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.

Thursday, July 12, 2018

How predictable is evolution?

Imagine 500 to 1,000 species of cichlids living in one of the African Great Lakes, one of the largest freshwater habitats in the world. The degree of complexity is unimaginable. Even the genealogical relationships between the cichlid species living in these lakes have only partially been resolved

For every two species of mammal there is one species of cichlid fish, which shows that biodiversity is distributed rather unevenly among animals. The question is why and to what extent can this be predicted? We know that a number of factors play a role in the evolution of this. One of them are ecological conditions, i.e. the number of different habitats and the similarity of ecological niches available. The demographic history of a population can also influence biodiversity. A higher level of genetic variation in a population is beneficial in a sense that it allows - given sufficient time - adaptation to more ecological niches. Quantifying all potential factors that contribute to biological diversity, even for only one group of animals, is not easy, not to mention that comparing mammals with a group of fish would be like comparing apples and oranges. 

A new study coming from the lab of my PhD supervisor, Axel Meyer shows some of the factors that contribute to recurrent patterns of diversity and similarity in cichlids. The colleagues aimed to determine factors that led to similar outcomes and thereby help to predict evolution. As any African Great Lake harbours an incredibly species diversity, the team studied a more simple system involving parallel species of Midas cichlids, which occur in two great lakes as well as in a chain of crater lakes in Nicaragua. 

The more similar the habitat of the crater lake is to that of the large source lake, the more similar the fish are to each other. Habitat seems to be more important than demographic criteria when it comes to predictability of diversity.  The data collected by the colleagues also shows that, compared to the source population, the morphology of all crater lake populations has diversified mostly in the same direction. Crater lake fish all very quickly evolved body shapes that are longer and more slender than those of their cousins from the bigger lakes. In addition body shapes of the crater lake populations correlate with the average depth of the lakes. It makes sense. The deeper a lake is, the more likely it is to provide various ecological niches, including in the deep open water. 

In summary parallel morphological divergence in allopatry and the propensity for diversification in sympatry across the entire Midas cichlid fish radiation is partly predictable and mostly driven by ecology.

Monday, July 9, 2018

(Post-) Weekend reads

Last Friday I was so invested in some data analysis that I forgot everything around me and that included my Friday blog post with weekend reading material. My apologies for that. Nevertheless, here are my weekly favourites for some throughout the week reading.

Efficient DNA extraction is fundamental to molecular studies. However, commercial kits are expensive when a large number of samples need to be processed. Here we present a simple, modular and adaptable DNA extraction ‘toolkit’ for the isolation of high purity DNA from multiple sample types (modular universal DNA extraction method or Mu-DNA). We compare the performance of our method to that of widely used commercial kits across a range of soil, stool, tissue and water samples. Mu-DNA produced DNA extractions of similar or higher yield and purity to that of the commercial kits. As a proof of principle, we carried out replicate fish metabarcoding of aquatic eDNA extractions, which confirmed that the species detection efficiency of our method is similar to that of the most frequently used commercial kit. Our results demonstrate the reliability of Mu-DNA along with its modular adaptability to challenging sample types and sample collection methods. Mu-DNA can substantially reduce the costs and increase the scope of experiments in molecular studies.

The monitoring of impacts of anthropic activities in marine environments, such as aquaculture, oil-drilling platforms or deep-sea mining, relies on Benthic Biotic Indices (BBI). Several indices have been formalised to reduce the multivariate composition data into a single continuous value that is ascribed to a discrete ecological quality status. Such composition data is traditionally obtained from macrofaunal inventories, which is time-consuming and expertise-demanding. Important efforts are ongoing towards using High-Throughput Sequencing of environmental DNA (eDNA metabarcoding) to replace or complement morpho-taxonomic surveys for routine biomonitoring. The computation of BBI from such composition data is usually being undertaken by practitioners with excel spreadsheets or through custom script. Hence, the updating of reference morpho-taxonomic tables and cross studies comparison could be hampered. Here we introduce the R package BBI for the computation of BBI from composition data, either obtained from traditional morpho-taxonomic inventories or from metabarcoding data. Its aim is to provide an open-source, transparent and centralised method to compute BBI for routine biomonitoring.

The degradation of freshwater ecosystems has become a common ecological and environmental problem globally. Owing to the complexity of biological communities, there remain tremendous technical challenges for investigating influence of environmental stressors (e.g. chemical pollution) on biological communities. High-throughput sequencing-based metabarcoding provides a powerful tool to reveal complex interactions between environments and biological communities. Among many technical issues, the clustering strategies for Operational Taxonomic Units (OTUs) which are crucial for assessing biodiversity of communities, may affect final conclusions. Here, we used zooplankton communities along an environmental pollution gradient in the Chaobai River in Northern China to test different clustering strategies, including non-clustering and clustering with varied thresholds. Our results showed that though the number of OTUs estimated by non-clustering strategies and clustering strategies with divergence thresholds of 99-97% largely varied, they were able to identify the same set of significant environmental and spatial variables responsible for geographical distributions of zooplankton communities. In addition, the ecological conclusions obtained by clustering thresholds of 99-97% were consistent with non-clustering strategies, where for all eight clustering scenarios we detected that species sorting predicted by environmental variables overrode dispersal as the dominant factor in structuring zooplankton communities. However, clustering with the divergence thresholds of <95% affected the environmental and spatial variables identified. We conclude that both newly developed non-clustering methods and traditional clustering methods with divergence thresholds ≥97% were reliable to reveal mechanisms of complex community-environment interactions, although different clustering strategies could lead to largely varied biodiversity estimates such as those for α-diversity.

Sediment bypass tunnels (SBTs) are guiding structures used to reduce sediment accumulation in reservoirs during high flows by transporting sediments to downstream reaches during operation. Previous studies monitoring the ecological effects of SBT operations on downstream reaches suggest a positive influence of SBTs on riverbed sediment conditions and macroinvertebrate communities based on traditional morphology-based surveys. Morphology-based macroinvertebrate assessments are costly and time-consuming, and the large number of morphologically cryptic, small-sized and undescribed species usually results in coarse taxonomic identification. Here, we used DNA metabarcoding analysis to assess the influence of SBT operations on macroinvertebrates downstream of SBT outlets by estimating species diversity and pairwise community dissimilarity between upstream and downstream locations in dam-fragmented rivers with operational SBTs in comparison to dam-fragmented (i.e., no SBTs) and free-flowing rivers (i.e., no dam). We found that macroinvertebrate community dissimilarity decreases with increasing operation time and frequency of SBTs. These factors of SBT operation influence changes in riverbed features, e.g. sediment relations, that subsequently effect the recovery of downstream macroinvertebrate communities to their respective upstream communities. Macroinvertebrate abundance using morphologically-identified specimens was positively correlated to read abundance using metabarcoding. This supports and reinforces the use of quantitative estimates for diversity analysis with metabarcoding data.

Metabarcoding is a popular application which warrants continued methods optimization. To maximize barcoding inferences, hierarchy-based sequence classification methods are increasingly common. We present methods for the construction and curation of a database designed for hierarchical classification of a 157 bp barcoding region of the arthropod cytochrome c oxidase subunit I (COI) locus. We produced a comprehensive arthropod COI amplicon dataset including annotated arthropod COI sequences and COI sequences extracted from arthropod whole mitochondrion genomes, the latter of which provided the only source of representation for Zoraptera, Callipodida and Holothyrida. The database contains extracted sequences of the target amplicon from all major arthropod clades, including all insect orders, all arthropod classes and Onychophora, Tardigrada and Mollusca outgroups. During curation, we extracted the COI region of interest from approximately 81 percent of the input sequences, corresponding to 73 percent of the genus-level diversity found in the input data. Further, our analysis revealed a high degree of sequence redundancy within the NCBI nucleotide database, with a mean of approximately 11 sequence entries per species in the input data. The curated, low-redundancy database is included in the Metaxa2 sequence classification software. Using this database with the Metaxa2 classifier, we performed a cross-validation analysis to characterize the relationship between the Metaxa2 reliability score, an estimate of classification confidence, and classification error probability. We used this analysis to select a reliability score threshold which minimized error. We then estimated classification sensitivity, false discovery rate and overclassification, the propensity to classify sequences from taxa not represented in the reference database. Our work will help researchers design and evaluate classification databases and conduct metabarcoding on arthropods and alternate taxa.

Honeydew produced from the excretion of plant-sucking insects (order Hemiptera) is a carbohydrate-rich material that is foraged by honey bees to integrate their diets. In this study, we used DNA extracted from honey as a source of environmental DNA to disclose its entomological signature determined by honeydew producing Hemiptera that was recovered not only from honeydew honey but also from blossom honey. We designed PCR primers that amplified a fragment of mitochondrial cytochrome c oxidase subunit 1 (COI) gene of Hemiptera species using DNA isolated from unifloral, polyfloral and honeydew honeys. Ion Torrent next generation sequencing metabarcoding data analysis assigned Hemiptera species using a customized bioinformatic pipeline. The forest honeydew honeys reported the presence of high abundance of Cinara pectinatae DNA, confirming their silver fir forest origin. In all other honeys, most of the sequenced reads were from the planthopper Metcalfa pruinosa for which it was possible to evaluate the frequency of different mitotypes. Aphids of other species were identified from honeys of different geographical and botanical origins. This unique entomological signature derived by environmental DNA contained in honey opens new applications for honey authentication and to disclose and monitor the ecology of plant-sucking insects in agricultural and forest landscapes.

Introduced species of mammals in New Zealand have had catastrophic effects on populations of diverse native species. Quantifying the diets of these omnivorous and predatory species is critical for understanding which native species are most impacted, and to prioritize which mammal species and locations should be targeted with control programmes. A variety of methods have been applied to quantify diet components in animals, including visual inspection of gut contents (Daniel 1973; Pierce and Boyle 1991), stable isotope analysis (Major et al. 2007; Carreon-Martinez and Heath 2010), and time-lapse video (Brown and Brown 1997; Dunlap and Pawlik 1996). Increasingly, DNA-based metabarcoding methods are being used (King et al. 2008; Soininen et al. 2009). These metabarcoding methods require a PCR step using primers that bind to highly conserved genomic regions (e.g. mitochondrial COI) to amplify specific regions for sequencing. This step introduces significant bias, primarily due to the lack of a universal primer set (King et al. 2008). Here we show that direct metagenomic sequencing using the Oxford Nanopore Minion allows rapid quantification of rat diets. Using a sample of rats collected from within 100km of Auckland, NZ, we show that these rats consume a wide variety of plant, invertebrate, vertebrate, and fungal taxa, with substantial differences in diet content between locales. We then show that, based on diet content alone, it is possible to pinpoint the sampling location of an individual rat within tens of kilometres. We expect that the rapidly increasing accuracy and throughput of nanopore-based sequencing, as well as increases in the species diversity of genomic databases, will soon allow rapid and unbiased assessments of animal diets in field settings.

Thursday, July 5, 2018

One decade of ZooKeys

One decade of ZooKeys - not bad at all. That means one decade of Open Access Taxonomy. Descriptions that are not hiding behind a paywall, a no-brainer if you ask me. How can we talk about democratization and equal access to information if a large part of the primary literature is still hidden to a substantial group of researchers simply because they or their institution can't afford a subscription. Especially for taxonomy that is pretty aggravating. So, my best wishes and congratulations to Pensoft (in particular Lubo) for this success story. 

The Pensoft blog has some more details. Here the first part:

So here we are, 10 years from that very first issue of ours published on a very special date – the 4th of July – and the result of a seemingly ordinary breakfast conversation between two respected entomologists, Prof Lyubomir Penev and Dr Terry Erwin, during the Entomological Society of America meeting in San Diego, USA, seven months earlier.

Then and there, under the California sun, an idea about a brand new taxonomic journal meant to revolutionise the scholarly publishing in zoology – in terms of both openness and technological innovation, was born. The rest, like they say, is history.

Ten years in, we stand as the most prolific open-access journal in zoology with a total of 4,103 published articles, 45 newly described animal families, 650 genera and 8977 species, authored by a total of 5,720 researchers coming from 131 different countries. We also take pride in having set an excellent example for the rest of the academic titles in Pensoft’s already extensive portfolio of open access journals.

Wednesday, July 4, 2018

Tour of Flanders video footage shows climate change impact on trees

Predicting how the timing of cyclic life history events, such as leafing and flowering, respond to climate change is of paramount importance due to the cascading impacts of vegetation phenology on species and ecosystem fitness. However, progress of this field is hampered by the relative scarcity, and geographic and phylogenetic bias, of longterm phenology datasets.

By analyzing nearly four decades of archive footage from the cycling Tour of Flanders, researchers from Ghent University have been able to detect climate change impacts on trees. Focusing on trees and shrubs growing around recognisable climbs and other landmarks along the route of this major annual road cycling race in Belgium, the colleagues looked at video footage from 1981 to 2016 obtained by a Flemish broadcaster. They visually estimated how many leaves and flowers were present on the day of the course (usually in early April) and linked their scores to climate data. 

They found that the trees had advanced the timing of leafing and flowering in response to recent temperature changes. Before 1990, almost no trees had grown leaves at the time of the spring race. After that year, more and more trees visible in the television footage, in particular magnolia, hawthorn, hornbeam and birch trees, were already in full leaf. These shifts were most strongly related to warmer average temperatures in the area, which have increased by 1.5°C since 1980.

Early-leafing trees can be good news for some species as they grow faster and produce more wood. However, their leaves also cast shadows. When trees flush earlier in the year, they shadow for a longer period of time, affecting other animals and plants, and even whole ecosystems. Some of the flowers growing under these trees may not be able to receive enough sunlight to bloom. As a result, insects can go without nectar and may struggle to find enough spots to sunbathe.

Phenology (the study of natural phenomena that recur periodically such as leafing and flowering) is mostly based on long-term observations and repeat photography, with data often being biased towards common species or geographical regions. In this study, archive footage allowed the researchers to use previously unexploited records of twelve tree species in the Flanders region in order to build long-term datasets of phenological responses.

Our method could also be used to collect data on other aspects important for ecological or evolutionary research, such as tree health, water levels in rivers and lakes, and the spread of invasive species. Only by compiling data from the past will we be able to predict the future effects of climate change on species and ecosystems.

Tuesday, July 3, 2018

Scale matters

Setophaga discolor - Credit: Julie Hart
Biodiversity is changing all around us and worldwide. Local species disappear and sometimes other species invade. Studying birds in the U.S. and worldwide, we show that patterns and implications of this ongoing change vary strongly with the scale.

A minor loss or gain of species richness or functional diversity at the local or county level can look like a major gain at the state or national level, and yet be a net loss when viewed at a global scale. Researchers at Yale University studied 50 years of data about nesting birds in North America and tracked biodiversity changes at different scales. They found significant differences in how much change had occurred, based upon how wide a geographic net they cast. In addition taxonomic diversity and functional diversity increased over all but the global scale. The larger the scale change in taxonomic diversity was higher than in functional diversity which suggests strong trait redundancy at those scales. Also, insectivorous birds (like the prairie warbler in the photo) showed the most drastic declines across all geographic scales, from local to continental.

Any reporting and interpretation of biodiversity change thus needs scale as a key qualifier. Better yet, researchers and practitioners of biodiversity science should adopt a multi-scale framework and consider all geographic scales simultaneously.

Friday, June 29, 2018

Weekend reads

Long weekend for all the Canadians which means more time to read and certainly no post on Monday.

Knowledge of community structure within an ecosystem is essential when trying to understand the function and importance of the system and when making related management decisions. Within the larger ecosystem, microhabitats play an important role by providing inhabitants with a subset of available resources. On coral reefs, cryptobenthic fishes encompass many groups and make up an important proportion of the biodiversity. However, these fishes are relatively small, exhibit extreme visual or behavioral camouflage, and, therefore, are often overlooked. We examined the differences in fish community structure between three common reef microhabitats (live hard coral, dead coral rubble, and sand) using ichthyocide stations in the central Red Sea. Using a combination of morphological and genetic (cytochrome oxidase I (COI) barcoding) techniques, we identified 326 individuals representing 73 species spread across 17 families, from fifteen 1 m2 quadrats. Fish assemblages in the three microhabitats were significantly different from each other. Rubble microhabitats yielded the highest levels of fish abundance, richness, and diversity, followed by hard coral, and then sand. The results show that benthic composition, even at a small scale, influences cryptobenthic communities. This study also provides new COI sequence data to public databases, in order to further the research of cryptobenthic fishes in the Red Sea region.

The cytochrome c oxidase subunit I (cox1) gene is the main mitochondrial molecular marker playing a pivotal role in phylogenetic research and is a crucial barcode sequence. Folmer's "universal" primers designed to amplify this gene in metazoan invertebrates allowed quick and easy barcode and phylogenetic analysis. On the other hand, the increase in the number of studies on barcoding leads to more frequent publishing of incorrect sequences, due to amplification of non-target taxa, and insufficient analysis of the obtained sequences. Consequently, some sequences deposited in genetic databases are incorrectly described as obtained from invertebrates, while being in fact bacterial sequences. In our study, in which we used Folmer's primers to amplify COI sequences of the crustacean fairy shrimp Branchipus schaefferi (Fischer 1834), we also obtained COI sequences of microbial contaminants from Aeromonas sp. However, when we searched the GenBank database for sequences closely matching these contaminations we found entries described as representatives of Gastrotricha and Mollusca. When these entries were compared with other sequences bearing the same names in the database, the genetic distance between the incorrect and correct sequences amplified from the same species was c.a. 65%. Although the responsibility for the correct molecular identification of species rests on researchers, the errors found in already published sequences data have not been re-evaluated so far. On the basis of the standard sampling technique we have estimated with 95% probability that the chances of finding incorrectly described metazoan sequences in the GenBank depend on the systematic group, and variety from less than 1% (Mollusca and Arthropoda) up to 6.9% (Gastrotricha). Consequently, the increasing popularity of DNA barcoding and metabarcoding analysis may lead to overestimation of species diversity. Finally, the study also discusses the sources of the problems with amplification of non-target sequences.

DNA metabarcoding is increasingly used in dietary studies to estimate diversity, composition, and frequency of occurrence of prey items. However, few studies have assessed how technical and biological replication affect the accuracy of diet estimates. This study addresses these issues using the European free-tailed bat Tadarida teniotis, involving high-throughput sequencing of a small fragment of the COI gene in 15 separate faecal pellets and a 15-pellet pool per each of 20 bats. We investigated how diet descriptors were affected by variability among (i) individuals, (ii) pellets of each individual, and (iii) PCRs of each pellet. In addition, we investigated the impact of (iv) analysing separate pellets versus pellet pools. We found that diet diversity estimates increased steadily with the number of pellets analysed per individual, with seven pellets required to detect ~80% of prey species. Most variation in diet composition was associated with differences among individual bats, followed by pellets per individual, and PCRs per pellet. The accuracy of frequency of occurrence estimates increased with the number of pellets analysed per bat, with the highest error rates recorded for prey consumed infrequently by many individuals. Pools provided poor estimates of diet diversity and frequency of occurrence, which were comparable to analysing a single pellet per individual, and consistently missed the less common prey items. Overall, our results stress that maximizing biological replication is critical in dietary metabarcoding studies, and emphasize that analysing several samples per individual rather than pooled samples produce more accurate results.

DNA metabarcoding is a technique used to survey biodiversity in many ecological settings, but there are doubts about whether it can provide quantitative results, i.e. the proportions of each species in the mixture as opposed to a species list. While there are several experimental studies that report quantitative metabarcoding results, there are a similar number that fail to do so. Here we provide the rationale to understand under what circumstances the technique can be quantitative. Basically, we simulate a mixture of DNA of S species with a defined initial abundance distribution. In the simulated PCR, each species increases its concentration following a certain amplification efficiency. The final DNA concentration will reflect the initial one when the efficiency is similar for all species; otherwise, the initial and final DNA concentrations would be poorly related. Although there are many known factors that modulate amplification efficiency, we focused on the number of primer-template mismatches, arguably the most important one. We used 15 common primers pairs targeting the mitochondrial COI region and the mitogenomes of ca. 1200 insect species. The results showed that some primers pairs produced quantitative results under most circumstances, whereas some other primers failed to do so. Many species, and a high diversity within the mixture, helped the metabarcoding to be quantitative. In conclusion, depending on the primer pair used in the PCR amplification and on the characteristics of the mixture analysed (i.e., high species richness, low evenness), DNA metabarcoding can provide a quantitative estimate of the relative abundances of different species.

Marine meiofauna comprises up to 22 phyla. Its morphological identification requires time and taxonomists' expertise, and molecular tools can make this task faster. We aim to disentangle meiofaunal diversity patterns at Araçá Bay by applying a model selection approach and estimating the effectiveness of metabarcoding (18S rDNA) and morphological methods for estimating the response of meiofauna diversity in small-scale interactions with environmental variables. A rarefaction curve indicated that ten samples were sufficient for estimating the total number of meiofauna OTUs in a tidal flat. In both approaches, richness was predicted by mean sand percentage, sediment sorting, and bacteria concentration. Nematode genera composition differed significantly between approaches, the result of taxonomic mismatch in the genetic database. The similarity between the model selected for diversity descriptors, the richness of nematode genera and meiofauna composition emphasized the utility of predictive models for metabarcoding estimates to detect small-scale interactions of these organisms.

Background: In light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community, but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies). Results: Using a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates. Conclusions: Sequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost effective, portable and universal approach for eukaryote DNA barcoding. Long rDNA amplicons scale up DNA barcoding by enabling the accurate recovery of taxonomic and phylogenetic diversity. However, bulk community analyses using long-read approaches may introduce biases and will require further exploration.

Background. Knowledge on the globally outstanding Amazonian biodiversity and its environmental determinants stems almost exclusively from aboveground organisms, notably plants. In contrast, the environmental factors and habitat preferences that drive diversity patterns for micro-organisms in the ground remain elusive, despite the fact that micro-organisms constitute the overwhelming majority of life forms in any given location, in terms of both diversity and abundance. Here we address how the diversity and community turnover of operational taxonomic units (OTU) of micro-organisms in soil and litter respond to soil physicochemical properties; whether OTU diversities and community composition in soil and litter are correlated with each other; and whether they respond in a similar way to soil properties. Methods. We used recently inferred OTUs from high-throughput metabarcoding of the 16S (prokaryotes) and 18S (eukaryotes) genes to estimate OTU diversity (OTU richness and effective number of OTUs) and community composition for prokaryotes and eukaryotes in soil and litter across four localities in Brazilian Amazonia. All analyses were run separately for prokaryote and eukaryote OTUs, and for each group using both presence-absence and abundance data. Combining these with novel data on soil chemical and physical properties, we identify abiotic correlates of soil and litter micro-organism diversity and community structure using regression, ordination, and variance partitioning analysis. Results. Soil organic carbon content was the strongest factor explaining OTU diversity (negative correlation) and pH was the strongest factor explaining turnover for prokaryotes and eukaryotes in both soil and litter. We found significant effects also for other soil variables, including both chemical and physical properties. The correlation between OTU diversity in litter and in soil was non-significant for eukaryotes and weak for prokaryotes, suggesting that diversity of in one substrate should not be used as a proxy for diversity in the other. The community compositions of both prokaryotes and eukaryotes were more separated for habitat type than for substrate (soil and litter). Discussion. In spite of the limited sampling (four localities, 39 plots), our results provide a broad-scale view of the physical and chemical correlations of soil and litter biodiversity in a longitudinal transect across the world’s largest rainforest. Our methods help to understand links between soil properties, OTU diversity patterns, and community composition and turnover. The lack of strong correlation between OTU diversity in litter and in soil suggests independence of diversity drives of these substrates and highlights the importance of including both measures in biodiversity assessments. Massive sequencing of soil and litter samples holds the potential to complement traditional biological inventories in advancing our understanding of the factors affecting tropical diversity.

Constructing networks has become an indispensable approach in understanding how different taxa interact. However, methodologies vary widely among studies, potentially limiting our ability to meaningfully compare results. In particular, how network architecture is influenced by the extent to which nodes are resolved to either taxa or taxonomic units is poorly understood. To address this, here we collate nine datasets of ecological interactions, from both observations and DNA metabarcoding, and construct networks under a range of commonly-used node resolutions. We demonstrate that small changes in node resolution can cause wide variation in almost all key metric values, including robustness and nestedness. Moreover, relative values of metrics such as robustness were seen to fluctuate continuously with node resolution, thereby potentially confounding comparisons of networks, as well as interpretations concerning their constituent ecological interactions. These findings highlight the need for care when comparing networks, especially where these differ with respect to node resolution.




Tuesday, June 26, 2018

Three opportunities to learn about metabarcoding

In case you are looking for ways to learn about metabarcoding, there are actually three different courses offered this year. All of them differ in approach, content focus, and venue but what they have in common is the fact that they provide participants with a comprehensive package that enables them make informed decisions when it comes to organizing experiments, field work and analytics.

This program will provide an overview of the state of current technology and the various platforms used. The course consists of a series of online lectures and research exercises introducing different aspects of metabarcoding and environmental DNA research. We will also touch on the suite of bioinformatics tools available for sequence analysis and data interpretation.

This course will focus on eDNA metabarcoding, however targeted single species detection and other alternatives will also be explored, as they can sometimes be suitable metabarcoding alternatives.

The lectures will cover different aspects of DNA metabarcoding. The bioinformatics practicals will introduce data analysis from raw sequences to basic ecological conclusions. The molecular ecology practical will present basic techniques for DNA extraction in the field and DNA amplification by PCR.

Monday, June 25, 2018

DNA barcoding for pollen forecasting

PollerGEN is a group of interdisciplinary researchers funded by NERC to understand grass pollen deposition. We aim to revolutionise the way that pollen is measured, model spatial and temporal deposition from different species of grass pollen and identify linkages to human health.

DNA barcoding of pollen is not a new invention. It is not easy either but has been shown to provide extremely valuable information, e.g. for understanding plant-pollinator interactionshoney bee foraging, or the characterization of honeybee pollen pellets. It should come to no surprise that researchers are also working on an application that intends to improve a forecasting system that has become more and more important for a large portion of the human population - pollen forecasting for hay fever and other allergic reactions.

At this point most forecasts are build using data from a network of pollen traps which operate throughout the main pollen seasons. These traps measure how many pollen grains are present on a daily basis and identifications of species are done using morphology-based methods. The latter is extremely challenging when it comes to species with very uniform appearance, e.g. grasses. However, the species identity often makes a big difference. It is fairly rare that somebody is allergic to all grass pollen but we are having difficulties to tell which pollen in the mix is the culprit.

PollerGen, a project run out of Bangor University wants to change this by using a DNA-based approach. 

The colleagues are now working on a way to detect airborne pollen from different species of allergenic grass. We’re also developing new pollen source maps, and modelling how pollen grains likely move across landscapes, as well as identifying which species are linked with the exacerbation of asthma and hay fever.

We’re going to be using a new UK plant DNA barcode library, as well as environmental genomic technologies to identify complex mixtures of tree and grass pollens from a molecular genetic perspective. By combining this information with detailed source maps and aerobiological modelling, we hope to redefine how pollen forecasts are measured and reported in the future.

We have just started the third year of pollen collection and hope to road test the combined forecasting methods over the next year. In the long run, our vision is to be able to provide specific pollen forecasts for grass, and unravel which species of grass pollen are most likely causing allergic responses. More broadly, we also want to provide information to healthcare professionals and charities, who can translate this information to help pollen allergy sufferers live healthier and more productive lives.

Pretty cool.