Wednesday, September 19, 2018

From the inbox: Eurytemora conference 2019

Dear colleagues

We are going to organize a conference on Eurytemora in Saint Petersburg.  We are planning to meet in spring (May 2019). It is the nicest season to visit SPb: good weather and not too much tourists. The conference will be held in the building of the Zoological Institute of the Russian Academy of Sciences situated in the very heart of SPb just opposite of the famous Hermitage museum.

Preliminary topic of our conference is: Use of molecular-genetic and morphological methods in taxonomy, phylogeny, biogeography and ecology of Eurytemora species. All oral presentations will be splitted in four sections mentioned in the conference title.

Among inviting speakers we are waiting Prof. Victor Alekseev (ZIN RAS), Prof. Carol Eunmi Lee (Wisconsin–MadisonUniv.), Prof. Sami Souissi (Lille1 Univ.) and some others. We are planning to publish proceedings of our conference as a special volume of an international journal. The conference should be not expensive for participants especially for students and young scientists. Your help in distribution of the information about our conference so far will be very much appreciated.

If the suggestion is interesting for you, please contact us until the first of December.

Sincerely,

Organizing committee
Kochanova Elena - Executive Secretary
Natalia Sukhikh - The chairmen of the local organizing comity

Zoological Institute of the Russian Academy of Sciences
Universitetskaya emb., 1, St.-Petersburg, 199034, Russia
(++7 812) 328-03-11

Thursday, September 13, 2018

Researcher in Environmental DNA (eDNA), Aarhus University

Researcher in Environmental DNA (eDNA), Aarhus University, Campus Roskilde, Denmark 1003793

The Department of Environmental Science at Aarhus University, Roskilde, Denmark invites applications for a four year researcher position in environmental DNA (eDNA) to strengthen and expand the research capability, and the science-based policy advisory activities at the department. The position is to be filled as soon as possible, subject to negotiation. 

The Department of Environmental Science 
The Department of Environmental Science is an interdisciplinary department under the Faculty of Science & Technology at Aarhus University. The expertise ranges from physics, chemistry, microbiology, molecular biology and mathematical modelling to social science, geography, economics and policy analysis. Both basic and applied research is conducted on some of the major challenges facing society, such as pollution and biodegradation, management of land, soil, water, air and biodiversity, protection of ecosystem services, climate change, and energy systems. Science-based policy advice within these areas is offered to ministries and other stakeholders. Currently, about 160 staff and PhD-students are working at the department. Further information can be found at http://www.envs.au.dk.

The selected candidate will be affiliated with the Section for Environmental Microbiology & Biotechnology and should contribute to the section's scientific environment. The section conducts research and science-based policy advise targeted towards the understanding of the fate and role of individual microorganisms in ecosystems and interactions between bacteria, vira, fungi, protozoa and microeukaryotes, especially in the terrestrial environment but also in aquatic and atmospheric environment and in mammal guts. Expertise within the section includes:

• Microbiology in air, soil, ice, rhizosphere, and animal gut 
• Microbial and microeukaryotic genetics and diversity 
• Microbial and protist ecology 
• Bacteriophages 
• eDNA analysis and use in environmental monitoring 
• Antibiotic resistance and pathogenicity 
• Biodegradation of persistent organic pollutants
• Plant-microbial interactions

Description of the position 
The selected candidate is expected to: 
• Contribute to ongoing research projects in eDNA for environmental monitoring with environmental genetics and bioinformatics and the establishment of this research area within the department.
• Further develop and consolidate the research area of environmental DNA of microbial eukaryotes in terrestrial environments within the department.
• Contribute to science-based policy advisory activities in the area of eDNA for environmental monitoring of terrestrial, aquatic and other relevant environments. This is foreseen to be in collaboration with Department of Bioscience as well as other departments at the faculty of Science and Technology within the ST center of eDNA for environmental monitoring (http://projects.au.dk/edna/) in order to unfold the major potential for integrating eDNA in environmental monitoring. 
• Actively contribute to attraction of external funding together with senior staff and also as PI. 
• The main responsibilities are research (including publication/academic dissemination duties) and science-based policy advisory activities.

Qualification requirements
• The position requires a background with skills of genetic analysis and bioinformatics as well as understanding of ecology; e.g. molecular biology and ecology, biotechnology, or microbial ecology supplemented with acquired competences of bioinformatics analysis. 
• Documented experience with bioinformatics and statistical analysis of eDNA data is required.
• Documented experience with analysis of eDNA, both at the molecular level including Next Generation Sequencing of metagenome and metatranscriptome as well as amplicon sequencing of targeted organisms.
• Applicants should as a minimum hold scientific qualifications equivalent to the PhD graduate level showing evidence of a strong and ambitious research oriented profile and an emerging publication record within eDNA research. 
• Experience from participation in applications for external funding as well as participation in international collaboration is required.
• It will be a benefit if the applicant is or will be able to apply for an ERC Starting Grant during the 4 year employment.
• Applicants should have excellent written and oral communication skills in English. 
• The candidate is expected to be enthusiastic about working in an interdisciplinary academic environment. 
The position, therefore, requires a highly motivated scientist who is comfortable working in teams.

We speak English on a daily basis at the department. However, to be able to conduct tasks related to policy advice, and to participate in administrative/social activities, it will be in the interest of an international candidate to be able to communicate in Danish. Foreign candidates are, therefore, expected to learn Danish. 

Work location
The place of work will be at Aarhus University, Campus Risø outside Roskilde, Denmark. Frederiksborgvej 399, 4000 Roskilde. The city of Roskilde (www.roskilde.dk) is located 35 km from Copenhagen. 

Further information 
Further information may be obtained by contacting Deputy Head of Department Dr. Anne Winding aw@envs.au.dk; +45 8715 8615 or Head of Section, Professor Lars H Hansen lhha@envs.au.dk; +45 8715 8588.

The area provides immediate access to a wealth of cultural and recreational pursuits. International candidates may find information concerning living and working in Denmark at www.workindenmark.dk. AU International Center offers a full range of services to make your transition to Denmark as smooth as possible (http://www.au.dk/en/internationalcentre/ias/), and the department will assist foreign candidates with finding a suitable place to live. 

Application procedure 
Shortlisting is used. This means that after the deadline for applications and with the assistance from the assessment committee chairman, and the assessment committee if necessary, the head of department selects the candidates to be evaluated. The selection is made on the basis of an assessment of who of the candidates are most relevant considering the requirements of the advertisement. All applicants will be notified within 6 weeks whether or not their applications have been sent to an expert assessment committee for evaluation. The selected applicants will be informed about the composition of the committee and will receive his/her assessment. Once the recruitment process is completed a final letter of rejection is sent to the deselected applicants, including the main considerations emphasized during the selection process. 

Formalities and salary range 

The application must be in English and include a curriculum vitae, degree certificate, a complete list of publications, a statement of future research plans and information about research activities, teaching portfolio and verified information on previous teaching experience (if any). Guidelines for applicants can be found here

Appointment shall be in accordance with the collective labour agreement between the Danish Ministry of Finance and the Danish Confederation of Professional Associations. Further information on qualification requirements and job content may be found in the Memorandum on Job Structure for Academic Staff at Danish Universities. (in Danish)

Salary depends on seniority as agreed between the Danish Ministry of Finance and the Confederation of Professional Associations.

All interested candidates are encouraged to apply, regardless of their personal background. Research activities will be evaluated in relation to actual research time. Thus, we encourage applicants to specify periods of leave without research activities, in order to be able to subtract these periods from the span of the scientific career during the evaluation of scientific productivity.

Aarhus University offers Relocation service to International researchers. You can read more about it here.

Friday, August 24, 2018

Weekend reads

A lot of papers have piled up in the past few weeks. Lots to recommend for thorough reading. Lets start with some that were written by colleagues at our institute:

PREMISE OF THE STUDY:
The detection of environmental DNA (eDNA) using high-throughput sequencing has rapidly emerged as a method to detect organisms from environmental samples. However, eDNA studies of aquatic biomes have focused on surveillance of animal species with less emphasis on plants. Pondweeds are important bioindicators of freshwater ecosystems, although their diversity is underestimated due to difficulties in morphological identification and monitoring.
METHODS:
A protocol was developed to detect pondweeds in water samples using atpB-rbcL and ITS2 markers. The water samples were collected from the Grand River within the rare Charitable Research Reserve, Ontario (RARE). Short fragments were amplified using primers targeting pondweeds, sequenced on an Ion Torrent Personal Genome Machine, and assigned to the taxonomy using a local DNA reference library and GenBank.
RESULTS:
We detected two species earlier documented at the experimental site during ecological surveys (Potamogeton crispus and Stuckenia pectinata) and three species new to the RARE checklist (P. foliosus, S. filiformis, and Zannichellia palustris).
DISCUSSION:
Our targeted approach to track the species composition of pondweeds in freshwater ecosystems revealed underestimation of their diversity. This result suggests that eDNA is an effective tool for monitoring plant diversity in aquatic habitats.

Characterisation of freshwater benthic biodiversity using DNA metabarcoding may allow more cost-effective environmental assessments than the current morphological-based assessment methods. DNA metabarcoding methods where sorting or pre-sorting of samples are avoided altogether are especially interesting, since the time between sampling and taxonomic identification is reduced. Due to the presence of non-target material like plants and sediments in crude samples, DNA extraction protocols become important for maximising DNA recovery and sample replicability. We sampled freshwater invertebrates from six river and lake sites and extracted DNA from homogenised bulk samples in quadruplicate subsamples, using a published method and two commercially available kits: HotSHOT approach, Qiagen DNeasy Blood & Tissue Kit and Qiagen DNeasy PowerPlant Pro Kit. The performance of the selected extraction methods was evaluated by measuring DNA yield and applying DNA metabarcoding to see if the choice of DNA extraction method affects DNA yield and metazoan diversity results. The PowerPlant Kit extractions resulted in the highest DNA yield and a strong significant correlation between sample weight and DNA yield, while the DNA yields of the Blood & Tissue Kit and HotSHOT method did not correlate with the sample weights. Metazoan diversity measures were more repeatable in samples extracted with the PowerPlant Kit compared to those extracted with the HotSHOT method or the Blood & Tissue Kit. Subsampling using Blood & Tissue Kit and HotSHOT extraction failed to describe the same community in the lake samples. Our study exemplifies that the choice of DNA extraction protocol influences the DNA yield as well as the subsequent community analysis. Based on our results, low specimen abundance samples will likely provide more stable results if specimens are sorted prior to DNA extraction and DNA metabarcoding, but the repeatability of the DNA extraction and DNA metabarcoding results was close to ideal in high specimen abundance samples.

Background
Macroinvertebrates such as non-biting midges (Chironomidae: Diptera) are important components of freshwater ecosystems. However, they are often neglected in biodiversity and conservation research because invertebrate species richness is difficult and expensive to quantify with traditional methods. We here demonstrate that Next Generation Sequencing barcodes (“NGS barcodes”) can provide relief because they allow for fast and large-scale species-level sorting of large samples at low cost.
Results
We used NGS barcoding to investigate the midge fauna of Singapore’s swamp forest remnant (Nee Soon Swamp Forest). Based on > 14.000 barcoded specimens, we find that the swamp forest maintains an exceptionally rich fauna composed of an observed number of 289 species (estimated 336 species) in a very small area (90 ha). We furthermore barcoded the chironomids from three surrounding reservoirs that are located in close proximity. Although the swamp forest remnant is much smaller than the combined size of the freshwater reservoirs in the study (90 ha vs. > 450 ha), the latter only contains 33 (estimated 61) species. We show that the resistance of the swamp forest species assemblage is high because only 8 of the 314 species are shared despite the close proximity. Moreover, shared species are not very abundant (3% of all specimens). A redundancy analysis revealed that ~ 21% of the compositional variance of midge communities within the swamp forest was explained by a range of variables with conductivity, stream order, stream width, temperature, latitude (flow direction), and year being significant factors influencing community structure. An LME analysis demonstrates that the total species richness decreased with increasing conductivity.
Conclusion
Our study demonstrates that midge diversity of a swamp forest can be so high that it questions global species diversity estimates for Chironomidae, which are an important component of many freshwater ecosystems. We furthermore demonstrate that small and natural habitat remnants can have high species turnover and can be very resistant to the invasion of species from neighboring reservoirs. Lastly, the study shows how NGS barcodes can be used to integrate specimen- and species-rich invertebrate taxa in biodiversity and conservation research.

The hyporheic zone, i.e. the ecotone between surface water and the groundwater, is a rarely studied freshwater ecosystem. Hyporheic taxa are often meiofaunal (<1 mm) in size and difficult to identify based on morphology. Metabarcoding approaches are promising for the study of these environments and taxa, but it is yet unclear if commonly applied metabarcoding primers and replication strategies can be used. In this study, we took sediment cores from two near natural upstream (NNU) and two ecologically improved downstream (EID) sites in the Boye catchment (Emscher River, Germany), metabarcoding their meiofaunal communities. We evaluated the usability of a commonly used, highly degenerate COI primer pair (BF2/BR2) and tested how sequencing three PCR replicates per sample and removing MOTUs present in only one out of three replicates impacts the inferred community composition. A total of 22,514 MOTUs were detected, of which only 263 were identified as Metazoa. Our results highlight the gaps in reference databases for meiofaunal taxa and the potential problems of using highly degenerate primers for studying samples containing a high number of non-metazoan taxa. Alpha diversity was higher in EID sites and showed higher community similarity when compared to NNU sites. Beta diversity analyses showed that removing MOTUs detected in only one out of three replicates per site greatly increased community similarity in samples. Sequencing three sample replicates and removing rare MOTUs is seen as a good compromise between retaining too many false-positives and introducing too many false-negatives. We conclude that metabarcoding hyporheic communities using highly degenerate COI primers can provide valuable first insights into the diversity of these ecosystems and highlight some potential application scenarios.

Metabarcoding of complex metazoan communities is increasingly being used to measure biodiversity in terrestrial, freshwater, and marine ecosystems, revolutionizing our ability to observe patterns and infer processes regarding the origin and conservation of biodiversity. A fundamentally important question is which genetic marker to amplify, and although the mitochondrial cytochrome oxidase subunit I (COI) gene is one of the more widely used markers in metabarcoding for the Metazoa, doubts have recently been raised about its suitability. We argue that (i) the extensive coverage of reference-sequence databases for COI, (ii) the variation it presents, (iii) the comparative advantages for denoising protein coding genes, and (iv) recent advances in DNA sequencing protocols argue in favour of standardising for the use of COI for metazoan community samples. We also highlight where research efforts should focus to maximise the utility of metabarcoding.

Resource variation along abiotic gradients influences subsequent trophic interactions and these effects can be transmitted through entire food webs. Interactions along abiotic gradients can provide clues as to how organisms will face changing environmental conditions, such as future range shifts. However, it is challenging to find replicated systems to study these effects. Phytotelmata, such as those found in carnivorous plants, are isolated aquatic communities and thus form a good model for the study of replicated food webs. Due to the degraded nature of the prey, molecular techniques provide a useful tool to study these communities. We studied the pitcher plant Sarracenia purpurea L. in allochthonous populations along an elevational gradient in the Alps and Jura. We predicted that invertebrate richness in the contents of the pitcher plants would decrease with increasing elevation, reflecting harsher environmental conditions. Using metabarcoding of the COI gene, we sequenced the invertebrate contents of these pitcher plants. We assigned Molecular Operational Taxonomic Units at ordinal level as well as recovering species-level data. We found small but significant changes in community composition with elevation. These recovered sequences could belong to invertebrate prey, rotifer inquilines, pollinators and other animals possibly living inside the pitchers. However, we found no directional trend or site-based differences in MOTU richness with elevational gradient. Use of molecular techniques for dietary or contents analysis is a powerful way to examine numerous degraded samples, although factors such as DNA persistence and the relationship to species presence still have to be completely determined. 

DNA metabarcoding is widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints limit most studies to marker lengths below 600 base pairs (bp). Longer sequencing reads of several thousand bp are now possible with third-generation sequencing. Increased marker lengths provide greater taxonomic resolution and allow for phylogenetic methods of classification, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most bioinformatics tools for DNA metabarcoding were designed for short reads and are therefore unsuitable. Here we used Pacific Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote SSU and LSU rRNA genes and the complete ITS region. We developed an analysis pipeline that reduced error rates to levels comparable to short-read platforms. Validation using a mock community indicated that our pipeline detected 98% of chimeras de novo. We recovered 947 OTUs from water and sediment samples from a natural lake, 848 of which could be classified to phylum, 397 to genus, and 330 to species. By allowing for the simultaneous use of three databases (Unite, SILVA, RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 non-fungal OTUs indicate that long-read DNA metabarcoding holds promise for studies of eukaryotic diversity more broadly.

BACKGROUND:
Taxonomic identification of plants and insects is a hard process that demands expert taxonomists and time, and it's often difficult to distinguish on morphology only. DNA barcodes allow a rapid species discovery and identification and have been widely used for taxonomic identification by targeting known gene regions that permit to discriminate these species. DNA barcode sequence analysis is usually carried out with processes and tools that still demand a high interaction with the user or researcher. To reduce at most such interaction, we proposed PIPEBAR, a pipeline for DNA chromatograms analysis of Sanger platform sequencing, ensuring high quality consensus sequences along with efficient running time. We also proposed a paired-end reads assembly tool, OverlapPER, which is used in sequence or independently of PIPEBAR.
RESULTS:
PIPEBAR is a command line tool to automatize the processing of large number of trace files. It is accurate as the proprietary Geneious tool and faster than most popular software for barcoding analysis. It is 7 times faster than Geneious and 14 times faster than SeqTrace for processing hundreds of barcoding sequences. OverlapPER is a novel tool for overlapping paired-end reads accurately that accepts both substitution and indel errors and returns both overlapped and non-overlapped regions between a pair of reads. OverlapPER obtained the best results compared to currently used tools when merging 1,000,000 simulated paired-end reads.
CONCLUSIONS:
PIPEBAR and OverlapPER run on most operating systems and are freely available, along with supporting code and documentation

Tuesday, August 21, 2018

From the inbox: Young researcher position

Here a call for a 30-month young researcher position in the InterReg AlpineSpace project “Eco-AlpsWater”, in the field of DNA biomonitoring of freshwater ecosystems, which will be hosted by Nico Salmaso, member of DNAqua-Net.


Thursday, July 19, 2018

Fishbase in trouble

Do you know Fishbase? If you - like me - are working in fish biology and anything relating to fish or if you are a serious fish hobbyist it is very likely that you came across Fishbase a number of times. It is a huge resource for fish names and life history information. Endless work has been put into this database but it has never been easy to find the money needed to run it especially to pay for the staff in Manila. I've visited them a couple of years ago and it is just incredible how much they've accomplished on a shoestring budget through their hard and dedicated work. The more concerned I became when I was told about this message from their website:

Dear FishBase users, We have just been informed that a major payment to support the FishBase encoders in 2018 will not be made. It comes at a time when use of FishBase produced a major scientific breakthrough with far-reaching practical implications. This leaves us with a funding gap of 200,000 US$ until end of 2018. We will now have to ask our expert staff [in the Philippines] to go on unpaid leave until we receive additional funding. This is an urgent call for your help: If FishBase is useful to you, please donate to bring us back on track.

Please spread the word and hopefully this call will reach somebody with big pockets who wants to help keeping this immensely valuable resource alive.

Tuesday, July 17, 2018

Temperature and mtDNA selection

Mitochondrial DNA (mtDNA) has traditionally been used in population genetic and biogeographic studies as a maternally-inherited and evolutionary-neutral genetic marker. However, it is now clear that polymorphisms within the mtDNA sequence are routinely non-neutral, and furthermore several studies have suggested that such mtDNA polymorphisms are also sensitive to thermal selection. 

A team of researchers from Japan, Australia, and the UK studied two naturally occurring mtDNA variants that are carried by fruit flies inhabiting the east coast of Australia. One of these variants is more common in the sub-tropical, northern part of the country, where temperatures are higher. The other fly variant is more common in the temperate, southern part, which tends to be colder. The colleagues collected flies from both sites and interbred them to get a series of populations with equally mixed genes. Each mixed population was subdivided into four subpopulations. Each of those wer maintained at different conditions, some at constant temperature(e.g. 19ºC and 25ºC respectively), some at fluctuating temperatures to simulate the thermal conditions at the two sites where the flies were collected. After three months, the researchers sequenced the mtDNA of flies from all these subpopulations.

In addition they also examined how the presence of bacteria such as Wolbachia, which commonly infects fruit flies, affects mtDNA selection. Some of the fly populations were treated with antibiotics to kill off any Wolbachia infections that they might be harboring.

The researchers found that in flies reared under warm laboratory conditions, one of the two mtDNA variants became more common than the other. The same mtDNA variant was also found to be widely prevalent in flies from the warmer northern parts of Australia. A similar pattern was observed with the other mtDNA variant in flies reared under cold laboratory conditions. However, researchers only observed this effect in populations where Wolbachia infections had been wiped out. Moreover, the variation patterns they observed in males didn't always match up with the ones in females.

The results show that temperature shapes how mtDNA varies in nature. They also suggest that additional factors, such as sex and infection with microbes, might also influence how the mitochondrial genome evolves in the wild. 

Our results strengthen the emerging view that intra-specific mtDNA variants are sensitive to selection, and suggest spatial distributions of mtDNA variants in natural populations of metazoans might reflect adaptation to climatic environments rather than within-population coalescence and diffusion of selectively-neutral haplotypes across populations.

Friday, July 13, 2018

Weekend reads

Back on track with the weekend read posts.  Maybe not as many as usual but still quite interesting to read.

Ecological and taxonomic knowledge is important for conservation and utilization of biodiversity. Biodiversity and ecology of fungi in Mediterranean ecosystems is poorly understood. Here, we examined the diversity and spatial distribution of fungi along an elevational gradient in a Mediterranean ecosystem, using DNA metabarcoding. This study provides novel information about diversity of all ecological and taxonomic groups of fungi along an elevational gradient in a Mediterranean ecosystem. Our analyses revealed that among all biotic and abiotic variables tested, host species identity is the main driver of the fungal richness and fungal community composition. Fungal richness was strongly associated with tree richness and peaked in Quercus-dominated habitats and Cistus-dominated habitats. The highest taxonomic richness of ectomycorrhizal fungi was observed under Quercus ilex, whereas the highest taxonomic richness of saprotrophs was found under Pinus. Our results suggest that the effect of plant diversity on fungal richness and community composition may override that of abiotic variables across environmental gradients.

Although insects dominate the terrestrial fauna, sampling constraints and the poor taxonomic knowledge of many groups have limited assessments of their diversity. Passive sampling techniques and DNA-based species assignments now make it possible to overcome these barriers. For example, Malaise traps collect specimens with minimal intervention while the Barcode Index Number (BIN) system automates taxonomic assignments. The present study employs Malaise traps and DNA barcoding to extend understanding of insect diversity in one of the least known zoogeographic regions, the Saharo-Arabian. Insects were collected at four sites in three countries (Egypt, Pakistan, Saudi Arabia) by deploying Malaise traps. The collected specimens were analyzed by sequencing 658 bp of cytochrome oxidase I (DNA barcode) and assigning BINs on the Barcode of Life Data Systems. The year-long deployment of a Malaise trap in Pakistan and briefer placements at two Egyptian sites and at one in Saudi Arabia collected 53,092 specimens. They belonged to 17 insect orders with Diptera and Hymenoptera dominating the catch. Barcode sequences were recovered from 44,432 (84%) of the specimens, revealing the occurrence of 3,682 BINs belonging to 254 families. Many of these taxa were uncommon as 25% of the families and 50% of the BINs from Pakistan were only present in one sample. Family and BIN counts varied significantly through the year, but diversity indices did not. Although more than 10,000 specimens were analyzed from each nation, just 2% of BINs were shared by Pakistan and Saudi Arabia, 4% by Egypt and Pakistan, and 7% by Egypt and Saudi Arabia. The present study demonstrates how the BIN system can circumvent the barriers imposed by limited access to taxonomic specialists and by the fact that many insect species in the Saharo-Arabian region are undescribed.

Fungal spores and mycelium fragments are particles which become and remain airborne and have been subjects of aerobiological studies. The presence and the abundance of taxa in aerobiological samples can be very variable and impaired by changeable climatic conditions. Because many fungi produce mycotoxins and both their mycelium fragments and spores are potential allergens, monitoring the presence of these taxa is of key importance. So far data on exposure and sensitization to fungal allergens are mainly based on the assessment of few, easily identifiable taxa and focused only on certain environments. The microscopic method used to analyze aerobiological samples and the inconspicuous fungal characters do not allow a in depth taxonomical identification. Here, we present a first assessment of fungal diversity from airborne samples using a DNA metabarcoding analysis. The nuclear ITS2 region was selected as barcode to catch fungal diversity in mixed airborne samples gathered during two weeks in four sites of North-Eastern and Central Italy. We assessed the taxonomic composition and diversity within and among the sampled sites and compared the molecular data with those obtained by traditional microscopy. The molecular analyses provide a tenfold more comprehensive determination of the taxa than the traditional morphological inspections. Our results prove that the metabarcoding analysis is a promising approach to increases quality and sensitivity of the aerobiological monitoring. The laboratory and bioinformatic workflow implemented here is now suitable for routine, high-throughput, regional analyses of airborne fungi.

Throughout the years, DNA barcoding has gained in importance in forensic entomology as it leads to fast and reliable species determination. High-quality results, however, can only be achieved with a comprehensive DNA barcode reference database at hand. In collaboration with the Bavarian State Criminal Police Office, we have initiated at the Bavarian State Collection of Zoology the establishment of a reference library containing arthropods of potential forensic relevance to be used for DNA barcoding applications. CO1-5P' DNA barcode sequences of hundreds of arthropods were obtained via DNA extraction, PCR and Sanger Sequencing, leading to the establishment of a database containing 502 high-quality sequences which provide coverage for 88 arthropod species. Furthermore, we demonstrate an application example of this library using it as a backbone to a high throughput sequencing analysis of arthropod bulk samples collected from human corpses, which enabled the identification of 31 different arthropod Barcode Index Numbers.

Environmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species-specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and "metabarcoding" have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high-throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.