Barcode Conference 2019 - Abstract submission open

Just received this:

Dear friends and colleagues,

It is a pleasure to announce that abstract submission for the upcoming Barcode of Life Conference now is open. For guidelines and access to the submission portal, please visit the conference website:  Abstract submission deadline is January 15, 2019

Please consider registering for the conference as soon as possible (space is limited). More information on how to register here.

We are eager to spread news about the conference to as many potential participants as possible. I would appreciate it if you forward this message in your network, and maybe point to the list of interesting session themes and outstanding plenary speakers.

Thank you and best wishes
Torbjørn

Global Taxonomy Initiative Forum

The Global Taxonomy Initiative Forum will take place on 16 November immediately before the opening day of the UN Conference on Biodiversity in Egypt. 

The provisional agenda is accessible at https://www.cbd.int/conferences/2018/parallel-meetings/The-Global-Taxonomy-Initiative-Forum and the notification to Parties and observers on this event can be found at https://www.cbd.int/doc/notifications/2018/ntf-2018-090-gti-en.pdf

The Forum will be captured and shared via video streaming on the CBD Facebook and other social media. It will feature several talks on DNA barcoding and taxonomy capacity building.

Professor/Associate Professor in Zoology (marine invertebrates) at Tromsø University Museum

Tromsø University Museum, UiT – The Arctic University of Norway, has a permanent position vacant as Professor/Associate professor in Zoology (marine invertebrates). The position is attached to the Department of Natural Sciences.

Further information about the position is available by contacting the Head of Department Torbjørn Alm or Museum Director Lena Aarekol.

The position’s affiliation

Tromsø University Museum has two academic departments, the Department of Cultural Sciences and the Department of Natural Sciences, with the additional units Polar Museum, MS Polstjerna and Tromsø Arctic-Alpine Botanic Garden. This position is at the Department of Natural Sciences, which is responsible for developing and maintaining scientific collections of natural objects (animals, plants, fossils and minerals) as well as public outreach, including the Tromsø Arctic-Alpine Botanical Garden. The Department has a permanent staff of 16, of which eight are in academic positions. At the moment, five PhD students, one post doc and two researchers are affiliated with the Department.

The Department is involved in research within the fields of ancient DNA, barcoding, taxonomy, phylogeography, ecology, palaeontology and biodiversity. The main focus is on northern Norway and the Arctic. The Department has well-established laboratory facilities for modern and ancient molecular biology, palynology, and palaeontology. For more information about the Tromsø University Museum’s activities, visit: https://en.uit.no/tmu .

The position’s field of research/field of work

Tromsø University Museum has a long tradition in research on various marine invertebrates. The successful applicant for the position is expected to carry out research in any taxonomic group of marine invertebrates. Relevant groups are those living in our preferred target region – along the coast of northern Norway and the Arctic. Applicants may focus their research in one or more of the fields biosystematics, biogeography, biodiversity, ecology and molecular genetics.

The position’s duties include research and research-based activities, collection management, dissemination and administration. We also encourage the teaching and supervision of Master and PhD students. This position will include responsibility for the marine invertebrate collections, both within and outside her/his own research field, and curation of other collections within zoology is expected.

The appointee is expected to participate actively in a broad range of museum outreach activities, such as public communications, media interactions and exhibitions. The appointee is furthermore expected to collaborate and seek collaboration within the institution as well as nationally and internationally. She/he is expected to be active in applying for external research funding.

Qualification requirements

The successful candidate is internationally recognized within the field, with scientific merits beyond doctoral level. Research experience within biosystematics/taxonomy of a relevant taxonomic group is required. Experience in museum work, especially scientific collections, is a further asset. A successful track record in applying for external funding and/or a high potential for external funding will be given emphasis when evaluating applicants. Personal suitability will also be evaluated.

For a position as associate professor, you should have a good publication record in terms of papers in peer-reviewed journals and other relevant international publication channels. Documented external funding, experience with research leadership and relevant collaboration with industry will be rated positively. As an associate professor, we expect you to aim at developing yourself further to a full professor.

For a position as professor, you should demonstrate international experience and have a strong publication record in terms of papers in peer-reviewed journals and other relevant international publication channels. You should document the ability to obtain external funding from relevant sources, and be able to initiate and lead research at a high international level. We will also assess outreach, network and teaching and supervision activities.

Applicants should submit a covering letter describing their interest in the position, including, but not restricted to, experience with natural history collections, the development of the zoological collection and future research plans.

The applicants must be able to document teaching qualifications in the form of university-level teaching seminars, other teaching education or through having developed a teaching portfolio. Alternatively, after carrying out an assessment of the applicant’s practical teaching skills, the committee may determine that this may be regarded as of equal value to formal teaching qualifications. For further information about requirements for teaching qualifications, refer to the website about Teaching Portfolios.

Applicants should have a good command of one of the Scandinavian languages and English. Applicants who do not have a good command of a Scandinavian language must be willing to learn Norwegian within a reasonable time period.

Working conditions

At UiT The Arctic University of Norway the allocation of working hours shall be flexible and allocated on a case by case basis.

As a general principle, every Professor and Associate Professor shall spend an equal amount of time on teaching and research and development work, after time spent on other duties has been deducted. As a norm the time resources spent on administrative duties constitutes 5 % for academic staff in this category of position.

Employees in permanent positions as Associate Professor have the right to apply for a paid sabbatical (research and development), cf. Guidelines for the allocation of R&D sabbatical (Only in Norwegian)

Moreover, applicants shall refer to the Guidelines for allocation of working hours in teaching and research positions (Only in Norwegian)

The successful applicant must be willing to engage himself/herself in the ongoing development of his/her discipline and the university as a whole.

The remuneration

The remuneration of a Professor is in accordance with the State salary scale code 1013 and for an Associate professor code 1011. A compulsory contribution of 2 % to the Norwegian Public Service Pension Fund will be deducted.

Assessment

An expert committee will assess the applicants. The applicants who are assessed as the best qualified, will be called to an interview. The interview shall among other things aim to clarify the applicant’s personal suitability for the position. A trial lecture may also be held.

For the professor position, it is particularly important that the applicant can document academic activity at a high level over the previous six years in a way that points towards to the continuation of such activity in the future.

Applicants bring original certificates/diplomas and reference letter (work) to a interview.

Interim appointment 

If there are no applicants who are clearly qualified for permanent appointment, a temporary appointment for a period of no more than three years may be made, cf. statsansatteloven § 9 and Section 6-5 (1) of the Act relating to universities and university colleges. A permanent appointment shall be made upon application before the three-year period elapses on the basis of a new evaluation. The appointment will become permanent if the applicant is found to be suitably qualified.

In the event of an interim appointment on the basis of lacking teaching qualifications, the applicant must document teaching qualifications through developing a teaching portfolio before the three-year period elapses. The appointment will become permanent in the event that the applicant is found to be suitably qualified.

Gender balance 

Women are encouraged to apply. UiT The Arctic University of Norway wishes to increase the proportion of females in senior research positions. In the event that two or more applicants are found to be approximately equally qualified, female applicants will be given priority.

Submit your application in www.jobbnorge.no

The application documents (mentioned below) must be enclosed in Jobbnorge within the expiration of the application deadline. Documents to be considered must be certified and translated into Scandinavian or English.

The application must include:

• Letter of application
• CV (containing a complete overview of education, supervised professional training and professional work)
• Certified copies of diplomas and reference letter
• References with contact information
• Documentation of English language proficiency
• Teaching portfolio
• Form for documentation of teaching qualifications (if you do not have a teaching portfolio)
• List of works and description of these

The list of works shall contain the following information:

• author(s), the work’s title
• for articles: the journal’s name and volume, the first and last page of the article, year of publication
• for publications: publisher, printer, year of publication, number of pages

Works- The applicant have to submit up to 10 works that are central to his/her production. Doctoral thesis is in this context regarded as one work.

In addition, the applicant shall provide a description of his/her scientific production stating which works he/she considers the most important and shall therefore be the main emphasis of the assessment. A brief description of the other listed works shall also be included to demonstrate depth of production. These descriptions shall be an attachment to the application.

My metabarcoding online course starts soon!

We're going into another round of the Metabarcoding Course I developed a few years back. I just finished updating its modules especially given how fast the field develops.

Date: November 12, 2018 to December 09, 2018

This 4-week, web-based course will provide an overview of the state of current technology and the various platforms used. The course consists of a series of online lectures, readings, videos, 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.

We tried to cover as much as possible given the online format and the limited time participants usually have available to do such training. The course is designed for four weekly instructional hours. 

If you are interested and sign up quickly (by end of the week) you would be eligible for a $100 early bird discount :-)

Special discount $100 (code: DNA2018)

From the inbox: Postdoctoral Researcher and PhD Positions at University of Oulu

The University of Oulu is an international scientific community, with 14,000 students and approximately 3,000 employees. The strengths of the University are wide multidisciplinary study/research interests and modern research and study environment, as well as good cooperation with international educational and research institutes. More information.

The following full-time jobs are open in the Insect Genomic Systematics Group at the University of Oulu, Finland:

1.     Postdoctoral Researcher

2.     PhD student

Project description: Taxonomy, and its central concept ‘species’ in particular, underpin all biological research and are important in numerous other ways for humanity. Despite its crucial role, taxonomy has long been suffering from a complication termed the ‘taxonomic impediment’. At the core of this impediment has been our inability to efficiently document the vast biodiversity of Earth with the available resources and methods. At the same time, the majority of Earth’s biodiversity awaits discovery. Emergent new and powerful methods in DNA sequencing technology have enabled the acquisition of breathtaking amounts of genomic data from organisms in straightforward and fast ways. In this project, genomic methods, particularly anchored hybrid enrichment (AHE), will be applied to develop novel approaches to define species boundaries under different evolutionary circumstances and with taxonomically challenging groups of species. The aim is to point the way ahead towards an entirely DNA-based taxonomy, and fostering the integration of taxonomic principles and practices into the genomic era. The primary model taxa will be Lepidoptera (butterflies and moths) and, to a lesser extent, Symphyta (sawflies). Sympatric, allopatric, parapatric, and asexual taxa will be considered, because the geographic relationship between species and mode of reproduction have great theoretical and practical effects on species delimitation. Influence of sexual pheromones, chromosomal re-arrangements, ecological differentiation and gene flow on species delimitation will also be considered. The research will be conducted with extensive national and international collaboration. The project is financed by Academy of Finland. Principal Investigator of the project is Marko Mutanen (University of Oulu, Finland).

Duties: The postdoctoral researcher will participate in material collection (incl. fieldwork), laboratory analyses, data analyses (incl. bioinformatics), manuscript preparation and supervision of the PhD student. She/he is also expected to teach at the department at most 5 % of working time. While studies will be conducted in a team, capability for independent working is expected. The PhD student will participate in 3 – 4 separate research studies. She/he will participate and is expected to gain good skills in fieldwork, laboratory analyses, data analyses, bioinformatics and scientific writing. She/he will be a leading author in at least two research papers. She/he will be supervised by the team leader and other senior core team members.

Qualification and selection criteria: The postdoctoral researcher must have a PhD in evolutionary biology, ecology, genetics or related area. The PhD degree must have been reached before the start of the contract. The postdoctoral researcher is expected to be highly motivated, committed to the project, and to have excellent English communication (writing and speaking) skills. Previous experience and knowledge of the study organisms, taxonomy, systematics, phylogenetics, genomic methods or bioinformatics is considered as an advantage. The PhD student must reach the MSc degree in biology (or similar) before the start of the contract. She/he is highly motivated and committed to finish her/his PhD studies during the funding period, and has good English communication skills. All above-mentioned experience and relevant skills are considered as an advantage.

Duration and terms of employment: The postdoctoral researcher: The earliest possible starting date is 1st of January, 2019. Optimally, the position is filled not later than 1st of March, 2019. The duration of the contract is 3 years (a short continuation to this is possible). The salary will be based on the levels 5 – 6 of the demand level chart for teaching and research staff of Finnish universities. In addition, a salary component based on personal work performance will be paid (maximum of 46.3 % of the job-specific component). The gross salary will be 3,100 – 3,600 euros/month. The PhD student will start earliest on 1st of January 2019, and not later than 1st of March 2019. The maximal duration is 3 years and 8 months. The last possible end date of the contract is 31st August 2022, so the delayed start will shorten the funding period. The salary will be based on the levels 2 – 4 of the demand level chart for teaching and research staff of Finnish universities. In addition, a salary component based on personal work performance will be paid (maximum of 46.3 % of the job-specific component). The gross salary will be about 2,300 – 2,500 euros/month. The six-month trial periods are applied in the beginning of employments. Healthcare is provided by the employer.

Inquiries: For further information, please contact Marko Mutanen (phone: +358 40 824 6749).

How to apply: Submit your application online latest on Thursday 1st of November 2018 at 24:00 (EET). The application must be submitted using the electronic application form with the following documents:

(1)   Motivation letter where the reasons for interest in this position are clearly indicated (max. 1 page). In the motivation letter, indicate clearly which position (PhD student or postdoctoral researcher) you apply.

(2)   CV formatted according to the guidelines of the Finnish National Board on Research Integrity (TENK)

(3)   List of publications formatted according to the guidelines of the Academy of Finland 

(4)   Contact information of at least two referees (recommended but not mandatory) and copy of the MSc or PhD degree as applicable.

Heineken Prize for Paul Hebert

The Royal Netherlands Academy of Arts and Sciences has awarded the 2018 Dr A.H. Heineken Prize for Environmental Sciences to Paul Hebert. Here the official announcement text plus a nice little video:

A catalogue for the library of life

Paul Hebert is receiving the Dr A.H. Heineken Prize for Environmental Sciences for his pivotal contribution to developing a genetic barcode capable of classifying every biological species on Earth. 

Paul Hebert is known as the 'father of DNA barcoding', a taxonomic method that uses a short section of DNA from a standardised region of the genome to identify different species, in the same way a supermarket scanner uses barcodes to identify purchases. 

Hebert first raised the possibility of such a method in 2000. He is now the Scientific Director of the International Barcode of Life Project (iBOL), in which researchers from 25 countries are attempting to assign barcodes to millions of species on Earth. 

The project has demonstrated that DNA barcoding can speed the discovery of new species and distinguish between separate species that used to be classified as one. Thanks to DNA barcoding, we now have a more precise way of measuring the number of species that inhabit a specific ecosystem and we can analyse complex food chains with much greater accuracy. 

The Barcode of Life Project is generating an impressive stream of data. Hebert’s research group is building digital systems to gather, store and analyse all this information and make it available to the community. The database now contains the genetic codes of approximately 600,000 species and is being used by almost 25,000 researchers worldwide. 

New methods are bringing the ultimate goal of the project ever closer: a comprehensive inventory of global biodiversity. If the project succeeds, its legacy will yield lasting benefits to humankind.

Yesterday was the official ceremony at the Royal Academy of Arts and Science of the Netherlands - time to congratulate Paul once again for receiving such a prestigious prize. Well deserved!

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

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 

Science and Technology refers to the Ministerial Order on the Appointment of Academic Staff at Danish Universities under the Danish Ministry of Science, Technology and Innovation. 

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.

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:

Finding the pond through the weeds: eDNA reveals underestimated diversity of pondweeds.

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.

Choice of DNA extraction method affects DNA metabarcoding of unsorted invertebrate bulk samples

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.

NGS barcoding reveals high resistance of a hyperdiverse chironomid (Diptera) swamp fauna against invasion from adjacent freshwater reservoirs

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.

A DNA metabarcoding protocol for hyporheic freshwater meiofauna: Evaluating highly degenerate COI primers and replication strategy

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.

Why the COI barcode should be the community DNA metabarcode for the Metazoa.

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.

DNA metabarcoding reveals changes in the contents of carnivorous plants along an elevation gradient

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. 

Long-read DNA metabarcoding of ribosomal RNA in the analysis of fungi from aquatic environments

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.

PIPEBAR and OverlapPER: tools for a fast and accurate DNA barcoding analysis and paired-end assembly

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

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.

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.

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.

Weekend reads

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

Tree species identity and diversity drive fungal richness and community composition along an elevational gradient in a Mediterranean ecosystem

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.

Insect diversity in the Saharo-Arabian region: Revealing a little-studied fauna by DNA barcoding

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.

DNA metabarcoding uncovers fungal diversity of mixed airborne samples in Italy

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.

DNA Barcoding in Forensic Entomology - Establishing a DNA Reference Library of Potentially Forensic Relevant Arthropod Species

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.

Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

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.

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.

(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.

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

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.

BBI: an R package for the computation of Benthic Biotic Indices from composition data

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.

Testing clustering strategies for metabarcoding-based investigation of community-environment interactions

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.

Ecological influence of sediment bypass tunnels on macroinvertebrates in dam-fragmented rivers by DNA metabarcoding

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.

A reference cytochrome c oxidase subunit I database curated for hierarchical classification of arthropod 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.

Entomological signatures in honey: an environmental DNA metabarcoding approach can disclose information on plant-sucking insects in agricultural and forest landscapes

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.

New tools for diet analyses: nanopore sequencing of metagenomic DNA from stomach contents to quantify diet in an invasive population of rats

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.