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Analysing the marine fauna of the North Sea: A molecular approach using various methods
Michael J. Raupach
© Wikipedia © Robin Moore
© Julian Gutt/AWI
Hotspots of species diversity
© Nordsee von oben
© Nordsee von oben
© Nordsee von oben
© Nordsee von oben
© Nordsee von oben© Wikipedia
North Sea
Hotspot of species diversity?
… not a stable sea …
© Wikipedia
Halpern et al. 2008
Global map of cumulative human impact across 20 ocean ecosystem types
Kaluza et al. (2010)
Routes, ports and centralities in the global cargo ship network
World Ocean Review 2010
Invasive species
Dr. Michael J. Raupach
Dr. Silke Laakmann
Dr. Thomas Knebelsberger
Dipl. Biol. Inga Mohrbeck
Dipl. Biol. Thorben Hofmann
Tech. Ass. Valeska Borges
founded by
AG Molecular Taxonomy of Marine Organisms (est. summer 2010)Senckenberg am Meer / Germany Centre of Marine Biodiversity Research
Goal: Validation and development of molecular methods for the identification
of the marine metazoan fauna of the North Sea
Barcoding
of the North Sea
Metazoa(COI, nuclear markers)
Concept:
“Pisces”(32 / 187)
Chaetognatha(1 / 5)
”Crustacea”(81 / 502)
”Polychaeta”(3 / 14)
Cnidaria(1 / 4)
Echinodermata(19 species / 81 specimens)
… in progress (BOLD) …
• Bryozoa• Pantopoda• Mollusca• Porifera (Sponge BoL)• Ascidia• Aves• Mammalia• …
0.01
Combining data:• Aves
• “Crustacea”
• Insecta
• Mollusca
• “Pisces“
Session Fish 1 (B41)Session Marine (C20)
Two case studies: Crustacea Fish
Potential pitfalls using CO1 …
• co-amplification of numts
• heteroplasmy
• presence of inherited endosymbionts (e.g. Wolbachia or Rickettsia)
> linkage disequilibrium with mtDNA
• no barcoding gap
• introgressive hybridization/incomplete lineage sorting
18S rDNA: V4
18S rDNA: V7
Supplementary nuclear markers: ribosomal expansion segments
Wuyts et al. 2001
285
1210
10
31
8
26
Ligia oceanica (Linne, 1767)
Why using supplementary nuclear markers? One example ...
© Wikipedia
5 mm
25 additional mutation steps!• K2P: 0 - 5.4%• split is also supported by 16S rDNA
MP Network (TCS): COI
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)
Barcoding
of the North Sea
Metazoa(COI, nuclear markers)
Analysis of
environmental
samples
www.tafi.org.au/zooplankton/index.html
Analysis of environmental samplesCase 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
Idea / Plans:
1. Inventory of species > buildup of a sequence library (COI, 18S rDNA)
Session Marine (C19)
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
18S rDNA: V1 + V2
Used markers:
Wuyts et al. 2001
Barcoding
of the North Sea
Metazoa(COI, nuclear markers)
Analysis of
environmental
samples
Design of rapid tests
for specific species
identification
Design of molecular test systems for specific species identificationCase 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 …
Specific species identification
Most important player
Barcoding
of the North Sea
Metazoa(COI, nuclear markers)
Analysis of
environmental
samples
Design of rapid tests
for specific species
identification
DNA-
and Tissue Bank
Long-term storage
of specimens and samples
Many different taxa – many different ways of handling
Work flow part I: specimen data acquisition
“Trust on proven, tested and simple systems”
Specimen information
• running number (unique code)
• specimen details
• sample information
• taxonomic lineage (WoRMS)
• collection data
• photography
• DNA datamodified BOLD excel sheets
identification by experts
specimen
Fixation(DESS, ethanol 96%)
sample
tissue sample
voucher collection
tissue bank
MOTU sequences and sequence data base
sequencing
DNA bank(QiaSafe)
work aliquot(freezer)
Work flow part II: molecular data acquisition
specimen DNA extract
DN
Aextraction
data base
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 • …
END