Biodiversity assessment of the North Sea fauna using DNA barcodes and oher molecular methods

1. Analysing the marine fauna of the North Sea:
A molecular approach using various methods
Michael J. Raupach

2. Hotspots of species diversity
© Julian Gutt/AWI
© Wikipedia © Robin Moore

3. Hotspot of species diversity?
© Nordsee von oben
North Sea © Nordsee von oben
© Nordsee von oben
© Nordsee von oben
© Wikipedia
© Nordsee von oben

4. © Wikipedia
… not a stable sea …

5. Global map of cumulative human impact across 20 ocean ecosystem types
Halpern et al. 2008

6. Routes, ports and centralities in the global cargo ship network
Kaluza et al. (2010)

7. Invasive species
World Ocean Review 2010

8. AG Molecular Taxonomy of Marine Organisms (est. summer 2010)
Senckenberg am Meer / Germany Centre of Marine Biodiversity Research
Dr. Michael J. Raupach
Dr. Silke Laakmann
Dr. Thomas Knebelsberger
Dipl. Biol. Inga Mohrbeck
Dipl. Biol. Thorben Hofmann
Tech. Ass. Valeska Borges
founded by
Goal:
Validation and development of molecular methods for the identification
of the marine metazoan fauna of the North Sea

9. Concept:
Barcoding
of the North Sea
Metazoa
(COI, nuclear markers)

10. Echinodermata
(19 species / 81 specimens) “Pisces”
(32 / 187)
Cnidaria
(1 / 4)
Chaetognatha
(1 / 5)
”Polychaeta”
(3 / 14)
0.01
… in progress (BOLD) …
• Bryozoa
• Pantopoda ”Crustacea”
• Mollusca (81 / 502)
• Porifera (Sponge BoL)
• Ascidia
• Aves
• Mammalia
•…

11. Combining data:
• Aves
• “Crustacea”
• Insecta
• Mollusca
• “Pisces“

12. Two case studies:
Crustacea Fish
Session Marine (C20) Session Fish 1 (B41)

13. Potential pitfalls using CO1 …
• introgressive hybridization/incomplete lineage sorting
• co-amplification of numts
• heteroplasmy
• presence of inherited endosymbionts (e.g. Wolbachia or Rickettsia)
> linkage disequilibrium with mtDNA
• no barcoding gap

14. Supplementary nuclear markers: ribosomal expansion segments
18S rDNA: V7
18S rDNA: V4
Wuyts et al. 2001

15. Why using supplementary nuclear markers? One example ...
10
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5 mm
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8 © Wikipedia
Ligia oceanica (Linne, 1767)

16. • K2P: 0 - 5.4%
• split is also supported by 16S rDNA 25 additional mutation steps!
MP Network (TCS): COI

17. 18S rDNA: V4, V7
> no variation (0%)
> evidence for gene flow on nuclear level > not two but only one species
(also supported by morphometric and morphological studies)

18. Barcoding Analysis of
of the North Sea environmental
Metazoa samples
(COI, nuclear markers)

19. Analysis of environmental samples
Case study: Zooplankton
Silke Laakmann and Inga Mohrbeck
Zooplankton > heterogeneous mix of different organisms,
including both small protozoans and large metazoans
of different life stages with a size between 80 – 500 µm
www.tafi.org.au/zooplankton/index.html

20. Idea / Plans:
1. Inventory of species > buildup of a sequence library (COI, 18S rDNA)
Session Marine (C19)

21. Idea / Plans:
1. Inventory of species > buildup of a sequence library (COI, 18S rDNA)
2. Seasonal survey of zooplankton communities to document shifts
in species composition using next-generation sequencing methods

22. Used markers:
18S rDNA: V1 + V2
Wuyts et al. 2001

23. Barcoding Analysis of
of the North Sea environmental
Metazoa samples
(COI, nuclear markers)
Design of rapid tests
for specific species
identification

24. Design of molecular test systems for specific species identification
Case study: commercial fish
Thomas Knebelsberger and Torben Hofmann
Thomas Knebelsberger
The next step: rapid molecular in situ species identification
Parallel Talk, Marine Barcoding, Napier 210, Friday, 14:15 to 14:30
Join it! I will …

25. Most important player
Specific species identification

26. DNA-
and Tissue Bank
Barcoding Analysis of
of the North Sea environmental
Metazoa samples
(COI, nuclear markers)
Design of rapid tests
for specific species
identification
Long-term storage
of specimens and samples

27. Many different taxa – many different ways of handling

28. Work flow part I: specimen data acquisition
“Trust on proven, tested and simple systems”
sample
Fixation
(DESS, ethanol 96%)
identification
by experts
specimen
Specimen information
• running number (unique code)
• specimen details
• sample information
• taxonomic lineage (WoRMS)
• collection data
• photography
• DNA data
modified BOLD excel sheets

29. Work flow part II: molecular data acquisition
data base
MOTU sequences
voucher collection and sequence data base
sequencing
extraction
DNA
tissue
sample
specimen DNA extract
work aliquot
(freezer)
tissue bank DNA bank
(QiaSafe)

30. Thanks to …
Wilko Ahlrichs • Rudi Amann • Jan Beermann • Olaf Bininda-Emonds •
Maarten Boersma • Luisa Borges • Dirk Brandis • Gary Carvalho • Filipe
Costa • Simon Creer • Nicole Dubilier • Christiane Eichner • Günther Fuchs •
Frank-Oliver Glöckner • Paul Hebert • Sabine Holst • Jonas Keiler •
Alexander Kieneke • Matthias Kloppmann • Florian Leese • Peter Lienau •
Alexandra Markert • Pedro Martinez-Arbizu • Ole Stan Møller • Hermann
Neumann • Thurid Otto • Stefan Richter • Ursula Siebert • Christoph
Schubart • Sabine Schückel • Ulrike Schückel • Myriam Schüller • Martin
Schulze-Dieckhoff • Dirk Steinke • Sven Thatje • Hilde van Pelt • Achim
Wehrmann • …

31. END