Archived project

ENVMETAGEN - Capacity Building at InBIO for Research and Innovation Using Environmental Metagenomics

Goal: The goal of the project is to expand the research and innovation potential of InBIO – Research Network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. The project aims to strengthen the research potential of human resources, lab facilities and next-generation genome sequencing equipment funded by a FP7 CAPACITIES project, supporting an emerging research line in environmental metagenomics for applications in biodiversity conservation, invasive species control, ecosystem services assessment, and environmental (bio)monitoring. The project will contribute to the regional and national smart specialization strategies, by developing tools and approaches to foster environmentally sustainable development, and strengthening innovation and knowledge transfer activities in close collaboration with local and global industrial partners, as well as with governmental agencies. In addition, the project will contribute to the advanced training of highly-qualified human resources, and to the communication, dissemination and exploitation of InBIO’s research and innovation. To achieve its objectives, EnvMetaGen will develop the following main activities: (i) Upgrade the research capacity and capability by expanding the human potential and fostering a critical mass of researchers with inter-disciplinary expertise in environmental metagenomics; (ii) Improve InBIO’s innovation potential and impact at the regional, national and European levels, by promoting the integration of cost-effective metagenomics approaches at the forefront of biodiversity, ecological and environmental research; and (iii) Raise the international and national network of collaborations and partnerships of InBIO and its industrial and governmental affiliates. These activities will contribute to better integrate InBIO with the European Research Area (ERA), thus increasing their level of participation in the Horizon 2020 program.

Date: 1 September 2015 - 31 December 2020

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Sonia Ferreira
added a research item
Background The Trichoptera are an important component of freshwater ecosystems. In the Iberian Peninsula, 380 taxa of caddisflies are known, with nearly 1/3 of the total species being endemic in the region. A reference collection of morphologically identified Trichoptera specimens, representing 142 Iberian taxa, was constructed. The InBIO Barcoding Initiative (IBI) Trichoptera 01 dataset contains records of 438 sequenced specimens. The species of this dataset correspond to about 37% of Iberian Trichoptera species diversity. Specimens were collected between 1975 and 2018 and are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources, Portugal) or in the collection Marcos A. González at the University of Santiago de Compostela (Spain). New information Twenty-nine species, from nine different families, were new additions to the Barcode of Life Data System (BOLD). A success identification rate of over 80% was achieved when comparing morphological identifications and DNA barcodes for the species analysed. This encouraging step advances incorporation of informed Environmental DNA tools in biomonitoring schemes, given the shortcomings of morphological identifications of larvae and adult Caddisflies in such studies. DNA barcoding was not successful in identifying species in six Trichoptera genera: Hydropsyche (Hydropsychidae), Athripsodes (Leptoceridae), Wormaldia (Philopotamidae), Polycentropus (Polycentropodidae) Rhyacophila (Rhyacophilidae) and Sericostoma (Sericostomatidae). The high levels of intraspecific genetic variability found, combined with a lack of a barcode gap and a challenging morphological identification, rendered these species as needing additional studies to resolve their taxonomy.
Pedro Sousa
added a research item
The dataset contains eight records of moth specimens (Lepidoptera, Ypsolophidae) collected in 2019 in continental Portugal. Specimens were detected and captured by direct search of the environment during the day, with adults’ hand-netted and larvae reared to adulthood, and by night using mercury-vapor lamps or UV light traps to attract the insects. Captured specimens were identified to species level and preserved in 96% ethanol or in dry storage. A tissue sample, a leg, was removed from each individual to be used for DNA extraction. The DNA barcoding of these specimens was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA barcode sequences were deposited in BOLD (Barcode of Life Data System) and GenBank online databases. DNA extracts are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).
Sonia Ferreira
added a research item
The orders Neuroptera and Raphidioptera include the species of insects known as lacewings and snake-flies, respectively. In Portugal, these groups account for over 100 species, some of which are very difficult to identify by morphological analysis. This work is the first to sample and DNA sequence lacewings and snakeflies of Portugal. A reference collection was built with captured specimens that were identified morphologically. DNA barcode sequences of 658 bp were obtained from 243 specimens of 54 species. The results showed that most species can be successfully identified through DNA barcoding, with the exception of seven species of Chrysopidae (Neuroptera). Additionally, the first published distribution data are presented for Portugal for the neuropterans Gymnocnemia variegata (Schneider, 1845) and Myrmecaelurus (Myrmecaelurus) trigrammus (Pallas, 1771).
Pedro Sousa
added a research item
The dataset contains 135 records of Hemiptera species collected from 2015 to 2019 in continental Portugal (Sousa et al., 2021). The species represented in the dataset, 90 in total, correspond to about 7.5% of the known true bugs diversity of continental Portugal, and contribute to the knowledge on the DNA barcodes and distribution of Portuguese Hemiptera. Specimens were captured during fieldwork by direct search of specimens or by sweeping the vegetation and stored in 96% ethanol. All specimens were identified to species level. A tissue sample, usually a leg, was collected from each individual, from which DNA was extracted. The DNA barcoding of these specimens was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA barcode sequences were deposited in BOLD (Barcode of Life Data System) online database. Preserved specimens and DNA extracts are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).
Sonia Ferreira
added a research item
The InBIO Barcoding Initiative (IBI) Hemiptera 01 dataset contains records of 131 specimens of Hemiptera. Most specimens have been morphologically identified to species or subspecies level and represent 88 species in total. The species of this dataset correspond to about 7.3% of continental Portuguese hemipteran species diversity. All specimens were collected in continental Portugal. Sampling took place from 2015 to 2019 and specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources. This dataset increases the knowledge on the DNA barcodes and distribution of 88 species of Hemiptera from Portugal. Six species, from five different families, were new additions to the Barcode of Life Data System (BOLD), with another twenty five species barcodes' added from under-represented taxa in BOLD. All specimens have their DNA barcodes publicly accessible through BOLD online database and the distribution data can be accessed through the Global Biodiversity Information Facility (GBIF). Eutettix variabilis and Fieberiella florii are recorded for the first time for Portugal and Siphanta acuta , an invasive species, previously reported from the Portuguese Azores archipelago, is recorded for the first time for continental Portugal.
Pedro Beja
added a research item
Freshwater macroinvertebrates provide valuable indicators for biomonitoring ecosystem change in relation to natural and anthropogenic drivers. DNA metabarcoding is an efficient approach for estimating such indicators, but its results may differ from morphotaxonomic approaches traditionally used in biomonitoring. Here we test the hypothesis that despite differences in the number and identity of taxa recorded, both approaches may retrieve comparable patterns of community change, and detect similar ecological gradients influencing such changes. We compared results obtained with morphological identification at family level of macroinvertebrates collected at 80 streams under a Water Framework Directive biomonitoring program in Portugal, with results obtained with metabarcoding from the ethanol preserving the bulk samples, using either single (COI-M19BR2, 16S-Inse01, 18S-Euka02) or multiple markers. Metabarcoding recorded less families and different communities compared to morphotaxonomy, but community sensitivities to disturbance estimated with the IASPT index were more similar across approaches. Spatial variation in local community metrics and the factors influencing such variation were significantly correlated between morphotaxonomy and metabarcoding. After reducing random noise in the dissimilarity matrices, the spatial variation in community composition was also significantly correlated across methods. A dominant gradient of community change was consistently retrieved, and all methods identified a largely similar set of anthropogenic stressors strongly influencing such gradient. Overall, results confirm our initial hypothesis, suggesting that morphotaxonomy and metabarcoding can estimate consistent spatial patterns of community variation and their main drivers. These results are encouraging for macroinvertebrate biomonitoring using metabarcoding approaches, suggesting that they can be intercalibrated with morphotaxonomic approaches to recover equivalent spatial and temporal gradients of ecological change.
Sonia Ferreira
added a research item
Ypsolopha milfontensis Corley & Ferreira, sp. n. is described from the coast of southwest Portugal where its hostplant is Ephedra fragilis Desf. It is closely related to Ypsolopha instabilella (Mann, 1866), but with some differences externally, in male and female genitalia and a significant difference in DNA barcode.
Pedro Sousa
added a research item
The dataset contains 38 records of Blattodea collected from 2005 to 2019 in continental Portugal. Specimens were identified to species level, except for a single record identified to genus level. Of the eight species in the dataset, seven are cockroaches and one is a termite. Specimens were detected during fieldwork by direct search and stored in 96% ethanol. A tissue sample (one leg), was collected from each individual, from which DNA was extracted. The DNA barcoding of these specimens was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA sequences were deposited in the Barcode of Life Data System (BOLD) and GenBank databases. Preserved specimens and associated DNA extracts are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).
Sonia Ferreira
added a research item
All species from continental Portugal of the dipterous families Limoniidae, Pediciidae and Tipulidae are presented including their distribution in Portugal and beyond. New records are added for the large majority of the species. In total 123 species are registered. Of these, 17 species of Limoniidae and 11 species of Tipulidae are recorded here for the first time for Portugal and two additional Limoniidae for continental Portugal. Tipula (Yamatotipula) intermedia Eiroa, 1990 stat. nov. is elevated to species rank from its original subspecies status and subsequent full synonymy with T. (Y.) lateralis Meigen, 1804. Two species of Limoniidae are removed from the list of species known to be present in Portugal.
Sonia Ferreira
added a research item
Oedemera (Oncomera) femoralis Olivier, 1803 (Coleoptera, Oedemeridae) is reported for the second time from Portugal nineteen years after the first record, considerably extending its known distribution in the country and in the Iberian Peninsula.
Pedro Beja
added an update
We are pleased to announce the 10th edition of TiBE - Trends in Biodiversity and Evolution Conference. This year, TiBE goes virtual!!
This is an annual meeting organized by CIBIO-InBIO, which aims to bring together senior researchers, post-graduate and graduate students working on the fields of biodiversity and evolutionary biology. In 2020, the aim of the meeting is to discuss cutting-edge findings in relevant topics related to metabarcoding and metagenomic techniques, and their application in ecological and environmental research, including biodiversity surveys, environmental assessment, ecosystem function and services, interaction networks and species autecology, among others.
TiBE2020 is organized by the CompBio and ApplEcol research groups, and it will be devoted to the methods and applications of METABARCODING AND METAGENOMICS in biodiversity, ecological and environmental research. At the same time, TiBE2020 will be the closing meeting of the ERA Chair project ENVMETAGEN – Capacity Building at InBIO for Research and Innovation Using Environmental Metagenomics, funded by the European Commission.
The meeting will discuss exciting developments associated with the advent of ever more powerful DNA sequencing technology, which are opening possibilities to explore the living world in ways that were unimaginable just a decade ago. During three afternoons, participants will discuss new opportunities, challenges and pitfalls of metabarcoding and metagenomic approaches, with examples provided by a wealth of case studies where these new techniques contributed decisively to improve our understanding of biodiversity and ecosystem patterns and processes. Moreover, participants will discuss the application of metabarcoding and metagenomics to foster new and more cost-efficient environmental assessment and monitoring programs. The conference will be held in an informal but stimulating scientific atmosphere, on an online platform that will foster networking opportunities and allows poster presentations. The program includes both plenary and short presentations from selected abstracts.
This is an excellent opportunity to present your work, and to interact and brainstorm with other members of the scientific community using metabarcoding and metagenomics for biodiversity, ecological and environmental research. Join us online this December!
PRELIMINARY PROGRAMME: The conference will be structured in three sessions, covering different topics. Each session will take place in the afternoon, starting at 2.30pm GMT.
Session 1: MOLECULAR SURVEYS OF BIODIVERSITY AND INVASIVE SPECIES
Keynote speaker: Mike Schwartz, National Genomics Center for Wildlife and Fish Conservation, USA Invited speaker: Sara Peixoto, University of Hull, UK (former CIBIO member) Invited speaker: Bastian Egeter, Nature Metrics, UK (former CIBIO member)
Session 2: NEXT GENERATION BIOMONITORING OF AQUATIC AND TERRESTRIAL ECOSYSTEMS
Keynote speaker: Agnès Bouchez, INRAE - Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, France Invited speaker: Filipa Martins, CIBIO-InBIO, Portugal Invited speaker: Sónia Ferreira, CIBIO-InBIO, Portugal
Session 3: UNDERSTANDING SPECIES INTERACTIONS IN COMPLEX ECOSYSTEMS
Keynote speaker: Tyler Kartzinel, Brown University, USA Invited speaker: Vanessa Mata, CIBIO-InBIO, Portugal Invited speaker: Joana Paupério, CIBIO-InBIO, Portugal
ABSTRACT GUIDELINES The conference will accept contributions in two formats: oral presentations and posters. Oral presentations will consist of 15 minutes’ presentations followed by 5 minutes for discussion.
Posters will be presented as a short (2 minutes) video in a dedicated platform, online, allowing interaction with other participants.
Only registered participants are allowed as presenting authors.
Please, download the abstract submission template here.
IMPORTANT DATES: Abstract submission opens: October 1st, 2020 Abstract submission deadline: October 27th, 2020 Deadline for registration: November 20th, 2020
REGISTRATION FEES CIBIO students (MSc and PhD) - free CIBIO members - 25€ Students from other Institutions* - 25€ Other participants - 50€ * must present evidence of status.
REGISTRATION FORM Registration information is available at: https://cibio.up.pt/tibe/details/tibe2020
ORGANIZING COMMITTEE Nuno Fonseca Pedro Beja Sandra Aresta
More information will be added to this page as we get closer to the event starting date. Please do not hesitate to disseminate this event to all your contacts.
 
Sonia Ferreira
added a research item
Stenolemus novaki Horváth, 1888 (Hemiptera, Reduviidae) is recorded from mainland Portugal for the second time, following a recent photographic record. A map synthesizing the knowledge about the species distribution in the Iberian Peninsula is included.
Luis P. da Silva
added a research item
Although sexual dietary differentiation is well known in birds, it is usually linked with significant morphological dimorphism between males and females, with lower differentiation reported in sexually monomorphic or only slightly dimorphic species. However, this may be an artifact of poor taxonomic resolution achieved in most conventional dietary studies, which may be unable to detect subtle intraspecific differentiation in prey consumption. Here, we show the power of multi‐marker metabarcoding to address these issues, focusing on a slightly dimorphic generalist passerine, the black wheatear Oenanthe leucura. Using markers from four genomic regions (18S, 16S, COI, and trnL), we analyzed fecal droppings collected from 93 adult black wheatears during the breeding season. We found that sexes were rather similar in bill and body features, though males had a slightly thicker bill and longer wings and tail than females. Diet was dominated in both sexes by a very wide range of arthropod species and a few fleshy fruits, but the overall diet diversity was higher for males than females, and there was a much higher frequency of occurrence of ants in female (58%) than male (29%) diets. We hypothesize that the observed sexual differentiation was likely related to females foraging closer to their offspring on abundant prey, while males consumed a wider variety of prey while foraging more widely. Overall, our results suggest that dietary sexual differentiation in birds may be more widespread than recognized at present and that multi‐marker DNA metabarcoding is a particularly powerful tool to unveiling such differences.
Pedro Sousa
added 9 research items
The dataset contains one record of the species Wesmaelius subnebulosus (Stephens, 1836) (Neuroptera, Hemerobiidae), a brown lacewing species, collected in continental Portugal in 2017. The specimens was detected and captured using a light-trap setup. A tissue sample, one leg, was removed to be used for DNA extraction. Sequencing of the 658 bp COI DNA barcode was conducted within the IBI project and the DNA sequence was submitted to BOLD (Barcode of Life Data System) database. The preserved specimen and DNA extract are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).
The dataset contains five records of the alderfly genus Sialis Latreille, 1803 (Megaloptera, Sialidae) collected in 2011 and 2015 in northern continental Portugal. Specimens were detected by direct search on vegetation and rocks around river streams and captured with a hand-net. Captured specimens were identified to species level and preserved in 96% ethanol. A tissue sample, a leg, was collected from each individual, from which DNA was extracted. The DNA barcoding of these specimens was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA barcode sequences were deposited in BOLD (Barcode of Life Data System) and GenBank online databases. DNA extracts are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).
The dataset contains eight records of moth specimens of the genus Ypsolopha collected from 2015 to 2017 in continental Portugal. Specimens were detected and captured by direct search of the environment during the day, with adults hand-netted and larvae reared to adulthood, and by night using mercury-vapor lamps to attract the insects. Captured specimens were identified to species level and preserved in 96% ethanol. A tissue sample, usually a leg, was collected from each individual, from which DNA was extracted. The DNA barcoding of these specimens was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA barcode sequences were deposited in BOLD (Barcode of Life Data System) and GenBank online databases. Preserved specimens and DNA extracts are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).
Pedro Beja
added 5 research items
The use of metabarcoding has grown exponentially in the study of animals’ diets. The accuracy and detail of this technique is invaluable for a better understanding of biotic interactions characterizing natural and human-dominated ecosystems, and thus to their conservation and sustainable use. However it has seldom been applied to passerine birds, though this order accounts for about half the bird species and encompasses a wide range of trophic niches. One of the problems is that many species have a wide trophic niche, feeding on both plants and on variety of arthropod orders, which is challenging for metabarcoding studies. For instance, a wide feeding spectrum may lead to methodological problems, due to primer selection and bias. Here we compare the effectiveness of metabarcoding against traditional microscopic analysis in evaluating the diet of the black wheatear Oenanthe leucura. This is a generalist passerine of conservation concern in Europe, occurring in semi-arid areas of Iberia and North Africa. It is known to feed on several berries, a large variety of arthropods and even small lizards. Here we apply a multi-marker approach, with different levels of taxonomical resolution (18S, 16S, trnL, and COI), as well as standard microscopic analysis of prey fragments, in order to assess the diet of this species. In general, genetic analysis was able to recover a higher number of taxa than microscopic analysis, with group specific primers showing also a higher taxonomic resolution. The COI primer used however, completely failed to amplify some invertebrate groups, showing the importance of using multiple markers when evaluating the diet of a species.
Filipa MS Martins
added 2 research items
DNA metabarcoding from the ethanol used to store macroinvertebrate bulk samples is a convenient methodological option in molecular biodiversity assessment and biomonitoring of aquatic ecosystems, as it preserves specimens and reduces problems associated with sample sorting. However, this method may be affected by errors and biases, which need to be thoroughly quantified before it can be mainstreamed into biomonitoring programs. Here we used 80 unsorted macroinvertebrate samples collected in Portugal under a Water Framework Directive monitoring program, to compare community diversity and taxonomic composition metrics estimated through morphotaxonomy vs. metabarcoding from storage ethanol using three markers (COI-M19BR2, 16S-Inse01, 18S-Euka02) and a multi-marker approach. A preliminary in silico analysis showed that the three markers were adequate for the target taxa, with detection failures related primarily to the lack of adequate barcodes in public databases. Metabarcoding of ethanol samples retrieved far less taxa per site (alpha diversity) than morphotaxonomy, albeit with smaller differences for COI-M19BR2 and the multi-marker approach, while estimates of taxa turnover (beta diversity) among sites were similar across methods. Using generalised linear mixed models, we found that after controlling for differences in read coverage across samples, the probability of detection of a taxon was positively related to its proportional abundance, and negatively so to the presence of heavily sclerotised exoskeleton (e.g., Coleoptera). Overall, using our experimental protocol with different template dilutions, the COI marker showed the best performance, but we recommend the use of a multi-marker approach to detect a wider range of taxa in freshwater macroinvertebrate samples. Further methodological development and optimisation efforts are needed to reduce biases associated with body armouring and rarity in some macroinvertebrate taxa.
Bastian Egeter
added a research item
Environmental DNA (eDNA) is increasingly used for biodiversity monitoring, particularly in aquatic systems. However, each step, from sample collection to bioinformatic analysis, can introduce biases and influence the reliability of results. While much effort has been put into the optimization of laboratory methods, less attention has been devoted to estimate the impacts of eDNA capture methods. To address this issue, water samples were collected at nine small ponds and puddles where up to 10 amphibian species occur, using precipitation, disc filters, and capsules. We focused on targeted detection of an amphibian species, Salamandra salamandra, and on the composition of the whole amphibian community. Species detection was performed using a novel qPCR assay for S. salamandra and high-throughput sequencing, combined with stringent versus relaxed PCR replication thresholds. Filtration techniques (disc filters and capsules) outperformed precipitation, generating a higher number of detections of S. salamandra and higher amounts of captured eDNA, while species detection was identical between disc filters and capsules. There were no significant differences between capture methods regarding amphibian community composition. The variation in detection success associated with capture methods was far higher than that associated with PCR replication, regardless of the detection method used. Our results highlight the importance of choosing a suitable capture method for eDNA studies and suggest that the choice of capture method outweighs the choice of detection method used. To the best of our knowledge, this is the first study to compare high-capacity capsules with common eDNA methods for water samples, such as precipitation and standard disc filters.
Pedro Sousa
added 3 research items
The dataset contains 11 records of Stag beetle (Lucanidae) species collected from 2015 to 2019 in continental Portugal. All specimens were identified to species level, and belong to 3 species of the Lucanidae (Coleoptera). Specimens were detected during fieldwork by direct search and stored in 96% ethanol. A tissue sample (a leg), was collected from each individual, from which DNA was extracted. The DNA barcoding of these specimens was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA barcode sequences available were deposited in BOLD (Barcode of Life Data System) online database. Preserved specimens and DNA extracts are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).
The dataset contains 63 records of Chiroptera species collected in continental Portugal. All specimens were identified to species level, for a total of 26 species belonging to three different families. Bat samples were collected under the scope of several projects spanning from 2005 to 2018. All bats were captured during mist-netting sessions or using harp-traps at roost exits. A non-lethal 3mm wing punch was collected from several individuals and stored in 96% ethanol from where DNA was extracted. The DNA barcoding of these samples was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA barcode sequences were deposited in BOLD (Barcode of Life Data System) online database. DNA extractions are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).
The dataset contains six records of Solifugae specimens collected from 2016 to 2018 in continental Portugal. All specimens were identified as Gluvia dorsalis (Latreille, 1817) that belongs to the Daesiidae family, and is the only Solifugae species known to occur in Portugal. Specimens were captured during fieldwork directed specifically for the sampling of Solifugae and stored in 96% ethanol. A tissue sample (a leg), was collected from each individual, from which DNA was extracted. The DNA barcoding of these specimens was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA barcode sequences were deposited in BOLD (Barcode of Life Data System) online database. Preserved specimens and DNA extracts are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).
Sonia Ferreira
added a research item
The advent and boom of DNA barcoding technologies have provided a powerful tool for the fields of ecology and systematics. Here, we present the InBIO Barcoding Initiative Database: Portuguese Bats (Chiroptera) dataset containing DNA sequences of 63 specimens representing the 25 bat species currently known for continental Portugal. For that, we sequenced tissues samples obtained in a vast array of projects spanning the last two decades. We added four new Barcoding Index Numbers (BINs) to existing Chiroptera barcodes on BOLD, two belonging to Myotis escalerai , one to Plecotus auritus and the other to Rhinolophus hipposideros . Surprisingly, one of the samples initially identified in the field as Myotis mystacinus turned out to be Myotis alcathoe , which represents the first record of this species for Portugal. The presence of Nyctalus noctula in Portugal was also genetically confirmed for the first time. This case study shows the power and value of DNA barcoding initiatives to unravel new data that may be hidden on biological collections.
Sonia Ferreira
added a research item
The use of DNA barcoding allows unprecedented advances in biodiversity assessments and monitoring schemes of freshwater ecosystems; nevertheless, it requires the construction of comprehensive reference collections of DNA sequences that represent the existing biodiversity. Plecoptera are considered particularly good ecological indicators and one of the most endangered groups of insects, but very limited information on their DNA barcodes is available in public databases. Currently, less than 50% of the Iberian species are represented in BOLD. The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of Iberian Plecoptera dataset contains records of 71 specimens of Plecoptera. All specimens have been morphologically identified to species level and belong to 29 species in total. This dataset contributes to the knowledge on the DNA barcodes and distribution of Plecoptera from the Iberian Peninsula and it is one of the IBI database public releases that makes available genetic and distribution data for a series of taxa. The species represented in this dataset correspond to an addition to public databases of 17 species and 21 BINs. Fifty-eight specimens were collected in Portugal and 18 in Spain during the period of 2004 to 2018. All specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources and their DNA barcodes are publicly available in the Barcode of Life Data System (BOLD) online database. The distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).
Sonia Ferreira
added 4 research items
The dataset contains 13 records of Scorpionflies (Panorpidae) species collected from 2008 to 2019 in continental Portugal. All captured specimens were identified as Panorpa meridionalis, so far the only species of Mecoptera known to exist in Portugal Specimens were detected and captured by direct search of the environment. Captured specimens were preserved in 96% ethanol. From each specimen one leg (tissue sample) was removed to be used for DNA extraction. Preserved specimens and DNA extracts are deposited in the within the InBIO Barcoding Initiative (IBI) collection at the CIBIO (Research Center in Biodiversity and Genetic Resources). Samples of each species were selected for DNA sequencing based on geographic provenance of the corresponding specimen. Sequencing of the 658 bp DNA barcodes was conducted within the InBIO Barcoding Initiative (IBI) and all DNA sequences were submitted to the Barcode of Life and GenBank databases.
The dataset contains 56 records of Earwigs (Dermaptera) species collected from 2006 to 2019 in continental Portugal. All captured specimens were identified to species level, belonging to ten of the fourteen species of Dermaptera present in Portugal. Specimens were detected and captured by direct search of the environment. Captured specimens were preserved in 96% ethanol. From each specimen one leg (tissue sample) was removed to be used for DNA extraction. Preserved specimens and DNA extracts are deposited in the within the InBIO Barcoding Initiative (IBI) collection at the CIBIO (Research Center in Biodiversity and Genetic Resources). Samples of each species were selected for DNA sequencing based on geographic provenance of the corresponding specimen. Sequencing of the 658 bp DNA barcodes was conducted within the InBIO Barcoding Initiative (IBI) and all DNA sequences were submitted to the Barcode of Life and GenBank databases.
The dataset contains 18 records of Stick insects (Phasmatodea) species collected from 2006 to 2019 in continental Portugal. All captured specimens were identified to species level, belonging to the two species of Phasmatodea present in Portugal. Specimens were detected and captured by direct search of the environment. Captured specimens were preserved in 96% ethanol. From each specimen one leg (tissue sample) was removed to be used for DNA extraction. Preserved specimens and DNA extracts are deposited in the within the InBIO Barcoding Initiative (IBI) collection at the CIBIO (Research Center in Biodiversity and Genetic Resources). Samples of each species were selected for DNA sequencing based on geographic provenance of the corresponding specimen. Sequencing of the 658 bp DNA barcodes was conducted within the InBIO Barcoding Initiative (IBI) and all DNA sequences were submitted to the Barcode of Life and GenBank databases.
Sonia Ferreira
added a research item
Following the discovery of a new species on the western coast of Portugal which is closely related to Apatetris mediterranella Nel & Varenne, 2012, the generic placement of both species is considered in relation to other genera within the Apatetris complex resulting in the description of a new genus, Mondeguina.
Sonia Ferreira
added a research item
The endemic Portuguese Cochylimorpha punctiferana (Ragonot, 1881) is resurrected from synonymy with C. discopunctana (Eversmann, 1844) based on clear differences in female genitalia and DNA barcode. A neotype is designated. Notas taxonómicas sobre Microlepidoptera de Portugal II. Cochylimorpha punctiferana (Ragonot, 1881) stat. rev., uma espécie negligenciada (Lepidoptera: Tortricidae) Resumo O endemismo português Cochylimorpha punctiferana (Ragonot, 1881) é removido da sinonímia com C. discopunctana (Eversmann, 1844) com base em marcadas diferenças tanto na genitália feminina, como no DNA barcode. Um neotipo é designado. PALAVRAS CHAVE: Lepidoptera, Tortricidae, reversão de sinonímia, Cochylimorpha, DNA barcoding, Portugal. Notas taxonómicas sobre Microlepidoptera de Portugal II. Cochylimorpha punctiferana (Ragonot, 1881) stat. rev., una especie descuidada (Lepidoptera: Tortricidae) Resumen El endemismo portugués Cochylimorpha punctiferana (Ragonot, 1881) es sacado de la sinonímia con C. discopunctana (Eversmann, 1844) con base a las diferencias tanto en la genitalia femenina, como en el código de barras ADN. Se designa un neotipo.
Sonia Ferreira
added a research item
Background The InBIO Barcoding Initiative (IBI) Diptera 01 dataset contains records of 203 specimens of Diptera. All specimens have been morphologically identified to species level, and belong to 154 species in total. The species represented in this dataset correspond to about 10% of continental Portugal dipteran species diversity. All specimens were collected north of the Tagus river in Portugal. Sampling took place from 2014 to 2018, and specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources. New information This dataset contributes to the knowledge on the DNA barcodes and distribution of 154 species of Diptera from Portugal and is the first of the planned IBI database public releases, which will make available genetic and distribution data for a series of taxa. All specimens have their DNA barcodes made publicly available in the Barcode of Life Data System (BOLD) online database and the distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).
Bastian Egeter
added 3 research items
Trophic networks in small isolated islands are in a fragile balance, and their disturbance can easily contribute toward the extinction vortex of species. Here, we show, in a small Atlantic island (Raso) in the Cabo Verde Archipelago, using DNA metabarcoding, the extent of trophic dependence of the Endangered giant wall gecko Tarentola gigas on endemic populations of vertebrates, including one of the rarest bird species of the world, the Critically Endangered Raso lark Alauda razae. We found that the Raso lark (27%), Iago sparrow Passer iagoensis (12%), Bulwer's petrel Bulweria bulwerii (15%), and the Cabo Verde shearwater Calonectris edwardsii (10%) are the most frequent vertebrate signatures found in the feces of the giant wall gecko. This work provides the first integrative assessment of their trophic links, an important issue to be considered for the long‐term conservation of these small and isolated island ecosystems. We show, using DNA metabarcoding, that one of the rarest passerine birds in the world, the Raso lark, confined to one of the smallest islands in Cabo Verde Archipelago (Atlantic Ocean), is the main vertebrate prey of one of the rarest and larger gecko of the world, the giant wall gecko. This clearly solves a gap in knowledge that will help to manage this fragile ecosystem and guide ongoing conservation actions to promote the survival of both species.
With less than 50 wild individuals restricted to Iran, Asiatic cheetah is one of the most globally endangered mammals. Here, we are aiming to: -Optimize protocol for cheetah detection based in non-invasive sampling (environmental metagenomics) -Estimate effective population size.-Assess population genetic structure. -Evaluate landscape connectivity between sub-populations. -Characterise diet composition (to evaluate potential conflicts with livestock raising).
Enhancing the role of the soil microbiota in plant phosphorus (P) and sulfur (S) supply through application of organic fertilizer could reduce dependencies on non-sustainable synthetic fertilizers. To compare the effects of organic/inorganic fertilizers on the soil microbiota, soil columns with Lolium perenne (ryegrass) were set up in a greenhouse and amended with an inorganic fertilizer, cattle slurry (organic), or urea (P- and S-free control). Ryegrass rhizosphere of the slurry treatment had significantly higher abundances of bacterial feeding nematodes, mycorrhizal colonization, cultivable heterotrophic bacteria, phosphonate- and sulfonate-utilizing bacteria, arylsulfatase activity, available P, and Variovorax asfA gene copies compared to the inorganic and urea treatments. Phosphomonoesterase activities, and gene abundances involved in organic P and S transformations (phoD, phoC, Burkholderia, and Polaromonas asfA) were similar in all treatments. Grass dry matter yield and shoot uptake of N, P, and S were significantly higher in the inorganic treatment compared to the urea and slurry treatments. Community compositions differed significantly between the three fertilizer treatments and included the bacterial, alkaline phosphomonoesterase-producing bacterial, fungal, AM fungal, and nematode communities. Bacteriodetes were found in higher relative abundance in the organic treatment, while Acidobacteria were more abundant in the urea and inorganic fertilizer treatments. These community shifts correlated significantly with grass dry matter yield, uptake of N, P, and S, mycorrhizal colonization, enzyme activities, abundances of bacteria, and bacterial feeding nematodes. We concluded that organic fertilization promoted soil microbes and nematodes which have the potential to support sustainable plant growth, provided that the overall nutrient requirements are met.
Aina Garcia-Raventós
added a research item
Conservation and sustainable management of aquatic ecosystems is a priority in environmental programs worldwide. However, these aims are highly dependent on the efficiency, accuracy and cost of existent methods for the detection of keystone species and monitoring of biological communities. Rapid advances in eDNA, barcoding and metabarcoding promoted by high-throughput sequencing technologies are generating millions of sequences in a fast way, with a promising cost reduction, and overcoming some difficulties of the traditional taxonomic approaches. This paper provides an updated broad perspective of the current developments in this dynamic field presented in the special session (SS) “The use of molecular tools in ecological and biodiversity assessment of aquatic ecosystems” of the XIX Congress of the Iberian Association of Limnology (AIL2018), held in Coimbra, Portugal. Developments presented are mainly focused on the Iberian Peninsula (Portugal and Spain, including Atlantic Macaronesian islands) but include studies in France, Germany, Finland, Russia (Siberia) and South America. The networks within which these researchers are involved are yet even broader, profiting from existing molecular facilities, and traditional taxonomic expertise, which can be viewed as a characteristic of this new research area. It was evident in the SS that the use of molecular tools is widespread, being used to study a diversity of aquatic systems, from rivers’ headwaters to estuaries and coastal lagoons, and volcanic, mountain and frozen lakes to hot springs. The organisms targeted are likewise varied and include fish, macroinvertebrates, meiofauna, microalgae such as diatoms and dinoflagellates, other protists, fungi, and bacteria (cyanobacteria and other). Some studies address the whole biodiversity (i.e., all species present independently of the taxonomic group) from environmental samples of water, biofilms and preservative solution from field samples (e.g., ethanol from macroinvertebrate samples). Great advances were acknowledged in the special session, namely in the use of metabarcoding for detecting hidden biodiversity, juvenile stages, low-abundance species, non-indigenous species and toxicity potential, and ultimately for ecological monitoring of diatoms and invertebrates. Yet, several drawbacks were highlighted and need further work, which include: taxonomic gaps in the reference databases (including gaps at species level and on intraspecific variability) or absence of public databases (e.g. for meiofauna), still high sequencing costs, the need of a substantial bioinformatics effort, difficulties in establishing the amount of environmental sample necessary for a good DNA extraction and the need for testing different genetic markers to obtain accurate results.
Sonia Ferreira
added a research item
14 species are added to the Portuguese Lepidoptera fauna and three species deleted, mainly as a result of fieldwork undertaken by the authors and others in 2018. In addition, second and third records for the country, new province records and new food-plant data for a number of species are included. A summary of recent papers affecting the Portuguese fauna is included.
Luis P. da Silva
added a research item
Metabarcoding studies are now common in ecology and dietary studies are no exception. Metabarcoding has been used to study the diet of an incredible range of animals and even the diet of carnivorous plants, yet incredibly few focuses on passerines. Among other advantages, metabarcoding allows an unprecedent level of taxonomic resolution of ingested items. Nonetheless, this technique has many technical caveats and biases, which are still not thoroughly assessed. The aims of this work were not only to describe in detail the diet of black wheatears (Oenanthe leucura) in Portugal, but also to evaluate the advantages and pitfalls of using metabarcoding in the study of generalist bird diets. In this study we used genetic identification of 115 bird droppings with 4 molecular markers. The method allowed the description of the diet with an unpreceded detail, detecting 552 different taxa. However, there were several problems associated with the technique, namely the bias of some molecular primers towards some taxonomic groups and the detection of secondary predation. In order to avoid some of the common problems of the methodology, we recommend the use of multiple molecular primers when targeting super-diverse taxonomic prey groups and to combine the information of all the primers used in a single dataset. In the case of the black wheatear, we found its diet to be extremely wide, with the presence of reptiles, numerous orders of invertebrates and several plant species. The animal component of the Black wheatear diet was dominated by Hymenoptera, mainly ants, while fruit ingestion was also very common, especially of ruderal plants. Our results also revealed the existence of dietary differences between males and females, with males having a more diverse diet, and females preying more often on ants than males. Metabarcoding proved to be a very powerful tool in providing a better and more detailed understanding of birds? diet. Nevertheless, careful planning according to the goals and subject of the study is needed when designing laboratory procedures and analysis of metabarcoding data.
Sonia Ferreira
added a research item
Following the description of Cacochroa rosetella Corley, 2018 it soon became clear that there was considerable confusion regarding the identity of Cacochroa permixtella (Herrich-Schäffer, 1854). In this paper the genus Cacochroa is revised and this confusion is resolved, a neotype is chosen for C. permixtella and nearly all records verified. Male and female genitalia of C. permixtella are remarkably different from those of the remaining species, which are here placed in Rosetea Corley & Ferreira, gen. nov. The distributions of the three species previously described in Cacochroa are clarified. Cacochroa permixtella has a distribution limited to Macedonia, Bulgaria, Greece and Turkey. Rosetea corfuella (Lvovsky, 2000), comb. nov., is recorded for the first time from Crete, Croatia, Macedonia, Turkey and Israel; the male of R. rosetella (Corley, 2018), comb. nov., is described for the first time and the species is recorded for the first time from Spain, France (mainland and Corsica), Italy (mainland and Sardinia), Greece (mainland and Crete), Croatia and Algeria. Rosetea sara sp. nov. is described from North Africa (Morocco and Tunisia). Male and female genitalia and DNA barcode data are presented for all four species.
Pedro Beja
added a research item
Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates.
Sonia Ferreira
added a research item
Depressaria infernella, a new species most similar to D. cinderella Corley, 2002 is described from the mountains of northeast Portugal and Sierra de Gredos in Spain. Photos of adults and male and female genitalia of the new species and D. cinderella are given. Morphological and genetic characteristics that demonstrate the distinctiveness of D. infernella from other species of Depressaria are presented.
Pedro Beja
added a research item
The application of DNA metabarcoding to dietary analysis of trophic generalists requires using multiple markers in order to overcome problems of primer specificity and bias. However, limited attention has been given to the integration of information from multiple markers, particularly when they partly overlap in the taxa amplified, and vary in taxonomic resolution and biases. Here we test the use of a mix of universal and specific markers, provide criteria to integrate multi-marker metabarcoding data and a python script to implement such criteria and produce a single list of taxa ingested per sample. We then compare the results of dietary analysis based on morphological methods, single markers, and the proposed combination of multiple markers. The study was based on the analysis of 115 faeces from a small passerine, the Black Wheatears (Oenanthe leucura). Morphological analysis detected far fewer plant taxa (12) than either a universal 18S marker (57) or the plant trnL marker (124). This may partly reflect the detection of secondary ingestion by molecular methods. Morphological identification also detected far fewer taxa (23) than when using 18S (91) or the arthropod markers IN16STK (244) and ZBJ (231), though each method missed or underestimated some prey items. Integration of multi-marker data provided far more detailed dietary information than any single marker and estimated higher frequencies of occurrence of all taxa. Overall, our results show the value of integrating data from multiple, taxonomically overlapping markers in an example dietary dataset.
Sonia Ferreira
added a research item
A new species Ypsolopha rhinolophi Corley is described from northern Portugal and south-east France. It resembles Y. alpella (Denis & Schiffermüller, 1775) and Y. lucella (Fabricius, 1775) but shows clear differences from both species in DNA barcode and in male and female genitalia. Male genitalia of Y. lucella are illustrated for the first time. The new species has been collected at light, reared from larvae on Quercus pyrenaica Willd. and recognised from DNA barcode fragments obtained from droppings of horseshoe bats.
Nuno A Fonseca
added an update
Bastian Egeter
added a research item
The decline of amphibians has been of international concern for more than two decades , and the global spread of introduced fauna is a major factor in this decline. Conservation management decisions to implement control of introduced fauna are often based on diet studies. One of the most common metrics to report in diet studies is Frequency of Occurrence (FO), but this can be difficult to interpret, as it does not include a temporal perspective. Here, we examine the potential for FO data derived from molecular diet analysis to inform invasive species management, using in-vasive ship rats (Rattus rattus) and endemic frogs (Leiopelma spp.) in New Zealand as a case study. Only two endemic frog species persist on the mainland. One of these, Leiopelma archeyi, is Critically Endangered (IUCN 2017) and ranked as the world's most evolutionarily distinct and globally endangered amphibian (EDGE, 2018). Ship rat stomach contents were collected by kill-trapping and subjected to three methods of diet analysis (one morphological and two DNA-based). A new primer pair was developed targeting all anuran species that exhibits good coverage, high taxonomic resolution, and reasonable specificity. Incorporating a temporal parameter allowed us to calculate the minimum number of ingestion events per rat per night, providing a more intuitive metric than the more commonly reported FO. We are not aware of other DNA-based diet studies that have incorporated a temporal parameter into FO data. The usefulness of such a metric will depend on the study system, in particular the feeding ecology of the predator. Ship rats are consuming both species of native frogs present on mainland New Zealand, and this study provides the first detections of remains of these species in mammalian stomach contents. K E Y W O R D S diet, Leiopelma, predation, primer, rat, trophic
Joana Paupério
added a research item
The overall goal of the EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics, and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package (WP) dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). This report provides an overview of best practices for analysing eDNA samples, focusing on vertebrate faeces, water samples and bulks of invertebrates. It covers all steps from DNA extraction to amplification and sequencing in high throughput platforms, while addressing the challenges identified in each step. Moreover, it describes protocols already optimised and currently under development at InBIO, with particular reference to the detection of single species, diet assessments and biodiversity assessments. These three different outputs are fully aligned with the three key application areas that have been targeted in EnvMetaGen for a wider application of environmental metagenomics, and on which the development of strategic triple helix initiatives will focus on. These will therefore contribute to mainstream this technology as a new cost-effective solution to tackle current challenges faced by business partners and other stakeholders, while fostering InBIO-Industry-Government relationships (WP5 - Strengthening the triple helix). The protocols described herein were developed in close connection to, and build on accomplishments of, the other WPs in the project, including: i) the recruitment of the ERA Chair team (WP2), ii) secondments and researcher exchanges through collaborations with international networks (WP3), iii) the enhancement of the computational infrastructure at InBIO (WP4), and iv) participation in and organization of workshops and conferences (WP6). Future directions in eDNA analyses at InBIO, and other potential laboratory pipelines for future optimisation are identified. Together, Deliverables D4.2-D4.5 (this document; Egeter et al. 2018; Ferreira et al. 2018; Galhardo et al. 2018) form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project, and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model.
Mafalda Galhardo
added a research item
The overall goal of the EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology - through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). These protocols are essential to the development of research projects in association with business partners and other stakeholders in key application areas identified in the project, and thus to the strengthening of InBIO triple-helix initiatives (InBIO-Industry-Government; WP5). This report (Deliverable D4.5) builds upon previous ones (Deliverables D4.2-D4.4, respectively Ferreira et al. (2018), Egeter et al. (2018), Paupério et al. (2018)) and provides an overview of the processing protocols for DNA sequence data generated by next-gen platforms within EnvMetaGen-affiliated projects. Deliverables D4.2-D4.5 form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model. This development was made possible through the recruitment of the ERA Chair team (WP2), secondments and Junior Researcher exchanges through the collaboration with international networks (WP3), an enhancement of computational infrastructure at InBIO (WP4) and participation of team members in workshops and conferences (WP6).
Bastian Egeter
added 2 research items
The overall goal of ERA Chair/EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). These protocols are essential for key application areas and to the development of research projects in association with business partners and other stakeholders, and thus to the strengthening of InBIO triple-helix initiatives (InBIO-Industry-Government; WP5). This report provides an overview of the current state of the art for collecting and preserving eDNA samples, with particular focus on vertebrate faecal samples, water samples and bulk invertebrate samples, which have been selected as key targets for the development of triple helix strategic initiatives (Task 5.3). The protocols already optimized and currently under development for the collection and preservation of eDNA samples are reported herein. Moreover, the future directions of sample collection and preservation at InBIO are discussed. This development was made possible through the recruitment of the ERA Chair team (WP2), secondments and Junior Researcher exchanges through the collaboration with international networks (WP3), an enhancement of computational infrastructure at InBIO (WP4) and participation of team members in workshops and conferences (WP6). Together, Deliverables D4.2-D4.5 (this document; Ferreira et al. 2018; Galhardo et al. 2018; Paupério et al. 2018) form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project, and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model.
The Sahara desert is the largest warm desert in the world and a poorly explored area. Small water-bodies occur across the desert and are crucial habitats for vertebrate biodiversity. Environmental DNA (eDNA) is a powerful tool for species detection and is being increasingly used to conduct biodiversity assessments. However, there are a number of difficulties with sampling eDNA from such turbid water-bodies and it is often not feasible to rely on electrical tools in remote desert environments. We trialled a manually powered filtering method in Mauritania, using pre-filtration to circumvent problems posed by turbid water in remote arid areas. From nine vertebrate species expected in the water-bodies, four were detected visually, two via metabarcoding, and one via both methods. Difficulties filtering turbid water led to severe constraints, limiting the sampling protocol to only one sampling point per study site, which alone may largely explain why many of the expected vertebrate species were not detected. The amplification of human DNA using general vertebrate primers is also likely to have contributed to the low number of taxa identified. Here we highlight a number of challenges that need to be overcome to successfully conduct metabarcoding eDNA studies for vertebrates in desert environments in Africa.
Sonia Ferreira
added a research item
The overall goal of ERA Chair/EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). These protocols are essential for key application areas and to the development of research projects in association with business partners and other stakeholders, and thus to the strengthening of InBIO triple-helix initiatives (InBIO-Industry-Government; WP5). This report provides an overview of the current state of the art for the development of best practice for building DNA reference collections of voucher specimens identified by specialised taxonomists, and how these practices are being implemented at InBIO. Building and organising reference collections of DNA sequences is an essential component of this task because eDNA studies require that DNA sequences recovered from the environment are compared to reference collections, which thus need to be developed following consistent and repeatable procedures. Such reference collections are likely to become a tool with significant relevance to the InBIO-Industry-Government triple-helix activities (WP5) by promoting the development of partnerships in all key areas: Monitoring of freshwater eDNA for species detection; Assessing natural pest control using faecal metagenomics; and Next-generation biomonitoring using DNA metabarcoding. Protocols described herein were developed in close connection and due to the accomplishment of the other WPs in the project, including: i) the recruitment of the ERA Chair team (WP2), ii) secondments researcher exchanges through collaborations with international networks (WP3), iii) the enhancement of the computational infrastructure at InBIO (WP4) and iv) participation and organization in workshops and conferences (WP6). Future directions in the improvement to enlarge the reference collection of DNA sequences of InBIO are identified. Together, Deliverables D4.2-D4.5 (this document; Egeter et al., 2018; Galhardo et al., 2018; Paupério et al., 2018) form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project, and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model.
Pedro Beja
added a research item
DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardisation efforts are still required to mainstream its application into biomonitoring programs. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenised bulk samples, which is time consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimise and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples, and thus including potential PCR inhibitors and non‐target organisms. We sampled macroinvertebrates at five sites, and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7‐14 than 1‐7 days after sampling, in terms of DNA concentration and integrity, taxa diversity, and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring. This article is protected by copyright. All rights reserved.
Sonia Ferreira
added a research item
DNA barcoding is increasingly used to identify species in a rapid and automated way, and may be particularly useful for morphologically similar larvae of aquatic insects. Identification of aquatic larvae of the alderfly genus Sialis Latreille, 1803 is troublesome due to limited morphological characters, though terrestrial adults are readily identifiable morphologically. This greatly limits our understanding of Sialis distribution and ecology, as adults are generally difficult to find and collect. In Portugal, only Sialis fuliginosa Pictet, 1836 has been previously collected, but there are numerous records of unidentified Sialis larvae. Based on morphology and DNA barcoding of the adult specimens recently collected in the country, we confirmed the presence of S. fuliginosa and in addition detected S. lutaria Linnaeus, 1758 and S. nigripes Pictet, 1865, which are reliably reported from Portugal for the first time. We found that DNA barcodes can unequivocally distinguish the three species, providing a basis to identify larval stages in future ecological and monitoring studies.
Pedro Beja
added an update
SAVE THE DATE: December 18th, 2018 | CIBIO-InBIO, Porto, Vairão
Assessment of biodiversity and ecological quality is of global importance in the monitoring of aquatic ecosystems, and is legally binding through the EU Water Framework Directive (WFD, 200/60/EC). High throughput DNA sequencing (HTS) has the potential to revolutionise the monitoring of ecosystems and biodiversity, but the uptake of molecular techniques in official monitoring programs at the national and regional scales has remained slow.
Organised by EnvMetaGen1, in close collaboration with DNAqua-Net2 and the EDP Biodiversity Chair, at CIBIO facilities in Vairão, Porto, this workshop will address the power of HTS techniques for assessing biodiversity and ecological quality in freshwater, transitional and marine waters, and provide information on how these techniques are starting to be used, in Portugal and across Europe and the USA, to address biomonitoring challenges, particularly those related to the WFD. A full day of conferences and networking where scientists, industry and government agencies will provide examples of best practices in the implementation of these techniques in different countries, such as the United Kingdom, Finland and Germany, allowing to nurture the discussion on what steps would be needed to improve their application and overcome technical problems, and how to address policy and regulatory issues.
Participation at the workshop is free of charge! Don´t miss this opportunity to gather with European experts, and acquire insight on how to implement these state-of-the-art molecular techniques on environmental monitoring programs. Confirmed speakers include @Florian Leese (University of Duisburg-Essen, Germany), @Ángel Borja (AZTI, Spain), @Bernd Hӓnfling (University of Hull, UK), @Rosetta Blackman (Eawag, Switzerland), @Jan Pawlowski (Université de Genève, Switzerland), @Kristian Meissner (Finnish Environment Institute, Finland), @Patricia Mergen (RMCA, Belgium), and @Taylor M Wilcox (University of Montana, USA), John Iwan Jones (Queen Mary University, UK), @Agnès Bouchez (INRA, France), @Filipe Costa (University of Minho), @João Pádua (Labelec), @Ana Filipa Filipe (CIBIO-InBIO).
More information and a full program will be given soon.
1 – EnvMetaGen, Capacity Building at InBIO for Research and Innovation Using Environmental Metagenomics, is an ERA Chair project funded by the European Commission (#668981)
2 – DNAqua-Net, Developing
 
Pedro Beja
added an update
Under the coordination of Sonia Ferreira , EnvMetaGen started collaborating with the Global Malaise Program (http://biodiversitygenomics.net/projects/gmp/) to barcode the arthropods sampled at two locations in Portugal using malaise traps. One trap has been set near the headquarters of CIBIO in the Campus Agrário de Vairão, and another one at the long term research site of Baixo Sabor (https://data.lter-europe.net/deims/site/lter_eu_pt_002). Samples will be takem weekly until January 2019.
The Global Malaise Program (GMP) is an international collaboration between the CBG and international contributors. With sampling sites across the world , the program represents the first step toward the acquisition of detailed temporal and spatial information on terrestrial arthropod communities across the globe. To date, over 1M specimens have been sequenced through the GMP, representing over 106K species. Over half (71K) of these species were new to the Barcode of Life Data Systems (BOLD).
This project will lead to a better knowledge of the arthropod fauna in Portugal as a whole, hopefully contributing to produce DNA Barcodes in a region still misrepresented in BOLD.
We will advertise new developments as they happen.
 
Pedro Beja
added an update
Professor Simon Jarman has accepted a new professional challenge and so he has resigned from his position as the holder of the ERA Chair in Environmental Metagenomics at CIBIO.
Therefore, we are opening a call to recruit a new ERA Chair holder. This position is advertised at:
We are accepting applications from researchers with expertise in this area from anywhere in the world.
 
Pedro Beja
added an update
The site of the EnvMetaGen project is now available online: http://inbio-envmetagen.pt/. Take a look for new, publications, and other project developments.
 
Pedro Beja
added an update
THE 7TH DNA METABARCODING SPRING SCHOOL
May 1-5 , 2017 | Porto, Portugal
The DNA Metabarcoding Spring School is now in its seventh edition, and this year it is co-organized by the metabarcoding.org Team and the CIBIO-InBIO/EnvMetaGen Project Team at Porto , Portugal.
The event will be held from May 1st to 5th, 2017 and at Parque Biológico de Gaia.
For further details and for registration information, please check the following link: http://metabarcoding.org/spip.php?article85
 
Sonia Ferreira
added 2 research items
Borkhausenia crimnodes Meyrick, 1912, a species described from Argentina, has been found resident in Beira Litoral, Portugal, constituting its first records in Europe. Borkhausenia intumescens Meyrick, 1921, described from South Africa, is shown to be synonymous with B. crimnodes, described from Argentina. COI barcode sequencing has shown that a Portuguese specimen has 100% similarity with specimens collected in South Africa. The origin of the Portuguese population remains unclear but it is likely to be connected with timber importation for the paper industry. Male and female genitalia of B. crimnodes type and the Portuguese specimens are illustrated and described.
Isotrias penedana Trematerra, 2013 was described from north Portugal based on males alone. Unidentified females were associated with the males using DNA barcoding, revealing sexual dimorphism in the species. Males and females differ in forewing shape, markings, and size, with females significantly smaller than males. The female is described and illustrated for the first time. We also document the species' occurrence in northern Spain.
Pedro Beja
added a research item
Understanding the drivers of long-term biodiversity change at local, regional and global scales requires detailed information on the diversity, composition and structure of biological communities, and on species interactions. For most species groups, this information is often scarce due to high biological diversity and limited taxonomic expertise. High throughput DNA sequencing is revolutionising the capacity to address this problem, by providing a relatively simple and inexpensive approach to identify virtually all sampled individuals from every species occurring in any area. Using as case study the LTER Sabor (NE Portugal), we explore in here the potential of this new technique, by describing an initiative for barcoding every species, and the application of metabarcoding for understanding drivers of biodiversity across scales. Preliminary biodiversity surveys have inventoried 2500 taxa, and we have completed the DNA barcoding of 1000 species of arthropods identified by expert taxonomists, but these figures are growing fast. Sequenced specimens are kept as vouchers for future reference, and DNA sequences are made freely available on the BOLD platform. The results obtained so far are unravelling cryptic diversity and allowing the establishment of a regional DNA barcoding reference database. Additionally, metabarcoding bulk samples of invertebrates (i.e., complex mixtures of species) are revealing community-level responses to land abandonment and how aquatic macroinvertebrates vary in relation to natural and anthropogenic drivers. Finally, analysis of trophic interactions based on metabarcoding are revealing complex links between bats and high-flying migratory moths, and are clarifying the role of semi-aquatic insectivore birds and mammals in aquatic food webs. Using these studies as examples, we make the case for a wider use of metabarcoding in the ILTER network.
Joana Paupério
added a research item
Background: Currently, the range of applications for DNA barcoding in biodiversity research is very wide and has surpassed purely academic goals. The development of services for public and private institutions is now paramount to tackle several societal challenges in diverse themes such as conservation biology, food security, and forensics. Yet, the applicability of the DNA barcoding approach is still undermined by the lack of taxonomic coverage in several biodiversity-rich regions. In particular, the Mediterranean basin is an underrepresented region in BOLD/GenBank, despite being a world biodiversity hotspot. Under the scope of the EDP Biodiversity Chair and an ERA-Chair in Environmental Metagenomics (capacity building EU projects), we propose to develop a DNA barcoding database covering all vertebrate and invertebrate taxa occurring in the Mediterranean basin. Results: We are assembling a network of taxonomists and optimising the analytical pipeline for barcoding a large number of voucher specimens. Each sequenced specimen will be identified by expert taxonomists, uniquely labelled, and then stored in Natural History Museums, CIBIO/InBIO, or private collections. Only taxonomically validated specimens will be uploaded in Barcoding.med and internationally-free databases like BOLD, constituting a source of development for services relating to biodiversity research. The first phase of this project is focusing on terrestrial and freshwater arthropods, particularly Plecoptera, Ephemeroptera, Odonata, Trichoptera, Orthoptera, Coleoptera, and Hymenoptera. Significance: By providing cost-efficient approaches for biodiversity assessments and monitoring, we aim to promote the use of DNA barcoding tools in public and private institutions, especially for providing services addressing current environmental legislation and international obligations (e.g., water directive, detection of invasive species), while also promoting research on ecosystem food webs. This initiative will also be used to develop tailor-suited services to state and private institutions, thus transferring skills and technology while also promoting capacity building at a regional level.
Pedro Beja
added an update
The project EnvMetaGen is opening one position for a Knowledge Transfer & Dissemination Officer.
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Pedro Beja
added an update
The project EnvMetaGen is opening two positions for research assistants.
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Pedro Beja
added an update
The project EnvMetaGen is opening three positions for post-doc researchers.
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Anyone interested and with the adequate level of expertise is welcome to apply!!
Pedro Beja
 
Pedro Beja
added a project goal
The goal of the project is to expand the research and innovation potential of InBIO – Research Network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. The project aims to strengthen the research potential of human resources, lab facilities and next-generation genome sequencing equipment funded by a FP7 CAPACITIES project, supporting an emerging research line in environmental metagenomics for applications in biodiversity conservation, invasive species control, ecosystem services assessment, and environmental (bio)monitoring. The project will contribute to the regional and national smart specialization strategies, by developing tools and approaches to foster environmentally sustainable development, and strengthening innovation and knowledge transfer activities in close collaboration with local and global industrial partners, as well as with governmental agencies. In addition, the project will contribute to the advanced training of highly-qualified human resources, and to the communication, dissemination and exploitation of InBIO’s research and innovation. To achieve its objectives, EnvMetaGen will develop the following main activities: (i) Upgrade the research capacity and capability by expanding the human potential and fostering a critical mass of researchers with inter-disciplinary expertise in environmental metagenomics; (ii) Improve InBIO’s innovation potential and impact at the regional, national and European levels, by promoting the integration of cost-effective metagenomics approaches at the forefront of biodiversity, ecological and environmental research; and (iii) Raise the international and national network of collaborations and partnerships of InBIO and its industrial and governmental affiliates. These activities will contribute to better integrate InBIO with the European Research Area (ERA), thus increasing their level of participation in the Horizon 2020 program.