Department of Evolutionary Biology Zoological Institute University of Copenhagen Ancient DNA in Sediments

Department of Evolutionary BiologyZoological InstituteUniversity of Copenhagen

Ancient DNA in Sediments

Ancient DNA Studies

DNA from Sediments

Sample Information

Samples Site Age range (B.P.)

Permafrost

1/02/0.5 Kolyma lowland, Plakhin Jar modern tundra soil

1/93/4.0 Kolyma lowland, Kon'kovaya river 10.425±45 yr

2/01/4.8 Laptev Sea coast, Cape Bykovskii 18.980±70 yr (8-9 kyr)

7/90/1.6 Kolyma lowland, Chukochia river 20-30 kyr

3/01/20.7 Laptev Sea coast, cape Svyatoi Nos 300-400 kyr

4/01/9.2 Laptev Sea coast, cape Svyatoi Nos 300-400 kyr

6/90/30.7 Kolyma lowland, Chukochia river 1.5-2.0 Ma

6/90/31.1 Kolyma lowland, Chukochia river 1.5-2.0 Ma

1/99/14.5 Beacon Valley, Antarctica 8.1 Ma

New Zealand

Cave sediment Clutha River 624±50 yr

Cells in the bacterial size range (about 107cells/ gww, average cell volume 0.03-0.05 µm3/cell)

Occasional fine rootlets (≥2 mm in diameter), seeds and small unidentifiable multicellular fragments

No bone/hair/identifiable animal

soft tissue

Microscopy

PCR Based Analyses

4 x 0.25gww soil FAST PREPDNA extraction/purificationPCR (“universal”/”specific”

primers for rbcL/mtDNA)CloningSequencing BLAST (GenBank)/phylogenetic

analysis

Precaution, Controls, Criteria

Special rotation-column coring method Spiking with bacterial Serratia marcescens Isolated, dedicated clean lab. Isolated ventilation system, UV-radiation,

flow hood Facemasks, gamma-sterilized glows, hats Removal of core surfaces Cleaning of reagents/tools: UV, HCL,

bleach, ultrafiltration Extraction/ PCR controls Cloning Independent reproducibility of results Phylogenetic criteria

Important!

Not previously worked with in the Copenhagen lab (at that stage):

plant rbcL DNA DNA from Arctic or NZ animals

(including megafauna) except for Reindeer mtDNA

Previously produced PCR products is a major source of contamination

Amplification Results

Plants (rbcL about 130 bp): PCR products up to 300-400 kyr

(including NZ cave site) No PCR products million year old samples

Animal (mtDNA 88-234 bp): PCR products up to 20-30 kyr (including

NZ cave site, only primers for bird mtDNA)

no PCR products 300-400 kyr and million year old samples

The results were independently confirmed in Oxford

Plant identifications (multiple GenBank sequences showing >96%

similarity to the clones; reproducibility confirmed by a bootstrap test )

Class or Sub-class =9

Order =22 Family =28

Liliopsida

Coniferopsida

Asteridae

Rosidae

CaryophyllidaeEudicotyledon

Bryidae

Polytrichopsida

Bryopsida

Poales

Liliales

Coniferales

Ericales

MalpighialesMyrtales

Malvales

Fagales

Fabales

Rosales

Brassicales

Caryophyllales

Lamiales

Asterales

Gentianales

Ranunculales

Rhizogoniales

Hypnales

Bryales

Polytrichales

Grimmiales

Pottiales

Cyperaceae

Poaceae

Liliaceae

Cupressaceae

Podocarpaceae

Ericaceae

Salicaceae

Flacourtiaceae

Onagraceae

Malvaceae

Nothofagaceae

Fabaceae

Rhamnaceae

Rosaceae

Brassicaceae

Caryophyllacae

Polygonaceae

Antirrhinaceae

Asteraceae

Campanulaceae

Rubiaceae

Papaveraceae

Rhizogoniaceae

Hylocomiaceae

Polytrichaceae

Grimmiaceae

Pottiaceae

Moraceae

Source of rbcL DNA

Chloroplast sequences are essentially absent from angiosperm pollen (Blanchard & Schmidt 1995)

The majority of the plant sequences must originate from locally deposited seeds, or somatic tissue such as the observed fine rootlets

mtDNA 16S (88-95 bp)Vombatus ursinusAJ304826

Dugong dugongAY075116

Homo sapiensAF382013

Oryctolagus cuniculusAJ001588

Lepus europaeusAJ421471

2 clones (10.4 kyr) - permafrost sediment100

100

Volemys kikuchiiAF348082

Dicrostonyx groenlandicusAY261992

54

93

92

75

63

68

72

95

Loxodonta africanaAF039436Loxodonta africanaAJ224821

99

96

97

Rhinoceros unicornisX97336Ceratotherium simumY07726

87

58

77

70

clone (19 kyr) - permafrost sediment

Lemmus lemmusAY261993




Mammuthus primigeniusAF154865

Mammuthus primigeniusZ54098

clone (10.4 kyr) - permafrost sediment




9 clones (10.4, 19, and 20-30 kyr) - permafrost sediment

Equus hemionus (Pleistocene)S65410

Equus hemionusZ18645

Equus asinusX97337

Equus caballusX79547

Equus sp. (Pleistocene)X86215





8 clones (19 kyr) - permafrost sediment

0.1

Rattus norvegicusAJ428514

Capricornis crispusU87029

Ovibos m

Control mtDNA region (124-129bp)

Vombatus ursinusAJ304826

Homo sapiensAF347015

Ozotoceros bezoarticusAF012572

Rangifer tarandus groenlandicusAF096441


100

98


Capricornis crispusAB055699

Ovibos moschatusAY261987

74

67

Bos taurusAB065127

Bison spp. (Pleistocene) CRS-DY-42AY261988


Bison spp. (Pleistocene) CRS-SY-2AF538947

70

96

78

83

94

clone (20-30 kyr) - permafrost sediment



0.

mtDNA cyt b sequences (A, 98 bp and B, 229 bp)


Loxodonta cyclotisAF132527


79

Elephas maximusAF132526

Elephas maximusD50844

100

Mammuthus primigeniusD50842





65

64

0




78

Elephas maximusAF132526

Elephas maximusY13886

90


Mammuthus primigeniusAF154864





63

94

59

3 clones (8-12 kyr) - permafrost sediment

0.

Control mtDNA region (202-203 bp)

clone (1-3 kyr) Tokerau Beach - sediment inside bone










clone (600 yr) Clutha River - cave sediment

















Euryapteryx curtusAY261989

Pachyornis elephantopusAY261990

Megalapteryx didinusAY261991

60

50

99

68

100

93

92

88

Control mtDNA region 234 bp

Psephotus varius

0.1

Nymphicus hollandicus

Cacatua roseicapilla

Nestor notabilis

Nestor meridionalis

Strigops habroptilus

Psephotus haematonotus

Barnardius barnardi

Barnardius zonarius

Northiella haematogaster

Cyanoramphus novaezelandiae

clone (600 yr Clutha River - cave sediment

100

100

56

68

63

81

66

100

94

Source of Animal mtDNA

UnknownDung is a possibility?

From Poinar et al. (2001)

Plant Sequence Diversity

(>96% similarity)

Frequency; Herbs, Shrubs, Mosses

Conclusions

Diverse ancient DNA directly from soil (even in the absence of obvious microfossils)

Change in plant diversity (following climate change)

Change in herb/shrub dominance Change in Poaceae and Cyperaceae

frequency(Pleistocene/Holocene boundary)

Megafauna present during LGM DNA better preserved in permafrost

than cave sediments Clutha River vegetation cover similar

to pre-human occupation of NZ even at 600 kyr

Perspectives

Combined with pollen records and fossil bones revealing Paleobiological change

Genetic information from archaeological records even in the absence of macrofossil evidence?

DNA damage analysis

• DNA in fossil remains is known to be degraded

• Unknown to a large extent what types of damages accumulate

• And especially what types of damages prevents amplification of DNA

DNA breaks

N

NH

O

O

CH3

N

NH

N

N

NH2

O

N

N

NH2

O

N

N

N

N

NH2

OO

O

OH

O-

O

P

OO

O

O-

O

P

OO

O

O-

O

P

OO

OH

O-

O

P

Clevage by depurinationand ß-elimination

Clevage of thephosphor backbone

Deaminationof Cytosine

Interstrand Crosslinks(Denaturation experiment)

DNA

DNAProtein

B

fICL = 0.43 + 0.49 (1 - e-0.0055 t)

R2 = 0.993

Age (kyr)

0 100 200 300 400 500 600

f ICL

0.0

0.2

0.4

0.6

0.8

1.0

fICL = 0.43 + 0.57(1-e-0.0034 · t)

k = 0.0034 kyr-1 = 1.1 x 10-13 s-1

r2 = 0.9684

Rate constants

Lesion type

Time

(kyr)

flesion

flesion

k*

(sec-1)

T½

(yr)

DSB 10.4 300-400 0.00013†

10.4 400-600

0.00037† < 3.7 x 10-17 > 8 x 108

SSB 10.4 0.00053‡

19 0.0016‡ 3.9 x 10-15 5.5x106

ICL 10.4 0.44

19 0.49

300-400 0.85

400-600 0.87

1.1 x 10-13 2.0 x 105

Conclusion

• DNA in permanently frozen sediments are degraded by alkylation and hydrolysis, producing single and double stranded breaks as well as interstrand crosslinks

• ICL accumulate more rapidly than SSB

• SSB is generated by depurination

• The observed damage pattern indicate that DNA degradation result from spontaneous rather than exogenous processes.

Perspectives

Repair of ancient DNA

Possible dating of sampels

Determination of spontaneous accumulation of DNA damages in cells

The work has been done by:

• Alan Cooper• Anders J. Hansen• Beth Shapiro• Carsten Wiuf• David A. Gilichinsky• David Mitchell• Eske Willerslev• Jonas Binladen• Lakshmi Paniker• M. Thomas P. Gilbert • Mike Bunce• Regin Rønn• Tina B. Brand

Department of Evolutionary

Biology, Zoological Institute, University of Copenhagen, Denmark

Henry Wellcome Ancient Biomolecules Centre, Department of Zoology, University of Oxford, UK

Department of Statistics, University of Oxford, UK

Soil Cryology Laboratory, Institute for PhysicoChemical and Biological Problems in Soil Science, Russian Academy of Sciences, Russsia

Department of Cariogenese, MDAnderson Cancer institute, UT

Beringia

Beringia Megafauna of the

Late Pleistocene

Arctic Dessert or Steppe?Why Megafauna got

Extinct?

Traditional Approach

Pollen analysesProblems:Variation in influx rates, long distance dispersal, no account for vegetative growth, problems of taxonomic identification

Vertebrate fossils Problems:Different preservation, dating beyond carbon age

Thoughts… Is it possible to address the paleo-

environment of Beringia by obtaining DNA directly from the permafrost sediments even in the absence of macrofossils?

Cold conditions is critical for the long-term preservation of DNA (Smith et al. 2002). If plant or animal DNA accumulates in sediments permafrost must provide ideal preservation conditions

Documents

Department of Evolutionary Biology Zoological Institute University of Copenhagen Ancient DNA in Sediments