dnaqua-net kick-off conference digital abstract...

DNAqua-Net Kick-Off Conference

Digital Abstract Book (EU COST Action CA15219)

Thanks to our sponsors!

Dear Conference Participants,

A warm welcome to the DNAqua-Net COST Action Kick-Off conference in Essen. We all are

aware of the increasing pressures on aquatic ecosystems. Several national and international

legal frameworks have shifted the focus on this problem and started to systematically assess

and monitor the quality of aquatic ecosystems. The process, however, focuses on only few

biological groups, is time-consuming and often limited in resolution. For many countries,

detailed species lists are even missing for most of the biota thus limiting the approach

substantially. Novel molecular tools can act as a game changer here. “OMICS- techniques”

such as metabarcoding or metagenomics have become increasingly powerful and cheaper over

the last 10 years and responses of biota to environmental stressors can be assessed and

monitored from micro- to macroorganisms. The potential of these innovative techniques,

however, is not used in large-scale assessment programs yet because of a lack of technical

agreement or conceptual issues. The aim of DNAqua-Net is to explore the potential of novel

DNA-based techniques and provide good practice solutions for their application in such

biomonitoring programs. In this conference and the first work group meetings directly after

the conference we will enter the debate and open the dialogue between academia, industry and

stakeholders from countries all around the world to find solutions on how to incorporate these

techniques into real-world biomonitoring programs.

Wishing you a great conference and good discussions,

(Florian Leese) (Agnès Bouchez) (Alexander M. Weigand)

Agenda overview

Monday – 6.3.17 Tuesday – 7.3.17 Wednesday – 8.3.17 Thursday – 9.3.17

MC2 meeting room S04 T01 A02 (Audimax building)

Conference room S04 T01 A01 (in Audimax)

Conference room S04 T01 A01 (in Audimax)

WG meetings various rooms, see daily details (in T02 R02/03, above Mensa)

am

coffee coffee coffee

09.00 - 09.20 Conference Opening

09.20 - 10.00 WG5 – Plenary 10.00 - 10.30 WG5 Session 10.30 - 11.15 coffee break

11.15 - 12.15 WG5 Session

09.30 - 10.10 WG3+4 – Plenary 10.10 - 10.40 WG3+4 Session 10.40 - 11.30 coffee break

11.30 - 12.30 WG3+4 Session

09.00 - 13.00

Morning Session WG1, 3, 5 (in parallel)

coffee

12.15 - 13.30

Lunch break 12.30 - 14.00

Lunch break 13.00 - 14.00

Lunch break

pm 14.00 - 18.00

MC2 meeting

13.30 - 14.10 WG2 – Plenary 14.10 - 15.25 WG2 Session 15.25 - 15.50 coffee break

15.50 - 16.30 WG1 – Plenary

16.30 - 18.00 WG1 Session

14.00 - 14.40 WG3+4 – Plenary

14.40 - 15.40 WG3+4 Session 15.40 - 16.30 coffee break 16.30 - 17.45 Open Session 17.45 - 18.00 Conference Closing

14.00 - 18.00

Afternoon Session WG2, 4 (in parallel)

19.00 -

conference dinner

Tuesday March 7th 2017

Time Speaker(s) Title

08:00 – Registration and Coffee

09:00 – 09:20 Florian Leese Conference Opening

Session WG5: DNA-based biomonitoring – Implementation Strategy & Legal issues Chairs: Patricia Mergen & Daniel Hering

09:20 – 10:00 Donald Baird - Mainstreaming metabarcoding in regulatory monitoring of ecosystems: mountaineering for beginners

10:00 – 10:15 Florian Leese - Considerations on the way of implementing DNA-based tools in biomonitoring programs

10:15 – 10:30 Agnès Bouchez - Diatom DNA metabarcoding & WFD : where are we?

10:30 – 11:15 Coffee Break

11:15 – 11:30 11:30 – 11:45 11:45 – 12:00 12:00 – 12:15

Naiara Rodriguez-Ezpeleta Didier Pont Iveta Matejusova Bernd Hänfling

- Improving marine management by implementing DNA-based monitoring and evaluation strategies

- Representativeness of Environmental DNA Metabarcoding signal in River Fish Biodiversity Assessment

- Development of monitoring strategies for marine invasive non-native species (INNS): Didemnum vexillum case study

- eDNA based meta-barcoding of fish communities in the UK

12:15 – 13:30 Lunch Break

Session WG2: DNA-based biomonitoring – Biotic Indices and Metrics Chairs: Jan Pawlowski & Maria Kahlert

13:30 – 14:10 Martyn Kelly - Implementation of a NGS-based phytobenthos tool for ecological assessment in the UK

14:10 – 14:25 14:25 – 14:40 14:40 – 14:55 14:55 – 15:10 15:10 – 15:25

Jan Macher Alfried Vogler Angel Borja Jens Boenigk Tristan Cordier

- Multiple agricultural stressor impacts on stream invertebrates: DNA barcoding extends the resolution of classical ecosystem assessment

- Measuring species turnover in aquatic macroinvertebrate communities using barcoding and metabarcoding

- Using metabarcoding to assess the status of bacteria and macroinvertebrates in marine waters: gAMBI and microgAMBI

- Potential of metatranscriptomics as indicator for ecosystem level diversity and function

- Marine biomonitoring: inferring biotic indices from eDNA metabarcoding data using supervised machine learning


Session WG1: DNA-based biomonitoring – DNA barcode references Chairs: Torbjørn Ekrem & Fedor Ciampor

15:50 – 16:30 Dirk Steinke - How it all begins – DNA reference libraries

16:30 – 18:00 Sónia Ferreira Matthias Geiger Kamil Hupalo Frédéric Rimet Michael T. Monaghan Ursula Eisendle-Flöckner

- Odonata Barcoding: The good, the bad and the ugly

- Overview of the German DNA barcoding initiatives and status of reference libraries for aquatic taxa

- First attempt to create DNA barcode reference library for freshwater gammarids of the Periadriatic Region

- R-Syst::diatom, a reference library for diatoms: overview, uses and perspectives

- Toward a European reference database of Ephemeroptera DNA barcodes

- Integrating free-living nematodes in freshwater assessment and monitoring programs?

18:10 and ~18:40 Busses leaving to Restaurant Dampfe. Please consider registering for bus tickets.

19:00 Conference Dinner (@Dampfe). Please consider registering for the conference dinner.

Conference Dinner

When? Start at 19:00

Where? Restaurant Dampfe

Heinrich-Brauns-Straße 9-15, 45355 Essen. Free bus transport will be available.

What? Free buffet, but drinks paid individually.

Wednesday March 8th 2017

Time Speaker(s) Title

Session WG3+4: DNA-based biomonitoring – Field and Lab Protocols / Data Analysis and Storage Chairs: Kat Bruce & Emre Keskin, Kessy Abarenkov & Diego Fontaneto

09:30 – 10:10 Kristy Deiner - Using Environmental DNA in Biomonitoring Aquatic Resources: Opportunities and Challenges

10:10 – 10:40 Elvira Mächler Valentin Vasselon

- Effects of sampling methods and environmental properties on the detection of macroinvertebrate eDNA

- Optimization of Diatom DNA Metabarcoding for Freshwater Biomonitoring: Application to Mayotte Streams Monitoring Network


11:30 – 12:30 Simon Creer Vasco Elbrecht Alain Franc Urmas Kõljalg

- Exploring the ecological relevance of eDNA in freshwater ecosystems

- Using DNA metabarcoding for stream ecosystem assessments: Opportunities and challenges

- Connecting metabarcoding with High Performance Computing: towards more accurate molecular based inventories

- Management of metabarcoding data using online platform PlutoF

12:30 – 14:00 Lunch Break

14:00 – 14:40 Andrew Mahon - The ‘next generation’ of molecular monitoring, surveillance, and quality assurance for aquatic systems: A North American perspective

14:40 – 15:40 Bettina Thalinger Micaela Hellström Jelger Herder Oliver Thronicker

- Standardized eDNA detection limits and sampling strategies in rivers

- Comparison of capture and storage methods for aqueous macrobial eDNA – advantage of enclosed filters

- Large comparison between WFD-monitoring fish and environmental DNA metabarcoding

- GenoTrail: A new tool for the monitoring of water distribution networks


Open Session

16:30 - 17:10 Lyubomir Penev - A new Metabarcoding and Metagenomics journal

17:10 - 17:45 Florian Leese - Wrapping up ideas of MC2 meeting and conference discussions

Closing remarks and Outlook

Thursday March 9th 2017: All day WG meetings

Morning Session: WG1, 3 and 5 in parallel

09:00 – 13:00

WG1 meeting in room T03 R02 D26 (max. 48 persons)


WG5 meeting in room T03 R02 D81/82 (max. 40 persons)

Afternoon Session: WG2 and 4 in parallel

14:00 – 18:00



Room Code at University Duisburg-Essen

T03 = building from outside, entrance 03. Green building on map below

R02 = within building, referring to the floor. R refers to red corridor, 02 means second floor

D = the floor with the D-rooms

Oral presentations

Oral Presentations (sorted by order of appearance)

Oral presentations Session WG5: DNA-based biomonitoring – Implementation Strategy and Legal Issues

Mainstreaming metabarcoding in regulatory monitoring of ecosystems:

mountaineering for beginners

D.J. Baird

Environment & Climate Change Canada@Canadian Rivers Institute, University of New Brunswick, Canada

Introducing a new technology into an established field of scientific practice is never easy. Although

DNA/RNA metabarcoding has been around for almost a decade, it is still struggling to make the

transition from a ‘method under development’ to a ‘trusted approach’. However, this lack of rapid

uptake on the regulatory side is hardly surprising, given the truly disruptive nature of the system

created by the use of DNA/RNA as units of biodiversity observation. In addition to significant, yet

surmountable obstacles such as incomplete barcode libraries and quantification difficulties, we face

more daunting challenges. As an ecologist, I continue to struggle with a central area of concern voiced

by biomonitoring scientists: how can observations made using traditional methods be related to new

metabarcoding results? At first glance, comparison studies seem simple: either collect matched

samples or develop methods using predefined mixtures of known taxonomic composition to make

direct comparisons. The reality is that these apparently straighforward approaches are fraught with

difficulty, and there is a danger that research efforts will lose momentum, and as a result, limit the

scope of our thinking in breadth of application. As a transformative technology, DNA/RNA

metabarcoding requires deep, clear thinking to support its application in what is arguably a highly

embedded, conservative area of applied science, where existing practices do not necessarily align well

with this new approach. In short, we should not be afraid to challenge openly the basis of old practices

in order to develop these new techniques to their full potential.


'You [should] never walk alone!'

Considerations on the way of implementing DNA-based tools in

biomonitoring programs

F. Leesea,b

, A. Beermanna,b

, V. Elbrechta,b

, J.N. Machera,b

, B. Peinerta,b

, E. Vamosa,b

, A.M. Weiganda,b

,

V. Zizkaa,b

a Aquatic Ecosystem Research, University of Duisburg-Essen, Germany b Centre for Water and Environmental Research, University of Duisburg-Essen, Germany

DNA-based approaches have the potential to act as a game-changer in environmental assessment and

biomonitoring. DNA barcoding and metabarcoding, eDNA screens, metagenomics and

metatranscriptomics offer a uniquely powerful toolbox to assess biodiversity and monitor its change

from the level of genes over species to functional ecosystem processes. While the global academic

community already makes use of these “OMICS”-technologies and has frequently demonstrated their

strengths (and weaknesses), no large-scale biomonitoring program, e.g. in the context of the Water

Framework Directive, applies these approaches systematically. The reasons cannot only be found in

slow transformation processes but are rather due to several inherent problems of the techniques and

their concerted applications.

In this presentation, we will first highlight the power of DNA-based approaches by presenting core

findings conducted on European and New Zealand faunal communities. These we will use then to

demonstrate the identified key limitations of these novel approaches and present – where available –

potential roadmaps to circumvent these. A recurring finding across projects is that collaborations

between labs and disciplines as well as between research and real-world monitoring are needed. This

openness maximises synergy to identify best practice solutions and helps to achieve what the COST

Action DNAqua-Net has been invented for: The Development of tools and roadmaps for the

implementation of powerful DNA-based approaches in real-world aquatic ecosystems. In Europe and

beyond.


Diatom DNA metabarcoding & WFD: Where are we?

A. Boucheza, A. Franc

b, P. Blancher

c, P. Chaumeil

b, J.M. Frigerio

b, F. Keck

a, L. Kermarrec

b,

O. Monnierd, Y. Reyjol

d, F. Salin

e, K. Tapolczai

a, V. Vasselon

a, F. Rimet

a

a UMR CARRTEL, INRA, Université Savoie Mont Blanc, 74200 Thonon-les-bains, France b UMR BIOGECO, INRA, Université de Bordeaux, 69 route d'Arcachon, 33610 Cestas, France c Asconit Consultants, Parc Scientifique Tony Garnier, 6-8 Espace Henry Vallée, 69366 Lyon Cedex 07, France d French National Agency for Water and Aquatic Environments, DAST, 5 sq. Félix Nadar, 94300 Vincennes, France e PGTB, Genome Transcriptome Facility of Bordeaux, INRA, 69 route d'Arcachon, 33610 Cestas, France

The best picture of an ecosystem health is provided by the biological characteristics of the

communities that live in it, in comparison to a reference state, free from anthropogenic pressures.

These are the founding ideas of the WFD. In France, the long-standing efforts to monitor water quality

have led to the development of bioassessment methods based on diatoms since the 1980s. These

methods have then been brought into compatibility with the WFD for freshwater.

The simultaneous development of DNA barcoding and of high-throughput DNA sequencing (HTS)

has led to the emergence of the idea of using such approaches to characterize environmental

communities. Since the 2010s, “proof of concept” as well as environmental scaling-up have shown

their potential to evaluate health status of rivers. Tools and tests have been progressively released for

applying diatom DNA metabarcoding in the context of bioassessment. Although many technical issues

have been enlightened for diatoms (e.g. DNA barcode, DNA extraction, Reference library), many are

still pending (e.g. DNA preservation, HTS technology, sequencing depth, quantification).

Different indicators can then be produced: WFD-friendly ones (community structure at species level,

comparison to reference state), or indicators better exploiting the big data from HTS. However,

taxonomical and ecological expertise of current stakeholders and comparability to historical data

should not be left out when implementing these indicators. Going towards WFD implementation of

DNA-based diatom indicators will also require standardized procedures (CEN standards) and

collective implementation with the numerous stakeholders (e.g. SMEs, academics, environmental

managers) at national and European levels.


Improving marine management by implementing DNA-based monitoring

and evaluation strategies

N. Rodriguez-Ezpeleta, E. Aylagas, J. Corell, A. Rey, I. Mendibil, Á. Borja, E. Bachiller, U. Cotano,

X. Irigoien

AZTI, Marine Research Division, Sukarrieta, 48395 Bizkaia, Spain

DNA metabarcoding, the taxonomic assignment of individuals from an environmental sample based

on their DNA sequences, could represent a faster, cheaper and more accurate alternative to

morphological identification for assessing the entire taxonomic composition of thousands of samples

simultaneously. Yet, before it can be routinely applied for biodiversity studies and assist

environmental management, this method needs to be benchmarked, calibrated and standardized. We

have applied DNA metabarcoding to i) study the global ocean plankton diversity, by analyzing

hundreds of samples collected during the Malaspina circumnavigation, ii) assess sea floor integrity, by

analyzing sediment macroinvertebrate samples used for coastal monitoring, iii) understand marine

food webs, by analyzing fish stomach contents and iv) detect invasive species introduced through

ballast water, by analyzing fouling, plankton and free DNA collected from commercial ports. For each

of these applications, we have developed sampling procedures, laboratory protocols and bioinformatic

pipelines and have compared metabarcoding derived results with traditional observations. Our studies

pinpoint steps of the process that are crucial for obtaining accurate assessments and provide guidelines

for the application of metabarcoding in marine monitoring and evaluation programs. Ultimately, our

results will assist the implementation of this technique within policies such as the Water and Marine

Strategy Framework Directives, the Ballast Water Management Convention, or the Common Fisheries

Policy.


Representativeness of Environmental DNA Metabarcoding signal in

River Fish Biodiversity Assessment

D. Ponta, R. Civade

b, A. Valentini

a, T. Dejean

a, P. Taberlet

c

a SPYGEN, Savoie Technolac—Bât. Koala, 17, Rue du Lac Saint-Andre, BP 274, Le Bourget-du-Lac Cedex 73375, France b Hydrosystems and Bioprocesses Research Unit, IRSTEA, Antony Cedex 92761, France (Present Address: Asconit

consultant, 6 espace Henry Vallée, 69007 LYON)

c Laboratoire d’Ecologie Alpine (LECA), CNRS - Univ. Grenoble Alpes, Grenoble 38000, France

In recent years, environmental DNA (eDNA) has drawn much attention due of its high potential for

the study of biodiversity and its low cost compared to traditional sampling. In particular, eDNA

metabarcoding (or multi-specific approach) allows the detection of a set of taxonomically related

species and in particular Teleostei and amphibians. The aim of this presentation - based on a

publication in Plos One (2016) and a PhD work (Civade, 2016 - is to test i) the ability of eDNA

metabarcoding pipeline approach defined by Valentini et al. (2016) to describe fish species diversity in

rivers by comparing classical eletrofishing and eDNA metabarcoding approach, and ii) the spatial

representativeness of eDNA metabarcoding samples. This study was conducted in partnership between

IRSTEA-SPYGEN-CNRS-ONEMA and in collaboration with EDF R & D and CEN-RA.

For the different study sites, the number of fish species detected by eDNA metabarcoding was well

above that obtained using electrofishing (approximately 50% additional species). Previous records on

the sampling sites show that almost all additional species detected using eDNA had already been

identified on the site. An experiment on a lake-river gradient provided initial results regarding the

eDNA detection distance. The lake species are detected in the outlet stream at a maximum of two

kilometres from the lake. In addition, eDNA can clearly separate fish assemblages that are few

kilometres apart. Finally, preliminary results show that, on several sites, there is a significant

relationship between eDNA signal intensity (number of reads) and relative abundances of species.


Development of monitoring strategies for marine invasive non-native species (INNS):

Didemnum vexillum case study

I. Matejusovaa, L. Brown

a, J. Graham

a, M. Gubbins

a, B. Turrell

a

a Marine Scotland Science, 375 Victoria Road, Aberdeen, United Kingdom

Invasive non-native species (INNS) pose a global threat causing both environmental as well as

economic damage to marine ecosystems. Impact of INNS, if well established, is often irreversible

and can lead to damage to natural habitats, including protected areas and sensitive species. Direct

costs associated with removal and eradication of INNS from an area and indirect cost to aquaculture

industry due to potential stock movement restrictions or treatment can be also very high. Therefore

necessary measures need to be taken to prevent their introduction and spread to maintain good

environmental status of the seas. There are obligations to report INNS under the EU Water

Framework Directive and the Marine Strategy Framework Directive as well as under national policies

such as the Wildlife and Natural Environment (Scotland) Act 2011 and Alien and Locally Absent

Species in Aquaculture (Scotland) Regulations 2014.

There is an urgent need to develop effective monitoring programme for early detection of invasive

tunicate Didemnum vexillum Kott, 2002 which has been causing issues worldwide. A project has been

initiated to develop eDNA sampling and storage protocols as well as efficient monitoring strategy and

sampling regimes based on estimates of species occupancy. Main advantages and drawbacks from the

single species- vs. DNA metabarcoding approach to detect D. vexillum in eDNA samples will be

assessed to maximize sensitivity of detection. Field work and aquarium studies are also ongoing to

evaluate eDNA degradation and surveillance efficacy before eDNA surveillance is trialled alongside

of the conventional physical observation of settled colonies.


eDNA based meta-barcoding of fish communities in the UK

B. Hänflinga, L.-L. Handley

a, D. Read

b, A. Duguid

d, G. Pierson

e, K. Walsh

f, I.J. Winfield

c

a Evolutionary and Environmental Genomics Group (@EvoHull), School of Environmental Sciences, University of Hull,

Cottingham Road, Hull, HU6 7RX b Centre for Ecology & Hydrology (CEH), Benson Lane, Crowmarsh Gifford, Wallingford, Oxfordshire, OX10 8BB c Lake Ecosystems Group, Centre for Ecology & Hydrology (CEH), Lancaster Environment Centre, Library Avenue,

Bailrigg, Lancaster, LA1 4AP d Scottish Environment Protection Agency, Strathearn House, Broxden Business Park, Lamberkine Drive, Perth, PH1 1RX e Environment Agency, Mance House, Arthur Drive, Hoo Farm Industrial Estate, Worcester Road, Kidderminster DY11

7RA f Environment Agency, Horizon House, Deanery Road, Bristol, BS1 5AH

Organisms continuously release DNA into their environments via shed cells, excreta, gametes and

decaying material. Analysis of this “environmental DNA” (eDNA) is revolutionising biodiversity

monitoring. eDNA outperforms many established survey methods for targeted detection of single

species. In a recent study we have shown that eDNA combined with high throughput sequencing can

be used to recover accurate qualitative and quantitative information about fish communities in large

lakes such as Windermere. The work which was funded by the UK environmental regulators was

motivated by the need to devise a fish classification tool for lakes to fulfil EU Water Framework

Directive obligations. The results to date suggest that an eDNA approach can provide the required

data for statutory monitoring. More recently the method has been applied to a large number of lakes

of varying size and trophic status in order to provide a data base for the development of such a tool.

Oral presentations Session WG2: DNA-based biomonitoring – Biotic Indices and Metrics

Implementation of a NGS-based phytobenthos tool for ecological assessment in the UK

M. Kellya, K. Walsh

b, R. Glover

c, D. Mann

d, S. Juggins

e, S. Sato

d, N. Boonham

c, T. Jones

f

a Bowburn Consultancy, 11 Monteigne Drive, Bowburn, Durham DH6 5QB, UK; b Environment Agency, Horizon House, Deanery Road, Bristol, BS1 5AH; c FERA, Sand Hutton, York, YO41 1LZ, UK; d Royal Botanic Gardens Edinburgh, 21a Inverleith Row, Edinburgh EH3 5LR, UK e Newcastle University, Newcastle NE1 7RU, UK; f Environment Agency, Higher Shaftsbury Road, Blandford Forum, Dorset DT11 8ST, UK

The UK has used diatoms for assessing the ecological condition of freshwaters since 1995. Since the

introduction of the Water Framework Directive, they have been used to fulfil obligations to assess

“phytobenthos” in rivers and lakes. From 2017 onwards, diatom identification and enumeration in

England will move from light microscopy to a new approach using Illumina MiSeq next generation

sequencing (NGS) to identify rbcl barcodes. A new version of the Trophic Diatom Index has been

developed, recognising that the basis for counting has changed from valves (= half of a silica cell

wall) to chloroplasts (which vary in number between species). The development of this new index

has provided many insights into how data produced by NGS data relate to natural populations.

In this talk, aspects of the WFD that affect the form of an assessment tool will be considered, with a

focus on patterns of spatial and temporal variation in the water body as a whole. These sources of

variation combine to influence the confidence with which an ecological status class can be expressed

and acted upon. Studies on variation at different scales, including both analytical and spatial and

temporal patterns, suggest that the relative contributions of each varies between sites, but that natural

variation within water bodies typically exceeds analytical variation for both light microscopic and

NGS analyses. Whilst analytical variation is lower for NGS than for light microscopy, this may not

result in substantial gains in sensitivity of assessments due to the influence of other spatial and

temporal factors working within water bodies.

Some broader lessons learnt (and being learned) during the transition from light microscopic to NGS

analyses will also be considered. These include the challenges in developing and maintaining barcode

libraries for microorganisms when the underlying taxonomy is still not fully understood, and in

maintaining higher-level ecological awareness in biologists whose only encounter with a site may be

via a spreadsheet.


Multiple agricultural stressor impacts on stream invertebrates:

DNA barcoding extends the resolution of classical ecosystem assessment

J.N. Machera, R. Salis

b, K. Blakemore

b, R. Tollrian

c, C. Matthaei

b, F. Leese

a

a Aquatic Ecosystem Research, University of Duisburg-Essen, Germany b University of Otago, New Zealand c Ruhr-University Bochum, Germany

Many freshwater ecosystems and their biodiversity are subject to multiple anthropogenic stressors.

Stressor responses are often studied using community measures and higher-order taxonomic groupings

as species identification is often difficult. However, species even in the same genus may have very

different ecological tolerances. DNA barcoding can improve species identification, provide information

on intraspecific genetic variation and thus population vulnerability and adaptability. In this study, we

sampled 43 stream sites in New Zealand spanning gradients of the agricultural stressors fine sediment

and nutrients. First, we used morphology- based data to investigate macroinvertebrate community

responses to stressors. Then, we used DNA- barcoding data (COI gene) to specifically test whether two

important indicator taxa, Deleatidium spp. (n=520) and Potamopyrgus spp. (n=305), consisted of

several unrecognized species differing in their responses to stressors. Finally, we tested whether

stressors impacted on intraspecific genetic diversity. While Potamopyrgus spp. consisted of only one

species, Deleatidium spp. consists of 12 molecularly identified species, whose stressor response profiles

differed significantly. Potamopyrgus antipodarum and one Deleatidium species showed higher

intraspecific genetic variation in stressed streams, while other species did not show any response in

molecular diversity. Overall, our results highlight the potential and importance of including genetic

methods in freshwater monitoring programs.


Measuring species turnover in aquatic macroinvertebrate communities

using barcoding and metabarcoding

A.P. Voglera,b

, C. Múrriac, K. Bruce

d, C. Andújar

a,b

a Imperial College London, Silwood Park Campus, Ascot, Berkshire, SL5 7PY, UK b Natural History Museum, Cromwell Road, London SW7 5BD, UK c Departament de Biologia Evolutiva, Universitat de Barcelona, Avinguda Diagonal 643, 08028-Barcelona, Spain; d NatureMetrics, Bakeham Lane, Egham, Surrey, TW20 9TY, UK

The monitoring of aquatic ecosystems requires well designed metrics for assessing local species

assemblages and a predictive framework for the responses to environmental change. Here we use two

case studies to show the utility of barcoding and their potential implementation of the Water Framework

Directive. First, standard cox1 barcoding was applied to macroinvertebrate communities of largely

pristine highland streams across Europe (Morocco to Sweden). This study demonstrates the great natural

turnover within and among biogeographical regions, which is influenced by a combination of historical

phylogeographical processes and local ‘environmental filtering’, resulting in huge natural differentiation

of freshwater streams. Second, we monitor whole-community turnover in a small river following an

insecticide spill, using metabarcoding with cox1 and nuclear rRNA genes, which shows the great change

of community composition. The key message is that (i) local species distributions vary greatly, and

monitoring schemes need to be set against solid knowledge of the local (basin-specific) community

composition; (ii) fast metabarcoding approaches are potentially powerful for assessing community

composition but require reference sets (possibly from standard DNA barcoding) for secure species

assignment; (iii) local disturbance has great impact on the composition of species diversity and functional

groups, but classical monitoring methods have limited resolution and make poorly founded assumptions

about functional group membership. Together, these studies illustrate the need for a phylogenetically

informed species reference system of key community components across Europe; based on combined

classical taxonomy, validated reference sequence libraries, and rapid surveys using metabarcoding and

metagenomics.


Using metabarcoding to assess the status of bacteria and macroinvertebrates

in marine waters: gAMBI and microgAMBI

A. Borjaa, E. Aylagas

a, N. Rodríguez-Ezpeleta

a

a AZTI, Marine Research Division, Pasaia, Spain

The Water Framework Directive aims to protect and restore aquatic ecosystems. Assessing the

ecological status of different biological elements is required. Hence, there is a demand of developing

tools in order to increase speed, accuracy and cost-efficiency of monitoring programs. We have

explored the application of metabarcoding for bacteria and macroinvertebrates in estuarine and coastal

waters, developing new biotic indices, based upon existing contrasted and highly used ones, such as the

AZTI’s Marine Biotic Index (AMBI).

We describe the application of metabarcoding in an ongoing monitoring program to confirm the

suitability, in a real context, of this technique using different variants of AMBI and gAMBI, on paired

samples collected from the same locations over multiple estuarine and coastal locations. The results

revealed that metabarcoding based accurate inferences of ecological status are possible and that

gAMBI succeeded in discriminating ecological status classes. Furthermore, compared to morphology

based inferences, metabarcoding is both more time and cost effective.

In addition, we have developed a new biotic index based on bacterial communities (microgAMBI).

Due to their high sensitivity to pollution and fast response to environmental changes, bacterial

assemblages could complement the information provided by benthic metazoan communities as

indicators of human-induced impacts. Using the relative abundance of putative indicator bacterial taxa,

microgAMBI was significantly correlated with a sediment quality index calculated on the basis of

pollutant concentrations. This new index based on bacterial assemblage composition can be a sensitive

tool for providing a fast environmental assessment and management.


Potential of metatranscriptomics as indicator

for ecosystem level diversity and function

J. Boenigka, L. Grossmann

a, J. Nuy

a, D. Beisser

a

a Department of Biodiversity, University of Duisburg-Essen, Germany

Inferring ecosystem functioning and ecosystem services through inspections of the species inventory is

a major aspect of ecological field studies. However, the presence of distinct species and groups of

organisms does not necessarily reflect their effective functional role in their respective ecosystems.

Ecosystem functions are often stable despite considerable species turnover. Assessing the response of

microorganisms to environmental changes might establish novel and more direct tools. Using

metatranscriptome analyses, we analyse 21 mainland European freshwater lakes from the Sierra

Nevada (Spain) to the Carpathian Mountains (Romania) and from northern Germany to the Apennines

(Italy) and covers an altitudinal range from 38 m above sea level (a.s.l) to 3110 m a.s.l. The dominant

eukaryotic taxa were green algae, ciliates, diatoms and chrysophytes. Dominant bacterial taxa were

Actinobacteria, Alphaproteobacteria, Bacteroidetes, Betaproteobacteria, Cyanobacteria and

Verrucomicrobia. Our analyses indicate that community composition is a good first proxy for the

analysis of ecosystem functions. However, differential gene regulation modifies the relative importance

of taxa in distinct pathways. Whereas taxon composition varies considerably between lakes, the

relative importance of distinct metabolic pathways is much more stable, indicating that ecosystem

functioning is buffered against shifts in community composition through a functional redundancy of

taxa. We further demonstrate that metatranscriptomes have the potential to reflect the relative

importance of different metabolic pathways. In particular, the relative abundance of distinct expressed

genes reflect environmental conditions and such indicator genes can be used for assessing ecosystem

quality.


Marine biomonitoring: inferring biotic indices from eDNA metabarcoding data using

supervised machine learning

T. Cordiera, P. Esling

b, J. Visco

c, A. Ouadahi

a, F. Lejzerowicz

a, J. Pawlowski

a

a Department of Genetics and Evolution, University of Geneva, Boulevard d’Yvoy 4, CH-1205 Geneva, Switzerland b IRCAM, UMR 9912, Université Pierre et Marie Curie, 4 place Jussieu, 75005 Paris, France c ID-Gene ecodiagnostics Ltd, 1228 Plan-les-Ouates, Switzerland

Biomonitoring is important for environmental impact assessment in marine environment. Traditional

benthic monitoring, based on macrofaunal inventories, involves the taxonomic identification of

thousands of manually sorted specimens, which is extremely time-consuming and taxonomic expertise

demanding. High-throughput sequencing technologies could make it more time and cost effective by

using DNA barcodes for species identification and automated pipeline for analysis of environmental

DNA (eDNA) data. However, most of eDNA sequences correspond to unassigned microbial or

meiofaunal taxa, which strongly limits their use for inferring biotic indices. To overcome these

limitations, we investigated the possibility of using supervised machine learning algorithms to build

predictive models in order to infer four commonly used biotic indices (AMBI, ISI, NSI, NQI1) from

eDNA metabarcoding data. We tested our approach on benthic foraminifera used as bioindicators of

organic enrichment impact of salmon farms in Norway. Our results show that machine learning tools

allow the building of accurate predictive models, leading to similar biotic indices values than the ones

derived from macrofaunal inventories. We argue that supervised learning approaches using eDNA

metabarcoding data could be used to overcome and even bypass the cost and time demanding morpho-

taxonomic inventories approach.

Oral presentations Session WG1: DNA-based biomonitoring – DNA reference libraries

How it all begins - DNA barcode reference libraries

D. Steinke

Centre for Biodiversity Genomics, University of Guelph, Canada

One of the main activities of the DNA Barcoding community in general and the DNAqua-Net group in

particular is to generate an open access library of reference barcode sequences which enables everyone

with the ability to obtain sequence information to identify specimens of various origin. Barcodes of

unidentified specimens can be compared with reference barcodes to find the matching species.

For over ten years we have already generated more than 5 Million barcodes for over 550,000 species.

Many of these species have been selected because they are of special interest to particular users who

need the ability to identify "their" species. However, as a result of our progress more and more we are

looking at the completion of larger reference libraries for particular taxonomic groups either world-wide

or more limited in range such as national libraries. Building such barcode reference libraries in a

systematic fashion can be challenging and comes with its own set of potential issues.

I will discuss the development of our workflows in the context of ongoing DNA barcode library

construction projects, emphasizing the importance of delineating a set of reference specimens to aid

investigations in cases of nomenclatural and DNA barcode discordance. I will also touch on available

tools and new technologies developed to simplify barcode library construction and parameterization.


Odonata Barcoding: The good, the bad and the ugly

S. Ferreiraa, A. Cordero-Rivera

b, J. Paupério

a, J. Veríssimo

a, F.M.S.Martins

a,c, V.A. Mata

a,c,

A. Muñoz-Meridaa, P.C. Alves

a,c, S. Jarman

a, P. Beja

a,d

a CIBIO/InBIO – Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do Porto, Vairão, 4485-

661 Vairão, Portugal b ECOEVO Lab, Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, EUE Forestal, Campus Universitario A

Xunqueira s/n, ES-36005 Pontevedra, Spain c Departamento de Biologia da Faculdade de Ciências da Universidade do Porto, Rua Campo Alegre, 4169-007 Porto,

Portugal d

CEABN, Instituto de Agronomia, Universidade de Lisboa /InBIO Laboratório Associado, Lisboa, Portugal

DNA barcoding is currently an essential tool in a vast array of ecological and conservation studies (e.g.

biodiversity monitoring, diet assessments). However, its applicability is still hampered by the lack of

comprehensive reference collections. This knowledge gap becomes greater in invertebrates, especially

from biodiversity hotspots like the Mediterranean Basin. Surprisingly, while dragonflies and

damselflies are one of the best studied insect groups, no comprehensive barcoding of the European

species has been made. These predatory insects are intimately connected to freshwater habitats, as their

larval phase is completed in the water, being particularly sensitive to changes in the aquatic

environment and constituting important bioindicators of ecosystem health.

Within InBIO Barcoding Initiative we barcoded more than 70 species of odonates, focusing mostly on

species from Iberian Peninsula. Genomic DNA was extracted and the barcoding mitochondrial COI

gene fragment (658 bp) was amplified. DNA barcodes were sequenced using either Sanger or high-

throughput sequencing (Illumina).

Our results exhibited a scenario that illustrates some of the challenges posed by insect identification

using DNA barcoding. While many species can be easily identified using the mitochondrial COI gene

fragment, this is not truth to all. Not all species possess a specific DNA barcode blocking the correct

assignment of taxonomic names to unidentified specimens. Other species possess multiple copies of

COI in the genome, impeding successful Sanger sequencing, which can be overcome using Next

Generation Sequencing. This data provides important insights into the diversity and taxonomy of

odonates and how to achieve a useful Barcode reference database.


Overview of the German DNA barcoding initiatives and status of

reference libraries for aquatic taxa

M. Geigera, B. Rulik

a, J. Moriniere

b

a Zoologisches Forschungsmuseum Alexander Koenig - Leibniz-Institut für Biodiversität der Tiere - Bonn, Germany b SNSB-Zoologische Staatssammlung, München, Germany

The great potential of DNA barcoding to enhance environmental assessments has led to the funding of

four major campaigns in Germany: Barcoding Fauna Bavarica (BFB), Freshwater Diversity

Identification for Europe (FREDIE), German Barcode of Life (GBOL), and Molecular Taxonomy of

Marine Organisms (DZMB). All initiatives included - or even focused - on aquatic organisms and BFB

and GBOL are still funded. Thus, the coverage with reference data (at least three taxonomically

verified DNA barcodes per species) is good to very good (>70% of the WFD species) in some taxa

(e.g., Amphipoda: 16/20; Coleoptera: 100/120; Ephemeroptera: 70/84; Odonata: 17/23; Pisces: 25/25;

Plecoptera: 15/19; Trichoptera: 136/157). However, in many real world situations, exemplified by a

study conducted in a River Rhine tributary, this will not be enough, given that the number of registered

species of these taxa for Germany is much higher. Thus, efforts should not be focused exclusively on

filling the reference libraries for taxa relevant for monitoring tasks, but target as many species as

possible. This endeavor would greatly benefit from more intense, pan European networking and the use

of common databases and interfaces, such as the check list option offered by BOLD. In addition,

examples from various taxa exhibiting morpho-molecular discrepancies (i.e. splitting or lumping)

demonstrate i) the importance of traditional taxonomic knowledge and expertise, in order to validate

reference data and solve taxonomic issues, but ii) also the necessity of a geographically broad sampling

scheme.


First attempt to create DNA barcode reference library for freshwater gammarids of the

Periadriatic Region

K. Hupaloa, T. Mamosa, W. Wrzesinskaa, T. Rewicza, A. Wysockab, R. Wattierc,

M. Grabowskia

a Department of Invertebrate Zoology and Hydrobiology, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland b Department of Genetics, University of Gdansk, Gdansk, Poland c Equipe Ecologie Evolutive, UMR CNRS 6282 Biogeosciences, Universite de Bourgogne, 6 Boulevard Gabriel, 21000 Dijon,

France

The Mediterranean Region is known as one of the most important biodiversity hotspots on the world

scale. However, majority of studies upon gammarid crustaceans of the region focused mostly on

marine species, leaving aside the freshwater fauna. During several sampling campaigns in years 2005-

2016, a very large collection of has been gathered from numerous inland and insular locations in the

Periadriatic region. Molecular analyses revealed extremely high level of cryptic diversity, largely

exceeding the number of morphospecies already described from the region, i.e. forty molecular species

of Gammarus balcanicus group, endemic species flock from the Lake Ohrid, over ten cryptic species in

Gammarus italicus species complex, ten molecular species of Gammarus from the Peloponnese and

high cryptic diversity of Echinogammarus in the Appenine Peninsula and Tunisia. The collected data

will greatly aid to reveal the phylogeographical affiliations and the time of the diversification of the

local gammarids and also help to update and revise the genus-level taxonomy. Creating a DNA barcode

reference library of the region is essential for planning reasonable and effective strategy preventing or

at least minimalizing the rapid loss of freshwater biodiversity, due to the extensive anthropogenic

degradation of the local freshwater ecosystems.


R-Syst::diatom, a reference library for diatoms:

overview, uses and perspectives

F. Rimeta, V. Vasselon

a, K. Tapolczai

a, A. Bouchez

a

a CARRTEL, INRA, Université de Savoie Mont Blanc, 74200, Thonon-les-Bains, France

Use of metabarcoding for ecological assessment using benthic diatoms is a promising approach but

there are several keys aspects which make it still premature to use it routinely at present. One of these

aspects is the reference libraries which have three potential limitations:

-1- taxonomical homogeneity

-2- data and metadata traceability

-3- the incomplete coverage of species

R-Syst::diatom (Rimet et al. 2016; http://www.rsyst.inra.fr/) is a reference library which was created in

2012 to answer these pitfalls. It links DNA-barcodes (18s and rbcl) to their taxonomical identifications.

Data come from two sources, a culture collection of freshwater algae and NCBI nucleotide database.

Before integrating data in the library, a curation procedure is followed every 6 months. Metadata, such

as sampling site, responsible of the different processing steps, photos, morphological features ... are

also available. Metadata necessary to keep in the library follow the requirement of a technical report

which was accepted by CEN (European Standardization Committee) last year and which is the result of

a collaborative work of several labs (B, CZ, D, F, FI, HR, UK, SE, SP).

But many taxa still lack in the library. Usually cell isolation and cultures are necessary to obtain

barcodes which is time consuming and many species are hardly cultivable. Using high throughput

sequencing data from ecological quality monitoring networks is an alternative technique which was

recently tested. We bet that in a near future this technique will be transferable to well trained diatom

technician to speed up the completion of the library.


Toward a European reference database of Ephemeroptera DNA barcodes

M.T. Monaghana, S. Rutschmann

a,b,c, J.-L. Gattolliat

d, M. Sartori

d

a Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany b University of Vigo, Vigo, Spain c University of Lausanne. Switzerland d Musée cantonal de zoologie, Lausanne, Switzerland

The mayflies (Ephemeroptera) are widely used as bioindicators in freshwater ecosystems. They are

taxonomically and ecologically diverse and most species are intolerant to many forms of habitat

degradation. They are also an ancient order, dating from the Carboniferous, and their age and

evolutionary diversification present particular challenges in the development and application of

reference sequences for DNA sequences. Here we present the results of a European-wide effort to

generate reference mtDNA COI sequences and taxonomic vouchers from the ca. 300 described

species. This included sampling from multiple areas within the range of widespread species and

sampling of topotypes where localities were known and sample collection was feasible. Because

several studies have documented the presence of multiple cryptic lineages within recognized species,

we used the coalescent-based gmyc-model approach to delineate species in a first step to assess

taxonomic-morphological congruence.


Integrating free-living nematodes in freshwater assessment and monitoring programs?

U. Eisendle-Flöckner

University of Salzburg, Division of Animal Structure and Function, Hellbrunnerstraße 34, 5020 Salzburg, Austria

In general, it might be widely accepted that free-living nematodes are essential to aquatic habitats’

functioning. But free-living nematodes seem to be better integrated in marine than in freshwater

environmental research. It is rather the rule then the exception to exclude nematodes from freshwater

habitat assessment and monitoring, although nematodes harbor running and standing freshwater bodies

widely independent from habitat size, geographic position (latitude, altitude) and hydrological regime.

Freshwater nematode communities reflect different environmental conditions but also their changes –

similarly as their macroinvertebrate counterparts. Environmental conditions include, for example,

impoverished habitat conditions due to organic enrichment, other pollutants and morphological

degradation, as well as succession in newly emerging or disturbed habitats.

Compared to macroinvertebrates, freshwater nematodes might offer some additional information on

habitat conditions and their changes and they might additionally be helpful to assess habitats (surface

and subsurface) where other invertebrates are less abundant, only sporadically distributed or even

missing. Lacking integration of freshwater nematodes in assessment and monitoring routine might

refer to (assumed) main problems such as time consuming sample processing and difficult taxonomy

and species determination. A database survey (GenBank, BOLD) on the other hand reveals that

nematode taxonomic genetic information is also still scarce. This might indicate little existing

taxonomic expertise in general, but also problems finding universal sequences for “barcoding”

nematodes. Further efforts need to be undertaken that these abundant, diverse and additionally

informative freshwater organisms can be reasonably integrated in freshwater assessment and

monitoring protocols based on genetic applications.

Oral presentations Session WG3+4: DNA-based biomonitoring – Field and Lab Protocols / Data Analysis and Storage

Using Environmental DNA in Biomonitoring Aquatic Resources:

Opportunities and Challenges

K. Deiner

Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY, USA

Over the last decade there has been increasing evidence that DNA shed into the environment can be

used to monitor aquatic species from across the tree of life. This approach to monitoring biodiversity

provides many opportunities in that it is non-invasive, requires very little field time, can be

standardized across taxonomic groups, and can be more time efficient after an initial development

phase. These attributes make using eDNA an ideal methodology for routine monitoring. However,

many challenges remain to be understood such as deciphering the spatial and temporal scale of

inference for a sample, and many field and lab protocol decisions that may influence the outcome of

detected species. In my talk I will explore both the opportunities and challenges this new tool has to

offer for monitoring aquatic resources.


Effects of sampling methods and environmental properties on the detection of

macroinvertebrate eDNA

E. Mächlera,b

, K. Deinerc, F. Altermatt

a,b

a Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Aquatic Ecology, Überlandstrasse 133,

CH-8600 Dübendorf, Switzerland b Department of Evolutionary Biology and Environmental Studies, University of Zurich, Winterthurerstr. 190, CH-8057

Zürich, Switzerland c Cornell University, Atkinson Center for a Sustainable Future, and Department of Ecology and Evolutionary Biology, Ithaca,

NY 14850 USA

While detection of rare and invasive amphibians and fishes in lake and river systems has been

effective and is already commonly implemented, much fewer studies have targeted macroinvertebrates

in aquatic systems. Macroinvertebrate-based indices are highly important for water quality

assessments. Therefore, we implemented single species detection with eDNA techniques and

compared it to the conventional kicknet-sampling method commonly applied in aquatic habitats to a

broad taxonomic array of macroinvertebrate species in river and lake systems. Our species-specific

eDNA method showed medium to very high consistency with the data from kicknet-sampling and was

able to detect both indicator and nonnative macroinvertebrates. However, concerns about accurate

detections using eDNA have been raised: Sampling methods, laboratory protocols as well as

environmental properties can affect eDNA-based detection of species. Therefore, it is crucial to

understand their effects on detectability. Thus, we firstly evaluated the effect of sampled water volume

(0.25 to 2 L) on the detection rate for three macroinvertebrate species. We found that all three species

were detected in all volumes of water. Surprisingly, however, only one species had a positive

relationship between an increased sample volume and an increase in the detection rate. Secondly, it is

speculated that UV-light as an environmental property could affect degradation and consequently

detection of eDNA. We experimentally tested the effect of different UV-light treatments on the

degradation and detectability of eDNA in mesocosms under semi-natural outdoor conditions.


Optimization of Diatom DNA metabarcoding for freshwater biomonitoring:

Application to Mayotte streams monitoring network

V. Vasselona, I. Domaizon

a, F. Rimet

a, K. Tapolczai

a, A. Bouchez

a

a CARRTEL, INRA, Université de Savoie Mont Blanc, 74200, Thonon-les-bains, France

DNA metabarcoding of benthic diatoms combined to high-throughput sequencing (HTS) is a

promising approach for water quality assessment. It provides the opportunity to be faster, cheaper and

to handle more samples than the classical approach based on microscopy. Hence, after optimizing

different key steps of metabarcoding process, we deployed this approach at the scale of a monitoring

network to evaluate the reliability of diatoms DNA metabarcoding compared to microscopy for

freshwater quality assessment.

Biofilms samples from the biomonitoring network of Mayotte streams were sampled in 2014 and 2015

and analyzed by both DNA metabarcoding and microscopy. Previous optimizations for diatoms

metabarcoding allowed to set-up appropriate DNA extraction method (GenElute; Vasselon et al.

2017), DNA barcode (rbcL gene; Kermarrec et al. 2013) and molecular reference libraries (R-

Syst::diatom; Rimet et al. 2016). Additionally, a correction factor (based on taxa biovolume),

developed in a previous study, was used to improve quantification of diatoms taxa in DNA

inventories. The optimized molecular data obtained afterwards were compared to morphological ones

based on diatom community structures, taxonomic inventories and water quality indices.

A significant correlation was observed between the diatom community structure (based on BrayCurtis

dissimilarity) obtained with DNA metabarcoding and microscopy. The congruence between molecular

and morphological taxonomic inventories was improved by the correction factor, remaining

differences were caused by either the absence of biovolume information for some taxa or the

incompleteness of the molecular reference database. Molecular and morphological quality indices

were however strongly correlated and allowed a good estimation of ecological quality status.


Exploring the ecological relevance of eDNA in freshwater ecosystems

S. Creera, M. Seymour

a, I. Bista

a, G.R. Carvalho

a, K. Walsh

b, M. Christmas

c, M. Hajibabaei

d,

D. Lalliase , Lofresh Consortium

f

a School of Biological Sciences, Bangor University, Bangor, United Kingdom b Environment Agency, United Kingdom c Environment Agency, Bristol, United Kingdom d Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada e Centre de Jouy-en-Josas, Unité GABI, Génétique en Aquaculture, INRA, France f School of Biological Sciences, Bangor University, United Kingdom

Over the past few years our perception of the term “environmental DNA” (eDNA) has subtly moved

away from the historical use of the term, that could refer to DNA derived from any environmental

source, to refer specifically to “free” eDNA or DNA derived from organisms without prior removal

from their substrate. Irrespective of specific terminology, using eDNA to detect the presence of a

broad range of organisms has had substantial social and economic impact, especially in freshwater

ecosystems. To date, the largest proportion of eDNA studies have employed forms of quantitative

PCR (qPCR) to assess the biodiversity of single species, but the potential for using simultaneous

species assessment methodologies such as metabarcoding and shotgun sequencing now offer

demonstrable opportunities for whole community assessments. Nevertheless, a large number of

questions still remain unanswered regarding the ecological relevance of eDNA in the wild. For

example, we need to identify precisely what different forms of eDNA are, what factors affect the

persistence of detectable fragments of eDNA and how eDNA traces relate to living communities in

space and time in both lentic and lotic ecosystems. Here, we will overview insights derived from

contemporary research from the laboratory to the ecosystem scale, in relation to the ecological

relevance of eDNA. Finally, we will explore emerging projects seeking to fill gaps in our current

knowledge and consider the future of eDNA analyses in relation to monitoring ecosystem health and

understanding biodiversity-ecosystem functioning relationships.


Using DNA metabarcoding for stream ecosystem assessments:

Opportunities and challenges

V. Elbrechta, E. Vamos

a, B. Peinert

a, J. Aroviita

b, K. Meissner

c, F. Leese

a,d

a Aquatic Ecosystem Research, Faculty of Biology, University of Duisburg-Essen, 45141 Essen, Germany b Finnish Environment Institute, Freshwater Centre, 90014 Oulu, Finland c Finnish Environment Institute, Freshwater Centre, 40500 Jyväskylä, Finland d Centre for Water and Environmental Research (ZWU) Essen, University of Duisburg-Essen, 45141 Essen, Germany

Accurate assessments of macrozoobenthic community composition are essential to monitor stream

health. Unfortunately, morphological identification of macrozoobenthic indicator taxa at larval stages

is often difficult, yet DNA barcoding has emerged as a powerful alternative allowing quick and

reliable identifications.

Here, we applied DNA metabarcoding using four different primer sets to identify macroinvertebrates

from 18 samples obtained from the Finnish stream monitoring programme. When compared with

morphological identification, metabarcoding performed better across all samples with detecting 57%

more taxa on average. All four primer sets, developed specifically for freshwater macrozoobenthos,

performed equally good. In addition, we showed that sample presorting into size classes increases taxa

detection rates and decreases the already low sequencing costs of about 110 € per sample up to 5 fold.

Finally, we explored modifications of the classical metabarcoding protocol to generate data with

haplotype level accuracy.

Despite these advantages there are several remaining challenges, e.g. gaps and/or incorrect entries in

reference databases as well as a lack of convergence in laboratory and bioinformatic pipelines. While

the latter aspects can be solved by molecular ecologists and bioinformatics, there is a growing need to

team-up with expert taxonomists and field biologists to describe newly identified species, assign

biological traits and taxonomically curate the still error-prone DNA barcoding databases. While we

here could show that metabarcoding is a suitable alternative to morphology-based identification of

macroinvertebrates, the full potential of DNA-based methods can only be unlocked in an

interdisciplinary network of researchers as well as of stakeholders.


Connecting metabarcoding with High Performance Computing:

Towards more accurate molecular based inventories

A. Franca,b

, J.M. Frigerioa,b

, P. Chaumeila,b

, E. Chancerela,c ,

F. Salina,c

, A. Bouchezd, F. Rimet

d,

M. Kahlerte

a UMR BIOGECO, INRA, Université de Bordeaux, 69 route d'Arcachon, 33610 Cestas, France

b PLEIADE Team, INRIA Bordeaux Sud-Ouest, 351, Cours de la Libération, 33405 Talence, France

c PGTB, Genome Transcriptome Facility of Bordeaux, INRA, Université de Bordeaux, 69 route d'Arcachon, 33610 Cestas,

France

d UMR CARRTEL, INRA, Université Savoie Mont Blanc, 74200 Thonon-les-bains, France

e SLU, Institutionen för vatten och miljö, Box 7050, 75007 UPPSALA, Sverige

Taxonomic assessment knowing a sequence of a given marker is standard. First a reference database is

built where the same marker is sequenced on a collection of well known specimen. Second, the

sequence of a query is mapped on the references, and the taxon of the best hit is transferred to the

query if it has a sufficiently high quality score. Many heuristics have been developed for

environmental samples because the limiting factor is the complexity of the computations. We have

built an as exact as possible algorithm which compares all queries with all references, selects a

neighborhood of a query in the reference database, and decides a taxonomic annotation, or not,

knowing the distances and taxa in the neighborhood. As it requires intensive computation, especially

for computing distances from SW score, we have parallelized the algorithm on HPC infrastructures

(Turing, French National HPC infrastructure, 16 384 CPUs) to keep calculations exact without

heuristics. We have applied this pipeline to (i) a set of ten environmental samples from Leman lake (ii)

a set of twenty environmental samples from Swedish rivers. We have compared the inventories built

this way with the ones with same data, built with Mothur. Our preliminary result is that Mothur yields

more false positive, and diagno-syst is more stringent (less false positive, some false negative). We are

currently porting this pipeline for public use on the cloud of European Grid Infrastructure (EGI),

within the project EOSCPilot. We present these results, and discuss possible perspectives.

Oral presentations Session WG3+4: DNA-based biomonitoring – Field and Lab Protocols / Data Analysis and

Storage

Management of metabarcoding data using online platform PlutoF

U. Kõljalga,b

, K. Abarenkovb, A. Zirk

b, K. Panksep

c, A. Savchenko

a

a 40 Lai Str., Institute of Ecology and Earth Sciences, University of Tartu, 51005 Tartu, Estonia b 46 Vanemuise Str., Natural History Museum, University of Tartu, EE51014 Tartu, Estonia c Centre for Limnology, Estonian University of Life Sciences, Rannu, Estonia

We will demonstrate online workflow for the metabarcoding data management. Workflow starts from

project planning by uploading project and protocol descriptions, project members, their access rights

and other associated data. Every project may have many subprojects. The next step is defining

sampling areas of polygon, transect or point type on the map. Hierarchical structure of sampling areas

and events can be set up. Sampling areas may include one or many samples. Habitat description and

associated traits can be linked to the sampling events and samples. The system accepts different

material sample types including water, soil, host-associated, etc. Depending on sample type different

traits can be attributed to the samples. For the water samples measurements on pH, temperature,

salinity, potassium, etc can be uploaded. Project leader may define different access rights to every

sampling area and sample if necessary. Metabarcoding studies allow uploading representative

sequences for every OTU from every sample. While raw data is recommended to be stored in SRA or

other repositories, OTU representative sequences and sample metadata can be stored in PlutoF making

these data easily searchable and accessible in standardized format. Links to raw data can be specified

and shown in project view. Storing OTU representative sequences and associated data in a relational

database makes it easy to: 1) export molecular and traits data in different formats needed by analysis

tools; 2) share data between projects and studies in order to run integrated analyses; 3) link OTU

based species with other types of taxon occurrences; 4) make OTUs available for the DNA-based

taxon keys; etc. Our online workflow uses the following global standards: DwC, MCL, MIxS, EML,

and can be accessed at https://plutof.ut.ee .

Oral presentations Session WG3+4: DNA-based biomonitoring – Field and Lab Protocols / Data Analysis and

Storage

The ‘next generation’ of molecular monitoring, surveillance,

and quality assurance for aquatic systems:

A North American perspective

A.R. Mahon

Department of Biology, Institute for Great Lakes Research, Central Michigan University

Detection of rare species (e.g., threatened and endangered, invasive) routinely relies upon the ability

to detect and monitor low densities of organisms with patchy distributions. This can be particularly

challenging in aquatic ecosystems, where even large organisms can be difficult to directly observe or

capture. North American has been fighting this battle with a number of species, including non-native

fishes and invertebrates throughout the Laurentian Great Lakes ecosystem. Traditional sampling

methods have proven to only be effective in a narrow range of habitats, principally shallow water with

slow water velocities, or moderate to high visibility. Additionally, monitoring established populations

of species and determining their population structure, origin, and size at low densities has proven

difficult. Genetic and genomic methods have the potential to overcome many of the constraints posed

by traditional aquatic monitoring and detection gear. However, despite the promise of DNA-based

monitoring methods, the adoption of these tools and their acceptance in decision-making frameworks

remains challenging. The current status of DNA-based tools for aquatic invasive species monitoring

in North America and the impediments to their effective translation into management contexts will be

discussed. Potential sources of uncertainty associated with molecular technologies, possibilities for

limiting that uncertainty, and the future use of these tools for invasive species surveillance in North

American waters will be presented.


Standardized eDNA detection limits and sampling strategies in rivers

B. Thalingera, D. Sint

a, D. Kirschner

a, C. Moritz

b, R. Schwarzenberger

b, J. Wanzenböck

c,

M. Traugotta

a Institute of Ecology, University of Innsbruck, Austria b ARGE Limnologie GesmbH, Hunoldstr. 14, 6020 Innsbruck c Research Institute for Limnology of the University of Innsbruck, Mondsee, Austria

Environmental DNA (eDNA) has been successfully used to detect rare species occurring in low

population densities as well as to identify dispersion ranges of invasive or endangered species. Even

though this molecular approach generally outperforms conventional methods of species detection and

identification like electrofishing or hand-searching, the diversity of the molecular methods itself leads

to variable DNA detection limits depending on the applied method. Especially when investigating

species occurring in low densities, knowledge of molecular detection limits and an optimized sampling

strategy are crucial for any eDNA-based work.

Here, we present a standardization method to determine the minimum amount of target DNA needed

for positive amplification in PCR and an optimized sampling strategy for the detection of remote

eDNA quantities in small to medium sized rivers and streams. In a field-experiment, species-specific

primers, standardized for successful target DNA amplification from 25 double strands, were used to

detect eDNA stemming from 200 g of caged fish in an Alpine, glacier-fed stream. At low discharge

situations fish-eDNA could be detected more than 1 km downstream. However, during phases with

turbid, high discharge, detections were only possible in close proximity to the source. For the detection

of species with low population densities it is thus advisable to sample sufficiently mixed water well

downstream of potential habitats during low discharge situations, but in their close proximity in case

of high runoff.


Comparison of capture and storage methods for aqueous macrobial eDNA

– advantage of enclosed filters

M. Hellströma,b,c

, A.R. Evansd, D. Halfmaerten

e, S.W. Knudsen

a, M.E. Sengupta

a, S.S.T. Mak

a,

E.E. Sigsgaarda, J. Spens

a,b,e

a Centre of GeoGenetics, Natural History Museum, Copenhagen, Denmark b Molecular Ecology and Fish Genetics Laboratory, University of Bangor UK

e Research Institute for Nature and Forest (INBO) Geraardsbergen, Belgium f Department of Veterinary Disease Biology, University of Copenhagen, Denmark

b Present address: AquaBiota Solutions Sweden/ Stockholm University, Sweden c Present address: Department of Ecology, Environment and Plant Sciences (DEEP) Stockholm University, Sweden f Swedish University of Agricultural Sciences, Umeå, Sweden

Aqueous environmental DNA (eDNA) is an emerging efficient non-invasive tool for species inventory

studies. To date the number of capture, storage and extraction methods are almost as diverse as the

number of scientists working on eDNA, making studies less comparable among each other. To

maximize performance of downstream quantitative PCR (qPCR) and next-generation sequencing

(NGS) applications, quality and quantity of the starting material is crucial, calling for optimized

capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA

capture/storage have tested precipitation and ‘open’ filters. However, practical ‘enclosed’ filters which

reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter

capsule (Sterivex GP polyethersulfone, pore size 0·22 µm, hereafter called SX) with commonly used

methods.

SX yielded higher amounts of total eDNA along with lower Cq-values than previously used capture

methods. DNA integrity of SX samples did not decrease significantly after two weeks of storage.

Adding preservative before storage improved SX results. Bottle necks are filtering in the field and

filtering very turbid waters.

We recommend SX filters as sampling macrobial eDNA. Preserved SX capsules may be stored at

room-temperature for at least two weeks without significant degradation. Reduced handling and less

exposure to outside stress compared with other filters may contribute to better eDNA results. SX

capsules enable eDNA sampling in remote and harsh field conditions as samples can be

filtered/preserved on site.

Recent examples of practical applications and results of the method will be presented.

http://ivs.ku.dk/english/


Large comparison between WFD-monitoring fish and environmental

DNA metabarcoding

J. Herdera, J. Kranenbarg

a, A. Valentini

b, T. Dejean

b

a Reptile, Amphibian and Fish Conservation the Netherlands (RAVON), Post Office Box 1413, 6501 BK Nijmegen,

Netherlands b SPYGEN, Savoie Technolac, 17 rue du Lac Saint-André, 73370 Le Bourget du Lac, France

The Water Framework Directive (WFD) is an European Union directive which commits European

Union member states to achieve good qualitative and quantitative status of all water bodies. This status

is determined by ecological and chemical criteria, including fish. Periodically monitoring of fish plays

a crucial role in the evaluation of the status of waterbodies.

In the Netherlands we have been working on eDNA-metabarcoding for fish in relation to the WFD

since 2012. In 2015 a very large study was carried out in which traditional fish monitoring for the

WFD was compared with the environmental DNA (eDNA) metabarcoding approach. On 55 locations

all over the Netherlands eDNA samples were collected in parallel to the traditional monitoring

following WFD protocols (electrofishing/sein nets/fykes etc).

The results are promising. Per location we found a (far) higher number of species with eDNA-

metabarcoding compared to the traditional WFD monitoring. In our presentation we will show the

outcomes for different water types (flowing water, linear stagnant water and lakes). We will present

species specific differences in detection with both methods. We discuss some unexpected detections

with eDNA-metabarcoding and present suggestions on how to deal with them.

Finally we will show that the eDNA-metabarcoding method was reproducible in terms of proportion

of eDNA-sequences per species (as well the field sampling as the analyses). Thereby showing

potential for producing reliable semi-quantitative results and the potential use of this approach for the

WFD.


GenoTrail: A new tool for the monitoring of water distribution networks

O. Thronickera, J. Rentsch

a, U. Szewzyk

a

a Blue Biolabs GmbH, Datalyze Solutions GmbH, TU Berlin, Germany

Genotrail is a software based analysis tool, which combines innovative data collection with effective

visualization options and enhances existing data sets with the results of genetic analyses. This

approach offers a completely new perspective on a range of sector-specific issues, simplifies the

processing of diverse inventory data and allows for a seamless evaluation of complex data sets.

While current microbiological monitoring systems in the water sector focus on discriminating between

E.coli and coliform bacteria, Genotrail integrates an identification of microorganisms at the genus

level, providing valuable information about root causes and underlying risks of hygienic

contamination.

When a contamination is detected in the distribution network a clear data visualization is of paramount

importance in the assessment and planning of corrective measures. Genotrail produces a

comprehensive graphical display of all relevant geo and genetic data, providing totally new insights

into geo-spatial relationships between discrete incidents. This service integrates seamlessly into

company-specific data infrastructure and enhances the visualization of pre-existing data sets. With the

help of flexible filter settings, any type of data can be processed effortlessly.

Genotrail is designed for water suppliers, veterinary and public health offices as well as healthcare

providers and environmental organizations. Since the unique integration of genetic information allows

for completely new insights during analysis of microbiological data, other applications in the

environmental sciences are already being evaluated.

Poster presentations

Poster Presentations (alphabetically sorted by first author surnames)


Taxonomy-free molecular diatom index

for high-throughput DNA biomonitoring of watercourses

L. Apothéloz-Perret-Gentila, A. Cordonier

b, F. Straub

c, J. Iseli

c, P. Esling

d, J. Pawlowski

a,e

a Department of Genetics and Evolution, University of Geneva, Switzerland b Water Ecology Service, Department of Territorial Management, Canton of Geneva, Switzerland c PhycoEco, La Chaux-de-Fonds, Switzerland d IRCAM, UMR 9912, Université Pierre et Marie Curie, Paris, France e ID-Gene Ecodiagnostics, 1228 Plan-les-Ouates, Switzerland

Current biodiversity assessment and biomonitoring are largely based on morphological identification

of selected bioindicator taxa. Recently, several attempts have been made to use eDNA metabarcoding

as an alternative tool. However, until now, most of applied metabarcoding studies were based on

taxonomic assignment of sequences that refers to morphospecies ecology. Usually, only a small

portion of metabarcoding data can be used due to limited reference database and lack of phylogenetic

resolution. Here, we investigate the possibility to overcome these limitations by using a taxonomy-free

approach that allows computing molecular index directly from eDNA data without any reference to

the morphotaxonomy. As a case study, we use the benthic diatoms index, commonly used for

monitoring the biological quality of rivers and streams. We analysed 87 epilithic samples from Swiss

rivers, which ecological status was established based on microscopic identification of diatom species.

We compared the diatom index inferred from eDNA data obtained with or without taxonomic

assignment. Our taxonomy-free approach gives promising results by providing a correct assessment in

77% of examined sites. The main advantage of our approach is that almost 95% of OTUs could be

used for index calculation, compared to 35% in the case of the taxonomic assignment approach. Its

main limitations are the under-sampling and need to calibrate the index on microscopic assessment of

diatoms communities. However, once calibrated, the taxonomy-free molecular index can be easily

standardized and applied in routine biomonitoring, as a complementary tool allowing fast and cost-

effective assessment of the biological quality of watercourses.


SYNAQUA: A French-Swiss program to modernize the environmental biomonitoring of

Lake Geneva and transboundary rivers

A. Boucheza, A. Cordonier

b, I. Domaizon

a, B.J.D. Ferrari

c , S. Jacquet

a, E. Lefrançois

d , A.L. Mazenq

e ,

A. Pawlowskaf, L. Perret-Gentil

g, F. Rimet

a, J.F. Rubin

h, E. Servoli

f, D. Trevisan

a, R. Vivien

c,

J. Pawlowskig

a UMR CARRTEL, INRA, Université Savoie Mont Blanc, 74200 Thonon-les-bains, France b Direction Générale de l'Eau, canton de Genève, avenue Sainte-Clotilde 25, CP 78, 1211 Genève 8, Switzerland c Centre Ecotox, Eawag/EPFL, EPFL-ENAC-IIE-GE, station 2, 1015 Lausanne, Switzerland d Asconit Consultants, Parc Scientifique Tony Garnier, 6-8 Espace Henry Vallée, 69366 Lyon Cedex 07, France e Asters, Conservatoire d’Espaces Naturels de Haute-Savoie, 84 route du Viéran, PAE de Pré Mairy, 74370 Pringy, France f ID-Gene Ecodiagnostics, c/o fondation Eclosion, 14 chemin des Aulx, 1228 Plan-les-Ouates, Switzerland g UNIGE, 30 quai Ernest Ansermet, 1211 Genève 4, Switzerland

h Fondation Maison de la Rivière, Chemin du Boiron 2, 1131 Tolochenaz, Switzerland

Rivers and lakes play a major role in the environment by providing not only habitat for fauna and

flora, but also improving the landscape and therefore the quality of life. The effectiveness of

environmental protection measures rests on the early and precise identification of pressure points,

which should be facilitated by environmental genomics. To implement measures to preserve or restore

aquatic areas, the SYNAQUA project proposes to use the genetic tools based on the recognition of

bio-indicator organisms present in the aquatic environment directly from their DNA. The method of

environmental DNA analysis, will be tested by Swiss and French scientists in collaboration with

public and private stakeholders on two groups of bioindicators commonly used for water quality

assessment: diatoms and oligochaetes.

The aim is to bring together scientists, environment managers as well as public and private partners in

Switzerland and France to develop and apply this DNA-based broadband approach to biomonitoring

of regional aquatic ecosystems. Collaboration between public and private stakeholders will help to

develop a robust and reliable tool that is tailored to their needs and will strengthen the current practice

of environmental diagnosis. The innovative tools that will be developed in synergy in France and

Switzerland should make it possible to respond to the need for better monitoring and protection of the

regional heritage of aquatic environments in a rapidly changing area subject to multiple pressures.


A DNA (meta)barcoding approach to assess changes in seabed ecosystems related to

human-induced pressures

D. Lisaa,|

, A. De Backera, S. Maes

a, G. Van Hoey

a, A. Haegeman

b, T. Ruttink

b, J. Wittoeck

a,

H. Hillewaerta, C. De Tender

a, K. Hostens

a

a Institute for Agricultural and Fisheries Research (ILVO), Animal Sciences Unit, Aquatic environment and quality,

Ankerstraat 1, 8400 Oostende, Belgium b Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Growth and Development, Caritasstraat 39,

9090 Melle, Belgium

| [email protected]

Biodiversity is influenced by a wide range of environmental and human-induced pressures. Physical

disturbance of the seabed is probably the best known pressure to affect benthic ecosystems. The aim of

this work is to evaluate the potential of DNA (meta)barcoding to assess human-induced changes in the

seabed ecosystem. We focus on two benthic ecosystem components: bacteria and macrobenthos, in

relation to sand extraction and dredge disposal in the Belgian part of the North Sea.

The bacterial community composition in the sediment was assessed by amplicon sequencing of the

V3-V4 fragment of the 16S rRNA gene using Illumina technology. Disturbance of the seabed due to

sand extraction activities on the Buiten Ratel sand bank revealed a significant impact on the bacterial

communities.

Macrobenthos is recognized as a good biological indicator of changes in marine ecosystems. Excellent

morphological identification tools exist, but accurate DNA-based species identification is lacking. COI

and 18S fragments of 70 macrobenthos species were amplified using several barcoding primers and

Sanger sequenced to evaluate which amplicon provides the best taxonomic resolution. First, we

performed amplicon sequencing of the V4 fragment of the 18S rRNA gene on DNA extracts of

individual species. In a following step, we used artificial mixtures of various species to assess the

effectiveness of the primers in detecting single species in mixed samples.

This work is ongoing and will be used to establish a complete North Sea DNA barcode reference

database for bacteria and macrobenthos, in close cooperation with other institutes along the North Sea.


The Norwegian Barcode of Life Network (NorBOL)

T. Ekrema,|

, I.G. Alsosb, A. Johnsen

c, E. Willassen

d, A.M. Aspaas

a , M.K. Føreid

b, K. Kongshavn

d,

G. Marthinsenc

a NTNU University Museum, Department of Natural History, Trondheim, Norway

b Tromsø University Museum, Tromsø, Norway

c University of Oslo, Natural History Museum, Oslo, Norway

d University Museum of Bergen, Department of Natural History, Bergen, Norway

| [email protected]

NorBOL (www.norbol.org) was formed in 2007 as a national network to 1) advance barcoding of

Norwegian and Arctic biodiversity, 2) raise funding, 3) curate barcode reference material, 4)

coordinate and initiate new barcoding projects, and 5) increase public awareness of DNA barcoding

and barcoding results in Norway. NorBOL is a regional node within iBOL, with a particular

responsibility for Polar Regions. NorBOL is coordinated by the NTNU University Museum in

Trondheim and connects 17 institutions, including all four major natural history museums as well as

all major biological research institutes in Norway. Despite strong support among research institutions,

substantial external funding was only first achieved in 2012 through a grant from the Norwegian

Biodiversity Information Centre. Further funding was obtained in 2014 from both the Research

Council of Norway and the Norwegian Biodiversity Information Centre. Since then, barcoding

progress of the Norwegian fauna, flora and fungi has increased and the Barcode of Life Data Systems

database currently holds more than 60 000 published sequence records (34 000 DNA barcodes) of

more than 11 700 species from Norway. The goal for NorBOL is to barcode 20,000 species by the end

of 2018. NorBOL currently targets barcoding of terrestrial arthropods, plants, lichens, fungi, marine

invertebrates, fish, and material from bioinventory projects supported by the Norwegian Taxonomy

Initiative. We collaborate with Swedish and Danish institutions on sampling of marine invertebrates,

earthworms and insects. As the standard barcodes have limited taxonomic resolution in many vascular

plant groups, we are engaged in testing if low coverage shotgun sequencing of herbarium material is

useful to obtain the full plant plastid genomes.


DNA metabarcoding on estuarine meiofauna:

testing the effect of sample’s amount and primer-pairs

M. Faisa,b

, S. Duartea, R. Sousa

a,c, M. Hajibabaei

d, C.A. Canchaya

b, F.O. Costa

a

a Centre of Environmental and Molecular Biology, University of Minho, Portugal b Department of Biochemistry, Genetics and Immunology, University of Vigo, Spain c Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Portugal d Centre for Biodiversity Genomics & Department of Integrative Biology, University of Guelph, Canada

In this study, we investigated the impact of a) the amount of sediment sample used for DNA extraction

and b) the primer-pairs used in PCR amplification, for the taxonomic profiling of estuarine meiofauna

by high-throughput sequencing. We tested different volumes of intertidal sediments (0.63, 2.50 and

10.0g) collected in the Lima estuary (Portugal), using MoBio® DNA Isolation kits. Three PCR

amplifications were performed, targeting V1-V2 (~400bp), V4 (~400bp) and V9 (~250bp) regions of

the 18S rDNA. Amplicons were sequenced in the Illumina-MiSeq platform and the resulting forward

and reverse reads were sorted-out using customized operating procedures in mothur. Afterwards,

processed sequences were OTU-clustered and aligned to the SILVA database using BLAST. Our

results showed that we recovered the highest number of OTUs in the 10.0g sediments for every

primer-pair used. Among these, we obtained the highest number of OTUs (>6000) with V4 primers

and, correspondingly, the highest number of OTUs assigned to taxa (>600), followed by V1-V2 and

V9. About 20% of sequences could not be taxonomically assigned. OTUs recovered using V9 primers

were the only dataset that approached saturation in the OTUs rarefaction curves. Therefore, the region

V4 appeared to recover the highest number of taxa, while the V9 the lowest. However, we also found

considerable differences in the number of OTUs for distinct meiofaunal groups detected using each

primer-pair. This suggested that the amount of starting material for DNA extraction and simultaneous

use of several target regions should be considered/optimized for comprehensive assessment of

estuarine meiofauna communities.


Designing optimized biotic indices using eDNA and metabarcoding

in a riverscape under multiple stressors

A.F. Filipea,b

, F. Martinsa, M.F. Magalhães

c, V. Hermoso

d,e, M. Monaghan

f, J. Paupério

a, S. Ferreira

a,

J. Veríssimoa, P.C. Alves

a,b, S. Jarman

a, P. Beja

a,b

a CIBIO/InBio – Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do Porto, Vairão, 4485-

661 Vairão, Portugal b CEABN/InBIO, Centro de Ecologia Aplicada “Prof Baeta Neves”, Instituto Superior de Agronomia, Universidade de

Lisboa, Lisboa, Portugal

c Ce3C, Centre for Ecology, Evolution and Environmental Changes, University of Lisboa Campo Grande, Bloco C2, 1749-

016 Lisboa, Portugal d Centre Tecnològic Forestal de Catalunya, Crta. Sant Llorenç de Morunys, Km 2. 25280, Solsona. Lleida, Spain e Australian Rivers Institute and Tropical Rivers and Coastal Knowledge, National Environmental Research Program

Northern Australia Hub, Griffith University, Nathan, Queensland, 4111, Australia f Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany

Currently environmental DNA (eDNA) and metabarcoding are potentially useful tools for monitoring

water ecological status. The ability of these tools to generate important data to inform managers is

now widely recognized, but its widespread application has been delayed by the lack of standardised

approaches, from field sample collections, to lab sequencing and bioinformatics. Specifically,

knowing how DNA traces vary under gradients of environmental conditions and multiple stressors

remain open questions.

Here we aim to illustrate the workflow developed under the FRESHING project to delineate optimized

water ecological indexes based on macroinvertebrates and using eDNA and metabarcoding tools. The

watercourses of Douro Basin (Spain and Portugal) were selected as a case study because they hold a

variety of climates, habitats, and human stressors. Firstly we will gather datasets of environmental

conditions and multiple stressors from online available datasets. Then we will use GIS and ordination

techniques to represent the environmental and multiple stressors gradients of the Douro Basin, and

further select eDNA sampling sites that represent the whole gradients in the basin. We will show how

this approach allows testing the sensitivity and reliability of biotic metrics that respond to gradients of

multiple stressors, in order to maximise the quality of information obtained while minimising the

costs. Our approach will allow to optimize biotic indices using eDNA and metabarcoding tools, so that

we can move to a new generation of freshwater monitoring plans applied at the riverscape scale.


Designing an optimized monitoring program for early detection of

invasive aquatic species by combining risk maps and eDNA

A.F. Filipea,b

, , M.F. Magalhãesc, V. Hermoso

d,e, M. Monaghan

f, F. Martins

a, J. Paupério

a, S. Ferreira

a,

J. Veríssimoa, P.C. Alves

a,b, S. Jarman

a, P. Beja

a,b


661 Vairão, Portugal b CEABN/InBIO, Centro de Ecologia Aplicada “Prof Baeta Neves”, Instituto Superior de Agronomia, Universidade de

Lisboa, Lisboa, Portugal

c Ce3C, Centre for Ecology, Evolution and Environmental Changes, University of Lisboa Campo Grande, Bloco C2, 1749-

016 Lisboa, Portugal d Centre Tecnològic Forestal de Catalunya, Crta. Sant Llorenç de Morunys, Km 2. 25280, Solsona. Lleida, Spain e Australian Rivers Institute and Tropical Rivers and Coastal Knowledge, National Environmental Research Program

Northern Australia Hub, Griffith University, Nathan, Queensland, 4111, Australia f Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany

Environmental DNA (eDNA) and metabarcoding are potentially useful tools for detecting rare species,

including threatened and recently introduced. While it is now clear these tools have the ability to

generate important data to inform managers, moving to an approach at the landscape scale has been

hindered due to the lack of standardised approaches, starting with field planning. This includes

knowing where and when these tools can be actually more efficient and cost effective than those

traditionally used.

Here we illustrate the workflow build under the FRESHING project to delineate an optimized

monitoring network of sites for the early detection of freshwater invasive species. For a list of

potentially new or spreading invaders, we propose to gather data in GIS layers of environmental

conditions, propagule pressures, and biotic relationships. Next, maps of habitat suitability (built using

species distribution modelling), pathways, and biotic facilitation will be built. We will then prioritise

the spatial allocation of a set of monitoring sites to maximise the capacity to early detect the presence

of multiple non-native species, while minimising costs of monitoring. The eDNA protocols for

detecting new occurrences will be implemented in these sites with high risk of invasion. The Douro

Basin (Spain and Portugal) will be used as a case study because there has been many intentional

aquatic introductions especially since the 1950s. With this workflow we intend to optimize the early

detection of non-natives so that management actions for the invasive can be rapidly implemented.


Species diversity and origin of Chironomidae from geologically young lake

and its old spring system

P. Gadawskia,|, B. Rossaro

c, V. Mereghetti

b, M. Grabowski

a, M. Montagna

b

a University of Lodz, Department Invertebrate Zoology and Hydrobiology, Banacha 12/16, 90-237 Lodz, Poland b University of Milan, Department of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy, Via

Celoria 2, 20133 Milano, Italy c University of Milan, Department of Food, Environmental and Nutritional Sciences, Via Celoria 2, 20133 Milano, Italy

| [email protected]

We used non-biting midges (Diptera; Chironomidae) as a model group to investigate the evolution of

semi-aquatic biota in the Skadar Lake system, a well-known hotspot of freshwater biodiversity

composed of the geologically very young lake Skadar (originated 1200 BP) and its old system of

springs (probably of Pliocene age, up to 5 Ma).

The following relevant evolutionary questions are addressed:

Q1) What is the biogeographic composition of the chironomid community in the Skadar Lake system?

Q2) Was the old spring system a source for colonization of the Skadar Lake?

Two basic tasks are specified. First is to assess the species composition in various habitats of this

almost unexplored basin. Second is to explore the fundamental processes shaping locally the

distribution of Chironomidae. These objectives will be achieved via DNA metabarcoding and the

high-throughput RAD sequencing. Chironomid larvae are very important for water quality

assessments and they remain underutilized since the current sorting techniques cannot deliver species-

level resolution at a reasonable cost. In addition, most of European species is described based only on

the adult male stage, leaving out larvae that would be most useful in bio-assessment. The development

of an accurate reference library of cytochrome oxidase subunit 1 (cox1) gene sequences for midges

inhabiting the Skadar Lake system, will be of great interest not only for basic science but also for the

aim of conservation purposes.

At present we already have cox1 gene sequences for 120 specimens (82 species). Pupal exuviae and

imago from expeditions performed in 2014 and 2015 were already identified to species level.

Taxonomic identification revealed that 904 pupal exuviae represents 102 species. Concerning to adult

forms 86 species were reported from almost 5500 identified individuals (100% of collection). In

almost all cases, obtained sequences confirmed taxonomic identifications.


Evaluating lab and field methods for monitoring invasive crayfish

based on environmental DNA (eDNA)

A.N. Geertsa, P. Boets

b,c, S. Van den Heede

a, P. Goethals

b, C. Van der heyden

a

a Hogeschool Gent / University College Ghent. Faculty of Science and Technology - Valentin Vaerwyckweg 1, 9000 Ghent,

Belgium b Ghent University. Faculty of Bioscience Engineering – Coupure links 653, 9000 Ghent, Belgium

c PCM Provincial Centre of Environmental Research. Godshuizenlaan 95, 9000 Ghent, Belgium

Environmental DNA (eDNA) techniques are becoming increasingly popular in conservation and

invasion biology, especially for monitoring and early detection of rare, endangered or invasive species.

An exponential increase in divergent methods regarding eDNA collection and analysis has been

observed, which leads to mixed success in detection efficiency in studies on aquatic invertebrates. In

this study, we tested and compared three DNA extraction methods using crayfish as model species.

Based on existing and newly developed primers we were able to identify two invasive crayfish species

(Procambarus clarkii and Orconectes limosus) from tissue and filtered eDNA samples from water of

aquaria kept in the laboratory as well as water from natural ponds. Both species could be positively

identified in field and laboratory samples, though effectiveness differed greatly. Results in our study

seemed to be highly dependent on primer choice, DNA extraction method, chosen species, and the

type of sample (tissue or filter). Our results showed the MasterPure extraction kit consistently

provided the most reliable results for tissue and for filtered eDNA samples for both species. The

results are promising, but also highlight the necessity for a standardized protocol for each step of the

eDNA process.


Microorganisms associated with the groundwater amphipod Crangonyx islandicus

R. Gudmundsdottira, B.K. Kristjánsson

b, V.Þ. Marteinsson

c, S. Pálsson

a

a University of Iceland b Holar University Collage c Matís ltd. - Icelandic Food and Biotech R&D

Two endemic subterranean freshwater amphipod species were recently discovered in Iceland,

Crangonyx islandicus and Crymostygious thingvallensis. These species are found in the groundwater

springs in lava fields within the volcanic active zone. Genetic patterns within C. islandicus show

different monophyletic groups and even cryptic species in different geographic areas which indicates

that the group has survived repeated glaciations in Iceland. Little is known of this groundwater

ecosystem and nothing is known about its other species or the food source of the amphipods. To

explore this system further we studied the micro-biotic community in the groundwater, the hyporheic

zone and in the amphipods by targeting 16S RNA for Bacteria and Archaea and 18S RNA for ciliates

using the Illumina MiSeq high throughput sequencing technique. Samples were obtained throughout

the distribution range of C. islandicus. Distinct species composition of bacteria and ciliates was found

in the amphipod samples while the amplification of the Archaea amplicons failed to yield results. The

results from the groundwater samples gives a good starting point for future research of this isolated

groundwater ecosystem within the volcanic zone of Iceland, which appears to be based on

chemoautotophic bacteria.


Two new endemic species of freshwater shrimps from the ancient Skadar Lake system

revealed with DNA barcoding

A. Jabłońskaa, T. Mamos

a, A. Zawal

b, V. Pesic

c, M. Grabowski

a

a Department of Invertebrate Zoology & Hydrobiology, University of Lodz, Poland b Department of Invertebrate Zoology and Limnology, University of Szczecin, Poland c Department of Biology, University of Montenegro, Montenegro

Skadar Lake system consists of lake, marshlands and numerous karst springs. It is also known as a

local biodiversity and endemism hotspot. However the whole system is recognised as being ancient,

recent studies revealed that the lake itself is geologically very young and it originated ca. 1200 years

ago with overflowing of the marsh and spring area by the Drin River. Accordingly, the local endemics

are associated rather with the springs than with the lake itself. During our recent studies upon aquatic

invertebrates of the lake system, we investigated also local shrimp species, classified based on

morphology as Atyaephyra desmaresti (Atyidae) and Palaemon antennarius (Palaemonidae). Results

of molecular studies with use of the COI barcode marker contradicted such classification and

suggested these may be two species new to science. In case of the Atyaephrya species, re-analysis of

morphological characters allowed to define traits differing it from other congeners. Phylogenetic

analysis based on the COI marker revealed that the new species is most closely related to the south-

Balkan A. stankoi and A. thyamisensis, from which it diverged probably in early Pliocene. The species

may be a relic of the old marshlands occupying the basin before the present lake. Genetic investigation

of the Palaemon species revealed that the Skadar Lake population is a sister cryptic species to P.

antennarius from the Apennine Peninsula. The two diverged probably in early Pleistocene.


Investigating the dispersal potential of the rheophilic Rhine sculpin

(Cottus rhenanus) in restored streams of the Emscher River catchment

from an eDNA perspective

G. Jacobs

a,|, T.-H. Macher

a,b, J.-N. Macher

a, F. Leese

a

a University of Duisburg-Essen, Aquatic Ecosystem Research, Germany b Ruhr-University Bochum, Germany | [email protected]

For over 4.5 billion Euros, the river Emscher is currently converted from an open sewage system to an

unpolluted low land stream. This provides an ideal test case for restoration ecology. The aim of our

study is to assess the potential of environmental DNA (eDNA) analyses to study restoration and

recolonization success of a fish species in the Emscher catchment (Germany, North Rhine-

Westphalia). As a suitable study object we chose the Rhine sculpin (Cottus rhenanus) – a small,

rheophile freshwater fishinhabiting streams in the northern River Rhine catchment. The Rhine sculpin

is an indicator species for stream structure and has a limited dispersal potential. Even small horizontal

barriers that can easily be surpassed by fish with greater migration potential such as trouts are

supposed to be severe migration barriers for the Rhine sculpin. To understand the impact of horizontal

stream barriers on recolonization success of the Rhine sculpin, eDNA will be used to detect the stream

upward migration of the resettled specimens in a restored stream. In a first step species-specific COI

primer-pairs are developed for the eDNA approach. The parallel second stage includes mapping of

stream morphology, in particular barriers in the restored stream and the design of a suitable sampling

strategy. The Rhine sculpin resettlement success is then monitored via eDNA samples from the

defined sample locations. The final step is a qualitative comparison of conventional (e.g. electronical

fishing, net fishing) and modern molecular monitoring strategies in a large-scale analysis of the

Emscher catchment.


REBIDA – Regional Database of Biodiversity

B. Kalamujić Stroila, L. Lasić

a, S. Dorić

a, J. Hanjalić

a, N. Pojskić

a

a University of Sarajevo-Institute for Genetic Engineering and Biotechnology, Zmaja od Bosne 8 (Kampus), 71000 Sarajevo,

Bosnia and Herzegovina

Successful solutions to serious problems regarding environmental protection, management and

research rely on the efficient use of comprehensive and reliable information on the nature that

surrounds us. Therefore, the availability of these information, as one of the most valuable assets, is

becoming ever more important in our society. A number of countries that successfully implement

conservation and revitalization projects have different types of databases on these resources, often

established and maintained at the national level. However, more often than not, transitional countries

lack any centralized, nationally funded database that could be used in decision making process. In such

cases, databases established in academic and scientific institutions could serve as valuable starting

points. REBIDA – Regional database of biodiversity is web oriented, open-access, searchable and

comprehensive database on wild natural resources of Bosnia and Herzegovina, established in the

Institute for Genetic Engineering and Biotechnology, University of Sarajevo. It is the only such

database in the country, consisting of three segments: tissue database, DNA database and digital

genetic database on plant, animal and human samples.


A short test on the impact of sequencing platform and marker

on marine macrobenthos identification

B.R. Leitea, C. Hollatz

a, J. Lobo

a,b, H. Froufe

c, C. Egas

c, F.O. Costa

a

a CBMA- Centre of Molecular and Environmental Biology, Biology Department, University of Minho, Campus de Gualtar,

4710-057 Braga, Portugal b MARE—Marine and Environmental Sciences Centre, Department of Environmental Sciences and Engineering, Faculty of

Science and Technology, Lisbon New University, 2829-516 Monte de Caparica, Portugal c Genoinseq, UC-Biotech, Center for Neurosciences and Ceel Biology, Biocant Park, 3060-197, Cantanhede, Portugal

In a previous study we used two mock marine macrobenthic communities (MC1 and MC2), each

containing 21 species from three phyla (Annelida, Arthropoda and Mollusca) in different proportions,

to test species detection success using 5 COI primer-pairs on a Roche-454 platform. Here we compare

detection success obtained for the same communities with an Illumina-MiSeq platform using a) one of

the 5 COI primers (ArF2/ArR5 – 310 bp), and b) a primer-pair targeting an alternative marker (V4

region of 18S rDNA – ca. 400 bp). BOLD and SILVA databases were used respectively for taxonomic

assignment of COI reads and 18S OTUs with ≥97% similarity. Compared to the 454, in MiSeq

platform the detection success increased in both communities (MC1: 43% vs. 52%; MC2: 43% vs.

62%), detecting 3 additional species in MC1 and 4 in MC2. Using the V4 region, taxonomic

assignments were possible for 22 and 63 OTUs in MC1 and MC2 respectively. OTUs were assigned to

three phyla in both communities, however species level identification was only attained for one

species (Lepidochitona cinerea). Some taxa were retrieved solely by V4, but a fair number of OTUs

could not be clearly linked to the taxa present in the MCs, including many assigned to algae phyla. In

summary, species detection rates can be improved considerably using deeper sequencing, whereas

COI performance in macrobenthic species detection remained superior to V4 recovery rates. This may

have been caused by lower species discrimination ability of V4, together with poor representation of

marine macrobenthic species in SILVA.


Impact of OTU-clustering on taxonomic richness estimation

in marine macrobenthic communities with known species composition

B.R. Leitea, C. Hollatz

a, J. Lobo

a,b, H. Froufe

c, C. Egas

c, F. O. Costa

a

a CBMA- Centre of Molecular and Environmental Biology, Biology Department, University of Minho, Campus de Gualtar,

4710-057 Braga, Portugal b MARE—Marine and Environmental Sciences Centre, Department of Environmental Sciences and Engineering, Faculty of

Science and Technology, Lisbon New University, 2829-516 Monte de Caparica, Portugal c Genoinseq, UC-Biotech, Center for Neurosciences and Cell Biology, Biocant Park, 3060-197, Cantanhede, Portugal

In a previous study, we used two mock communities (MC1 and MC2), both composed by 21

macrobenthic species, to test the amplification success of 5 COI primer-pairs on a Roche-454

platform. Species detection was made through direct similarity search of individual reads against

BOLD. Here we investigated the impact of OTU-clustering and different clustering thresholds on the

assessment of the taxonomic composition of these experimentally contrived communities. The number

of OTUs produced for a range of clustering thresholds, between 0% and 15% p-distance, was assess

using a 0.5% step. As expected, there was a large variation in the number of OTUs as a function of the

threshold. The optimal threshold for each primer-pair / mock assemblage was determined through a

published graphical method, and was set up at ≥ 97% for subsequent analyses. A representative

sequence of each OTU was then used to search BOLD for taxonomic assignments using 97%

similarity. Compared to the known number of macrobenthic species present in the community (21),

the OTU clustering resulted in an excess of retrieved taxonomic units in all primer-pairs (range 31-

169), except ArF2/ArR5 in MC1. This implies that frequently multiple OTUs were produced for the

same species (e.g. 4 OTUs for Patella aspera). Using communities of known composition we

observed that OTU clustering procedures, even when optimized, might still result in considerable

overestimation of the taxonomic richness. Therefore, to avoid potential artifacts and dispense OTU-

clustering, more effort must be allocated to completion of reference libraries.


Karstic salt wedge estuaries –

an ideal environment for phytoplankton life strategies and species ecology testing

Z. Ljubešića, M. Šimunović

b, B. Gašparović

c, J. Godrijan

c, M. Gligora Udovič

a

a University of Zagreb, Faculty of Science, Department of Biology, Rooseveltov trg 6, 10 000 Zagreb, Croatia b Croatian Agency for the Environment and Nature, Radnička cesta 80, 10000 Zagreb, Croatia c Ruđer Bošković Institute, Division for Marine and Environmental Research, Bijenička 54, Zagreb, Croatia

River Krka is karstic river situated in the central part of the eastern Adriatic coast. The geomorphology

and hydrology of the Krka River enables expansion of freshwater phytoplankton which develops in the

Lake Visovac. The river inflows in the Adriatic Sea forming salt wedge estuary including expansion

Lake Prokljan. Cyclotella species belong to codon B, Reynolds FG, and have a wide tolerance to

various environmental parameters, allowing them to dominate in mesotrophic systems. Therefore, it

provides great potential for quality assessments. The phylogenetic analyses of the Cyclotella showed it

can be split into several genera and C. ocellata to be assigned as type species of a new diatom genus,

Pantocsekiella. Species Pantocsekiella ocellata is a variable species in terms of its morphology and

ecology, therefore a number of studies have been performed to delineate its phenotypic plasticity. P.

ocellata is considered as a species complex together with C. krammeri, P. rossii, P. tripartita, C.

kuetzingiana, P. polymorpha and P. comensis. It is the main descriptor of the diatom community in

Lake Visovac, with accompanying P. polymorpha and Discostella pseudostelligera. In Lake Visovac

P. ocellata reached the highest abundances in July and August (2 x 106 cells L

-1). In the estuary, in

Lake Prokljan P. ocellata was found also dominating the halocline and brackish layer reaching

abundances up to 7,4 x 105 cells L

-1 (reaching up to 98% of the total diatom community). The salinity

stress experiment showed that 42% of P. ocellata cells survive higher salinities after 12h of exposure.

http://www.irb.hr/eng/Research/Divisions/Division-for-Marine-and-Environmental-Research


Effect of eDNA filtration strategies on metabarcoding success

of freshwater macroinvertebrate communities

M. Majanevaa, O. H. Diserud

b, E. Boström

a, S. Eagle

c, M. Hajibabaei

c, T. Ekrem

a

a Department of Natural History, NTNU University Museum, Norwegian University of Science and Technology, NO-7491

Trondheim, Norway b Norwegian Institute for Nature Research (NINA), P.O. Box 5685, Sluppen, NO-7485, Trondheim, Norway

c Biodiversity Institute of Ontario, University of Guelph, 579 Gordon St Guelph, ON N1G 1Y2, Canada

Identification of freshwater macroinvertebrate communities based on metabarcoding of environmental

DNA (eDNA) offers new opportunities for biodiversity assessments. Filtering of water to capture

eDNA is superior to alternative techniques and filtering water in the field is an advantage compared to

transport of water for logistical reasons. Appropriate filter preservation is then crucial for maximum

DNA recovery and sample replicability. Here, we report results from the comparison of two filter

types and four different filter preservation approaches as well as the effect of pre-filtration on DNA

yield, number of recovered OTUs and metazoan community composition, using eDNA collected from

a river and a lake ecosystem. Based on our results, 0.45-µm mixed cellulose ester filters yield higher

concentration of DNA and number of OTUs than 0.20-µm polyethersulfone filters, and pre-filtration

negatively affects the DNA yield and OTU number. Ethanol is a poor preservative for filters as these

samples recovered fewer OTUs and a more variable community composition than other approaches.

Preservation in extraction buffer, dry or cooled on ice resulted in differences in DNA yield and

numbers of OTUs, but no significant differences in community composition. Overall, pre-filtration

affects the community composition more than filter type or filter preservation.


DNA barcode reference library of gammarids

from the Carpathian biodiversity and endemism hotspot

T. Mamosa, D. Copilaş-Ciocianu

b, c, R. Wattier

d, F. Čiampor

e, Z. Čiamporová-Zaťovičová

e,

A. Petrusekb, M. Grabowski

a

a Department of Invertebrate Zoology and Hydrobiology, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland b Department of Ecology, Faculty of Science, Charles University in Prague, 12844 Prague, Czech Republic c Department of Biology and Chemistry, West University of Timișoara, 300115 Timisoara, Romania d Equipe SAMBA, UMR CNRS 6282 Biogéosciences, Université de Bourgogne Franche Comté, 6 Boulevard Gabriel, 21000

Dijon, France e Institute of Botany, Slovak Academy of Science, Dúbravská cesta 9, 845 23 Bratislava, Slovakia

The Carpathian Arch has a complex geological history, spanning millions of years of Alpine

orogenesis and associated Paratethys regression. Recent studies showed that the Carpathians are

characterized by very rich biodiversity and high level of endemism. Amphipod crustaceans of the

family Gammaridae are among the most diverse animal groups inhabiting aquatic ecosystems of the

area. They are also commonly used as bioindicators and model organisms in ecotoxicological studies.

On the other side, gammarids have been recently reported for high level of cryptic diversity within

conventionally recognized morphospecies. Thus, there is a need for a wide-scale assessment of the real

level of diversity within this group and its spatial distribution over the area. We have amplified the

COI barcoding marker from over 700 individuals collected on ca. 130 sampling sites covering the

whole Carpathian Arch. By using several methods of molecular species delimitation, including the

barcode index number (BIN), and tools for spatial mapping of genetic diversity we have revealed that

nearly all morphospecies living in the area are in fact complexes of cryptic species, mostly endemics

with narrow distribution ranges. Most of the diversity is centered in the Southern Carpathians, yet the

northern region of the Arch included probably local glacial refugia for numerous Gammarus lineages.


Modelling the influence of the physical-chemical parameters on Brillouin evenness index

using machine learning tree methods

A. Naumoskia, G. Mirceva

a

a Ss Cyril and Methodius University in Skopje, Skopje, Republic of Macedonia

In this research work we present computer models using methods and techniques that build models

only measured data. These models are used to reveal the complex relationship between the abiotic

factors of the environment and the biodiversity indices represented with the evenness indices. By

modelling this relationship, we aim to provide better understanding the influence of the abiotic factors

on the diatom community structure found in aquatic ecosystems. Several decision tree methods were

taken into account in order to model the relationship using data obtain for 16 months’ period of 17

abiotic factors and 116 diatom species. Later the biological information of the 116 diatom species is

represented with four Evenness indices. We analyse the obtained models along two dimensional axes;

one based on the models performance and the other is the quality of the obtained knowledge from the

models. The model performance is done by comparing the model accuracy for each method of all four

evenness indices. This metric of how much the model is good is not the only and the most important

factor of ecological modelling. Another factor is the quality and accurate knowledge gained from the

model. Typical rule obtained from the model will be in IF-THEN form, for example: IF Temperature >

17 and Conductivity < 230 and Nitrate > 2.13 THEN Brillouin_Evenness = 0.78. Based on the results,

we based our future work on using other machine learning method in order to improve the method

accuracy and quality of the knowledge.


Biodiversity modelling of the abiotic influence

on the Shannon diatom evenness index

A. Naumoskia, G. Mirceva

a

a Ss Cyril and Methodius University in Skopje, Skopje, Republic of Macedonia

Data analytics have become one of the first steps in the path of investigating the complex relationship

between the involved actors in ecological modelling. One branch of data analytics, machine learning

methods, are trying to build models using measured ecological data. These models aim to reveal the

relationship between abiotic factors and the biological information represented with the bio-diversity

indices. In this direction, these models will try to represent the influence of the stress factors on the

organisms’ community structure. The hieratical-tree like structure of the model provide easy

interpretability of the results. In this research we will use measurement data that reflect the influence

of 17 abiotic factors (temperature, conductivity, nitrates, potassium, zinc and etc.) on 116 difference

diatom species over 16 months’ period. Before the methods generate the required model, the measured

data is pre-processed by converting the biological information into biodiversity indices. The obtained

models are analysed based on their performing model accuracy for each biodiversity index and also by

the quality of the obtain knowledge from them. This is done by comparing the obtained knowledge

from the models and the existing knowledge from the literature. Typical knowledge obtained from the

models is in IF-THEN form, for example: IF Conductivity > 198 and Temperature < 24 and Zinc >

7.65 THEN Shannon_Evenness = 0.24. As future work, we find useful to improve the method and

models accuracy, quality and the interpretability of the models by using different type of method

heuristic.


Illumina data (16S and 18S) of plankton from Lake Tovel, Italy:

Two years of investigation

U. Oberteggera, M. Pindo

b, D. Albanese

c, C. Donati

c, G. Flaim

a

a Department of Sustainable Agroecosystems and Bioresources, Research and Innovation Centre, Fondazione Edmund Mach,

Via E. Mach 1, 38010 S. Michele all’Adige (TN), Italy b Department of Genomics and Advanced Biology, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach

1, 38010 S. Michele all’Adige (TN), Italy c Computational Biology, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele

all’Adige (TN), Italy

Microbial pro- and eukaryotes hold a key role in aquatic ecosystem functioning. Yet, their diversity in

freshwater lakes, particularly in mountain lakes, is relatively unknown. We sampled plankton in two

basins of Lake Tovel (altitude 1178 m a.s.l.) at different depths (0-4 m in the shallow basin and 0-20

and 30-35 m in the deep basin). We analysed genetic diversity (V3-V4 region of the 16S rRNA and

V4-V5 region of the 18rRNA) in 2014 and 2015 using MICCA (http://micca.org/) as pipeline for

processing amplicon sequencing data. We discuss the challenges encountered in metagenomic data-

analyses and compare the metagenomic data to microscopic identification of the planktonic

community. Seasonal and spatial patterns were investigated at different taxonomical levels.

Preliminary analysis indicated that both basins despite their close spatial proximity are clearly

distinguished based on sequencing data.


Rivers biomonitoring in Cyprus: Past, Present, Future

A. Papatheodouloua, b

, I. Tziortzisc, G. Dörflinger

c, C. Demetriou

c, M. I. Vasquez

a

a Department of Environmental Science and Technology, Cyprus University of Technology b I.A.CO Environmental and Water Consultants Ltd c Water Development Department- Ministry of Agriculture, Rural Development and Environment

The island’s biomonitoring scheme, currently involves 47 river catchments in 6500km2 total area.

Approximately 14% of the total length of the river water bodies has perennial, 49% intermittent and

37% ephemeral flow. A monitoring network for the Biological Quality Elements (BQEs) of the Water

Framework Directive was developed in 2007, to assess -by the means of dedicated biotic indices- the

status of the water bodies. This expanded over the years and currently is comprised of 70 stations in

159 river water bodies. The BQEs being monitored -for composition and abundance- include benthic

invertebrates, phytobenthos (diatoms) and macrophytes. The fish fauna of the island is very poor (with

only 2 indigenous species and 1 naturalised among many alien species), hampering the design of an

assessment method based on fish; nevertheless the information gained from fish monitoring provides

valuable complementary data. The national assessment method uses the STAR Intercalibration

Common Metric Index for benthic invertebrates, the Specific Polluosensibility Index for

phytobenthos, the Biological Macrophyte Index for Rivers and the Multimetric Macrophyte Index for

macrophytes. The latter has been specifically developed for intermittent rivers based on local data.

This presentation focuses on the distribution and abundance of benthic invertebrates taxa as indicators

of water bodies’ health. So far -to our knowledge- no comprehensive list of the benthic invertebrates

taxa involved in biomonitoring has been published. Acknowledging this need, we herewith present the

results of the ecological assessment of the river water bodies for the past decade. The applicability and

purpose of use of environmental DNA sampling and analysis is also elaborated.


InBIO Barcoding Initiative: building a reference database for freshwater insects

using Next-Generation Sequencing

J. Paupérioa, S. Ferreira

a, F.M.S. Martins

a,b, J. Veríssimo

a, A. Cordero-Rivera

c, J. Garrido-González

d,

L. Martine, J Martínez

e, M.A. González

e, J.M. Tierno de Figueroa

f, J.M. Grosso-Silva

g,

L. Quagliettaa,h

, A. Muñoz-Meridaa, M.T. Monaghan

i, A.F. Filipe

a,h, P.C. Alves

a,b, S. Jarman

a,

P. Bejaa,h


661 Vairão, Portugal b Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto,

Portugal c ECOEVO Lab, Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, EUE Forestal, Campus Universitario

A Xunqueira s/n, ES-36005 Pontevedra, Spain d Departamento de Ecoloxía e Bioloxía Animal, Faculdad de Biología, Universidade de Vigo, Vigo, Spain e Dpto. Zoología, Antropología Física & Genética, Facultad de Biología. Universidad de Santiago de Compostela

R/Lope Gómez de Marzoa, s/n. Campus Vida, Santiago de Compostela, Spain f Departamento de Zoología, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, Granada, Spain g Museu de História Natural e da Ciência, Universidade do Porto, Porto, Portugal h CEABN/InBIO, Centro de Ecologia Aplicada “Prof Baeta Neves”, Instituto Superior de Agronomia, Universidade de

Lisboa, Lisboa, Portugal i Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany

Freshwater biomonitoring is traditionally performed with methods that rely on morphology and

taxonomy, and that are labour-intensive and time consuming. DNA-metabarcoding enables the

molecular identification of organisms and with continued optimization is expected to have high

applicability for biodiversity monitoring. The extent of its application is however dependent upon the

existence of comprehensive reference collections and corresponding DNA sequence databases.

Unfortunately, these are still lacking for many invertebrate groups, especially regarding areas

considered biodiversity hotspots, like the Mediterranean region. In this context, InBIO is developing a

DNA barcoding database focusing on Iberian aquatic insects. More than 750 specimens of seven

insect orders were already analysed: Coleoptera, Ephemeroptera, Hemiptera, Megaloptera, Odonata,

Plecoptera and Trichoptera. Genomic DNA was extracted from each specimen and the mitochondrial

COI gene fragment (658 bp) was amplified in two overlapping fragments and sequenced using high-

throughput sequencing (Illumina). Most species could be easily identified using the targeted DNA

fragment but some cases of low divergence between species were detected. Cryptic diversity was

found in several situations, mostly when comparing specimens from Iberia and central Europe, but in

some circumstances, even within Iberian Peninsula specimens. Furthermore, by using high-throughput

sequencing techniques nuclear copies of COI fragments were recovered in some species. These

sequences are also likely to be detected in eDNA metabarcoding studies, and should therefore be

documented and databased. We expect InBIO’s reference collection to become a fundamental tool for

long-term and large-scale monitoring of aquatic freshwater ecosystems in the Iberian Peninsula.


FreshBase: The UK freshwater genomic reference library

B. Price a, A. Briscoe

a

a Natural History Museum, London

Freshwater habitats account for less than 1% of the world’s water, yet may contain 10% of the world’s

biodiversity. While the human impact on freshwater ecosystems is widely accepted, our actual impact

on freshwater biodiversity is largely unknown. Specimen identifications based solely on morphology

do not scale efficiently when used in applied fields such as biodiversity or water quality monitoring.

This has provided a niche which molecular methods are beginning to fill, more recently with the use of

high throughput sequencing of bulk samples and eDNA.

While molecular based approaches to identification are proving to be rapid, robust, cost effective and

repeatable, their widespread use is currently hampered by: (1) the lack of a comprehensive sequence

reference library and (2) the rapid evolution of molecular methods, precluding standardization.

Approximately 1/3 of the 5000 freshwater macro-invertebrate species in the UK are represented in

public barcode repositories. By partnering with academic and agency end users the FreshBase project

aims to build a comprehensive, open access reference library, based around expertly identified

specimens and high quality DNA extracts housed in perpetuity at the Natural History Museum,

London. By archiving the specimens, DNA extracts and genomic libraries this voucher collection will

provide a gold standard resource as molecular methods advance. Although initially targeting standard

barcode fragments, FreshBase aims to include mitogenome, transcriptome and whole genome datasets

in future, while integrating with existing sustainable online data sharing and archiving platforms to

enable to widest possible re-use of this data.


Early detection of invasive aquatic Ponto-Caspian hydrobionts based on eDNA

T. Rewicza, T. Mamos

b, M. Grabowski

b, D. Strapagiel

c, R. Wattier

d, A. Burzyński

e, J. Grabowska

f,

K. Bącela-Spychalskab

a Laboratory of Microscopic Imaging and Specialized Biological Techniques, University of Lodz, Banacha 12/16, 90-237

Lodz, Poland b Department of Invertebrate Zoology and Hydrobiology, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland c Biobank Lab, Department of Molecular Biophysics at The University of Lodz, Poland d Equipe Ecologie Evolutive, UMR CNRS 6282 Biogeosciences, Universite de Bourgogne, 6 Boulevard Gabriel, 21000

Dijon, France e Institute of Oceanology, Polish Academy of Sciences, Powstańców Warszawy 55, 81-712 Sopot, Poland f Department of Ecology and Vertebrate Zoology, University of Lodz, ul. Banacha 12/16, 90-237 Łódź, Poland

Alien species represent a major threat to conservation management on both continental and global

scale. Invasions of the Ponto-Caspian species are among the oldest and most severe to aquatic

ecosystems of European and North American ecosystems, bringing significant economic and

environmental damage. Detecting alien invasive species at the early stages of introduction or when

they populations have low densities is a key to control their populations by implementing eradication

procedures, or stop the further spreading.

The study will be focused on Ponto-Caspian exotic community consisted of three taxonomically

distinct groups: dreissenid mussels (Dreissena polymorpha, D. bugensis), amphipods (Chelicorophium

curvispinum, Chaetogammarus ischnus, Echinogammarus trichiatus, Pontogammarus robustoides,

Obesogammarus crassus, Dikerogammarus villosus, D. haemobaphes) and gobiid fishes (Neogobius

melanostomus, N. fluviatilis, Babka gymnotrachelus, Proterorhinus semilunaris) inhabiting Polish

inland waters (Vistula, Oder rivers and Masurian Lakeland) as a model system. The proposed research

objectives are entirely comprehensive, and requires the use of variety of techniques and methods.

Within the project proper eDNA sampling, extraction protocols, experiments, laboratory and fields

tests will be designed and performed. Constituent part of the project will be: mitochondrial genomes

sequencing (D. villosus, D. polymorpha - unpublished, D. bugensis, P. robustoides, D. haemobaphes -

assembled). The metagenomes will be used for: molecular marker selection and primers design (qPCR

based on Taqman probes). Results of this project will provide tools necessary for comprehensive

detection of Ponto-Caspian invasive species in European freshwaters.


New tools for assessing and predicting emerging contaminants effects

in the marine environment

A. Rodríguez Romeroa, J. Blasco

b, C. Michán

c, J. Alhama

c, M. Hampel

d,e, J.R. Viguri

a

a Green Engineering & Resources Research Group (GER), Department of Chemistry and Process & Resources Engineering,

ETSIIT, University of Cantabria, Avda. de los Castros s/n, 39005 Santander, Cantabria, Spain

b Instituto de Ciencias Marinas de Andalucia (CSIC), Campus Universitario Río San Pedro s/n, 11510 Puerto Real, Cadiz,

Spain

c Department of Biochemistry and Molecular Biology, University of Córdoba, 14071 Córdoba, Spain

d Department for Physical Chemistry, Faculty of Marine and Environmental Sciences, University of Cadiz, Campus

Universitario Río San Pedro s/n, 11510 Puerto Real, Cadiz, Spain eAndalusian Center of Marine Science and Technology (CACYTMAR), Campus Universitario Río San Pedro s/n, 11510

Puerto Real, Cadiz, Spain

European marine ecosystems are subjected to many economic activities. The maintenance of their

function and ecosystem services is of central importance, for preservation and conservation of their

environmental quality and biodiversity. One of the main impacts of the human activity in marine

ecosystems, is chemical contamination. Among the chemical compounds, emergent contaminants have

received a large attention due to the lack of information about their effects.

The multidisciplinary research group is focused on establishing the relationships between three

emergent contaminant groups (nanoparticles: cadmium quantum dots and silver; pharmaceuticals:

anti-inflammatory, antibiotics and anti-depressives; and microplastics: polystyrene) and their sub

lethal effects by the design and application of multispecies/microcosms assays by using two marine

phytoplankton species (Phaeodactylum tricornutum and Nanochloropsis gaditana), a crustacean

(Artemia sp), a bivalve (Scrobicularia plana), and the microbiome from waters and sediments,

exposed to single and mixtures chemicals at ecological relevance concentrations.

The research project, in the first step of develop, will create and apply massive analytical

methodologies, including different meta-omics (metagenomics, metatranscriptomics and

metaproteomics), which will allow the development of new tools and the identification of unbiased

biomarkers. Furthermore, the study of physiological, biochemical and functional responses in the

selected organisms will be performed. Finally, a modeling approach will be applied to predict the

biological effects of these contaminants and determine links across the food web in order to improve

marine environmental risk assessment, getting a better protection, management and sustainability of

marine environment.


Persistence and quantification of environmental DNA in experimental river systems:

Implications for molecular biodiversity assessment

M. Seymoura, I. Durance

b, J. Cosby

c, G. Carvalho

a, J. Colbourne

d, NERC LOFRESH Consortium,

S. Creera

a School of Biological Sciences, Bangor University, Bangor, United Kingdom, b Cardiff University, Cardiff, United Kingdom, c Centre for Ecology and Hydrology (CEH), Bangor, United Kingdom, d University of Birmingham, Birmingham, United Kingdom

Accurately quantifying biodiversity is paramount for understanding ecosystem functioning and

environmental assessments. With concern regarding anthropogenic impacts on environmental stability

and the high level of expertise required for traditional sampling, molecular biodiversity identification,

using environmental DNA (eDNA), offers a non-invasive, rapid and cost-effective method to assess

biodiversity. While eDNA analyses have become increasing prevalent in biodiversity assessment, little

is known regarding the persistence of eDNA under different environmental conditions, especially in

temporally and spatially variable systems such as rivers. Utilizing a unique set of upland artificial

stream channels, located in mid-Wales, we assessed the temporal degradation of eDNA in relation to

environmental variation, including pH, temperature and DOC. For each experimental stream, we

added quantified concentrations of eDNA sourced from five taxonomically distant species, including

Daphnia magna, Salmo salar, Salmo trutta, Ephemera danica and Anguilla anguilla. Sampling,

including on-site filtration and preservation, occurred periodically over 48 hours to reflect the

transport time of eDNA traveling the length of natural river drainages. Quantification of sampled

DNA was done using qPCR and analyzed using mixed effect models. We observed strong temporal

patterns of eDNA detection that also varied interspecifically and related to environmental variation

and initial eDNA concentration. These findings are important as they indicate the need to not only

consider eDNA degradation, but also the local environmental conditions and target species groups.

Additionally, these findings point towards the ability of eventually using targeted molecular

approaches to quantify biodiversity.


Long-Term Natural Experiments in Ecology and Evolution:

Yeasts from Low O2 and High CO2 Mofette Environments

N. Šibanca,b

, P. Zalarc, H. J. Schroers

d, J. Zajc

e, J. P. Sampaio

f, I. Maček

a,b

a University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia b University of Primorska, Faculty of Mathematics, Natural Sciences and Information Technologies (FAMNIT), Glagoljaška

8, 6000 Koper, Slovenia c University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia d Agricultural Institute of Slovenia, Hacquetova 17, p.p.2553, 1001 Ljubljana, Slovenia e National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia f Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia Centro de Recursos Microbiológicos, 2825-114

Caparica, Portugal

Locally extreme ecosystems have proven to be useful for natural long-term experiments in microbial

ecology, giving an insight into much-needed processes of adaptation and acclimation of natural

communities to abiotic stress. An inventory of cultivable yeasts from soil and water mofettes (natural

CO2 springs) near Stavešinci in Slovenia is presented where CO2 of geological origin and ambient

temperature reaches the soil surface, resulting in temporary and spatially stable high CO2 and low O2

environment. In total 142 yeast strains were isolated from meadow soil, ground-water and forest pond

water. They were affiliated to 6 basidiomycetous and 19 ascomycetous species from 15 genera.

Cryptococcus podzolicus, Meyerozyma guilliermondii, newly described species Occultifur mephitis,

Rhodotorula graminis (basidiomycetes), and Wickerhamomyces (ascomycetes) predominated in high

CO2 exposed soils. High CO2 exposed water accommodated the ascomycetous yeasts Candida

sophiae-reginae, Candida vartiovaraae, and Pichia fermentans. Our data on mofette ecosystems show

that locally extreme environments could further serve as a powerful tool to study slow ecological and

evolutionary processes that normally require long-term observations and experiments to study them.


Biodiversity monitoring of South-Mediterranean aquatic ecosystems using benthic

macroinvertebrates: Comparison between traditional and genomic methods

V. Specchiaa,|

, G. Marinia, L. Mazzotta

a, A. Franco

a, S. Janzen

b, M. Lanzo

a, M. Pinna

a,+

a Department of Biological and Environmental Sciences and Technologies, University of Salento, S.P. Lecce-Monteroni -

73100 Lecce, Italy b School of GeoSciences, University of Edinburgh, EH9 3JN Edinburgh, UK | [email protected] + [email protected]

The conservation of biodiversity is fundamental to ensure the ecosystem stability, functions and

services. Transitional waters (TWs) support a remarkable biodiversity but the growing of

anthropogenic pressures threats their ecosystem health, induces to act new conservation strategies and

biomonitoring plans. So that new genomic approaches are also emerging as strategic and useful tools

for the assessment of ecological status.

Macroinvertebrates are good ecological indicators for TWs, but very few studies have highlighted that

both descriptors and ecological indicators can depend by sampling method and effort. The

bioassessment with macroinvertebrates is time consuming and requires high costs. Moreover the use

of more than one sampling technique can be require to collect both bottom and vagile fauna.

Our research aims to evaluate the influence of sampling methods - box-corer and litterbag, and of

habitat type - prairie and unvegetated, on descriptors and indicators. The experiment was run in a

protected Mediterranean shallow lagoon dominated by marine waters.

Biodiversity and ecological indicators of macroinvertebrates were measured and compared among

sampling techniques and habitats and the relevance of using more than one sampling technique to

obtain a better description of biodiversity and ecological status was highlighted. Anyway the

transferring of this methodology to biomonitoring programmes is highly limited by time consuming

and costs. The future goal of our research is to apply molecular tools to record the entire biodiversity

of macroinvertebrates in Southern-Italy aquatic ecosystems, both in reference and perturbed

conditions. In particular, we will use DNA barcoding and eDNA analysis for this purpose.


Comparison of eDNA and conventional techniques for monitoring species diversity

of boulder reefs in Danish waters

P.A. Stæhra, S. Santos

b, L.H. Hansen

b, S. Lundsteen

a, L. Haraguchi

a, K. Dahl

a, M.P. Ávila

a,c,

A. Windingb,|

a Aarhus University, Department of Bioscience, Aarhus University, Roskilde, Denmark b Aarhus University, Department of Environmental Sciences, Aarhus University, Roskilde, Denmark c Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte,

Brazil

| [email protected]

Metabarcoding of environmental DNA (eDNA) from seawater samples at a transect along a boulder

reef in Smålandsfarvandet, Denmark was used to test if biological material containing DNA from the

reef significantly contributed to the integrated pool of DNA extracted from the water column above.

This was compared with conventional sampling techniques of composition and abundance of benthic

flora and fauna using divers and with microscopic identification of planktonic organisms. Sampling

was done at 6 stations in August 2015 and at 7 stations August 2016. Profiles of temperature, salinity

and oxygen showed that sampling over the reef was done in oxygen rich, well mixed water column at

all stations.

Based on the operational taxonomic units (OTU) derived from metabarcoding, eDNA identified a

higher taxonomic richness (~300) compared to benthic reef survey (~45) and microscopic (~75)

methods, and a higher diversity, but less evenly distributed taxonomic composition. Dissimilarity

analysis showed a gradient in sampling stations going up current for the diver based reef sampling,

whereas the eDNA and microscopic techniques suggested communities of a more random distribution

along the transect. Changes in taxa richness, diversity and evenness along the transect were

comparable between the benthic reef survey and eDNA technique, despite a large influence of

planktonic DNA.

Our study suggests that eDNA techniques can provide valuable additional information to conventional

diversity monitoring of boulder reefs in Danish waters given careful consideration of sampling and

data processing. However, more detailed analysis and further validation is needed before eDNA can

replace traditional monitoring techniques.


eDNA monitoring an outbreak of crayfish plague in Norway –

Snapshots of invasion, infection and extinction

D. Stranda,b

, S.I. Johnsenc, J. Rusch

a, S.W. Knudsen

d, S. Agersnap

d, W. Brenner Larsen

d,

P. Rask-Møllerd, T. Vrålstad

a

a Norwegian Veterinary Institute

b Norwegian Institute for Water Research c Norwegian Institute for Nature Research d The Natural History Museum of Denmark

The use of environmental DNA (eDNA) for detection and quantification of aquatic organisms is a

rapidly growing field with a great potential for streamlined inventory- and monitoring purposes. The

TARGET-project aims at implementing eDNA approaches for monitoring the red-listed Astacus

astacus and its major threats.

In 2014-2016, we followed an outbreak of crayfish plague in the Norwegian Halden watercourse after

an illegal introduction of Pacifastacus leniusculus, comparing classical surveillance of crayfish plague

(caged noble crayfish) with eDNA water monitoring. Water (~5 L) was filtered on-site through glass

fiber filters, and each sample was analysed using species specific qPCR assays for the crayfish plague

pathogen Aphanomyces astaci, and the freshwater crayfish species A. astacus and P. leniusculus.

eDNA of the three species was successfully detected and A. astaci was detectable in the water before

the caged crayfish succumbed due to the disease. The infection source (signal crayfish), represented by

a scarce population of P. leniusculus in the southern part of the lake, was detected at trace levels.

Furthermore, eDNA from noble crayfish was readily detected and increased in quantity during the

mortalities, before decreasing to trace levels about 4-8 weeks after the outbreak.

We are currently working on transferring the qPCR assays to the droplet digital PCR platform and will

also present some preliminary comparisons and results from this work.

Our study demonstrates an efficient and non-invasive approach for a combined eDNA monitoring of

native crayfish and its threats (invasive crayfish and crayfish plague) from the same water samples.


Under the canopy:

Community-wide effects of invasive algae in marine protected areas

revealed by metabarcoding

X. Turona, O.S. Wangensteen

b, E. Cebrian

c, C. Palacín

d

a Center for Advanced Studies of Blanes (CEAB-CSIC), Blanes (Girona), Spain b Ecosystems and Environment Research Centre, School of Environment and Life Sciences, University of Salford, Salford,

UK c Institut d’Ecologia Aquàtica, Universitat de Girona, Campus Montilivi, Girona, Spain d Department of Animal Biology and Institute for Research on Biodiversity (IRBio), University of Barcelona, Barcelona,

Spain

We analysed with multigene (18S and COI) metabarcoding the effects of the proliferation of three

invasive seaweeds on rocky littoral communities in Spanish National Parks. The algae chosen were

Caulerpa cylindracea and Lophocladia lallemandii in the Mediterranean, and Asparagopsis armata in

the Atlantic. These algae are landscape-dominant forms where they proliferate, and we studied their

effects on the underlying communities composed of the meiobenthos and small macrobenthos,

separated in two size fractions through sieving. A new semiquantitative treatment of metabarcoding

data is introduced. Metazoa was the most diverse group for both markers, followed by Stramenopiles

(18S) and Archaeplastida (COI). For both genes Arthropoda, Annelida and Cnidaria were the most

diverse metazoan Phyla. The results were dependent on the alga considered; while size fraction was a

significant factor in all cases, the presence of the invasive alga had a significant effect on the

understory communities for Lophocladia lallemandii and Asparagopsis armata but not for Caulerpa

cylindracea. Likewise, there was no consistent effect of the invasive algae in changes of MOTU

richness and diversity of the communities, which varied in magnitude and direction depending on the

alga considered. Both markers generated widely different biodiversity estimates, with the number of

MOTUs obtained for COI being an order of magnitude higher than for the shorter and less variable

18S fragment analysed. Our results showed that metabarcoding can be used in invasion biology to

assess the less conspicuous, but not least important, effects of the presence of dominant invasive

seaweeds in sublittoral communities.


eDNA based monitoring of the invasive American Crayfish, Procambarus clarkii,

in Andesian lakes (Ecuador)

C. Van der heydena, L. Riascos

d, A.N. Geerts

a, T. Oña

d, J.M. Cevallos

e, W. Vanden Berghe

g, h,

F. Volckaerti, J. Bonilla

e, f, P. Goethals

b, E. Velarde

d, P. Boets

b,c

a Hogeschool Gent / University College Ghent. Faculty of Science and Technology, Valentin Vaerwyckweg 1, 9000 Ghent,

Belgium b Ghent University. Faculty of Bioscience Engineering, Coupure links 653, 9000 Ghent, Belgium

c PCM Provincial Centre of Environmental Research. Godshuizenlaan 95, 9000 Ghent, Belgium d Universidad Tècnica del Norte, Avenida 17 de julio 5-21 y Gral. José María Cordova, Codigo postal 199, Ibarra, Ecuador e Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnológicas del Ecuador CIBE, Campus

Gustavo Galindo Km 30.5 vía perimetral, P.O. Box 09-01-5863, Guayaquil-Ecuador f Escuela Superior Politécnica del Litoral, ESPOL, Facultad de Ciencias de la Vida, Campus Gustavo Galindo Km 30.5 vía

perimetral, P.O. Box 09-01-5863, Guayaquil-Ecuador g Ghent University. Faculty of Sciences, Department of Biochemistry & Biotechnology, L-GEST, Campus Heymans,

Ottergemsesteenweg 460, 9000 Gent h University of Antwerp, Department of Biomedical Sciences, Campus Drie Eiken, Universiteitsplein 1, 2610 Wilrijk,

Belgium i Laboratory of Biodiversity and Evolutionary Genomics, Charles Deberiotstraat 32 - box 2439, 3000 Leuven, Belgium

Since 1989, Procambarus clarkii has been found in Ecuador and is caught in the Yahuarcocha lake, a

tropical, high altitude lake located in the Andes in the northern part of the country, by the local

fisherman for consumption. This American crayfish is an alien species that invaded lake Yahuarcocha,

thereby disturbing the autochthonous aquatic communities. However, little is known about its

distribution throughout Ecuador and monitoring is important in order to plan elimination actions.

Therefore, a molecular method based on environmental DNA (eDNA) for monitoring macro-

invertebrates was developed and was used to assess the distribution of the American crayfish,

Procambarus clarkii. Twenty one sites in Yahuarcocha lake were sampled; the samples were tested on

the presence of DNA of the American crayfish. For this, the water was filtered, eDNA was extracted

and subsequently processed by PCR. Results were visualised with agarose gel electrophoresis. The

molecular results were compared to catchment based distribution patterns obtained via traditional

sampling.

As good results were obtained in Yahuarcocha, two other Andesian lakes were sampled and tested for

the presence of the American crayfish. The results indicate that the method is easy to use and can

provide important information on the occurrence of alien species in the tropics.

This research was funded through a South Inititiative (ZEIN2014Z150) by VLIR-UOS.


Quantitative Diatom metabarcoding:

A correction factor inferred from cell biovolume

V. Vasselona, A. Bouchez

a, M. Corniquel

a, S. Jacquet

a, F. Rimet

a, K. Tapolczai

a, I. Domaizon

a

A CARRTEL, INRA, Université de Savoie Mont Blanc, 74200, Thonon-les-bains, France

DNA metabarcoding combined to high-throughput sequencing (HTS) is a promising approach for

freshwater biomonitoring using benthic diatoms. This molecular approach is becoming faster, cheaper

and allows handling more samples than the classical morphological one. rbcL gene has been proven

suitable as a barcode for diatoms providing qualitatively relevant taxonomic inventories; however,

optimizations are still required for quantifying relative species abundances. We hypothesized that rbcL

copy number is correlated to diatom cell biovolume (as it is the case for 18S gene) and a correction

factor that account for cell biovolume is applicable to optimize taxa quantification.

An experiment was carried out using 8 pure diatom cultures chosen for their contrasted biovolumes.

The 18S and the rbcL copy numbers were both correlated to diatom cell biovolume, but only rbcL

gene followed a linear model applicable to the 8 species, thus allowing to define a correction factor.

The efficiency of the correction factor was tested on 5 mock communities composed by different

proportions of DNA from the 8 species. The 5 mock communities were sequenced using HTS by

targeting the rbcL barcode, and the obtained proportions of DNA reads were corrected using the

defined model. The correction made molecular inventories closer to the morphological ones. This

correction factor was finally validated on environmental DNA metabarcoding data (biofilms) and was

shown to reduce differences between molecular and morphological water quality indices. Our results

show that the correction factor is effective for correcting quantitative biases in diatoms metabarcoding

studies, improving the resulting water quality assessment.


Ecological risk assessment of microcystins on aquatic organisms:

Determination of MIC-LR, RR and YR in water

G. Vukovića, M. Đukić

a, V. Bursić

b, A. Popović

b, D. Marinković

b, A. Petrović

b, I. Ivanović

b

a Institute of Public Health of Belgrade, Bulevar despota Stefana 54A, Serbia b University of Novi Sad, Faculty of Agryculture, Dositeja Obradovića 2, Novi Sad, Serbia

Microcystins (MICs) are potential toxins produced worldwide by cyanobacteria (commonly called

"blue-green algae"), during bloom events. The literature data indicate that phosphatases inhibition is a

well recognized effect of MICs as well as oxidative stress in aquatic animals. However, it is not fully

understood why and how MICs exposure can lead to an excessive formation of reactive oxygen

species (ROS) that culminate in oxidative damage. The first step is the detection of MICs. Due to that

fact the liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS)

method has been validated for the determination of three MCs (Microcystin-LR, RR and YR)in water.

Validation method makes quantification of MICs with nodularin as an internal standard. The method

was confirmed on proficiency testing LGC AQ519, Z-score =0.3. The limit of quantitation (LOQ) for

all analytes studied was set experimentally to be 0.1 g/L (considering the regulated MRL of 1.0

g/L) with the limit of detection (LOD) of 0.002 g/L. A sample preparation method involving solid-

phase extraction (Bond Elut Plexa, Agilent, USA) gives satisfactory recoveries and precisions. During

2016 sixty-one samples were analysed. The detection of microcystin-RR was confirmed in five

samples in the range of 0.001 to 0.04 g/L. Only one sample contained the concentration of MIC over

the MRL, the concentration of dmMIC-RR was 1.15 g/L.


Metabarcoding of eDNA from Suspended Particulate Matter (SPM)

for fish population monitoring

F.-F. Wegea, C.A. Diaz Navarette

a, G. Böhle

a, M. Kohler

b, M. Teigeler

a, C. Schäfers

a, H. Rüdel

a,

E. Eilebrechta, J. Koschorreck

c

a Fraunhofer IME, Department of Ecotoxicology, Auf dem Aberg 1, 57392 Schmallenberg, Germany b Fraunhofer IME ScreeningPort, Schnackenburgallee 114, 22525 Hamburg, Germany c Federal Environment Agency (UBA), Bismarckplatz 1, 14193 Berlin, Germany

The evaluation of fish populations and biodiversity in aquatic environments is usually performed by

time- and cost-intensive studies, and often presents only a snap-shot of the actual fish population. To

date, the conventional method used for this purpose is electro fishing. However, monitoring of rare

species is often unsuccessful with this method. Sampling of so called environmental DNA (eDNA)

allows for non-invasive species determination. Cellular material carrying the genomic information of a

large number of species might be present in the aquatic environment as fish continuously release

bodily components (e.g. excrements, mucus, scales).

In this study we used metabarcoding to analyse the whole species community. DNA was extracted

from suspended particulate matter (SPM), sampled at different riverine sampling points in Germany.

We tested different PCR primer pairs, which all bind to conserved regions of the genome. These

regions for example encode genes for CytB, 12S, and COI. The final approach, resulting in reliable

and repeatable results, was a nested PCR approach with newly designed primers, amplifying a

fragment of the COI gene. The PCR products were analyzed by Next Generation Sequencing (NGS).

The results allowed the identification and discrimination of fish species. The data obtained were used

for comparisons with fish monitoring data from electro fishing.

With the described approach, we developed a method to reliably identify and discriminate fish species,

allowing fish monitoring in a non-invasive way. By using archived SPM samples of the German ESB

as starting material, this method allows retrospective monitoring of fish populations.


When DNA barcoding fails.

A case of intergeneric mtDNA introgression in freshwater Balkan amphipods

W. Wrzesinskaa, K. Hupalo

a, A. Jablonska

a, T. Mamos

a, A. Zawal

b, V. Pesic

c, M. Grabowski

a

a Department of Invertebrate Zoology and Hydrobiology, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland b Department of Invertebrate Zoology & Limnology, Center of Molecular Biology & Biotechnology, University of Szczecin,

Poland c Departmentof Biology, University of Montenegro, Montenegro

DNA barcoding has proven to be an effective tool in providing molecular tags for animal species and

even, although not without a debate, in species delimitation. However, the tool is far from being

perfect. For example, the species having very recent common ancestor may still share mtDNA

haplotypes. There are also reports of mitochondrial genome introgressions as result of secondary

contact between closely related species. Much rarer are, though, the mtDNA introgressions in case of

species belonging to different genera, with significant morphological differences and long divergence

history. Here we report such an introgression of mtDNA from Laurogammarus scutarensis, a

freshwater amphipod endemic to the ancient spring system of the Skadar Lake in the Balkan

Peninsula, to Echinogammarus thoni, a species with much wider distribution around the Adriatic Sea

encompassing both the fresh and brackish waters. The two species diverged ca. 7-9 Ma, in Miocene,

while the introgression occurred very recently, not earlier than 0.5 Ma, in late Pleistocene.We

reconstruct the historical demography of L. scutarensis in order to provide historical framework for

understanding the introgression event. Our findings put also in doubt the currently established

taxonomy of Echinogammarus and validity of establishing Laurogammarus as a new genus.


Evaluation and optimization of DNA metabarcoding of aquatic invertebrates

V. Zizkaa, B. Peinert

a, F. Leese

a

a Aquatic Ecosystem Research, University of Duisburg-Essen, Germany

DNA metabarcoding is increasingly used to study and monitor biodiversity of aquatic ecosystems.

However, even if the method is well published, it still needs optimization and standardization to

become established as a widely applied tool for biodiversity determination. Implemented in the GBOL

II (German Barcode of Life II) project we work on refining steps of metabarcoding with the main

focus on laboratory protocols.

(1) During the homogenization of bulk samples, which is currently an inevitable step of

metabarcoding, large specimens contribute more DNA to the homogenate than smaller ones, leading

to biased results. To circumvent this, time-consuming size sorting of bulk samples prior to DNA

extraction and PCR was implemented. As an attempt to reduce this time-consuming step, we extract

DNA directly from the fixative (ethanol). Thereby, ethanol from two phases of the fixation process is

studied: (a) ethanol into which the (living) specimens have been transferred immediately after

collection and (b) ethanol in which (dead) specimens have been stored. The latter is exposed to three

different treatments (ultrasonification, shaking, freezing) to increase the amount of free DNA in the

ethanol. Fixative is then filtered, DNA is extracted from the filters and processed after a standard

metabarcoding protocol.

(2) The picking of specimens from collected bulk samples in the field is time consuming and can lead

to overlooking of up to 30 % of species. We want to assess if/how the sampled substrate effects

sequencing results for macrozoobenthos. Inclusion of substrate material might lead to the detection of

otherwise overlooked taxa or to an increased inhibition of laboratory processes, e.g. through detection

of untargeted taxa (i.e. bacteria, macrophytes).

(3) Two different PCR protocols for metabarcoding including the usage of “Fusion”-primers are

compared. The two methods are tested for primer bias and their consistency. Furthermore approaches

are compared to the process of sample indexing using a standard Illumina Kit. Results will also help to

reduce PCR problems by primer bias and provide assumptions about the number of replicates, which

are necessary for reliable metabarcoding data.

Data and analyses are intended to contribute to simplify and specify steps in metabarcoding for

biodiversity assessment. All methodological steps are evaluated for two stream ecosystems in western

Germany, the Sieg and the Emscher.

List of participants



Surname Firstname Affiliation Country

Abarenkov KESSY University of Tartu Estonia

Bacela-Spychalska KAROLINA University of Lodz Poland

Bailet Berangere BONNIE Swedish University of Agricultural Sciences Sweden

Baird DONALD Environment and Climate Change Canada Canada

Beentjes KEVIN Naturalis Biodiversity Center The Netherlands

Beermann ARNE University of Duisburg-Essen Germany

Beja PEDRO CIBIO-InBIO Porto University Portugal

Boenigk JENS University of Duisburg-Essen Germany

Boets PIETER Provincial Centre of Environmental Research Belgium

Boggero ANGELA CNR - Institute of Ecosystem Study Italy

Borja ÁNGEL AZTI Spain

Bouchez AGNÈS INRA CARRTEL France

Brandsegg HEGE Norwegian Institute for Nature Research Norway

Bruce KAT Nature Metrics United Kingdom

Brys REIN INBO (Research Institute for Nature and Forest) Belgium

Bursic VOJISLAVA University of Novi Sad Serbia

Buttigieg PIER LUIGI AWI, Helmholtz-Zentrum für Polar- und Meeresforschung Germany

Carlsson JENS University College Dublin Ireland

Castro Paupério JOANA CIBIO-InBIO Porto University Portugal

Čiampor FEDOR Plant Science and Biodiversity Centre SAS Slovakia

Cordier TRISTAN University of Geneva Switzerland

Costa FILIPE University of Minho Portugal

Costache MARIETA University of Bucharest Romania

Creer SIMON Bongor University United Kingdom

Csabai ZOLTÁN University of Pécs Hungary

Darschnik SONJA Ruhr University Bochum Germany

Deiner KRISTY Cornell University United States of America

Devriese LISA ILVO Belgium

Diaz Navarette CECILIA Frauenhofer Institute IME Germany



Domaizon ISABELLE INRA France

Duarte SOFIA University of Minho Portugal

Duncan WILLIE Scottish Environment Protection Agency United Kingdom

Eckartz-Vreden GABRIELE Landesamt für Natur, Umwelt und Verbraucherschutz NRW Germany

Egeter BASTIAN CIBIO-InBIO Porto University Portugal

Eisendle-Flöckner URSULA University of Salzburg Austria

Eisinger MICHAEL University of Duisburg-Essen Germany

Ekrem TORBJØRN NTNU University Museum Norway

Elbrecht VASCO University of Duisburg-Essen Germany

Fais MARIA University of Minho Portugal

Fazi STEFANO CNR-IRSA Italy

Ferreira SÓNIA CIBIO-InBIO Porto University Portugal

Filipe ANA FILIPA CIBIO-InBIO Porto University Portugal

Fleituch TADEUSZ Institut of Nature Conservation Polish Academy of Sciences Poland

Fontaneto DIEGO Institute of Ecosystem Study Italy

Forster DOMINIK Technical University of Kaiserslautern Germany

Fossøy FRODE Norwegian Institute for Nature Research Norway

Franc ALAIN INRA France

Gadawski PIOTR University of Lodz Poland

Geerts AURORA HoGent Belgium

Geiger MATTHIAS Zoologisches Forschungsmuseum Alexander Koenig Germany

Gerner NADINE Emschergenossenschaft Germany

Grabowski MICHAL University of Lodz Poland

Gudmundsdottir RAGNHILDUR University of Iceland Iceland

Guðrandsson JÓHANNES Marine and Freshwater Institute Iceland

Halfmaerten DAVID INBO (Research Institute for Nature and Forest) Belgium

Hänfling BERND University of Hull United Kingdom

Hahn HANS JÜRGEN Institut für Grundwasserökologie IGÖ GmbH Germany

Hellström MICAELA AquaBiota Solutions / Stockholm University Sweden



Herder JELGER RAVON (Reptile Amphibian Fish Conservation Netherlands) The Netherlands

Hering DANIEL University of Duisburg-Essen Germany

Hermida ANTÓN AllGenetics & Biology Spain

Hershkovitz YARON Tel Aviv University Israel

Hupalo KAMIL University of Lodz Poland

Jabłońska ALEKSANDRA University of Lodz Poland

Jacobs GUNNAR Emschergenossenschaft Germany

Jarman SIMON CIBIO-InBIO Porto University Portugal

Jones JOHN River Communities Group United Kingdom

Kahlert MARIA Swedish University of Agricultural Sciences Sweden

Kalamujić Stroil BELMA University of Sarajevo Bosnia and Herzegovina

Kasapidis PANAGIOTIS Hellenic Centre for Marine Research Greece

Keck FRANÇOIS Swedish University of Agricultural Sciences Sweden

Kelly MARTYN Bowburn Consultancy United Kingdom

Kelly-Quinn MARY University College Dublin Ireland

Keskin EMRE Ankara University Turkey

King LYDIA Limnologie-Phykologie-Diatomologie Germany

Klein ROLAND Universität Trier Germany

Kochems REBECCA Technical University of Kaiserslautern Germany

Kõljalg URMAS University of Tartu Estonia

Koschorreck JAN Umweltbundesamt Germany

Leese FLORIAN University of Duisburg-Essen Germany

Leite BARBARA University of Minho Portugal

Ljubešić ZRINKA University of Zagreb Croatia

Lorenz ARMIN University of Duisburg-Essen Germany

Macek IRENA University of Ljubljana Slovenia

Macher JAN University of Duisburg-Essen Germany

Mächler ELVIRA Eawag Aquatic Research Switzerland

Mahon ANDY Central Michigan University United States of America



Majaneva MARKUS NTNU University Museum Norway

Mamos TOMASZ University of Lodz Poland

Mann DAVID Royal Botanic Garden Edinburgh United Kingdom

Marcel RÉMY Aquabio United Kingdom

Marečková MARKÉTA Crop Research Institute Czech Republic

Martins FILIPA CIBIO-InBIO Porto University Portugal

Matejusova IVETA Marine Scotland Science United Kingdom

Meissner KRISTIAN Finnish Environmental Institute Finland

Mergen PATRICIA Royal Museum for Central Africa Belgium

Merzi THOMAS TOTAL S.A. France

Monaghan MICHAEL IGB Berlin Germany

Moritz CHRISTIAN ARGE Limnologie GesmbH Austria

Múrria CESC University of Barcelona Spain / Catalonia

Mynott SEBASTIAN Applied Genomics United Kingdom

Naumoski ANDREJA Ss. Cyril and Methodius University, Skopje Mazedonia

Navodaru ION Institutul National de Cercetare-Dezvoltare Delta Dunarii Romania

Nijssen DAVID Bundesanstalt für Gewässerkunde Germany

Obertegger ULRIKE FEM-CRI Italy

Padisák JUDIT Institute of Environmental Science Hungary

Pálsson SNÆBJÖRN University of Iceland Iceland

Panksep KRISTEL Estonian University of Life Science Estonia

Pawlowska ALINA ID-Gene Ecodiagnostics Switzerland

Pawlowski JAN University of Geneva Switzerland

Peinert BIANCA University of Duisburg-Essen Germany

Penev LYUBOMIR Pensoft Publishers Bulgaria

Petrusek ADAM Charles University Czech Republic

Pfannkuchen MARTIN Ruder Boskovic Institute Croatia

Piggott JEREMY University of Otago New Zealand

Pinna MAURIZIO University of Salento-Lecce Italy



Podraza PETRA Ruhrverband Germany

Pont DIDIER SPYGEN France

Price BEN Natural History Museum United Kingdom

Rewicz TOMASZ University of Lodz Poland

Reyjol YORICK Onema / AFB France

Rimet FRÉDÉRIC INRA France

Rinkevich BARUCH National Institute of Oceanography Israel

Rodriguez-Ezpeleta NAIARA AZTI Spain

Rotter ANA National Institute of Biology Slovenia

Rulik BJÖRN Zoologisches Forschungsmuseum Alexander Koenig Germany

Rusch JOHANNES Norwegian Veterinary Institute Norway

Salmaso NICO Fondazione Mach-Istituto Agrario di S. Michele all'Adige Italy

Schenk JANINA Universität Bielefeld Germany

Segurado PEDRO Universidade de Lisboa Portugal

Servos MARCEL University of Duisburg-Essen Germany

Seymour MATHEW Bangor University United Kingdom

Siemensmeyer TOBIAS Institut für Grundwasserökologie IGÖ GmbH Germany

Simakova ULYANA Russian Academy of Sciences Russia

Sint DANIELA University of Innsbruck Austria

Siposova DARINA Plant Science and Biodiversity Centre SAS Slovakia

Sivertsgård ROLF Norwegian Institute for Nature Research Norway

Specchia VALERIA University of Salento-Lecce Italy

Speksnijder ARJEN Naturalis Biodiversity Center The Netherlands

Steinke DIRK University of Guelph Canada

Stoeck THORSTEN University of Kaiserslautern Germany

Strand DAVID Norwegian Veterinary Institute/Norwegian Institute for Water Research Norway

Šulčius SIGITAS Nature Research Centre Lithuania

Tapolczai KALMAN INRA France

Thalinger BETTINA University of Innsbruck Austria



Thronicker OLIVER Blue Biolabs GmbH Germany

Traugott MICHAEL University of Innsbruck Austria

Trobajo ROSA IRTA Spain

Turon XAVIER Centre for Advanced Studies of Blanes Spain

Vamos EDITH University of Duisburg-Essen Germany

Van der heyden CHRISTINE HoGent Belgium

van der Hoorn BERRY Naturalis Biodiversity Center The Netherlands

van Kuijk TIEDO Naturalis Biodiversity Center The Netherlands

Varbiro GABOR Centre for Ecological Research, Hungarian Academy of Sciences Hungary

Vasquez MARLEN Cyprus University of Technology Cyprus

Vasselon VALENTIN INRA France

Vierna JOAQUÍN AllGenetics & Biology Spain

Viguri JAVIER University of Cantabria Spain

Vitecek SIMON University of Vienna Austria

Vogler ALFRIED Imperial College London & Natural History Museum United Kingdom

Vrålstad TRUDE Norwegian Veterinary Institute Norway

Vyverman WIM Ghent University Belgium

Wattier REMI Université de Bourgogne France

Weigand ALEXANDER University of Duisburg-Essen Germany

Weigand HANNAH University of Duisburg-Essen Germany

Wenne ROMAN Institute of Oceanology, Polish Academy of Sciences Poland

Winding ANNE Aarhus University Denmark

Winking CAROLINE Emschergenossenschaft Germany

Wörner MANUEL Universität Trier Germany

Wrzesinska WERONIKA University of Lodz Poland

Zimmermann JONAS Botanic Garden and Botanical Museum Berlin Germany

Zizka VERA University of Duisburg-Essen Germany

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