Tuesday, April 25, 2017

Tuesday reads

Happy DNA Day! Has been a little while since the last post but I have been busy with a lot of different tasks and working myself into new projects. Here is hope that things will go back to normal slowly. Lots of interesting reads today starting with two of my own.

The School Malaise Trap Program (SMTP) provides a technologically sophisticated and scientifically relevant educational experience that exposes students to the diversity of life, enhancing their understanding of biodiversity while promoting environmental stewardship. Since 2013, the SMTP has allowed 15,000 students at 350 primary and secondary schools to explore insect diversity in Canadian schoolyards. Students at each school collected hundreds of insects for an analysis of DNA sequence variation that enabled their rapid identification to a species. Through this hands-on approach, they participated in a learning exercise that conveys a real sense of scientific discovery. As well, the students contributed valuable data to the largest biodiversity genomics initiative ever undertaken: the International Barcode of Life project. To date, the SMTP has sequenced over 80,000 insect specimens, which includes representatives of 7,990 different species, nearly a tenth of the Canadian fauna. Both surprisingly and importantly, the collections generated the first DNA barcode records for 1,288 Canadian species.

To date the global initiative to barcode all fishes, FISH-BOL, has delivered barcodes for approximately 14,400 of the 30,000 fish species; there is still much to do to attain its ultimate goal of barcoding all the world’s fishes. One strategy to overcome local gaps is to initiate short but intensive efforts to collect and barcode as many species as possible from a small region – a barcode ‘blitz’. This study highlights one such event, for the marine waters around Lizard island in the Great Barrier Reef (Queensland, Australia). Barcode records were obtained from 983 fishes collected over a two-week period. The resulting dataset comprised 358 named species and another 13 species that presently can only be reliably identified to genus level. Overall, this short expedition provided DNA barcodes for 13% of all marine fish species known to occur in Queensland.

The difficulty of censusing marine animal populations hampers effective ocean management. Analyzing water for DNA traces shed by organisms may aid assessment. Here we tested aquatic environmental DNA (eDNA) as an indicator of fish presence in the lower Hudson River estuary. A checklist of local marine fish and their relative abundance was prepared by compiling 12 traditional surveys conducted between 1988–2015. To improve eDNA identification success, 31 specimens representing 18 marine fish species were sequenced for two mitochondrial gene regions, boosting coverage of the 12S eDNA target sequence to 80% of local taxa. We collected 76 one-liter shoreline surface water samples at two contrasting estuary locations over six months beginning in January 2016. eDNA was amplified with vertebrate-specific 12S primers. Bioinformatic analysis of amplified DNA, using a reference library of GenBank and our newly generated 12S sequences, detected most (81%) locally abundant or common species and relatively few (23%) uncommon taxa, and corresponded to seasonal presence and habitat preference as determined by traditional surveys. Approximately 2% of fish reads were commonly consumed species that are rare or absent in local waters, consistent with wastewater input. Freshwater species were rarely detected despite Hudson River inflow. These results support further exploration and suggest eDNA will facilitate fine-scale geographic and temporal mapping of marine fish populations at relatively low cost.

We report the discovery of a non-native gammarid, Gammarus fossarum (Koch, 1836) (Crustacea, Amphipoda), in UK rivers. Gammarus fossarum is a common freshwater gammarid in many parts of mainland Europe, but was previously considered absent from the UK. Gammarus fossarum was detected in a number of UK rivers following DNA metabarcoding of a mini-barcode region of the COI gene in macroinvertebrate kick samples, and environmental DNA (eDNA) from water and sediment samples. Subsequent morphological analysis and standard DNA barcoding showed that the species could be reliably identified and separated from Gammarus pulex (Linnaeus, 1758), the most dominant and widespread native freshwater gammarid in the UK. Our data demonstrate extensive geographical coverage of G. fossarum in the UK, spanning distant river catchments. At present there is no data to confirm the likely origin of G. fossarum’s introduction. Subsequent re-examination of historic archive material shows the species to have been present in the UK since at least 1964. This study is among the first to demonstrate the potential of eDNA metabarcoding for detection of new non-native species. 

Consumption of frog legs is increasing worldwide, with potentially dramatic effects for ecosystems. More and more functioning frog farms are reported to exist. However, due to the lack of reliable methods to distinguish farmed from wild-caught individuals, the origin of frogs in the international trade is often uncertain. Here, we present a new methodological approach to this problem. We investigated the isotopic composition of legally traded frog legs from suppliers in Vietnam and Indonesia. Muscle and bone tissue samples were examined for δ15N, δ13C, and δ18O stable isotope compositions, to elucidate the conditions under which the frogs grew up. We used DNA barcoding (16S rRNA) to verify species identities. We identified three traded species (Hoplobatrachus rugulosus, Fejervarya cancrivora and Limnonectes macrodon); species identities were partly deviating from package labeling. Isotopic values of δ15N and δ18O showed significant differences between species and country of origin. Based on low δ15N composition and generally little variation in stable isotope values, our results imply that frogs from Vietnam were indeed farmed. In contrast, the frogs from the Indonesian supplier likely grew up under natural conditions, indicated by higher δ15N values and stronger variability in the stable isotope composition. Our results indicate that stable isotope analyses seem to be a useful tool to distinguish between naturally growing and intensively farmed frogs. We believe that this method can be used to improve the control in the international trade of frog legs, as well as for other biological products, thus supporting farming activities and decreasing pressure on wild populations. However, we examined different species from different countries and had no access to samples of individuals with confirmed origin and living conditions. Therefore, we suggest improving this method further with individuals of known origin and history, preferably including samples of the respective nutritive bases.

Biological diversity is depleting at an alarming rate. Additionally, a vast amount of biodiversity still remains undiscovered. Taxonomy has been serving the purpose of describing, naming, and classifying species for more than 250 years. DNA taxonomy and barcoding have accelerated the rate of this process, thereby providing a tool for conservation practice. DNA barcoding and traditional taxonomy have their own inherent merits and demerits. The synergistic use of both methods, in the form of integrative taxonomy, has the potential to contribute to biodiversity conservation in a pragmatic timeframe and overcome their individual drawbacks. In this review, we discuss the basics of both these methods of biological identification- traditional taxonomy and DNA barcoding, the technical advances in integrative taxonomy, and future trends. We also present a comprehensive compilation of published examples of integrative taxonomy that refer to nine topics within biodiversity conservation. Morphological and molecular species limits were observed to be congruent in ~41% of the 58 source studies. The majority of the studies highlighted the description of cryptic diversity through the use of molecular data, whereas research areas like endemism, biological invasion, and threatened species were less discussed in the literature.

Effective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next-generation sequencing (NGS) is a high-throughput technology that has the potential to modernise vector surveillance. When combined with DNA barcoding, it is termed 'metabarcoding'. The aim of our study was to establish a metabarcoding protocol to characterise pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269 bp section of the mitochondrial cytochrome c oxidase subunit I (COI) locus. Additionally, a 67 bp region of the RRV E2 gene was amplified from synthesised cDNA to screen for RRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using a DNA barcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species and RRV in every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species and RRV from a single mosquito. The primers used for amplification, number of PCR cycles, and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches. 

The potential of DNA barcoding approaches to identify single species and characterize species compositions strongly depends on the marker choice. The prominent "Folmer region", a 648 basepair fragment at the 5' end of the mitochondrial CO1 gene, has been traditionally applied as a universal DNA barcoding region for metazoans. In order to find a suitable marker for biomonitoring odonates (dragonflies and damselflies), we here explore a new region of the CO1 gene (CO1B) for DNA barcoding in 51 populations of 23 dragonfly and damselfly species. We compare the "Folmer region", the mitochondrial ND1 gene (NADH dehydrogenase 1) and the new CO1 region with regard to (i) speed and reproducibility of sequence generation, (ii) levels of homoplasy and (iii) numbers of diagnostic characters for discriminating closely related sister taxa and populations. The performances of the gene regions regarding these criteria were quite different. Both, the amplification of CO1B and ND1 was highly reproducible and CO1B showed the highest potential for discriminating sister taxa at different taxonomic levels. In contrast, the amplification of the "Folmer region" using the universal primers was difficult and the third codon positions of this fragment have experienced nucleotide substitution saturation. Most important, exploring this new barcode region of the CO1 gene identified a higher discriminating power between closely related sister taxa. Together with the design of layered barcode approaches adapted to the specific taxonomic "environment", this new marker will further enhance the discrimination power at the species level.

Monday, April 24, 2017

From the inbox: Postdoc @ Kartzinel Lab at Brown

Position Title: Postdoctoral Research Associate in Environment and Society: Molecular Ecology/Conservation Biology

Location: Department of Ecology and Evolutionary Biology; Institute at Brown for Environment and Society, Brown University, Providence, Rhode Island, USA

Position Description: The Kartzinel Lab at Brown University is seeking a postdoctoral research associate in molecular ecology and conservation biology to collaborate on studies of the ecology and evolution of animal diets, especially in African savannas (http://www.kartzinellab.com/). The postdoctoral project will emphasize conceptual and analytical advances in our understanding of plant-herbivore, predator-prey, and/or host-microbe interaction networks using DNA- based analyses. These results will be placed in the context of manipulative field experiments, broad geographic gradients, and different land use histories in order to illuminate the biological processes that determine population and community dynamics in a changing world. The postdoc will have ample opportunities to integrate both field and lab-based research on wildlife in Kenya, and other potentially relevant field sites. The Department of Ecology and Evolutionary Biology and Institute at Brown for Environment and Society are home to a diverse community of scholars and world-class scientific resources.

Qualifications: The successful candidate will have a recent Ph.D. and relevant experience in molecular ecology, such as environmental DNA analysis, microbiome analyses, DNA (meta) barcoding, metagenomics, or related approaches. A strong interest in community ecology and conservation is required, and prior field experience is a plus. Responsibilities include: helping develop and coordinate laboratory and field research activities; data management; analysis of ecological and molecular data; publication of manuscripts and dissemination of results. Candidates should demonstrate strong communication skills and an ability to work both independently and collaboratively with groups from diverse backgrounds.

Applicants should submit: (1) a cover letter describing research interests, qualifications, and motivations, (2) a CV, and (3) contact information for three references. Applications will be reviewed starting May 12, 2017, and accepted until the position is filled. The ideal start date is September 2017, but flexible. The initial appointment will be for one year with an opportunity for extension based on satisfactory performance. Please contact Tyler Kartzinel directly with any questions.

Equal Employment Opportunity Statement: Brown University is committed to fostering a diverse and inclusive academic global community; as an EEO/AA employer, Brown considers applicants for employment without regard to, and does not discriminate on the basis of, gender, race, protected veteran status, disability, or any other legally protected status.

Tuesday, April 18, 2017

Metabarcoding and Metagenomics journal

Not too long ago I posted a call for participation at a questionnaire which solicited feedback and suggestions for a new open access journal focusing on metabarcoding and metagenomics. Today, just a few months later the journal out in the open and ready for submission:

Metabarcoding and Metagenomics (MBMG) is an innovative open access journal which facilitates the publication of articles on metabarcoding and metagenomics both in basic and applied context. The journal welcomes submissions representing all stages of the research cycle: data, models, methods, workflows, software, perspectives, opinions, and conventional research articles. The journal will consider manuscripts for publication related (but not limited) to the following topics: Environmental MBMG, Microbial MBMG, Applied MBMG (biomonitoring, quarantine, environmental assessment, nature conservation, eDNA, species invasions and others), and other emerging fields related to MBMG. Submissions of bioinformatic approaches to MBMG (algorithms, software) are also encouraged.

So, if you are currently working on a metabarcoding or metagenomics study and looking for a home for your publication you might want to consider this new option.

Thursday, April 13, 2017


7th International Barcode of Life Conference, 20 – 24 November 2017 at Kruger National Park


The African Centre for DNA Barcoding (ACDB), the University of  Johannesburg (UJ), International Barcode of Life Project (iBOL), and the Department of Environmental Affairs (DEA) are proud to announce and welcome delegates to our hosting of the 7th International Barcode of Life Conference, 20 – 24 November 2017. This is the first time that this event will be held on the African continent. The venue for the hosting of this prestigious event will be the Nombolo Mdhluli Conference Centre, 

The major theme of the conference is exploring mega-diverse biotas with DNA barcodes. A series of presentations and workshops will focus on the use of DNA to understand diversity patterns and ecological processes in species-rich and complicated ecosystems. The conference also provides a 
general forum for presentations, posters, and discussion on the wider field of DNA barcoding.

Delegates are encouraged to register as soon as possible as space is limited.

Abstracts should be submitted here before or on 28th April 2017.

More information at:

Website: http://dnabarcodes2017.org
Facebook: @DNABarcodes2017
Twitter: @DNABarcodes

Wednesday, April 12, 2017

Wednesday reads

Two days delay and a lot to go through but I think I came up with a list of quite interesting reads:

Phlebotomine sand flies are haematophagous dipterans of primary medical importance. They represent the only proven vectors of leishmaniasis worldwide and are involved in the transmission of various other pathogens. Studying the ecology of sand flies is crucial to understand the epidemiology of leishmaniasis and further control this disease. A major limitation in this regard is that traditional morphological-based methods for sand fly species identifications are time-consuming and require taxonomic expertise. DNA metabarcoding holds great promise in overcoming this issue by allowing the identification of multiple species from a single bulk sample. Here, we assessed the reliability of a short insect metabarcode located in the mitochondrial 16S rRNA for the identification of Neotropical sand flies, and constructed a reference database for 40 species found in French Guiana. Then, we conducted a metabarcoding experiment on sand flies mixtures of known content and showed that the method allows an accurate identification of specimens in pools. Finally, we applied metabarcoding to field samples caught in a 1-ha forest plot in French Guiana. Besides providing reliable molecular data for species-level assignations of phlebotomine sand flies, our study proves the efficiency of metabarcoding based on the mitochondrial 16S rRNA for studying sand fly diversity from bulk samples. The application of this high-throughput identification procedure to field samples can provide great opportunities for vector monitoring and eco-epidemiological studies.

Translating the vast amounts of molecular barcodes from global surveys of microbial eukaryotes into ecological insight depends critically on a well-curated reference database with adequate taxonomic coverage. In this respect, the choanoflagellates resemble other eukaryotic lineages: reasonable coverage at higher taxonomic levels, but missing diversity at the species level. The acanthoecid (loricate) choanoflagellates are well-characterized morphologically, with over 115 species described, but less than 10% with any sequence data. Because lorica shape is species-specific, the acanthoecids represent an opportunity to link morphological with molecular data within a lineage of eukaryotes. To match morphospecies to sequences, we sampled the Kattegat and the Isefjord in Denmark in September 2014 and February 2015. We identified 45 morphospecies and sequenced ribosomal DNA of nine previously unsequenced species, roughly doubling the number of acanthoecid species with sequence data, including the first data representing five genera: Bicosta, Calliacantha, Cosmoeca, Crinolina and Pleurasiga. Our phylogenetic analysis is mainly congruent with morphology-based systematics. Five of the newly sequenced species match a previously unidentified barcode from Tara Oceans, providing access to the global distribution of species isolated from Danish waters. One species, Calliacantha natans, is the second most globally abundant choanoflagellate present in Tara Oceans. Our project translating new ribosomal DNA sequences to distributions of described species on a global scale supports the approach linking morphology to molecular barcodes for microbial eukaryote ecology.

Almost all plants in nature harbour fungi in their roots but the knowledge on distribution and the underlying principles of assemblage is still poorly developed for the root-associated fungi. In this study we analysed the root endophytic fungal communities associated with switchgrass, rosette grass, and pitch pine in the acidic, oligotrophic pine barrens ecosystem. A total of 434 fungal isolates were obtained from 600 root segments of 60 plant samples. DNA barcoding and morphological analyses identified 92 fungal species, which belong to 39 genera in six classes. Compared to other ecosystems, the pine barrens has a higher proportion of Leotiomycetes. The fungal community associated with pitch pine was significantly different from those associated with the grasses, while less difference was found between those associated with the two grasses. Our results suggest that edaphic factors and host specificity play a role in shaping root endophytic fungal community. This study also corroborates our previous finding that plant roots in the pine barrens are a rich reservoir of novel fungi.

The introduction of exotic species can have serious consequences for marine ecosystems. On the shores of the Cantabrian Sea (North of Spain) there are no routine examinations of seaweeds that combine molecular and morphological methods for early detection of exotic species making it difficult to assess in the early stages their establishment and expansion processes as a result of anthropogenic activities (e.g., shipping and/or aquaculture).
In this work we used both morphological identification and molecular barcoding (COI-5P and rbcL genes) of red algae collected in Asturias, Bay of Biscay (Gijón and Candás harbours) and from the University of Oviedo's herbarium samples.
The results confirmed the presence of exotic Asian seaweeds Pachymeniopsis gargiuli and Grateloupia turuturu Yamada on Cantabrian Sea shores. Several individuals of these species were fertile and developing cystocarps when collected, underlining the risk of possible expansion or continued establishment. This study constitutes the first report of the Asian P. gargiuli in this area of the Bay of Biscay.
Here the presence of the exotic species of the Halymeniales P. gargiuli is confirmed. We hypothesize that this species may have been established some time ago as a cryptic introduction with G. turuturu in Galician shores. The detection of these species on the shores of the Cantabrian Sea is relevant since introductions of Pachymeniopsis species could have been overlooked on other European coasts, probably mixed with G. turuturu and P. lanceolata. Our results confirm one new alien seaweed species that has been detected using molecular methods (COI-5P region and rbcL genes barcoding) on North Atlantic shores: the Asian native P. gargiuli. This demonstrates that routine screening for early detection of exotic algae in the Cantabrian Sea can be used for risk assessment. Genetic barcoding should be done using both rbcL gene and COI-5P regions since, although COI-databases are still poorer in sequences and this inhibits successful outcomes in Grateloupia-related species identifications, it is nonetheless a useful marker for species-level identifications in seaweeds.

The difficulty of censusing marine animal populations hampers effective ocean management. Analyzing water for DNA traces shed by organisms may aid assessment. Here we tested aquatic environmental DNA (eDNA) as an indicator of fish presence in the lower Hudson River estuary. A checklist of local marine fish and their relative abundance was prepared by compiling 12 traditional surveys conducted between 1988–2015. To improve eDNA identification success, 31 specimens representing 18 marine fish species were sequenced for two mitochondrial gene regions, boosting coverage of the 12S eDNA target sequence to 80% of local taxa. We collected 76 one-liter shoreline surface water samples at two contrasting estuary locations over six months beginning in January 2016. eDNA was amplified with vertebrate-specific 12S primers. Bioinformatic analysis of amplified DNA, using a reference library of GenBank and our newly generated 12S sequences, detected most (81%) locally abundant or common species and relatively few (23%) uncommon taxa, and corresponded to seasonal presence and habitat preference as determined by traditional surveys. Approximately 2% of fish reads were commonly consumed species that are rare or absent in local waters, consistent with wastewater input. Freshwater species were rarely detected despite Hudson River inflow. These results support further exploration and suggest eDNA will facilitate fine-scale geographic and temporal mapping of marine fish populations at relatively low cost.

Monday, April 10, 2017

Student Travel Bursaries for African and South African Students

Happy to pass on some important conference related information:

Dear Delegates, 

We are pleased to announce that there are Travel Bursaries available for African and South African students to attend the upcoming 7th International Barcode of Life Conference in November 2017.

To apply and for more information, please go to to this link.

We encourage all African students to apply - please note the application deadline is 01 May 2017.

Good luck!

With best wishes, 

7th iBOL Local Organizing Committee

Monday, April 3, 2017

Monday reads

Happy April! Another week of light posting passed and I am afraid there will be one more of the same. A number of things got in the way despite the fact that there is no shortage of things to blog about. In short - I am too busy. Here is hope that this will change in the near future. And now to this weeks selection:

DNA-barcoding is a rapidly developing method for efficiently identifying samples to species level by means of short standard DNA sequences. However, reliable species assignment requires the availability of a comprehensive DNA barcode reference library, and hence numerous initiatives aim at generating such barcode databases for particular taxa or geographic regions. Historical museum collections represent a potentially invaluable source for the DNA-barcoding of many taxa. This is particularly true for birds and mammals, for which collecting fresh (voucher) material is often very difficult to (nearly) impossible due to the special animal welfare and conservation regulations that apply to vertebrates in general, and birds and mammals in particular. Moreover, even great efforts might not guarantee sufficiently complete sampling of fresh material in a short period of time. DNA extracted from historical samples is usually degraded, such that only short fragments can be amplified, rendering the recovery of the barcoding region as a single fragment impossible. Here, we present a new set of primers that allows the efficient amplification and sequencing of the entire barcoding region in most higher taxa of Central European birds and mammals in six overlapping fragments, thus greatly increasing the value of historical museum collections for generating DNA barcode reference libraries. Applying our new primer set in recently established NGS protocols promises to further increase the efficiency of barcoding old bird and mammal specimens.

We foresee a new global-scale, ecological approach to biomonitoring emerging within the next decade that can detect ecosystem change accurately, cheaply, and generically. Next-generation sequencing of DNA sampled from the Earth’s environments would provide data for the relative abundance of operational taxonomic units or ecological functions. Machine-learning methods would then be used to reconstruct the ecological networks of interactions implicit in the raw NGS data. Ultimately, we envision the development of autonomous samplers that would sample nucleic acids and upload NGS sequence data to the cloud for network reconstruction. Large numbers of these samplers, in a global array, would allow sensitive automated biomonitoring of the Earth’s major ecosystems at high spatial and temporal resolution, revolutionising our understanding of ecosystem change.

Anthropogenic activities are having devastating impacts on marine systems with numerous knock-on effects on trophic functioning, species interactions and an accelerated loss of biodiversity. Establishing conservation areas can not only protect biodiversity, but also confer resilience against changes to coral reefs and their inhabitants. Planning for protection and conservation in marine systems is complex, but usually focuses on maintaining levels of biodiversity and protecting special and unique landscape features while avoiding negative impacts to socio-economic benefits. Conversely, the integration of evolutionary processes that have shaped extant species assemblages is rarely taken into account. However, it is as important to protect processes as it is to protect patterns for maintaining the evolutionary trajectories of populations and species. This review focuses on different approaches for integrating genetic analyses, such as phylogenetic diversity, phylogeography and the delineation of management units, temporal and spatial monitoring of genetic diversity and quantification of adaptive variation for protecting evolutionary resilience, into marine spatial planning, specifically for coral reef fishes. Many of these concepts are not yet readily applied to coral reef fish studies, but this synthesis highlights their potential and the importance of including historical processes into systematic biodiversity planning for conserving not only extant, but also future, biodiversity and its evolutionary potential.

Determining the ecosystem function of high-order predators is critical for evaluation of food web interactions. Insectivorous birds are abundant predators in many ecosystems yet because they forage upon small taxa, it remains largely unknown whether birds are providing ecosystem services in the form of pest control or disservices by preying upon predaceous arthropod species. We extracted DNA from noninvasive fecal samples of adult and nestling Western Bluebirds (Sialia mexicana) in California vineyards. Using universal arthropod-specific primers, we sequenced prey items via massively parallel sequencing on the Illumina MiSeq platform. Bluebirds consumed a broad diet comprising 66 unique arthropod species from 6 orders and 28 families. Aedes sp. (mosquitoes: Culicidae), a previously unknown prey, was the most common item recovered, occurring in 49.5% of the fecal samples. Ectoparasitic bird blowfly (Protocalliphora) DNA was found in 7% of adult and 11% of nestling samples, presenting clear evidence of active feeding by the avian hosts on adult or larval ectoparasites. Herbivorous insects, primarily from the orders Hemiptera and Lepidoptera, represented over half (56%) of the prey items in bluebird diets. Intraguild predation (consumption of predator or parasitoid arthropods) represented only 3% of adult and nestling dietary items. Diets of adults were significantly different from nestlings as were diets from birds sampled in different vineyard blocks. Sex, date, number of young, and individual bird (based on resampled individuals) were all insignificant factors that did not explain diet variability. Nestling age was a significant factor in explaining a small amount of the variability in dietary components. In addition, our analysis of subsampling larger fecal samples and processing them independently revealed highly dissimilar results in all 10 trials and we recommend avoiding this common methodology. Molecular scatology offers powerfully informative techniques that can reveal the ecosystem function and services provided by abundant yet cryptic avian foragers.