DNA barcoding-at-sea on Polarstern ANTXXXIV-1DNA barcoding-at-sea on Polarstern ANTXXXIV-1

TEAM DNA (left to right): Christina Folker, Paola Batta Lona, Leo Blanco Bercial, Lisa Nigro, Rob Jennings, Ann Bucklin, CJ Sweetman

Zooplankton BarcodesSargasso Sea & Eastern Atlantic

GroupSpecies Known

Individuals Submitted

Species Submitted

DNA Extracted

Individuals Barcoded

Species Barcoded

Individuals Barcoded

Species Barcoded

CNIDARIA Hydromedusae 842 0 0 0 0 0 12 8 Siphonophora 160 0 0 0 0 0 50 50 Scyphozoa 161 0 0 0 0 0 5 5CRUSTACEA 0 Amphipoda 400 42 27 31 9 9 22 17 Copepoda 2,000 686 162 298 26 15 96 46 Euphausiidae 86 144 23 79 16 6 23 11 Ostracoda 169 540 77 161 99 45 156 58 Other crustacea xx 54 6 4 0 0 10 8MOLLUSCA 0 Gastropoda 144 225 44 114 53 20 96 37 Cephalopoda 370 19 16 15 12 11 23 20Nemertea 99 3 1 1 1 1 3 2ANNELIDA 0 Polychaeta 110 0 0 0 0 0 5 3CHAETOGNATHA 93 67 18 33 12 5 28 11

TOTAL 4,634 1,780 374 736 228 112 529 310

Both Cruises to DateEastern Atlantic

DNA Barcoding Steps & Errors

1) Identify specimen

2) Extract DNA

3) PCR mtCOI barcode

4) Sequence DNA

5) Edit / align sequences

6) Validate barcode

7) Submit to database

Wrong specimen - specimen incorrectly identified - specimen incorrectly labeled

No DNA - bug dead too long before preservation - DNA cooked under microscope - DNA cooked during sample shipment

PCR fails - PCR primers don’t match species - PCR protocol not optimal for species - too much or too little DNA

Sequence fails - mis-priming in sequencing reaction

Incorrect sequence - pseudogene of mtCOI - duplication / translocation of mtCOI - contamination with exogenous DNA - contamination from symbiont, gut

Eastern Atlantic Barcodes

228 individuals, 112 speciesNeighbor Joining algorithm, Kimura-2-Parameter distances, Bootstrap 1000X

Amphipods 9 ind, 9 spp

Ostracods

99 ind, 45 spp, 65%

Gastropods 46 ind, 17 spp

Copepods26 ind, 15 spp, 95%

Gastropods 7 ind, 3 spp, 100%

Cephalopods12 ind, 11 spp, 72%

Nemerteans1 ind, 1 sp

Euphausiids 16 ind, 6 spp

Chaetognaths 12 ind, 5 spp, 96%

0.1

99

100

95100

Zooplankton BarcodesSargasso Sea & Eastern Atlantic

529 individuals, 310 speciesNeighbor Joining algorithm, Kimura-2-Parameter distances

0.1

Copepods96 ind, 46 spp, 98%

Gastropods77 ind, 27 spp

Gastropods19 ind, 10 spp, 99%

Cephalopods 23 ind, 20 spp, 99 %

Polychaetes 5 ind, 3 spp, 99%

Nemerteans3 indiv, 2 spp

Amphipods 22 ind, 17 spp, 61%

Ostracods 156 ind, 58 spp, 99%

Other Crustacea 10 ind, 8 spp, 75%

Siphonophores50 ind, 50 spp

Hydromedusae 12 ind, 8 spp

Scyphzoa 5 ind, 5 spp

Chaetognaths 28 ind, 11 spp, 90%

Euphausiids23 ind, 11 spp, 85%

99

100

98

Barcoding Euphausiids

193 individuals, 40 speciesNeighbor Joining algorithm, Kimura-2-Parameter distances, bootstrapped 1000X

N. atla

ntica(

15)

N. micropsN. tenella(4)

N. gracilis(3)N. megalops(4)

N. difficilis(5)

T. monacantha(6)

S. abbreviatum(4)

S. carinatum(5)

S. elongatum(4)

S. longicorne(3)S. affine(3)

S. suhmii(6)

T. longipes(2)T. ocu l atu m

T. spinifera(6)

N. s

exsp

inos

um (4

) N

. fle

xipe

s(4)

T. o

rient

alis

(3)

S. m

axim

um(3

) B

. am

blyo

ps

T. o

btusif

rons(5

)

T. aequalis(16)

N. australis(2)

N. simplex

M. norvegica

T. gregaria(3)

T. macrura(3)

T. tricuspidata(3)

E. tenera(6) E. lammellibrachea(7)

E. sibogae(3) E. distinguenda(9)

E. gibboides(6)

E. brevis(4)

E. eximia(2)

E. brevis(14)

E. mutica(2)

E. americana(2)

E. krohnii(14)

0.05

Euphausia

Thysanoessa

Nyctiphanes

Meganyctiphanes

Thysanopoda

Stylocheiron

Nematobrachion

Nematoscelis

Bentheuphausia

Tessarobrachion

98

99

99

100% bootstrap

Barcoding Taxonomic Experts

Martin AngelOstracods

Francesc Pagès (dec.)Siphonophores

Janet Bradford-Grieve Sigrid SchieldCopepods

Russ HopcroftGastropods

Uwe PiatkowskiCephalopods

Dhugal LyndseyAmphipods

Annelies Pierrot-BultsChaetognaths

Peter Wiebe Fred BuccholtzEuphausiids