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