richness of soft‐bodied, Lepidoptera prey species, a taxonomic group that has been largely underrepresented in previous trout dietary studies that used traditional morphological techniques. • This study demonstrates the applicability of dietary DNA metabarcoding for the detection and species‐level identification of arthropods found in freshwater fish lavage samples and highlights the importance of taxonomically precise techniques when attempting to better understand trophic interactions within freshwater communities.
... Organisms will leave traces of DNA in their surrounding environment so that is possible to sample water to detect rare species or suspected invaders at the invasion front, and reconstruct past communities from sediment analyses (Bohmann et al., 2014;Secondi et al., 2016;Thomsen and Willerslev, 2014). Using this approach, the diet of an individual can also be attained from scat, regurgitates or stomach contents (Côt e et al., 2013;Jo et al., 2016;Thomsen and Willerslev, 2014). The data can then be used to elucidate the diet or prey range of invaders (Ward and Ramón-Laca, 2013;Witczuk et al., 2013), or whether invaders may become novel prey for native predators (Gorokhova, 2006), and hence determine any unobserved trophic interaction. ...
... The data can then be used to elucidate the diet or prey range of invaders (Ward and Ramón-Laca, 2013;Witczuk et al., 2013), or whether invaders may become novel prey for native predators (Gorokhova, 2006), and hence determine any unobserved trophic interaction. For example, molecular analyses of the stomach contents of invasive brown trout S. trutta in Tasmania, Australia, indicated that the species consumes 44 unique taxa, showing a greater accuracy than visual inspection alone (Jo et al., 2016). However, while molecular approaches may provide additional taxonomic resolution, there still may be clear advantages of using multiple analytical techniques, for example, to distinguish between different life stages of prey groups that may require subtly different foraging methods. ...
Article
When invasive species become integrated within a food web, they may have numerous direct and indirect impacts on the native community by creating novel trophic links, and modifying or disrupting existing ones. Here we discuss these impacts by drawing on examples from freshwater ecosystems, and argue that future research should quantify changes in such trophic interactions (i.e. the links in a food web), rather than simply focusing on traditional measures of diversity or abundance (i.e. the nodes in a food web). We conceptualise the impacts of invaders on trophic links as either direct consumption, indirect trophic effects (e.g. cascading interactions, competition) or indirect nontrophic effects (e.g. behaviour mediated). We then discuss how invader impacts on trophic links are context-dependent, varying with invader traits (e.g. feeding rates), abiotic variables (e.g. temperature, pH) and the traits of the receiving community (e.g. predators or competitors). Co-occurring invasive species and other environmental stressors, such as climate change, will also influence invader impacts on trophic links. Finally, we discuss the available methods to identify new food web interactions following invasion and to quantify how invasive species disrupt existing feeding links. Methods include direct observations in the field, laboratory trials (e.g. to quantify functional responses) and controlled mesocosm experiments to elucidate impacts on food webs. Field studies which use tracer techniques, such as stable isotope analyses, allow diet characterisation of both invaders and interacting native species in the wild. We conclude that invasive species often drastically alter food webs by creating and disrupting trophic links, and future research should be directed particularly towards disentangling the effects of invaders from other environmental stressors.
... However, Sanger sequencing can only be applied in situations where only one host plant species is likely to be found in the gut contents or feces of the herbivore (Lim et al., 2017), which is not applicable to generalist insects, which are likely, have a mix of host plant DNA in their guts. Cloning is a useful method to enable dietary analysis of polyphagous animals (Jo et al., 2016) but its labor-intensive (Zeale et al., 2011). Furthermore, the cost increases substantially as the number of samples requiring Sanger sequencing increases (Vesterinen et al., 2013;Yang et al., 2016). ...
Article
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Interactions between plants and insects are among the most important life functions for all organism at a particular natural community. Usually a large number of samples are required to identify insect diets in food web studies. Previously, Sanger sequencing and next generation sequencing (NGS) with short DNA barcode were used, resulting in low species‐level identification, meanwhile the costs of Sanger sequencing are expensive for metabarcoding together with more samples. Here, we present a fast and effective sequencing strategy to identify larvae of Lepidoptera and their diets at the same time without increasing the cost on Illumina platform in a single HiSeq run, with long‐multiplex‐metabarcoding (COI for insects, rbcL, matK, ITS and trnL for plants) obtained by Trinity assemble (SHMMT). Meanwhile, Sanger sequencing (for single individuals) and NGS (for polyphagous) were used to verify the reliability of SHMMT approach. Furthermore, we show that SHMMT approach is fast and reliable, with most high‐quality sequences of five DNA barcodes of 63 larvae individuals (54 species) recovered (full length of 100% COI gene and 98.3% plant DNA barcodes) using Trinity assemble (up size to 1015 bp). For larvae diets identification, 95% are reliable, other 5% failed because their guts were empty actually. The diets identified by SHMMT approach are 100% consistent with the host plants that the larvae were feeding on during our collection. Our study demonstrates that SHMMT approach is reliable and cost‐effective for insect‐plants network studies. This will facilitate insect‐host plants studies that generally contain a huge number of samples. This article is protected by copyright. All rights reserved
... DNA barcoding techniques have increased the reliability of identifying specific taxonomic groups of organisms at both species and genus level [14], and environmental DNA techniques have enabled the detection of elusive species in various environments [15,16]. Genomic approaches have also been used to understand trophic ecology, particularly biological interaction, for both aquatic habitat environments and food webs by collecting information from food material found in gut contents and the excrement of various organisms and this helps to overcome the existing limitations of food source analyses, which were usually based on visual analysis [17][18][19][20]. ...
Article
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We designed an experiment to analyze the gut content of Rotifera based on DNA barcoding and tested it on Asplanchna sp. in order to ensure that the DNA extracted from the rotifer species is from the food sources within the gut. We selected ethanol fixation (60%) to minimize the inflow effects of treated chemicals, and commercial bleach (the final concentration of 2.5%, for 210 s) to eliminate the extracellular DNA without damage to the lorica. Rotifers have different lorica structures and thicknesses. Therefore, we chose a pretreatment method based on Asplanchna sp., which is known to have weak durability. When we used the determined method on a reservoir water sample, we confirmed that the DNA fragments of Chlorophyceae, Diatomea, Cyanobacteria, and Ciliophora were removed. Given this result, Diatomea and cyanobacteria, detected from Asplanchna, can be considered as gut contents. However, bacteria were not removed by bleach, thus there was still insufficient information. Since the results of applying commercial bleach to rotifer species confirmed that pretreatment worked effectively for some species of rotifers food sources, in further studies, it is believed to be applicable to the gut contents analysis of more diverse rotifers species and better DNA analysis techniques by supplementing more rigorous limitations.
... This study represents an important step toward harnessing eDNA for the targeted detection of freshwater pearly mussels. Although traditional sampling methods will continue to be important for collecting population monitoring data such as abundance, demographic structure, sex ratios, and fecundity, eDNA offers a valuable complement to traditional sampling, as has already been demonstrated in other taxonomic groups ( Jo et al., 2015;McKee et al., 2015). Through these and other innovative applications, adopting eDNA as a tool to complement or augment traditional sampling and habitat occupancy models could improve detection sensitivity, increase knowledge of extant species distributions, and enhance the effectiveness of conservation efforts. ...
Conference Paper
Background: Environmental DNA (eDNA) detection can be used to infer species presence by using species-specific markers to screen DNA from water samples. This technique is increasingly being used to track the spread of invasive fish species but has largely not been applied to other taxonomic groups. However, the low impact of eDNA sampling on aquatic habitats makes the method ideal for sampling sensitive populations. Here we report on research that aims to quantify the opportunities and limitations of eDNA for the detection of rare, endangered organisms. Our study focuses on the detection of native freshwater mussel (Unionidae) species at risk using species-specific qPCR markers. We will use a multi-species approach to develop highly specific markers for each of the five closely related species. Results: To date, species-specific qPCR primer-probe sets have been developed, validated under controlled conditions, and implemented across the species' ranges to map occurrences. The eDNA-derived species occurrences will be compared with existing distribution maps to evaluate the efficacy of the method. Significance: eDNA shows promise as an effective tool for the targeted detection of closely related at-risk invertebrates; however, a thorough knowledge of the detection thresholds and limitations of the eDNA tools is crucial for meaningful interpretation of results and the eventual use of eDNA for management applications.
... This study represents an important step toward harnessing eDNA for the targeted detection of freshwater pearly mussels. Although traditional sampling methods will continue to be important for collecting population monitoring data such as abundance, demographic structure, sex ratios, and fecundity, eDNA offers a valuable complement to traditional sampling, as has already been demonstrated in other taxonomic groups (Jo et al., 2015;McKee et al., 2015). ...
Article
Documenting the occurrence and habitat occupancy of rare aquatic species is an ongoing challenge for conservation. Characterization of environmental DNA (eDNA) from bulk water samples has emerged as a powerful tool to infer species presence or absence without the need to observe or handle organisms. Previous eDNA studies have yet to develop species-specific markers that target taxa with many potentially sympatric confamilials. Forty-one freshwater pearly mussel species (Unionidae) are found in southern Ontario, Canada, with many of these listed as threatened, endangered, or of conservation concern; however, locating populations for protection can be challenging owing to morphological crypsis and species scarcity. Species-specific eDNA markers were developed to target four unionid species. Following in silico and in vitro validation, markers were validated in the field by comparing eDNA results from water samples to detections based on quadrat sampling. Target species were detected by eDNA sampling at all sites where they had previously been located by quadrat sampling. The paired sampling design showed that species-specific markers can be designed even within speciose families, and that eDNA detection of mussels is at least as sensitive as quadrat sampling. Furthermore, detection probabilities were not affected by sampling depth, and eDNA concentrations were positively correlated with mussel densities. These findings confirm that eDNA assays are a valuable complement to traditional methods for locating and managing imperilled unionid populations.
... The bones were then frozen in liquid nitrogen and homogenized manually using a mortar and pestle. Each bone was totally homogenized, and DNA extraction and amplification procedures were conducted, as described by Jo et al. (2016). ...
Thesis
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Industrial development has caused biodiversity loss. To preserve biodiversity, studies about wildlife management has been fast developing. Wildlife management deals with invasive species eradication, endangered species protection, human-carnivore conflict, and crop damage, among others. In South Korea, there are two semi-aquatic mammals (Nutria, Myocastor coypus and Eurasian otter, Lutra lutra). Nutria is an invasive species and the Eurasian otter is an endangered species in South Korea. To manage the species, the first things to understand are their habitats, distribution, and status. During the course of my Ph.D, my studies aimed to: (1) identify the distribution, spread, and habitat preferences of nutria invading the lower Nakdong River in South Korea, in order to facilitate current control and eradication endeavors, (2) determine the food habits of nutria using stable isotope analysis, (3) assess the impact of environmental policies on the re-establishment of the Eurasian otter through a literature review, (4) trace conservation activities for the otter in South Korea by reviewing newspapers from 1962–2010, (5) define distribution, habitats, and status of the otters in Nakdong River basin by applying ecological stream health assessment indices, (6) know advancement patterns in association with environmental factors, based on self-organizing map and diffusion kernel, and, finally, (7) identify prey items of the otters at the small swimmer fish dominant sites using DNA barcoding. The studies highlights are: (1) Nutria populations were recorded in 45 of 236 sites investigated. The distribution patterns revealed that the spread of nutria from farming sites has mainly proceeded along rivers via tributaries. Important factors associated with the establishment of new populations were food availability, levees with suitable burrow materials, and slow water flow. The winter climate was also important, and the total number of days below -4℃ significantly affected nutria occurrence. To date, efforts to control or locally eradicate nutria populations have had little success due to insufficient budgets and the lack of coordinated management activities between local governments. To improve the efficiency of nutria eradication programs, local governments must establish an integrated and coordinated strategy that is overseen by a single, national agency. (2) With regard to diet, isotope ratios suggested that coypu primarily fed on aquatic vegetation. Coypus appear to make more use of the heavier nitrogen isotope in hind-leg muscle during winter, presumably associated with muscle tissue metabolism contributing to weight loss. During winter, these higher metabolic requirements together with the decreased availability of aquatic vegetation suggest that baiting near waterways in winter could be an effective method to control invasive coypu populations. (3) The Korean government implemented a policy in the 1980’s to promote the coexistence of nature conservation and development. We hypothesized that two distinct improvements (water quality improvement and forest development) in the environment enabled the distribution of otters to expand significantly. Overall, forest development in basins was the most important factor positively influencing the otter population. (4) Since 2000, the number of newspaper articles about conflicts between otters and urban development, and the roles of otters in encouraging eco-tourism have increased significantly. About 12% of grid squares with otters were reported only in newspaper articles, mostly in urban areas. High citizen interest could be the basis for understanding the ecological importance of human-carnivore coexistence. Otters serve as a good example for establishing conservation strategies and citizen education for effective conservation. (5) Habitat quality was investigated using the stream health assessment program (SHAP). We hypothesized that higher SHAP indices would indicate better habitat for otters. Benthic macro-invertebrate and otter population indices were highly related. Otter population indices provided better habitat information compared with presence-absence data. Stream health assessment indices had the highest correlation with otter distribution. (6) Otter populations recovered rapidly in the southern peninsula of Korea. Two geographically separate populations (N and S) were identified by the diffusion kernel method. Otter population densities, environmental factors, and sample locations were effectively integrated according to the geo-SOM. Overall associations between otter populations and environmental factors were observed strongly for population establishment during the course of dispersal. Anthropogenic factors (e.g., distance to factory) were overall influential in the dispersal of otter populations while food sources (e.g., number of fish individuals) were preferred at the frontlines of advancement. (7) Because the dominant Cyprinidae are small and fast swimmers at the study sites, otters fed more on frogs and benthic fishes. In a comparison between fish fauna and consumed items, in more than half of sites (62.5 %), all fishes of the fauna at the sites failed to match all fishes consumed, although the number of samples were limited. In addition, most food items coincidentally present in fish fauna were not the dominant fish species at the sites. Otter protection could be established on a large scale according to their large home range. The results of my studies revealed the importance of governance and of highly improved public conservation awareness for wildlife management. Habitat condition is mainly based on food availability for both species. According to public efforts, nutria populations have decreased and otter populations have greatly recolonized in South Korea. Further studies will include fine scales of human and otter coexistence, genetic approaches for populations, and theoretical interactions between the environment and two species.
... The hypervariable region across the COI gene can categorize incognizable animal samples into different taxa by sequencing and comparing the results with reference databases that contain millions of species gene information. However, the amplification products of classical versatile primers (LCO1490 and HCO2198) have a length of 658 bp, which is likely to cause degradation or breakage (Jo et al., 2016, Huber et al., 2009). In addition, the longer the distance between forward and reverse primers, the better the required integrity of the prey target gene sequence and the lower the identification efficiency. ...
Article
Full-text available
Spiders are the most common and predominant predators in terrestrial ecosystems. The predatory behavior of spiders affects the energy flow across the food web within an ecosystem. Traditiaonal methods for analyzing spider diets such as field observation, anatomy and faeces analysis are not suitable for spider experiments due to spiders’ special dietary behavior. The molecular method based on the specific primers of prey DNA seems to be inefficient either in spite of its wide application in diet analysis. As the next-generation sequencing (NGS) technology becomes prevalent in many different areas, several cases of the NGS-based analysis of mammal diets have been published. This study analyzed the diet differences of Pardosa pseudoannulata (Araneae: Lycosidae) in four habitats (a wetland, a tea plantation, an alpine meadow and a paddy field) by using the NGS technology, combined with the DNA barcode method. The results suggested that the Pardosa pseudoannulata feed on a broad range of prey, and 7 orders and 24 families of insects were detected in the four investigated habitats. Moreover, it is found that the diet diversity of Pardosa pseudoannulata is greatly influenced by their living environments and seasons. In a nutshell, this study established an NGS-based methodology for spider diets analysis, and the results provided some basic materials to inform the protection and utilization of the Pardosa pseudoannulata as a potential eco-friendly predator against pests.
... De plus, les résultats obtenus à partir des séquences ADN pourront être comparés aux contenus stomacaux et à des mesures d'isotopes stables. Cette dernière perspective de recherche s'est avérée concluante pour étudier le régime alimentaire des larves de krill[473], des poissons[231,272], des pingouins[229] ou des macareux[57]. Enpratique, la technique du métabarcoding se décompose en quatres étapes majeures : (i) l'extraction d'ADN de l'échantillon, (ii) l'amplication du marqueur par une réaction en chaîne par polymérase (PCR) à l'aide d'un couple d'amorces, (iii) le séquençage, et (iv) l'identication des amplicons lorsque la banque de référence le permet. ...
Thesis
Le terme plancton désigne l'ensemble des organismes dérivant au grès des courants marins. On distingue le plancton végétal et principalement photosynthétique, "le phytoplancton", du plancton animal hétérotrophe, "le zooplancton". Au cours des dernières décennies, de nombreuses études ont documenté une croissance de l'abondance et de la distribution spatiale du zooplancton gélatineux à travers diverses régions. Même si le terme "gélification" des océans doit être utilisé avec beaucoup de précaution, des régions comme la mer Méditerranée montre une constante augmentation des méduses au cours de ces 40 dernières années. L'espèce Pelagia noctiluca (Forsskål, 1775) est considérée comme étant la méduse la plus abondante du bassin méditerranéen depuis les années 70. Du fait de leur présence massive dans cette région, il est primordial d'évaluer précisément l'impact de P. noctiluca à la fois sur les cycles biogéochimiques et sur la structuration des écosystèmes pélagiques. Pour cela, les deux processus majeurs de transfert de matière dans l'écosystème doivent être étudiés : la séquestration de carbone via la pompe biologique et le transfert de carbon au travers des réseaux trophiques. Cette thèse s'articule autour de trois axes majeurs: (i) réaliser un premier bilan de l'export de carbone organique particulaire (POCtotal) et dissous (DOC) en mer Méditerranée, (ii) construire un modèle écophysiologique de P. noctiluca pour déterminer la contribution de cette méduse à la pompe biologique, et (iii) évaluer le niveau trophique de P. noctiluca et son potentiel impact sur les niveaux trophiques inférieurs.
... Despite the wide applicability of DNA barcoding in many taxa, the use of DNA barcoding for the identification of unknown specimens in real-world conditions has not been extensively surveyed. Previous DNA barcoding studies have heavily relied on invasive tissue samples, most of which having employed one or two analytical methods for species identification in biodiversity conservation (Ardura et al. 2010;Gonçalves et al. 2015), food traceability (Quinto et al. 2016;Wong and Hanner 2008), diet analyses (Côté et al. 2013;Jo et al. 2015), bird strikes (Dove et al. 2008;Waugh et al. 2011) and biosecurity etc. (Armstrong and Ball 2005;González-Varo et al. 2014). There are, however, inherent limitations and complementary advantages in these DNA barcoding analyses (DeSalle et al. 2005;van Velzen et al. 2012). ...
Article
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Owning to advantages over traditional species identification methods, DNA barcoding is suggested to be a promising tool in conservation research. However, the use of DNA barcoding to accurately identify unknown samples in conservation practices has not been well documented in the literature. To illustrate this issue, we implemented a survey of endangered birds and mammals in China based on mitochondrial Cytochrome c Oxidase subunit I (COI) gene. We included mostly confiscated specimens and non-invasive samples while concealing species information to simulate real-world scenarios of identification. In total, 47 avian and 39 mammalian specimen were re-identified by sequential analyses of online species assignment, genetic distances, phylogenetic reconstruction, and diagnostic nucleotide method. With this multiple analyses approach, 82 individuals were accurately assigned to the species level and four individuals to the genus level. 78.72% of the avian specimen and 87.18% of mammalian specimen identifications were consistent with morphological classification. Among those inconsistent with morphological classification, we identified several potential errors including misidentification based on morphology and mislabelling that may have occurred while combining results from different analytical methods. Our case study not only enriches the barcode database, but also reports a successful application of DNA barcoding identification to conservation practices, which could effectively facilitate species identification of unknown samples in conservation practices in the future.
... Biological monitoring contributes to the understanding of complex ecosystem structures and functions in targeted systems [1]. Accordingly, it is crucial in detecting and assessing environmental changes in order to ensure proper management and conservation of complex ecosystems [2]. ...
Article
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The nonlinearity and complexity of coastal ecosystems often cause difficulties when analyzing spatial and temporal patterns of ecological traits. Environmental DNA (eDNA) monitoring has provided an alternative to overcoming the aforementioned issues associated with classical monitoring. We determined aquatic community taxonomic composition using eDNA based on a meta-barcoding approach that characterizes the general ecological features in the Gwangyang Bay coastal ecosystem. We selected the V9 region of the 18S rDNA gene (18S V9), primarily because of its broad range among eukaryotes. Our results produced more detailed spatial patterns in the study area previously categorized (inner bay, main channel of the bay and outer bay) by Kim et al. (2019). Specifically, the outer bay zone was clearly identified by CCA using genus-level identification of aquatic organisms based on meta-barcoding data. We also found significant relationships between environmental factors. Therefore, eDNA monitoring based on meta-barcoding approach holds great potential as a complemental monitoring tool to identify spatial taxonomic distribution patterns in coastal areas.
... (Table 4). 한편, 18S rRNA 영역은 분 류 해상도를 목 (Order)-과 (Family) 수준으로 제한하는 반면, 28S rRNA 및 mtCOI은 속 (Genus)-종 (Species) 수준의 해상 도를 갖는다는 연구 결과가 있다 (Pearman et al., 2014;Hirai et al., 2015b;Harvey et al., 2016) (Symondson, 2002;Carreon-Martinez et al., 2011;Jo et al., 2016;Oh et al., 2020a). ...
... However, contrary to our expectations, the direct effect of the non-native predatory fish, measured here as the proportion of galaxiids in the stomach content of non-native predatory fish, was confirmed using both visual inspection and DNA barcoding analyses in a subset of the studied lakes (Jo et al. 2016). This effect seemed stronger in the turbid and the humic lakes than in the clear-water lakes, indicating that predation on galaxiids occurred despite the reduced visibility. ...
Article
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Interaction between non-native predatory fishes and native galaxiids (Pisces: Galaxiidae) shapes food web structure in Tasmanian lakes, Inland Waters, ABSTRACT Non-native fish invasions threaten native fauna and ecosystem functioning, not least in isolated island lakes. In Tasmania, where the native fish are mostly galaxiids, 9 non-native freshwater fish species have been introduced over the past 150 years, with uncertain ecological outcomes. We evaluated the effects of non-native predatory fishes (NNPF) and various environmental and biological variables on the trophic niche of native fish (galaxiids) and potential cascading effects. We analysed Layman's food web metrics based on both stable isotope (δ 15 N and δ 13 C) values and fish stomach contents in 14 shallow Tasmanian lakes along a NNPF abundance gradient. The food web metrics calculated were (1) range of δ 13 C (CR) and δ 15 N (NR) centroid distance (CD) and (2) standard ellipse area. Our results showed that NNPF relative abundance in the fish catch per unit effort was negatively related to the galaxiids' trophic niche metrics (e.g., CR G , NR G , and CD G), trophic position, and the pelagic contribution to the diet. Moreover, the proportion of galaxiids in the diet of NNPF was higher in turbid lakes. The zooplankton standard ellipse area was negatively correlated with the pelagic contribution to the NNPF diet, and NNPF relative abundance was positively correlated with the maximum body size of calanoid copepods. While our results suggest a negative effect of NNPF on the trophic niche of galaxiids, the cascading effect on phytoplankton biomass was weak. Non-native predatory fish affect native fish prey, and the outcome of these interactions should be considered for conservation purposes, particularly for island lakes, such as those in Tasmania
... Trophic functioning of protected versus unprotected areas could be examined in new ways as eDNA offers the potential to accurately quantify producers or invertebrates at the base of the food chain as easily as the large fishes, which currently receive the bulk of research attention (Cinner et al., 2016Edgar et al., 2014;Martin et al., 2017;Mora et al., 2011). Stomach content analysis using eDNA has also been shown to be more accurate than visual analysis and can provide much needed data on trophic interactions ( Jo et al., 2016). The higher taxonomic resolution that can be achieved using eDNA compared to conventional methods can further open up the way for far more specific spatial planning than is currently possible (Table 1). ...
Chapter
Marine management areas provide a key tool for efforts towards sustainable development, reconciling socio-economic goals with those for biodiversity conservation. Decisions about where and when to establish spatial management areas in the oceans are currently hampered by the uncertainties of incomplete, or overly general, information about biodiversity. The analysis of environmental DNA (eDNA) provides a potentially powerful tool to overcome this lack of data in the future. Here we present directions to develop robust approaches to integrate eDNA and spatial planning processes, aiming to provide guidance to underpin tool development. The potential of eDNA use in conservation is widely recognised, although direct applications almost exclusively focus on detection of invasive or threatened species and not spatial management decisions. The implementation of broader interaction between the fields of conservation science and eDNA analysis could create substantial benefits to biodiversity conservation and management. In particular, eDNA analysis can provide information on biodiversity over spatial-temporal scales that are currently prohibitive in spatial planning studies. Here, we provide an overview of how eDNA is currently used in conservation practice, in addition to understanding its limitations and benefits within the context of spatial planning. With the goal to harness rapid technological developments in both molecular and conservation sciences, we provide a horizon scan of the future of eDNA analysis and its application to inform biodiversity conservation in a rapidly changing world.
... to identify prey to the species level for predatory mammals (Gillet et al., 2015) and birds (Wong et al., 2015). For fishes, DNA-based techniques have been used to study diet using stomach or gut contents (Smith et al., 2005;Jo et al., 2013Jo et al., , 2016Moran et al., 2016) and faeces (Corse et al., 2010;Taguchi et al., 2014) and often provide better resolution than conventional stomach-content analysis. ...
Article
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In this study, the results of conventional stomach-content analysis are compared with the recent DNA metabarcoding approach on faeces to identify fish species consumed by non-native European catfish Silurus glanis in the Garonne River (south-western France), with a special emphasis on anadromous prey. Fourteen prey species were identified in the stomach contents or faeces, including four anadromous fish species. Despite higher intestine than stomach emptiness, more species were identified through faecal analysis (11 of 14) than through stomach-content analysis (five of 14) suggesting that DNA metabarcoding on faeces is an efficient, non-intrusive technique to study the diet of predatory fishes.
... Applying DNA sequence-based techniques to gut-content identification has recently increased identification resolution, particularly in marine ecosystems [19][20][21][22][23][24]. However, relatively few studies have used DNA barcoding for gut-content analysis in complex freshwater ecosystems [25][26][27] although this technique has been recognized as a promising tool for studying food-web interactions [28]. ...
Article
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Chironomidae larvae play an important role in the food chain of river ecosystems in Korea, where it is dominant. However, detailed information on the diet of Chironomidae larvae are still lacking. The purpose of this study was to identify the gut contents of 4th instar larvae of a Chironomidae inhabiting four large-scale weirs (Sejong Weir, Juksan Weir, Gangjeong-Goryeong Weir, and Dalseong Weir) using a DNA meta-barcoding approach. We found that dominant Operational Taxonomic Unit (OUT) was assigned to Paractinolaimus sp. (Nematoda), and the sub-dominant OTU was assigned to Dicrotendipes fumidus (Chironomidae). The most common OTUs among the individuals included phytoplankton, such as Tetrahymena sp., D. armatus, Pseudopediastrum sp., Tetradesmus dimorphus, Biddulphia tridens, and Desmodesmus spp. We calculated the selectivity index (E’) and provided scientific evidence that Chironomidae larvae have a significant preference (E’ > 0.5) for Desmodesmus armatus, E. minima, and T. dimorphus, while it does not show preference for other species found in its gut. Differences in physico-chemical factors, such as water quality, nutrients, Chl-a, and carbon concentrations, resulting from anthropogenic impacts (i.e., construction of large-scale weirs) as well as the particle size of prey organisms (small-sized single cell) and effects of chemicals (chemokinesis) could affect the feeding behavior of Chironomidae larvae.
... The eDNA approaches have also been used to clarify and understand systematic ecology, particularly biological trophic interaction in both aquatic habitat environments and food webs by collecting information from food sources found in gut contents of species and the excrement of lived organisms. This helps to overcome unidentified limitations of food source analyses, which were based on microscopic analysis [8][9][10][11]. At present, it is necessary to develop a method to separate pure gut content from target organisms for a wide range of applications of DNA technology in food source identification. In addition, the most fundamental methodology is to produce a framed "blocking primer", which removes the DNA of the target species from the target gut contents. ...
Article
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Food chains and food webs describe the structure of communities and their energy flows, and they present interactions between species. Recently, diverse methods have been developed for both experimental studies and theoretical/computational studies on food webs as well as species interactions. They are effectively used for various applications, including the monitoring and assessment of ecosystems. This Special Issue includes six empirical studies on food chains and food webs as well as effects of environmental factors on organisms in aquatic ecosystems. They confirmed the usefulness of their methods including isotope, DNA-barcoding with gut contents, and environmental DNA for biological monitoring and ecosystem assessment.
... Even our data clearly indicated this limitation, wherein diet species added in the mock community (at equal extracted DNA concentration) were represented by skewed read counts. Some alternatives, such as using the number of sequence variants for every taxon as an index of prey biomass (Jo et al. 2016) or minimum number of individuals consumed (Corse et al. 2017), have been suggested. However, for the mock communities, we retrieved multiple variants (prior to HTS filtering) for every taxon although DNA from just one individual was added, making these approximations unreliable. ...
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After suffering a massive decline (~ 99%) in numbers caused by feeding on livestock carcasses containing the nephrotoxic drug diclofenac, critically endangered Gyps vultures now persist in low numbers in the Indian subcontinent. Most are concentrated near National Parks, likely due to availability of wild carcasses free from toxic veterinary drugs. Quantification of vulture diets is critical to test this hypothesis. We describe a validated “field-to-benchtop-to-desktop” metabarcoding workflow for assessing species- and sex-specific diet of these obligate scavengers from non-invasively collected faecal samples. Seven metabarcodes were designed to simultaneouslsy identify the vulture species, sex and diet. Amplicons generated from multiplex PCRs were indexed and sequenced on an Illumina Miseq platform. We included controls and three replicates per sample to establish a series of non-arbitrary thresholds to filter the sequence data and eliminate cross-contamination, PCR/sequencing errors and false positives. This strategy enabled identification of species and sex for all samples and diet-specific sequences with high taxonomic resolution for 97% of samples. Just four (two for diet, one each for vulture species and sexing) metabarcodes were sufficient to meet the objectives. This preliminary analysis suggests that domestic livestock is the most frequently consumed diet item across samples from inside and outside protected habitats. Our method provides a rapid and reliable tool for describing large-scale variation in consumption of domestic versus wild species in the diet of these scavengers, paving the way for a better understanding of the role protected areas play in persistence and recovery of the remaining Gyps vultures in the wild.
... Our dataset may be used to reconstruct geographical patterns or seasonal/depth distributions of coral larvae as well as to evaluate coral spawning without the need to spend hours diving at night-time. Another possible application dataset may be related to the study of gut and faecal contents of corallivorous animals in order to evaluate the composition and diversity of their coral diet, as already performed for carnivorous predators (Leray et al. 2013;Jo et al. 2015). This offers the potential for improving our understanding of feeding behaviour of these animals and estimate which coral taxa are preferentially selected. ...
Article
Scleractinian corals (i.e. hard corals) play a fundamental role in building and maintaining coral reefs, one of the most diverse ecosystems on Earth. Nevertheless, their phylogenies remain largely unresolved and little is known about dispersal and survival of their planktonic larval phase. The small subunit ribosomal RNA (SSU rRNA) is a commonly-used gene for DNA barcoding in several metazoans, and small variable regions of SSU rRNA are largely adopted as barcode marker to investigate marine plankton community structure worldwide. Here, we provide a large sequence dataset of the complete SSU rRNA gene from 298 specimens, representing all reef coral families and a total of 106 genera. The secondary structure was extremely conserved within the order with few exceptions due to insertions or deletions occurring in the variable regions. Remarkable differences in SSU rRNA length and base composition were detected between and within acroporids (Acropora, Montipora, Isopora, and Alveopora) compared to the other corals. V4 and V9 regions seem to be promising barcode loci because variation at commonly used barcode primer binding sites was extremely low and their levels of divergence allowed to distinguish families and genera. A time-calibrated phylogeny of Scleractinia is provided and mutation rate heterogeneity is demonstrated across main lineages. The use of this dataset as a valuable reference resource to investigate aspects of ecology, biology, molecular taxonomy, and evolution of scleractinian corals is discussed. This article is protected by copyright. All rights reserved.
... Recently, advancements in molecular biology have made diet analysis of some hematophagous species possible. DNA metabarcoding has been used to characterize the diet of a wide range of animals including ticks (Gariepy et al., 2012), leeches (Schnell et al., 2012), mosquitoes (Reeves et al., 2016), fishes (Schreier et al., 2016;Jo et al., 2016), bats (Walker et al., 2016), birds (Merkes et al., 2014), cats (Shehzad et al., 2012), and whales (Ford et al., 2016). In medical leech (Hirudo medicinalis), bloodmeal was found to contain host DNA for at least 4 months after its last feeding and 21 of 25 leeches tested from wild populations yielded host DNA (Schnell et al., 2012). ...
Article
Sea lamprey (Petromyzon marinus) are invasive in the Laurentian Great Lakes, parasitize large-bodied fishes, and therefore are the focus of an international control program. However, damage caused by sea lamprey to modern day fish stocks remains uncertain because diet analysis of juvenile sea lamprey has been challenging; they feed on blood and are difficult to randomly sample in the lakes. Here, both challenges were addressed by showing that DNA metabarcoding of fecal material can be used to identify the diet of actively feeding juvenile sea lamprey, and can also be used to determine what non-feeding adult sea lamprey captured in streams fed on while para-sitizing fish. Fecal samples from juvenile sea lamprey that were feeding on lake trout in northern Lake Huron overwhelmingly contained lake trout (Salvelinus namaycush) DNA (90%), while smaller percentages contained lake whitefish (Coregonus clupeaformis; 5%) and longnose sucker (Catostomus catostomus; 5%) DNA. Fecal samples from adult sea lamprey captured from a tributary to northern Lake Huron overwhelmingly contained longnose and white sucker DNA (Catostomus spp.; 80%), while a smaller percentage contained lake trout DNA (10%). Diet composition of adult sea lamprey sampled in the tributary (Black Mallard Creek) was more diverse than juvenile diet composition. DNA metabarcoding suggests that Catostomus spp. may be an important host fish in northern Lake Huron for sea lamprey prior to spawning. Future research could investigate how diet varies across years and lakes and the prevalence and sources of DNA contamination. Application of DNA metabarcoding for diet assessment may be practical for identifying populations of invasive sea lamprey that feed on highly valued fishes and help guide restoration of lampreys worldwide.
... Using only macroscopic diet analysis, could in this sense, cause incorrect diet determination, if including samples from unintended predators. Diet analyses can provide information not only on food preferences of individuals and their fitness level 57 , but can also contribute to the description of biodiversity in the foraging area, especially in the case of generalist omnivores 30,31 . However, results on diet composition can be influenced by the detection probabilities of different diet components connected to the chosen methodology, i.e. macroscopic vs. molecular 34,35 . ...
Article
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With an accelerating negative impact of anthropogenic actions on natural ecosystems, non-invasive biodiversity assessments are becoming increasingly crucial. As a consequence, the interest in the application of environmental DNA (eDNA) survey techniques has increased. The use of eDNA extracted from faeces from generalist predators, have recently been described as “biodiversity capsules” and suggested as a complementary tool for improving current biodiversity assessments. In this study, using faecal samples from two generalist omnivore species, the Eurasian badger and the red fox, we evaluated the applicability of eDNA metabarcoding in determining dietary composition, compared to macroscopic diet identification techniques. Subsequently, we used the dietary information obtained to assess its contribution to biodiversity assessments. Compared to classic macroscopic techniques, we found that eDNA metabarcoding detected more taxa, at higher taxonomic resolution, and proved to be an important technique to verify the species identification of the predator from field collected faeces. Furthermore, we showed how dietary analyses complemented field observations in describing biodiversity by identifying consumed flora and fauna that went unnoticed during field observations. While diet analysis approaches could not substitute field observations entirely, we suggest that their integration with other methods might overcome intrinsic limitations of single techniques in future biodiversity surveys.
... Of particular relevance in aquatic ecosystems are studies investigating diet ecology and trophic interactions between organisms [155], where in short time passed from Sanger sequencing to study this topic [150] to metabarcoding [170], the structuring and dynamics of plankton communities [156,157], marine benthic biomonitoring [158], and freshwater invertebrate water quality assessment [159]. As well, gut contents can lead to the discovery of unknown biodiversity, as demonstrated in marine and freshwater fishes [150,171]. COI barcode reference libraries for animals have now become standard resources for DNA metabarcoding applications, and have been recommended as the standard metabarcode for metazoans [160]. Environmental DNA (eDNA) metabarcoding techniques have also further transformed biodiversity research by extending metabarcoding to include indirect sequencing of animal communities via their trace DNA [161]. ...
Article
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Since their inception, DNA barcodes have become a powerful tool for understanding the biodiversity and biology of aquatic species, with multiple applications in diverse fields such as food security, fisheries, environmental DNA, conservation, and exotic species detection. Nevertheless, most aquatic ecosystems, from marine to freshwater, are understudied, with many species disappearing due to environmental stress, mostly caused by human activities. Here we highlight the progress that has been made in studying aquatic organisms with DNA barcodes, and encourage its further development in assisting sustainable use of aquatic resources and conservation.
... When using molecular methods for species identification and delimitation, generally from the variation in the cytochrome c oxidase subunit I (COI) gene (widely applied for freshwater animals [13][14][15][16]), most studies involve evaluation of genetic distances between populations and application of species delimitation algorithms such as jMOTU, GMYC, and ABGD, which diagnose the difference between intra-and inter-specific variability by statistical criteria [17][18][19]. Although these methods have been widely tested in aquatic taxa [5,[20][21][22][23][24], only a few studies use them in a comparative setting, i.e., simultaneously on several co-distributed freshwater species from the same area. ...
Article
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Comparative phylogeography has become a powerful approach in exploring hidden or cryptic diversity within widespread species and understanding how historical and biogeographical factors shape the modern patterns of their distribution. Most comparative phylogeographic studies so far focus on terrestrial and vertebrate taxa, while aquatic invertebrates (and especially freshwater invertebrates) remain unstudied. In this article, we explore and compare the patterns of molecular diversity and phylogeographic structure of four widespread freshwater copepod crustaceans in Eu-ropean water bodies: the harpacticoids Attheyella crassa, Canthocamptus staphylinus and Nitokra hiber-nica, and the cyclopoid Eucyclops serrulatus, using sequence data from mtDNA COI and nuclear ITS/18S rRNA genes. The three taxa A. crassa, C. staphylinus and E. serrulatus each consist of deeply diverged clusters and are deemed to represent complexes of species with largely (but not completely) non-overlapping distributions, while in N. hibernica only little differentiation was found, which may however reflect the geographically more restricted sampling. However, the geographical patterns of subdivision differ. The divisions in A. crassa and E. serrulatus follow an east-west pattern in Northern Europe whereas that in C. staphylinus has more of a north-south pattern, with a distinct Fennoscandian clade. The deep mitochondrial splits among populations of A. crassa, C. staphylinus and E. serrulatus (model-corrected distances 26-36%) suggest that divergence of the lin-eages predate the Pleistocene glaciations. This study provides an insight into cryptic diversity and biogeographic distribution of freshwater copepods.
... Avian endoparasite communities can be investigated by using metabarcoding approach among birds [79]. Hidden biodiversity can be discovered through the use of complementary monitoring of fish diet based on DNA barcoding, which is helpful to understand the complex ecosystem functions [80]. An overall biodiversity monitoring was evaluated by analyzing the quantitative trophic interactions among sympatric carnivores from three assemblages in the Mountains of Southwest China [74]. ...
Article
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Diet analysis is a critical content of animal ecology and the diet analysis methods have been constantly improving and updating. Contrary to traditional methods of high labor intensity and low resolution, the next generation sequencing (NGS) approach has been suggested as a promising tool for dietary studies, which greatly improves the efficiency and broadens the application range. Here we present a framework of adopting NGS and DNA metabarcoding into diet analysis, and discuss the application in aspects of prey taxa composition and structure, intra-specific and inter-specific trophic links, and the effects of animal feeding on environmental changes. Yet, the generation of NGS-based diet data and subsequent analyses and interpretations are still challenging with several factors, making it possible still not as widely used as might be expected. We suggest that NGS-based diet methods must be furthered, analytical pipelines should be developed. More application perspectives, including nutrient geometry, metagenomics and nutrigenomics, need to be incorporated to encourage more ecologists to infer novel insights on they work.
... DNA metabarcoding), now allows for hundreds of complex samples to be processed in parallel . Although powerful, the greatest drawbacks associated with high-throughput techniques lie in the up-front costs and the computational complexity of analysis (Jo et al. 2016). However, the cost of sequencing continues to decrease-particularly the persample costs when highly multiplexed-and open-source software is available for the analysis of many prey types (Bolyen et al. 2019, Palmer et al. 2018. ...
Article
Identifying the composition of avian diets is a critical step in characterizing the roles of birds within ecosystems. However, because birds are a diverse taxonomic group with equally diverse dietary habits, gaining an accurate and thorough understanding of avian diet can be difficult. In addition to overcoming the inherent difficulties of studying birds, the field is advancing rapidly, and researchers are challenged with a myriad of methods to study avian diet, a task that has only become more difficult with the introduction of laboratory techniques to dietary studies. Because methodology drives inference, it is important that researchers are aware of the capabilities and limitations of each method to ensure the results of their study are interpreted correctly. However, few reviews exist which detail each of the traditional and laboratory techniques used in dietary studies, with even fewer framing these methods through a bird-specific lens. Here, we discuss the strengths and limitations of morphological prey identification, DNA-based techniques, stable isotope analysis, and the tracing of dietary biomolecules throughout food webs. We identify areas of improvement for each method, provide instances in which the combination of techniques can yield the most comprehensive findings, introduce potential avenues for combining results from each technique within a unified framework, and present recommendations for the future focus of avian dietary research.
... This study represents an important step toward harnessing eDNA for the targeted detection of freshwater pearly mussels. Although traditional sampling methods will continue to be important for collecting population monitoring data such as abundance, demographic structure, sex ratios, and fecundity, eDNA offers a valuable complement to traditional sampling, as has already been demonstrated in other taxonomic groups (Jo et al., 2015;McKee et al., 2015). ...
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1. Documenting the occurrence and habitat occupancy of rare aquatic species is an ongoing challenge for conservation. Characterization of environmental DNA (eDNA) from bulk water samples has emerged as a powerful tool to infer species presence or absence without the need to observe or handle organisms. 2. Previous eDNA studies have yet to develop species-specific markers that target taxa with many potentially sympatric confamilials. Forty-one freshwater pearly mussel species (Unionidae) are found in southern Ontario, Canada, with many of these listed as threatened, endangered, or of conservation concern; however, locating populations for protection can be challenging due to morphological crypsis and species scarcity. 3. Species-specific eDNA markers were developed to target four unionid species. Following in silico and in vitro validation, markers were validated in the field by comparing eDNA results from water samples to detections based on quadrat sampling. 4. Target species were detected by eDNA sampling at all sites where they had previously been located by quadrat sampling. 5. The paired sampling design showed that 1) species-specific markers can be designed even within speciose families, and 2) eDNA detection of mussels is at least as sensitive as quadrat sampling. Furthermore, detection probabilities were not affected by sampling depth, and eDNA concentrations were positively correlated with mussel densities. 6. These findings confirm that eDNA assays are a valuable complement to traditional methods for locating and managing imperilled unionid populations.
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Bigeye (Lampris megalopsis) and smalleye (L. incognitus) opah are pelagic fishes that inhabit the North Pacific Subtropical Gyre. Previous studies examined opah diet using traditional morphological techniques to identify prey items, finding opah prey primarily on fishes and squids in the epipelagic and mesopelagic zones. Many of the challenges regarding traditional observational techniques stem from the rigor of identifying fragments of soft-bodied organisms and highly digested prey. This study uses dDNA (diet DNA) metabarcoding of stomach contents to illustrate how a genetic approach may complement traditional morphological identification of prey to increase the number of organisms detected and identify degraded prey to species level. To better understand the sexual dimorphism observed in opah species, diet was compared between male and female opah and as well as between opah of varying sizes. Stomachs from bigeye and smalleye opah were obtained from fish caught on deep-set tuna longline vessels and landed in Hawai‘i. Stomachs were dissected, and undigested organisms were removed and identified morphologically. The remaining stomach contents were homogenized and subsampled for DNA extraction. Extracted DNA was amplified at the cytochrome c oxidase subunit I (COI) and 12S regions and sequenced on an Illumina MiSeq. This study did not find any significant differences in prey community between male and female opah, but larger opah were found to have consumed more deep-dwelling prey species that inhabit depths greater than 500 m. Furthermore, this study found that metabarcoding enables the detection of recently consumed prey items to species level even when an individual’s stomach contains no distinguishable prey. This study detected never-before-seen prey items and provides evidence that dDNA metabarcoding can expand the diversity of identified prey. Incorporating dDNA metabarcoding into diet studies contributes additional life history information for highly migratory pelagic predators for which feeding cannot be directly observed.
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The main objective of this work was to develop and validate a robust and reliable 'from benchtop-to-desktop' metabarcoding workflow to investigate the diet of invertebrate-eaters. We applied our workflow to fecal DNA samples of an invertebrate-eating fish species. A fragment of the COI gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of non-arbitrary thresholds established from control samples and from molecular replicates in order to address the elimination of cross-contamination, PCR/sequencing errors and mistagging artifacts. This resulted in a conservative and informative metabarcoding dataset. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semi-quantitative statistic in our diet study, the Minimum Number of Individuals (MNI), which is based on the number of distinct variants in each sample. The metabarcoding approach described in this paper may guide future diet studies that aim to produce robust datasets associated with a fine and accurate identification of prey items. This article is protected by copyright. All rights reserved.
Article
DNA-barcoding approaches to estimate the diet compositions of grazing animals have received significant attention, and particularly when combined with next-generation sequencing, these techniques have substantially improved in recent years. In this study, the identity and species composition of plant material ingested by Mongolian sheep were estimated through the use of 350 bp ITS2 gene sequences of the vegetation found in fecal samples. Four diets were formulated using varying amounts of eight plant species that are common in the grasslands of northern China. Sixteen Mongolian sheep were taken from pastures and randomly assigned to four groups, and each group received one of four diets. Each sheep was randomly assigned to one of 16 confinement pens and fed its respective diet for 12 consecutive days. Fecal samples were removed from each pen from days 7–12, preserved, and composited for each pen. All herbage species included in the daily diets were detected in each fecal sample, with the exception of Phragmites australis. Moreover, 12 additional different plant species were retrieved from feces of the experimental sheep. The obtained data provided preliminary support for the use of the ITS2 barcode to determine which plants were consumed. Moreover, the proportions of the herbage DNA sequences recovered from sheep feces and those of the herbage masses in the daily diets did not completely match. These results indicate that the non-Gramineae DNA sequences amplified with ITS2 primers (including those of Chenopodium album, Artemisia scoparia, Artemisia tanacetifolia, and Medicago sativa) far exceeded those of the Gramineae species (including Leymus chinensis and Puccinellia distans), which constitute the largest share of the experimental diets. A significant positive correlation (Spearman’s ρ = 0.376, P = 0.003) between the actual herbage mass proportions in the experimental diets and the herbage-DNA-sequence proportions provided sufficiently favorable support for the further investigation of DNA barcoding for the quantification of plants in feces. A significant regression coefficient was found between the relative DNA-sequence proportions of L. chinensis (R² = 0.82, P < 0.0001), P. distans (R² = 0.64, P = 0.0017), and C. album (R² = 0.98, P < 0.0001) and their respective herbage mass proportions. The quantitative relationship can be expressed by the linear-regression equations y = 0.90x – 0.22, y = 0.98x – 0.03, and y = 5.00x – 0.25, respectively. Thus, these results demonstrate that dietary-DNA-barcoding methods exhibited potential in providing valuable quantitative information regarding food-item components. However, it should be noted that this explorative data needs to be further improved by using additional genes and by creating a sophisticated reference database, thus enhancing both quality and accuracy of the obtained results.
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In South Korea, the Eurasian otter (Lutra lutra (Linnaeus, 1758)), a semi-aquatic carnivore, is found mainly in lower order streams that tend to have a low abundance of preferred prey fish species. To investigate the relationship between resource use and availability, we used DNA barcoding to identify otter diet items in 24 otter spraints (faeces) from 16 sites along the Nakdong River basin from 4 to 6 June 2014. At these sites fish availability was assessed using scoop nets and casting nets. Fish formed the bulk of otter diet, which included also frogs, mammals, and reptiles. By DNA barcoding (success rate: 72.38%), we identified 79 prey items from 105 bone remains. The diet comprised mostly fish, but frogs, mammals, and reptiles were also identified. The fish fauna and otter diet composition differed significantly. Across the study sites, members of the Cyprinidae dominated in netted samples, but occurred less frequently in otter diet. Because most Cyprinidae are fast swimmers, otters also fed on benthic fishes and frogs, suggesting limited foraging flexibility in otters and specialization on more slowly moving prey.
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The trophic ecology of lake charr Salvelinus namaycush morphotypes from small and large lakes within their native and introduced ranges is reviewed over the past 50 years. The lake charr is an apex predator in most habitats it occupies, where it plays a significant role in defining food webs. While often considered piscivores, lake charr feed on a range of aquatic prey throughout their life history, including zooplankton, benthic invertebrates, and fish, as well as terrestrial insects, mammals, birds, amphibians, and reptiles. Lake charr diets that vary within morphotypes among lakes and among sympatric morphotypes reflect differences in habitat use, prey availability, and individual preferences. Temporal variability in diet can result from seasonal prey pulses, thermal barriers, and long-term prey dynamics. Lake charr adapt quickly to consume invasive prey fishes, and often decimate native prey fishes and other piscivores in lakes into which they are introduced. Salient research topics in lake charr trophic ecology include: (1) how best to quantify spatial and temporal trophic niche space; and, (2) how changing environmental conditions, such as invasive species and lake warming, will influence lake charr feeding and broader lake food-web dynamics.
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Feeding ecology explains predator’s preference to some preys over others in their habitat and their competitions thereof. The subject, as a functional and applied biology, is highly neglected and, in case of fish a uniform and consistent methodology is absent. The currently practiced methods are largely centered on mathematical indices and highly erroneous due to non-uniform outcomes. Therefore, it requires a relook into the subject to elucidate functional contributions to make it more comparable and comprehensive science. In this article, approachable methodological strategies have been forwarded in three hierarchical steps viz. food occurrence, feeding biology and interpretative ecology. All these steps involve wide ranges of techniques, within a scope of ecology but not limited to, and traverse from narrative to functional evolutionary ecology. The first step is an assumption-observation practice to assess food of fish, followed by feeding biology that links morphological, histological, cytological, bacteriological or enzymological correlations to preferred food in the environment. Interpretative ecology is the higher level of analysis, where the outcomes are tested and discussed against evolutionary theories. A description of possible pedagogics on methods of feeding ecological studies has also been forwarded.
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Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
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DNA metabarcoding enables efficient characterization of species composition in environmental DNA or bulk biodiversity samples, and this approach is making significant and unique contributions in the field of ecology. In metabarcoding of animals, the cytochrome c oxidase subunit I (COI) gene is frequently used as the marker of choice because no other genetic region can be found in taxonomically verified databases with sequences covering so many taxa. However, the accuracy of metabarcoding datasets is dependent on recovery of the targeted taxa using conserved amplification primers. We argue that COI does not contain suitably conserved regions for most amplicon-based metabarcoding applications. Marker selection deserves increased scrutiny and available marker choices should be broadened in order to maximize potential in this exciting field of research.
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Understanding predator-prey interactions is a major challenge in ecological studies. In particular, the accurate identification of prey is a fundamental requirement in elucidating food-web structure. This study took a molecular approach in determining the species identity of consumed prey items of a freshwater carnivorous fish (largemouth bass, Micropterus salmoides), according to their size class. Thirty randomly selected gut samples were categorized into three size classes, based on the total length of the bass. Using the universal primer for the mtDNA cytochrome oxidase I (COI) region, polymerase chain reaction (PCR) amplification was performed on unidentified gut contents and then sequenced after cloning. Two gut samples were completely empty, and DNA materials from 27 of 28 gut samples were successfully amplified by PCR (success rate: 96.4%). Sequence database navigation yielded a total of 308 clones, containing DNA from 26 prey items. They comprised four phyla, including seven classes, 12 orders, and 12 families based on BLAST and BOLD database searches. The results indicate that largemouth bass show selective preferences in prey item consumption as they mature. These results corroborate a hypothesis, presence of ontogenetic diet shift, derived through other methodological approaches. Despite the practical limitations inherent in DNA barcoding analysis, high-resolution (i.e., species level) identification was possible, and the predation patterns of predators of different sizes were identifiable. The utilization of this method is strongly recommended for determining specific predator-prey relationships in complex freshwater ecosystems.
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A comprehensive, but simple-to-use software package for executing a range of standard numerical analysis and operations used in quantitative paleontology has been developed. The program, called PAST (PAleontological STatistics), runs on standard Windows computers and is available free of charge. PAST integrates spreadsheettype data entry with univariate and multivariate statistics, curve fitting, time-series analysis, data plotting, and simple phylogenetic analysis. Many of the functions are specific to paleontology and ecology, and these functions are not found in standard, more extensive, statistical packages. PAST also includes fourteen case studies (data files and exercises) illustrating use of the program for paleontological problems, making it a complete educational package for courses in quantitative methods.
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We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements our RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
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Prompt and accurate methods for assessing the species composition of given areas are indispensable in addressing the rapid loss of biodiversity. Here, we propose a method for the surveillance of fish species composition in freshwater using environmental DNA as species markers. First, the applicability of the method was demonstrated through aquarium experiments. DNA was extracted from 120 ml aquarium water, and the degenerated primers targeting the fish mitochondrial cytochrome b gene were used for amplification. PCR-amplified fragments were analysed by random cloning, and all species reared in the aquarium were detected. Next, this method was applied to natural freshwater environments. Water samples were collected from three sites in the Yura River, Japan; DNA was concentrated from 2 l of environmental water, and then amplified and cloned. Up to four species of fish were detected by sequencing 47 randomly selected clones from a single water sample. Overall, the results were consistent with previous knowledge of fish habitat utilisation. Using this method, the surveillance of fish species composition can be conducted less laboriously than with traditional methods.
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Approximately 34 alien fish species have established populations in Australian freshwaters with a further 53 native fish species translocated within the country. Twelve pathways of human‐assisted dispersal for freshwater fish have been identified in Australia, and each is discussed with examples given. The major pathway has been the aquarium or ornamental fish industry, with 22 of the 34 alien species originating from this source. Management actions to date have largely focused on eradication or mitigation, with little national or interstate coordination of efforts. Future management actions are suggested, with priorities being public and industry education and the development of coordinated strategies based on pest management principles.
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Introduction The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. Results We first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. Conclusions The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.
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The US Long Term Ecological Research (LTER) Network enters its fourth decade with a distinguished record of achievement in ecological science. The value of long-term observations and experiments has never been more important for testing ecological theory and for addressing today's most difficult environmental challenges. The network's potential for tackling emergent continent-scale questions such as cryosphere loss and landscape change is becoming increasingly apparent on the basis of a capacity to combine long-term observations and experimental results with new observatory-based measurements, to study socioecological systems, to advance the use of environmental cyberinfrastructure, to promote environmental science literacy, and to engage with decisionmakers in framing major directions for research. The long-term context of network science, from understanding the past to forecasting the future, provides a valuable perspective for helping to solve many of the crucial environmental problems facing society today.
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Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days.
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1.Fish of the family Galaxiidae are restricted to the southern hemisphere where they occupy a diverse array of habitats ranging from over 2000 m in elevation to sea level. Some species are diadromous and, hence, freshwater, estuarine and marine habitats are used during their life-cycle; other species complete their entire life-cycles in freshwater environments.2.Tasmania has a diverse galaxiid fauna that accounts for 64% of native freshwater fish species found on the island. The Tasmanian galaxiid fauna is characterized by high species richness (5 genera and 16 species), endemism (11 species), restricted distributions, and non-diadromous life histories (11 species).3.The galaxiid fauna of Tasmania has significant conservation status with 69% of species considered ‘threatened’. The conservation status of the fauna is recognized at State, national and international levels.4.The key threats to galaxiids in Tasmania are exotic species, hydrological manipulations, restricted distributions, general habitat degradation and exploitation of stocks.5.Although work has recently been undertaken to conserve and manage Tasmanian galaxiid populations, the fauna is still thought to be imperilled. Knowledge gaps that need to be addressed include the biology and ecology of most species (e.g. reproductive biology, life histories, habitat use and requirements) and impacts of habitat manipulations, as well as mechanisms and impacts of interactions with exotic species. Techniques to monitor accurately the status of galaxiid species and their populations need to be developed and the coexistence of some galaxiids with introduced salmonids should also be examined. Copyright © 2005 John Wiley & Sons, Ltd.
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Sampling benthic macroinvertebrates in large rivers has several limitations, arising not only from the selectivity of traditional sampling gears but also from difficulty in capturing organisms that inhabit the deeper zones and high current velocities. Considering the importance of benthic macroinvertebrates as a food resource for fishes, the sampling restrictions in sediment collection done by dredges, and the importance of surveying benthos biodiversity, the objective of this study was to evaluate the stomach contents of five commonly-occurring invertivorous fish species as a means of complementing a benthic macroinvertebrate inventory. Three sampling campaigns (fish and benthic macroinvertebrate) were conducted in a reach of the Araguari River (Minas Gerais, Brazil), approximately 9km long and 90m wide. Astyanax altiparanae Garutti & Britski 2000, Leporinus friderici (Bloch 1794), Leporinus amblyrhynchus Garavello & Britski 1987, Iheringichthys labrosus (Lütken 1874) and Pimelodus maculatus Lacepède 1803 were the fish species collected and studied. To determine benthic macroinvertebrate taxonomic richness, a total of 54 Van Veen sediment samples were obtained. We compared lists of the benthic taxa found in fish stomachs with those from the sediment samples. The differences in the taxonomic composition of the benthic macroinvertebrate communities between the sediment samples and each fish species stomachs contents were assessed through NMDS and ANOSIM analyses, using a Sorensen similarity index with the presence/absence of taxa data. Independent of sampling period, additional benthic macroinvertebrate families or classes were provided by identifying fish stomach contents. We found a total of 30 taxa in this study, including 5 unique taxa (or 17% of the total) in the sediment samples, 9 unique taxa (30%) in the stomach samples, and 16 taxa (53%) common to both. The NMDS and ANOSIM analyses showed a significant separation between Van Veen sediment samples and two fish species stomach contents—L. amblyrhynchus and P. maculatus. These results indicate that fish can be used as additional samplers and are an efficient method to complement the benthic taxonomic inventory obtained through traditional sediment sampling techniques in large areas, as river segments and catchments. KeywordsStomach contents–Samplers–Benthos–Large rivers–Trophic interactions
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The contributions of environmental DNA to ecology are reviewed, focusing on diet, trophic interactions, species distributions and biodiversity assessment. Environmental DNA has the potential to dramatically improve quantitative studies in these fields. Achieving this, however, will require large investments of time and money into developing the relevant databases, models, and software.
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The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.
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Freshwater ecosystems are among the most endangered habitats on Earth, with thousands of animal species known to be threatened or already extinct. Reliable monitoring of threatened organisms is crucial for data-driven conservation actions but remains a challenge owing to nonstandardized methods that depend on practical and taxonomic expertise, which is rapidly declining. Here, we show that a diversity of rare and threatened freshwater animals--representing amphibians, fish, mammals, insects and crustaceans--can be detected and quantified based on DNA obtained directly from small water samples of lakes, ponds and streams. We successfully validate our findings in a controlled mesocosm experiment and show that DNA becomes undetectable within 2 weeks after removal of animals, indicating that DNA traces are near contemporary with presence of the species. We further demonstrate that entire faunas of amphibians and fish can be detected by high-throughput sequencing of DNA extracted from pond water. Our findings underpin the ubiquitous nature of DNA traces in the environment and establish environmental DNA as a tool for monitoring rare and threatened species across a wide range of taxonomic groups.
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Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research.
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DnaSP is a software package for the analysis of DNA polymorphism data. Present version introduces several new modules and features which, among other options allow: (1) handling big data sets (∼5 Mb per sequence); (2) conducting a large number of coalescent-based tests by Monte Carlo computer simulations; (3) extensive analyses of the genetic differentiation and gene flow among populations; (4) analysing the evolutionary pattern of preferred and unpreferred codons; (5) generating graphical outputs for an easy visualization of results. Availability: The software package, including complete documentation and examples, is freely available to academic users from: http://www.ub.es/dnasp
Book
Submerged macrophytes have been the object of intensive research, and a large body of literature exists on their growth, reproduction, and physiology. Several studies have focused on the interactions between submerged macrophytes and other autotrophic components and the impact of the plants on the dynamics of nutrients, dissolved organic and inorganic carbon, oxygen, and pH. Comparatively few studies have dealt with the ability of submerged macrophytes to modulate the structure and dynamics of pelagic and benthic food webs. Recently, however, the amount of research into the structuring role of submerged macrophytes in food webs has markedly increased, and the results obtained so far suggest that sub­ merged macrophytes are of significant importance for the food web interactions and environmental quality of lakes, even at relatively low areal plant coverage. For example, plants affect the interactions between predacious, planktivorous, and benthivorous fish and between fish and invertebrates, including key organisms such as large zooplankton and snails. Changes in these interactions in turn may have cascading effects on the entire food web in both the pelagial and the littoral zone. To provide a forum for discussion of recent results in this growing field of research and to define future research needs, a workshop was held on 16 to 20 June, 1996, at the Freshwater Centre in Silkeborg, Denmark. The present book is a result of the workshop. It is divided into three parts.
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The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
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A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
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A single solution reagent is described for the determination of phosphorus in sea water. It consists of an acidified solution of ammonium molybdate containing ascorbic acid and a small amount of antimony. This reagent reacts rapidly with phosphate ion yielding a blue-purple compound which contains antimony and phosphorus in a 1:1 atomic ratio. The complex is very stable and obeys Beer's law up to a phosphate concentration of at least 2 μg/ml.The sensitivity of the procedure is comparable with that of the stannous chloride method. The salt error is less than 1 %.
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The perception that primary production in lakes is positively related to phosphorus loading is based almost entirely on studies of phytoplankton. This is partly because benthic and pelagic habitats in lakes are often treated as separate ecosystems, the processes of which can be evaluated independently. However, light and nutrients often limit primary producers in both benthic and pelagic habitats. We tested the hypothesis that reductions in light associated with increases in phytoplankton could cause compensatory decreases in benthic algal (periphyton) primary production. We monitored production of periphyton on sediments (epipelon), periphyton on wood (epixylon), and phytoplankton in four lakes in upper Michigan, USA, from 1991 to 1995. During the summers of 1993-1995, we stimulated phytoplankton production in three of the lakes by fertilizing with nitrogen and phosphorus (N:P ≥ 25 by atoms) at rates between 0.3 and 2.0 mg P· m-3· d-1. The response of periphyton to fertilization was substratum specific: epixylon increased with fertilization, but epipelon decreased. However, when area-specific production was extrapolated to the whole-lake scale, epixylon never constituted >4% of benthic primary production. Thus, the decline in epipelic production dominated the benthic response to fertilization. We also estimated whole-lake (epipelon + phytoplankton) primary production. Epipelic algae constituted 50-80% of whole-lake primary production at ambient nutrient levels. However, only 10-40% of primary production was benthic at the highest fertilization rates. The increase in whole-lake primary production caused by water column fertilization was greatly overestimated when we did not include the compensatory decline in epipelic algae as they were shaded by increases in phytoplankton concentrations.
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Across the globe, regulatory bodies are moving towards an ecosystem-based approach to management (EAM) of oceans. As part of this directive, criteria have been outlined for defining ecologically and biologically significant areas and marine protected areas. One of the first steps in identifying either is the development of an inventory of species' spatial distributions and the identification of habitats used by different life history stages of these species. Many of these data have been gleaned from research vessel trawl surveys that only sample a slice of the ecosystem. Here, for the first time, food habits data collected from a trawl survey were integrated with the original trawl data to improve estimates of species richness, distribution, and habitat associations with the objective of providing additional information for an EAM. The inclusion of stomach content data in creased the number and size range of species ob served: its greatest value was in describing invertebrate and small finfish species richness. We found differences in the spatial patterns and the influence of environmental factors on species richness be tween stomach and trawl samples. We conclude that the addition of stomach data can en rich the definition process for spatial management tools, adding dimensions that would otherwise be missed. This has important implications for EAM since we may not be using all of the resources at our disposal.
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The Barcode of Life Data System (BOLD) is an informatics workbench aiding the acquisition, storage, analysis and publication of DNA barcode records. By assembling molecular, morphological and distributional data, it bridges a traditional bioinformatics chasm. BOLD is freely available to any researcher with interests in DNA barcoding. By providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation in the global sequence databases. Because of its web-based delivery and flexible data security model, it is also well positioned to support projects that involve broad research alliances. This paper provides a brief introduction to the key elements of BOLD , discusses their functional capabilities, and concludes by examining computational resources and future prospects.
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Ecological understanding of the role of consumer-resource interactions in natural food webs is limited by the difficulty of accurately and efficiently determining the complex variety of food types animals have eaten in the field. We developed a method based on DNA metabarcoding multiplexing and next-generation sequencing to uncover different taxonomic groups of organisms from complex diet samples. We validated this approach on 91 faeces of a large omnivorous mammal, the brown bear, using DNA metabarcoding markers targeting the plant, vertebrate, and invertebrate components of the diet. We included internal controls in the experiments and performed PCR replication for accuracy validation in post-sequencing data analysis. Using our multiplexing strategy, we significantly simplified the experimental procedure and accurately and concurrently identified different prey DNA corresponding to the targeted taxonomic groups, with ≥60% of taxa of all diet components identified to genus/species level. The systematic application of internal controls and replication was a useful and simple way to evaluate the performance of our experimental procedure, standardize the selection of sequence filtering parameters for each marker data, and validate the accuracy of the results. Our general approach can be adapted to the analysis of dietary samples of various predator species in different ecosystems, for a number of conservation and ecological applications entailing large-scale population level diet assessment through cost effective screening of multiple DNA metabarcodes, and the detection of fine dietary variation among samples or individuals and of rare food items. This article is protected by copyright. All rights reserved.
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Careful attention to pH, alkalinity, the neutralizing buffer, reaction vessels, and dilution factors have proved neccessary. This modified method is acceptable for samples from fresh to oceanic waters, is accurate for organic compounds tested, has a maximum capability of 40 micrometre nitrogen in undiluted samples, and has a mid-range precision of + or -2%. -from Authors
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From December 2003 to September 2004, benthic macroinvertebrates (BM), fishes, water and sediment were collected quarterly at six stations in two streams of the upper Sao Francisco River basin, south-eastern Brazil. We evaluated the ecological conditions, habitat diversity, water quality, composition and structure of BM communities, as well as the food habits of the local fish fauna. By applying a protocol for rapid characterization of ecological conditions and habitat diversity, three of the sampled localities were classified as "pristine" while the others stations were considered "altered". A well oxygenated water with near neutral pH and low electric conductivity (< 0.01 mS/cm) and nutrient concentrations (< 0.08 mg/l total P and < 0.90 mg/l total N) was found for both streams. Sediment analysis revealed the dominance of medium, fine and very fine sand fractions in all sampled areas. The organic matter content in the sediment was higher in the lotic and well preserved area. We found 45 BM taxa, and Chironomidae (68%), Oligochaeta (10%) and Elmidae (8.5%) showed the highest abundances. From the stomach contents analysis of 13 fish species, 26 BM taxa were found, including four that were not collected in the sediment samples, being Chironomidae the dominant group (> 60%). Our results show that human activities such as forest clearing, agriculture and cattle rising have altered the habitat diversity in freshwater ecosystems in a process that affects the aquatic biota and thus the food availability to the fish fauna. The results also highlight the importance of the fish stomach contents analysis as a complementary tool in BM inventories.
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This paper investigates the extent to which there is hierarchical information about area-relationships contained in the distributions of Australian vertebrates. Distributions of genera, their included species, and their included subspecies (when appropriate) were coded present/absent for 14 areas of endemism. The data were then analysed cladistically and most parsimonious area-cladograms constructed; bootstrap consensus trees were used to assess the strength of the cladistic signal. Distributions of birds and mammals were found to be nearly identical in their hierarchical pattern, and snakes shared the same general pattern of area-relationships. Frogs and lizards exhibited greater differences but were still congruent in some respects. This congruence calls for a general explanation. It is proposed that either the pattern can be explained in terms of (1) true area-relationships (vicariance), in which case biogeographic noise, including redundant distributions and widespread taxa do not disturb historical signal; (2) constraints on processes or events that lead to congruent histories of cosmopolitanism; or (3) a combination of both.
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The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
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Large-scale monitoring schemes are essential in assessing global mammalian biodiversity, and in this framework, leeches have recently been promoted as an indirect source of DNA from terrestrial mammal species. Carrion feeding flies are ubiquitous and can be expected to feed on many vertebrate carcasses. Hence, we tested whether fly-derived DNA analysis may also serve as a novel tool for mammalian diversity surveys. We screened DNA extracted from 201 carrion flies collected in tropical habitats of Côte d'Ivoire and Madagascar for mammal DNA using multiple PCR systems and retrieved DNA sequences from a diverse set of species (22 in Côte d'Ivoire, four in Madagascar) exploiting distinct forest strata and displaying a broad range of body sizes. Deep sequencing of amplicons generated from pools of flies performed equally well as individual sequencing approaches. We conclude that the analysis of fly-derived DNA can be implemented in a very rapid and cost-effective manner and will give a relatively unbiased picture of local mammal diversity. Carrion flies therefore represent an extraordinary and thus far unexploited resource of mammal DNA, which will probably prove useful for future inventories of wild mammal communities.
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1. The annual input, contribution to the diet of salmonids, and quantitative input of terrestrial invertebrates to four reaches with contrasting forest (n=2) and grassland riparian vegetation (n=2) were compared in a Japanese headwater stream. 2. The annual input of terrestrial invertebrates falling into the forest reaches (mean±1 SE=8.7×10³±0.3×10³ mg m⁻² year⁻¹) was 1.7 times greater than that in the grassland reaches (5.1×10³±0.8×10³ mg m⁻² year⁻¹), with clear seasonality in the daily input of invertebrates in both vegetation types. The daily input, however, differed between the vegetation types only in summer, when it rose to a maximum in both vegetation types. 3. Fish biomass also differed among the seasons in both vegetation types, being less in the grassland reaches. The contribution of terrestrial invertebrates to the salmonid diet in the forest and grassland reaches was 11 and 7% in spring, 68 and 77% in summer, 48 and 33% in autumn, and 1 and 1% in winter, respectively. The prey consumption rate of fish, which was similar between the vegetation types, increased with stream temperature and was highest in summer. Terrestrial invertebrates supported 49% (mean±1 SE=5.3×10³±0.4×10³ mg m⁻² year⁻¹) of the annual, total prey consumption (10.9×10³±1.7×10³ mg m⁻² year⁻¹) by salmonids in the forest and 53% (2.0×10³±0.3×10³ mg m⁻² year⁻¹) (3.8×10³±0.6×10³ mg m⁻² year⁻¹) in the grassland reaches. 4. Salmonids were estimated to consume 51 and 35% of the annual total (falling plus drift) input of terrestrial invertebrates in the forest and grassland reaches, respectively. The input of terrestrial invertebrates by drift, however, was almost equal to the output in both vegetation types, suggesting that the reach-based, in-stream retention of terrestrial invertebrates almost balanced these falling in. 5. Difference in the riparian vegetation, which caused spatial heterogeneity in the input of terrestrial invertebrates, could play an important role in determining the local distribution of salmonids.
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The objective of this study was to demonstrate the importance of chironomid (Diptera, Insecta) larvae as food resource for Leporinus amblyrhynchus Garavello & Britski, 1987 (Anostomidae, Characiformes) and Pimelodus maculatus Lacépède 1803 (Pimelodidae, Siluriformes) and to call attention for the importance of the trophic relation between fish and chironomids assemblages in Miranda reservoir in Araguari River (Minas Ge- rais State, Brazil), focusing on the influence of the dam on the structure and taxonomic composition of chironomid assemblage. A total of 318 stomach contents of L. amblyrhynchus and P. maculatus were checked of which 108 had chironomid larvae. The number of chironomid larvae observed in stomach contents was higher for L. amblyrhynchus (86.3%) than for P. maculatus (52.7%). The recorded taxonomic richness of chironomid larvae in the stomach contents, suggest that these two fish species invest considerable time and energy feeding on chironomid larvae, thus consisting an important food resource. The recorded data constitute important additional information on the biology of the two studied fish species.
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The ratio of the number of observed taxa to that expected to occur in the absence of human-caused stress (O/E) is an intuitive and ecologically meaningful measure of biological integrity. We examined how O/E ratios derived from stream invertebrate data varied among 234 unimpaired reference sites and 254 test sites potentially impaired by past logging. Data were collected from streams in three montane ecoregions in California. Two sets of River Invertebrate Prediction and Classification System (RIVPACS) predictive models were built: one set of models was based on near-species taxonomic resolution; the other was based on family identifications. Two models were built for each level of taxonomic resolution: one calculated O and E based on all taxa with probabilities of capture (Pc) > 0; the other calculated O and E based on only those taxa with Pc ≥ 0.5. Evaluations of the performance of each model were based on three criteria: (1) how well models predicted the taxa found at unimpaired sites, (2) the degree to which O/E values differed among unimpaired reference sites and potentially impaired test sites, and (3) the degree to which test site O/E values were correlated with independent measures of watershed alteration. Predictions of species models were more accurate than those of family models, and predictions of the Pc ≥ 0.5 species model were more robust than predictions of the Pc ≥ 0 model. O/E values derived from both species models were related to land use variables, but only assessments based on the Pc ≥ 0.5 model were insensitive to naturally occurring differences among streams, ecoregions, and years.
Article
SUMMARY 1. Pelagic and epipelic microalgal production were measured over a year in a pre-defined area (depth 0.5 m) in each of two lakes, one turbid and one with clear water. Further estimates of epiphytic production within reed stands were obtained by measuring production of periphyton developed on artificial substrata. 2. Total annual production of phytoplankton and epipelon was 34% greater in the turbid lake (190 g C m−2 year−1) than in the clearwater lake (141 g C m−2 year−1). However, the ratio of total production to mean water column TP concentration was two fold greater in the clearwater lake. 3. Phytoplankton accounted for the majority of the annual production (96%) in the turbid lake, while epipelic microalgal production dominated (77%) in the clear lake. The relative contribution of epipelic algae varied over the year, however, and in the turbid lake was higher in winter (11–25%), when the water was relatively clear, than during summer (0.7–1.7%), when the water was more turbid. In the clearwater lake, the relative contribution of epipelon was high both in winter, when the water was most clear, and in mid-summer, when phytoplankton production was constrained either by nutrients or grazing. 4. Compared with pelagic and epipelic primary production, epiphytic production within a reed stand was low and did not vary significantly between the lakes. 5. The study supports the theory of a competitive and compensatory trade-off between primary producers in lakes with contrasting nutrient concentrations, resulting in relatively small differences in overall production between clear and turbid lakes when integrating over the season and over different habitats.
Article
With nearly one quarter of mammalian species threatened, an accurate description of their distribution and conservation status is needed [1]. For rare, shy or cryptic species, existing monitoring methods are often prohibitively expensive or unreliable. The problem is particularly acute in tropical forests, where a disproportionate number of species are listed by IUCN as 'data deficient'[2], due to the difficulty of monitoring with conventional approaches. This presents serious obstacles to conservation management. We, here, describe a new screening tool, the analysis of mammalian DNA extracted from haematophagous leeches. By demonstrating that PCR amplifiable mammalian blood DNA survives for at least four months post feeding in haematophagous Hirudo spp. leeches, we hypothesise that most wild caught adult leeches will contain DNA traces of their last blood meal. We subsequently demonstrate the efficacy of the method, by testing it in situ using terrestrial Haemadipsa spp. leeches caught in a tropical Vietnamese rainforest setting, and identify cryptic, rare and newly discovered mammalian species. We propose that DNA from leeches represents a quick, cost-effective and standardised way to obtain basic data on mammalian biodiversity and species occupancy, facilitating efficient use of limited conservation resources.
Article
A single solution reagent is described for the determination of phosphorus in sea water. It consists of an acidified solution of ammonium molybdate containing ascorbic acid and a small amount of antimony. This reagent reacts rapidly with phosphate ion yielding a blue-purple compound which contains antimony and phosphorus in a 1:1 atomic ratio. The complex is very stable and obeys Beer's law up to a phosphate concentration of at least 2 μg/ml.The sensitivity of the procedure is comparable with that of the stannous chloride method. The salt error is less than 1 %.RésuméUne méthode spectrophotométrique est décrite pour le dosage du phosphate dans l'eau de mer, an moyen de molybdate d'ammonium, en présence d'acide ascorbique et d'antimoinc. Il se forme rapidement un composé violet bleu, renfermant antimoine et phosphore dans un rapport atomique de 1:1.ZusammenfassungBeschreibung einer Methode zur Bestimmung von Phosphat in Mecrwasser mit Hilfe von Ammoniummolybdat in Gegenwart von Ascorbinsäure und Antimon. Der gebildete blau-violette Komplex wird spektrophotometrisch gemessen.
Article
Biological monitoring has failed to develop from simple binary assessment outcomes of the impacted ⁄ unimpacted type, towards more diagnostic frameworks, despite significant scientific effort over the past fifty years. It is our assertion that this is largely because of the limited information content of biological samples processed by traditional morphology-based taxonomy, which is a slow, imprecise process, focused on restricted groups of organisms. We envision a new paradigm in ecosystem assessment, which we refer to as ‘Biomonitoring 2.0’. This new schema employs DNA-based identification of taxa, coupled with high-throughput DNA sequencing on next-generation sequencing platforms. We discuss the transformational nature of DNA-based approaches in biodiversity discovery and ecosystem assessment and outline a path forward for their future widespread application.
Article
Biological monitoring has failed to develop from simple binary assessment outcomes of the impacted/unimpacted type, towards more diagnostic frameworks, despite significant scientific effort over the past fifty years. It is our assertion that this is largely because of the limited information content of biological samples processed by traditional morphology-based taxonomy, which is a slow, imprecise process, focused on restricted groups of organisms. We envision a new paradigm in ecosystem assessment, which we refer to as ‘Biomonitoring 2.0’. This new schema employs DNA-based identification of taxa, coupled with high-throughput DNA sequencing on next-generation sequencing platforms. We discuss the transformational nature of DNA-based approaches in biodiversity discovery and ecosystem assessment and outline a path forward for their future widespread application.
Article
A new method for the isolation of intracellular and extacellular DNA from a range of sediment types has been developed. This method is based upon the direct lysis of cells in the sediment, extraction of released DNA from the sediments and its subsequent purification by CsCL-EtBr gradient centrifugation and/or hydroxyapatite chromatography. Yields of 26 μg intracellular DNA and 1 μg extracellular DNA have been obtained per gram of sediment.
Article
DNA molecules originating from animals and plants can be retrieved directly from sediments and have been used for reconstructing both contemporary and past ecosystems. However, the extent to which such 'dirt' DNA reflects taxonomic richness and structural diversity remains contentious. Here, we couple second generation high-throughput sequencing with 16S mitochondrial DNA (mtDNA) meta-barcoding, to explore the accuracy and sensitivity of 'dirt' DNA as an indicator of vertebrate diversity, from soil sampled at safari parks, zoological gardens and farms with known species compositions. PCR amplification was successful in the full pH range of the investigated soils (6.2 ± 0.2 to 8.3 ± 0.2), but inhibition was detected in extracts from soil of high organic content. DNA movement (leaching) through strata was evident in some sporadic cases and is influenced by soil texture and structure. We find that DNA from the soil surface reflects overall taxonomic richness and relative biomass of individual species. However, one species that was recently introduced was not detected. Furthermore, animal behaviour was shown to influence DNA deposition rates. The approach potentially provides a quick methodological alternative to classical ecological surveys of biodiversity, and most reliable results are obtained with spatial sample replicates, while relative amounts of soil processed per site is of less importance.
Article
We present here a new version of the Arlequin program available under three different forms: a Windows graphical version (Winarl35), a console version of Arlequin (arlecore), and a specific console version to compute summary statistics (arlsumstat). The command-line versions run under both Linux and Windows. The main innovations of the new version include enhanced outputs in XML format, the possibility to embed graphics displaying computation results directly into output files, and the implementation of a new method to detect loci under selection from genome scans. Command-line versions are designed to handle large series of files, and arlsumstat can be used to generate summary statistics from simulated data sets within an Approximate Bayesian Computation framework.