TECHNICAL ADVANCES

Using next-generation sequencing for molecularreconstruction of past Arctic vegetation and climate

J. H. SØNSTEBØ,* L. GIELLY,† A. K. BRYSTING,‡ R. ELVEN,* M. EDWARDS,§ J. HAILE,¶**

E. WILLERSLEV,¶ E. COISSAC,† D. RIOUX,† J. SANNIER,* P. TABERLET† and C. BROCHMANN*

*National Centre for Biosystematics, Natural History Museum, University of Oslo, P.O.Box 1172 Blindern, NO-0318 Oslo,

Norway, †Laboratoire d’Ecologie Alpine, UMR UJF-CNRS 5553, Universite Joseph Fourier, 38041 Grenoble, France, ‡Centre for

Ecological and Evolutionary Synthesis, Department of Biology, University of Oslo, P.O.Box 1066 Blindern, NO-0316 Oslo,

Norway, §School of Geography, University of Southampton, Room 2019 Shackleton Building, Highfield, Southampton, SO17 1BJ,

UK, ¶Centre for Ancient Genetics, University of Copenhagen, Universitetsparken 15, DK-2100, Denmark, **Department of

Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

Abstract

Palaeoenvironments and former climates are typically inferred from pollen and macrofossil

records. This approach is time-consuming and suffers from low taxonomic resolution and

biased taxon sampling. Here, we test an alternative DNA-based approach utilizing the P6

loop in the chloroplast trnL (UAA) intron; a short (13–158 bp) and variable region with highly

conserved flanking sequences. For taxonomic reference, a whole trnL intron sequence data-

base was constructed from recently collected material of 842 species, representing all wide-

spread and ⁄ or ecologically important taxa of the species-poor arctic flora. The P6 loop alone

allowed identification of all families, most genera (>75%) and one-third of the species, thus

providing much higher taxonomic resolution than pollen records. The suitability of the P6

loop for analysis of samples containing degraded ancient DNA from a mixture of species is

demonstrated by high-throughput parallel pyrosequencing of permafrost-preserved DNA

and reconstruction of two plant communities from the last glacial period. Our approach

opens new possibilities for DNA-based assessment of ancient as well as modern biodiversity

of many groups of organisms using environmental samples.

Keywords: ancient DNA, Arctic Flora, chloroplast, climate change, DNA barcoding sensu lato,

environmental sample

Received 16 September 2009; revision received 14 December 2009, 27 January 2010; accepted 4 February 2010

Introduction

Many species may face extinction because of the current

climate warming (Thomas et al. 2004). Bioclimatic models

can be used to estimate relationships between species

distribution and climate and to predict future range

shifts and extinction risks (Araujo & Rahbek 2006). How-

ever, improved knowledge of historical species distribu-

tions and climates is needed for backward testing of

models before applying them to future climate scenarios

(Guisan & Thuiller 2005).

Past plant communities are traditionally inferred

from identification of fossil pollen or macrofossils, but

the taxonomic sampling and resolution is limited, partic-

ularly in the Arctic (Lamb & Edwards 1988). An alterna-

tive approach is molecular community analyses which

utilize information from ancient DNA accumulated in

soil (Willerslev et al. 2003). Here, traces in environmental

samples can be identified to species, genera or family

using diagnostic variation in a suitable DNA region,

which may be termed ‘DNA barcoding sensu lato’ (Valen-

tini et al. 2009). This approach has, for example, been

used to identify boreal tree species from silty ice at the

bottom of the Greenland Ice Sheet, showing that southern

Greenland was covered with forest between 450 000 and

800 000 years ago (Willerslev et al. 2007).

Traditionally, identification of organisms in environ-

mental (e.g. soil) samples containing DNA from a mix-

ture of organisms is time-consuming and expensiveCorrespondence: Christian Brochmann, Fax: +47 22 85 18 35;

E-mail: christian.brochmann@nhm.uio.no

� 2010 Blackwell Publishing Ltd

Molecular Ecology Resources (2010) doi: 10.1111/j.1755-0998.2010.02855.x

because the different amplified DNA fragments have to

be cloned into bacteria. Often, thousands of clones have

to be Sanger sequenced to detect the diversity of organ-

isms of interest. However, recently, new sequencing tech-

nologies have become available that bypass the cloning

step, which makes them ideal for analysis of mixed DNA

in environmental samples.

Molecular community analysis, especially of ancient

DNA, is not only dependent on a robust system with

primers amplifying over a wide range of taxa but also

on targeting a very short DNA region (<200 bp) that is

sufficiently variable and can be amplified from highly

degraded DNA (Willerslev & Cooper 2005; Taberlet

et al. 2007). Previously, Hajibabaei et al. (2006) found

that a short DNA region within the mitochondrial gene

cytochrome c oxidase I was effective to identify species

of wasps and moths. In plants, earlier studies of

ancient DNA have used parts of the rbcL chloroplast

gene (Poinar et al. 1998; Hofreiter et al. 2000; Willerslev

et al. 2003), but this system usually only enables identi-

fication to the family level (Willerslev et al. 2003). As

an alternative, Taberlet et al. (2007) suggested using a

part of the trnL (UAA) intron, the P6 loop, to identify

plant species from highly degraded DNA. In their anal-

ysis of modern DNA from 123 arctic species, it was

possible to distinguish close to 50% of the species with

the P6 loop only (Taberlet et al. 2007). This suggests

that the P6 loop may serve as a suitable marker for

reconstructing historical plant communities in species-

poor floras such as in the Arctic, which also provides

unique conditions for ancient DNA preservation in per-

mafrost soils.

Here, we assess the usefulness of the P6 loop for

reconstructing the composition of past arctic plant com-

munities from a mixture of ancient DNA sequences pre-

served in permafrost soils. We expand the taxonomic

reference database of Taberlet et al. (2007) to include all

widespread and ⁄ or ecologically important species

occurring in the Arctic (842 species). As our main pur-

pose is to develop a system for identification of species

from permafrost soil samples, the taxonomic resolution

of the P6 loop was compared to the maximum resolu-

tion that can be obtained from pollen analysis. To com-

pare the level of resolution with other DNA regions, the

chloroplast DNA barcoding regions proposed by Chase

et al. (2007) were also sequenced for several genera

which showed low or medium variation in the P6 loop.

We also used a bioinformatic approach to identify alter-

native regions based on all available complete cpDNA

genomes and to evaluate their potential usefulness. The

P6 loop approach was then tested by high-throughput

pyrosequencing on 454 Genome Sequencer FLX (Roche)

(Margulies et al. 2005) of dated samples from permafrost

soils.

Material and methods

Construction of the taxonomic reference database

Leaves from herbarium specimens or silica-dried leaf

samples of 856 species deposited at the Natural History

Museum in Oslo were analysed (Appendix S1). These

species represent 84 families and 289 genera following

the taxonomy of the Panarctic Flora checklist (Elven

2008). For most species, two specimens (in some cases

representing two subspecies) were selected from differ-

ent parts of the species distribution area.

Sequencing of the whole trnL intron was conducted in

parallel in our laboratories in Grenoble and Oslo and

merged into a single database. DNA extraction and poly-

merase chain reaction (PCR) of the trnL intron were per-

formed as reported for the arctic plant dataset in Taberlet

et al. (2007) with the following modification: PCR was

performed in volumes of 10 lL in Oslo and 25 lL in Gre-

noble; the concentration of the primers was 0.5 lM

instead of 1 lM; and the number of PCR cycles was 30 in

Oslo and 35 in Grenoble.

PCR for the other cpDNA regions was as follows:

trnH-psbA spacer – PCR was performed in volumes of

10 lL containing 2.5 mM MgCl2, 160 lM of each dNTP,

0.4 lM of each primer, 0.1 lg ⁄ mL bovine serum albumin

(BSA; Roche Diagnostic, Basel, Switzerland) and 0.4 U

of AmpliTaq DNA Polymerase (Applied Biosystems).

The trnH-psbA spacer was amplified with primers

trnHGUG and psbA (Sang et al. 1997). matK, rpoB and

rpoC1 – PCR was performed in volumes of 10 lL accord-

ing to the protocols of the Royal Botanic Gardens, Kew

(http://www.kew.org/barcoding/protocols.html). The

primers used for amplifying these three markers are also

available on this website. The matK region was ampli-

fied with the updated forward primer X and the reverse

primer 5, and the rpoB and the rpoC1 regions were

amplified by the forward primer 1 and the reverse pri-

mer 3.

Following the PCR amplification, PCR products were

diluted four times (in Oslo only) and purified with Exo-

SAP-IT (USB Corporation) according to the manufac-

turer’s instructions to remove excess primers and

deoxynucleotide triphosphates. Sequencing was per-

formed on both strands as described by Taberlet et al.

(2007), but in volumes of 10 lL containing 0.5 lM of the

amplification primer and 1 lL of the PCR products (in

Oslo only). For trnH-psbA, matK, rpoB and rpoC1, the

sequencing reactions underwent 30 s at 95 �C and 35

cycles of 30 s at 94 �C, 15 s at 50 �C and 4 min at 60 �C.

Excess dye terminators were removed by spin-column

purification. Sequencing reactions were electrophoresed

on an ABI PRISM�3100 Genetic Analyzer (Applied Bio-

systems) using either 80-cm capillaries and POP-4�

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2 T E C H N I C A L A D V A N C E S

polymer for 3 h (Oslo) or 36-cm capillaries and POP-

7�polymer for 1 h (Grenoble).

Data analysis

PCR of the P6 loop (primers g and h; Taberlet et al. 2007)

and the whole trnL intron (after adding the c and d prim-

ers (Taberlet et al. 2007) to the sequences) was simulated

on the whole taxonomic reference database using the

electronic PCR software ecoPCR (Taberlet et al. 2007).

This was performed to identify mismatch in the g and h

primer sequences and to identify to which taxonomic

unit each sequence could be identified, e.g. whether a

sequence represented a single species or a group of spe-

cies. The ecoPCR software counts the maximum mis-

match of primers (maximum mismatch of three bases

was allowed), minimum and maximum length of the

amplified sequence and retrieves taxonomic data from

the analysed entries. We also performed local Blast

searches on the database for each sequence to record dif-

ferent P6 loop and whole trnL intron haplotypes and

intraspecific variation based on the two (or in a few cases

three or four) sequences from each species (Appen-

dix S1).

Variation in the DNA barcoding regions matK, rpoB,

rboC1 and trnH-psbA was compared with the variation in

the P6 loop and the whole trnL intron by counting the

number of indels (indels covering several characters were

scored as one) and substitutions and calculating the per-

centage variability. The distribution of the variation

along the matK region and the trnL intron was visualized

using the FINGERPRINT software (Lou & Golding 2007; see

Fig. 2).

Tracking other potentially suitable regions based on allavailable complete cpDNA genomes

We used a bioinformatic approach to identify other

potentially suitable regions for analysis of degraded

DNA, taking advantage of the 110 currently available

complete cpDNA genomes (Streptophyta complete

cpDNA genomes). A potentially suitable region was

defined as a short and variable mid-region flanked by

two conserved regions that will be the target sequences

of the amplification primers. For finding all potentially

suitable regions, we first identified all conserved regions

of at least 18 bp among all the cpDNA genomes. Then,

we paired these regions to generate a set of all available

mid-regions shorter than 200 bp. Each of these mid-

regions (potential barcodes sensu lato) was evaluated for

its coverage (proportion of species potentially amplified

by the primer pair) and for its specificity (proportion of

species unambiguously identified among the species

potentially amplified).

Pollen resolution

The maximum potential taxonomic resolution that can

be obtained from pollen data was assessed using two

palynological keys (Fægri & Iversen 1964; Moore et al.

1991).

Permafrost analysis

Two permafrost samples were collected from an expo-

sure of late-Quaternary, ice-rich, silty sediments on the

Mayn River, Southern Chukotka, Russia (64�17¢N;

171�15¢E; A. Sher & E. Willerslev, unpublished). The sam-

ples were radiocarbon dated at the Oxford University

Accelerator Unit. Total DNA was extracted from approxi-

mately 6 g of sediment from each permafrost sample

using a combined Sergey Bulat protocol (Bulat et al. 2000)

and Cambio PowerMax Soil kit protocol, which employs

a silica clean-up method. The DNA extracts underwent

amplification of the P6 loop in a 50-lL volume containing

2 U AmpliTaq� Gold DNA Polymerase (Applied Biosys-

tems, Foster City, CA), 10 mM Tris-HCl, 50 mM KCl,

2 mM MgCl2, 0.2 mM of each dNTPs, 0.2 lM of each pri-

mer g and h (Taberlet et al. 2007), and 0.008 mg of BSA

and 1-lL DNA extract as template. PCR cycling condi-

tions were as follows: 10 min at 95 �C (enzyme activa-

tion, manufacturer’s condition), 15 cycles of 30 s at 95 �C

and 30 s at 50 �C followed by 40 cycles of 30 s at 95 �C

and 30 s at 60 �C. Elongation steps were omitted because

of the short length of the target sequences. DNA was

amplified with ‘fusion’ primers: primers g and h were

modified by the addition of a specific 6-base pair-long tag

and the sequencing primer at the 5¢ end (Jarvie 2007) to

facilitate parallel sequencing of multiple homologous

samples (Valentini et al. 2009). The sequences obtained

by pyrosequencing were subsequently compared to the

P6 loop database to assign them to the particular arctic

taxa that fully matched in lengths and sequences using

the software ecoPCR (Taberlet et al. 2007).

Results

Resolution of the P6 loop and the whole trnL intron

A total of 842 species was successfully sequenced for the

whole trnL intron. Two or more samples were sequenced

for each of 709 of these species (Appendix S1). Fourteen

species that were attempted, but not successfully

sequenced, are also listed in Appendix S1. The ecoPCR

analysis showed that the P6 loop varied in length from

13 bp to 158 bp (excluding primer target sites) and that

the primers g and h were generally well conserved, with

a maximum of two and three mismatches, respectively.

Ninety-eight per cent of the primer sequences exhibited a

� 2010 Blackwell Publishing Ltd

T E C H N I C A L A D V A N C E S 3

maximum of one mismatch. The mismatches were not

located close to the 3¢ end of primer (Appendix S2).

The ecoPCR analysis further showed that a total of

33.5% of the species and 77.1% of the genera could be

identified by the P6 loop. Among the identified species,

10.3% were represented by a single species in each family

(Fig. 1a). Based on the whole trnL intron, 59.5% of the

species and 98.5% of the genera could be identified

(Fig. 1b). All families were unambiguously identified

with both the P6 loop and the whole trnL intron. The

level of resolution (both in terms of species- and genus

resolution) of the P6 loop varied among families (Fig. 1a).

Only few species could be identified in the Salicaceae

and Papaveraceae. The P6 loop resolution was also low

in the Poaceae and Asteraceae, with only about 10% of

the species and 20–40% of the genera identified. In other

(a)

(b)

(c)

Fig. 1 Power of the P6 loop (a), the trnL

intron (b) and pollen (c) for discrimination

between genera and species in plant fami-

lies with more than two genera or five spe-

cies included in the taxonomic reference

data base. Black and grey histograms

show the percentages of discriminated

species and genera, respectively. Numbers

in parentheses refer to number of species

and number of genera analysed.

� 2010 Blackwell Publishing Ltd

4 T E C H N I C A L A D V A N C E S

important arctic families, such as the Cyperaceae, Brass-

icaceae, Caryophyllaceae, Ranunculaceae and Saxifraga-

ceae, the species resolution was 25–50%, but in these

families, all genera could be unambiguously identified

(Fig. 1a).

In several genera, for which the P6 loop provided

overall low species resolution, we could discriminate

among intrageneric groupings of species. In Ranunculus,

for example, the 13 species represented six identifiable

taxonomic sections, and in Pedicularis, five species

belonged to the P. sudetica aggregate that could be sepa-

rated from all other species (Appendix S1).

The intraspecific variation identified by the P6 loop

was generally low. Variation was found in 80 of the 709

species that were represented by two or more accessions.

This was usually length variation in poly(A) ⁄ poly(T)

regions (32 species), variation at single-nucleotide sites

(28 species) or indels of variable lengths (20 species)

(Appendix S1). The intraspecific variation did not usu-

ally affect the species resolution, except that the variation

in the length of poly(A) ⁄ poly(T) regions sometimes was

not species specific (particularly in Vaccinium).

Resolution of other molecular markers sequenced

The other molecular markers matK, trnH-psbA, rpoC1 and

rpoB (Chase et al. 2007) were sequenced for 24

(FN668453-FN668476), 22 (FN668431-FN668452), 33

(FN668504-FN668536) and 29 (FN668477-FN668503) spe-

cies, respectively, belonging to six different genera (App-

endix S3). Amplification was unsuccessful in one genus

each for matK, rpoB and trnH-psbA (Appendix S3). The

markers varied considerably in level of resolution among

genera, as was observed for the whole trnL intron and for

the P6 loop. matK and trnH-psbA were most variable,

similar to or more variable than the whole trnL intron

(Appendix S3). When combining sequences from all gen-

era, the variation appeared distributed evenly through-

out the whole regions, except in the trnL intron, where

clusters of conserved characters were found around the

variable P6 loop (illustrated for matK vs trnL in Fig. 2).

Other potentially suitable cpDNA regions identifiedusing a bioinformatics approach

Using a bioinformatics approach, quality of a barcode

region can be estimated measuring its coverage, the abil-

ity of the primer pair to amplify a large number of taxa

and measuring its specificity as its capacity to discrimi-

nate between taxa (Ficetola GF, Coissac E, Zundel S, Riaz

T, Bessiere J, Taberlet P, Pompanon F unpublished).

These indices allow for relatively evaluating the utility of

the different regions as barcodes useable for degraded

DNA. Following these criteria, Table 1 lists the 37 poten-

tially most suitable regions identified from 110 complete

cpDNA genomes of Streptophyta. Three of them were

most promising based on their combination of high cov-

erage and high specificity (1, 3, and 16). The first was

located in the 16S gene and was the most specific one

(72% unambiguously identified species), but this region

was so long (average 166.2 bp) that many species will

probably remain undetected in a matrix of highly

degraded DNA. The second region, which represented

the P6 loop flanked by the g and h primers used in this

study, was less specific (61% unambiguously identified

species), but it was shorter (average 43.2 bp) and had

better coverage. The third region (no 16) corresponded to

the first part of the trnL intron between primer c and the

reverse of primer g. This region had high coverage and

was short; however, its specificity was lower than that of

the P6 loop. In our arctic trnL intron database, this region

identified only 14% of the species and 43% of genera as

Fig. 2 Variation of the two chloroplast regions trnL intron (above) and matK (below) in 25 species. Location of primers g and h flanking

the P6 loop is shown for the trnL intron. Each line represents one character (bp), and the colour indicates the level of variation; black lines

for invariable characters and white lines for characters with four different states. Different shades of grey indicate intermediate variabil-

ity. Arrows indicate gaps in the alignment.

� 2010 Blackwell Publishing Ltd

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0.95

550.

5262

135

128.

1

26T

CT

TT

CG

GC

TA

AC

CT

AG

CT

CC

AA

TG

CC

CC

GA

AA

AC

C13

4826

105

0.95

550.

5263

136

129.

1

27G

CT

AA

CC

TA

GC

CT

CC

TC

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AA

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4833

107

0.97

560.

5256

129

122.

1

28G

GC

TA

AC

CT

AG

CC

TC

CT

CT

CC

AA

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CC

CC

GA

AA

AC

C13

4832

107

0.97

560.

5257

130

123.

1

29C

GG

CT

AA

CC

TA

GC

CT

CC

TT

CC

AA

TG

CC

CC

GA

AA

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C13

4831

107

0.97

560.

5258

131

124.

1

30A

GA

TG

AG

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CT

CT

CC

TA

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GG

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GC

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CA

GC

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T10

9125

104

0.94

540.

5211

117

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GA

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GA

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� 2010 Blackwell Publishing Ltd

6 T E C H N I C A L A D V A N C E S

compared to 34% and 77%, respectively, with the P6

loop.

Resolution from fossil pollen morphology

Pollen morphology can theoretically distinguish all fami-

lies included in our database, although some of them

(e.g. in the Ericales) are difficult to distinguish based on

fossil pollen because of poor preservation of morpho-

logical characters. Furthermore, the resolution at lower

taxonomic level varies considerably among families

(Fig. 1c). We estimated that the maximum resolution

based on well-preserved fossil pollen was identification

of 46.5% of the genera and only 10.2% of the species. In

the major arctic families Cyperaceae, Poaceae, Salicaceae,

Brassicaceae and Caryophyllaceae, little resolution can be

achieved from fossil pollen, and species-level resolution

is often difficult in the Saxifragaceae as well. However,

some important genera (e.g. Artemisia and Populus) are

identifiable based on pollen, but not on the P6 loop

(Appendix S1).

The permafrost soil samples

The two permafrost samples were dated to 22 960 ± 120

(OxA-15348) and 15 810 ± 75 (OxA-14930) uncalibrated

radiocarbon years B.P. Pyrosequencing of the two sam-

ples yielded haplotypes representing 47 and 18 different

molecular taxonomic units, respectively, the former iden-

tified from 1983 P6 loop sequences obtained in the 454

run (Table 2). Among the 47 taxonomic units from the

oldest sample, 20 could be identified to species, 19 to spe-

cies group or genus and eight only to family. Among the

18 units from the youngest sample, seven could be iden-

tified to species, six to species group or genus and five

only to family. The taxonomic composition differed sub-

stantially between the two samples. For the oldest sam-

ple, almost half of the sequences obtained from the 454

run were identified as Bistorta vivipara and a quarter of

them as Equisetum sp. In the youngest sample, sequences

of Trollius sp., Festuca sp. and Valeriana sp. occurred with

highest frequencies.

Discussion

This study represents the first implementation of P6 loop

analysis for palaeoecological reconstruction based on

ancient DNA. We have clearly demonstrated the power

of this approach. The system is very robust with highly

conserved primer sites, permitting potential amplifica-

tion of all gymnosperms and angiosperms in a DNA

extract from environmental samples containing a mixture

of different species. PCR can be carried out efficiently,

even with highly degraded DNA, as demonstrated here

Table 2 Lists of molecular taxonomic units based on DNA from

the two Chukotkan sediment samples, showing the molecular

taxonomic units identified and their frequency of occurrence

(% of sequences obtained in the 454 run)

%

22 960 ± 120 years BP

Bistorta vivipara 47.25

Equisetum arvense ⁄ E. fluviatile ⁄ E. sylvaticum 24.31

Salix sp. ⁄ Chosenia arbutifolia ⁄ Populus balsamifera 4.74

Armeria scabra 3.03

Thymus oxyodontus 2.77

Lagotis glauca 2.17

Asteraceae 1* 1.87

Avenella flexuosa 1.77

Aconogonon alaskanum ⁄ A. ocreatum ⁄ A. tripterospermum 1.36

Rumex sp. 1.31

Packera sp. ⁄ Senecio sp. 0.96

Poaceae 1† 0.96

Ranunculus acris ⁄ R. subborealis ⁄ R. turneri 0.81

Festuca sp. 0.76

Hulteniella integrifolia 0.66

Saxifraga hirculus 0.55

Trientalis europaea 0.45

Asteraceae 2‡ 0.40

Valeriana capitata ⁄ V. officinalis agg. 0.35

Myosotis alpestris 0.30

Asteraceae 3§ 0.30

Empetrum sibiricum ⁄ E. subholarcticum 0.30

Anthoxanthum nipponicum 0.25

Crepis chrysanta 0.25

Saxifraga bracteata ⁄ S. cernua ⁄ S. hyperborea ⁄S. radiata ⁄ S. rivularis

0.20

Papaver sp. 0.15

Elymus sp. ⁄ Leymus sp. 0.15

Trollius sp.¶ 0.15

Koeleria asiatica ⁄ Trisetum spicatum 0.15

Pedicularis oederi 0.10

Viola biflora 0.10

Claytonia arctica ⁄ C. scammaniana 0.10

Sanguisorba officinalis 0.10

Vaccinium uliginosum 0.10

Calamagrostis sp. 0.10

Potentilla sp. 0.10

Pulsatilla patens 0.05

Beckmannia syzigachne 0.05

Cardamine pratensis 0.05

Trisetum sibiricum 0.05

Vaccinium alaskense ⁄ V. myrtillus 0.05

Castilleja elegans ⁄ C. hyperborea 0.05

Deschampsia sp. 0.05

Parrya arctica ⁄ P. nudicaulis 0.05

Astragalus alpinus ⁄ A. umbellatus 0.05

Thalictrum alpinum ⁄ T. minus ⁄ T. sparsiflorum 0.05

Caltha arctica ⁄ C. palustris 0.05

15 810 ± 75 years BP

Trollius sp.¶ 27.87

Festuca sp. 16.34

Valeriana capitata ⁄ V. officinalis agg. 14.91

Asteraceae 1* 13.69

� 2010 Blackwell Publishing Ltd

T E C H N I C A L A D V A N C E S 7

with ancient DNA preserved in frozen sediments, and by

Valentini et al. (2009) for diet analysis based on DNA in

animal faeces. The system does not depend on time-con-

suming cloning and is well designed for the next-genera-

tion sequencing technology. It thus permits large-scale

and fast analysis. If the goal is to obtain about 2000 reads

per sample, a total of about 200 samples can be

sequenced in a single run with the current technology

available on the 454 GS FLX (Roche, Basel, Switzerland).

Full analysis can be carried out within few weeks as soon

as a taxonomic reference database is available, here con-

structed for the bulk of the arctic flora. However, a large

number of trnL sequences are already available in Gen-

Bank (about 28 000), facilitating development of similar

reference databases also for other floras and purposes.

We have demonstrated the potential for reconstruc-

tion of past plant communities from DNA preserved

in permafrost with the P6 loop. Our focus on a single,

relatively species-poor flora enabled resolution of most

genera (77%) and many species (33%) with this very

short DNA region. This would not be possible if all plant

species occurring worldwide were considered. Taberlet

et al. (2007) found that 19% of the species and 41% of the

genera could be identified in an electronic PCR analysis

of more than 11 000 P6 loop sequences from GenBank.

The approach might result in even higher resolution if

restricted to smaller areas within the Arctic. For example,

42% of the species and 89% of the genera currently

occurring in the isolated arctic archipelago of Svalbard

(for information about the Svalbard Flora, see http://

svalbardflora.net/index.php?id=1) can be identified using

the P6 loop only, but it cannot be excluded that other

species may have occurred in this archipelago in earlier

time periods.

We have shown that the proportion of genera and spe-

cies identifiable with the P6 loop is considerably higher

than with pollen analyses, based on the maximum poten-

tial resolution inferred from two frequently used palyno-

logical keys. However, in some cases, genera or species

can be identified palynologically but not with the P6

loop. Furthermore, the low pollen production in insect-

pollinated plants tends to result in poor representation of

such species in the fossil record (although very high pol-

len counts may improve the diversity in pollen records;

see Cwynar (1982) and Anderson et al. (1994)). Prolific

wind-pollinated genera (particularly Betula and Alnus)

are over-represented and further skew pollen assemblage

composition. In addition, wind-dispersed pollen can be

transported over long distances and is difficult to distin-

guish from locally produced pollen. DNA-based flora

reconstruction from soil may thus give a more accurate

picture of the local species assemblage, as do detailed

plant macrofossil studies (Goetcheus & Birks 2001; Kie-

nast et al. 2008).

Among the alternative chloroplast regions we

sequenced here (matK, rpoB, rpoC1, trnH-psbA), only

matK and trnH-psbA had similar or more variation than

the whole trnL intron. Previously, difficulties with ampli-

fication of the trnH-psbA intron in some genera have been

reported (e.g. Lahaye et al. 2008), suggesting that this

region is not suitable for sequencing of environmental

samples. In coding genes, e.g. matK, mutations occur

frequently over the whole region, resulting in lack of

potentially conserved internal primer sites (see Fig. 2 for

a graphical presentation of the variation in matK).

Thus, the alternative regions sequenced here were less

suitable than the P6 loop for our specific purpose of

reconstructing past vegetation based on degraded DNA

from samples containing a mixture of different species.

This conclusion was supported by our bioinformatic

analysis of all available complete Streptophyta cpDNA

genomes, demonstrating that the P6 loop of the trnL

intron is the best candidate region for analysing highly

degraded template DNA (no 3 in Table 1). It is the short-

est one among the 37 potential cpDNA regions identified,

Table 2 Continued

%

Salix sp. ⁄ Chosenia arbutifolia ⁄ Populus balsamifera 8.98

Bistorta vivipara 4.95

Alchemilla glomerulans 4.91

Larix cajanderi ⁄ L. dahurica ⁄ L. laricina 2.99

Pulsatilla patens 1.72

Koeleria asiatica ⁄ Trisetum spicatum 1.48

Hulteniella integrifolia 0.88

Asteraceae 3§ 0.66

Viola biflora 0.29

Asteraceae 2‡ 0.11

Vaccinium alaskense 0.11

Gentianopsis barbata ⁄ G. detonsa 0.07

Omalotheca norvegica 0.04

*Eurybia sibirica, Solidago multiradiata, S. virgaurea, Symphyotri-

chum pygmaea.

†Alopecurus magellanicus, Anthoxanthum arcticum, A. monticola,

Arctagrostis latifolia, Beckmannia syzigachne, Hyalopoa lanatiflora,

Milium effusum, Poa abbreviata, P. arctica, P. eminens, P. glauca,

P. hartzii, P. laxa, P. palustris, P. paucispicula, P. pratensis,

P. pseudoabbreviata, P. sibirica, Vahlodea atropurpurea.

‡Hieracium alpinum, H. sect. Sylvatica, Lactuca sibirica, Saussurea

alpina, S. tilesii, Taraxacum croceum, T. Lacerum.

§Achillea alpina, A. millefolium, Arctanthemum arcticum, Artemisia

arctica, A. arctisibirica, A. borealis, A. frigida, A. furcata, A. glomera-

ta, A. kruhsiana, A. tilesii, Chrysanthemum mongolicum, Tanacetum

bipinnatum.

¶We refer to this haplotype as Trollius sp.: only the European

T. europaeus and none of the Siberian Trollius species is included

in our current database. However, all trnL sequences of 12 spe-

cies of Trollius, including T. europaeus and two of the Siberian

arctic species, deposited in GenBank have identical P6 loops.

� 2010 Blackwell Publishing Ltd

8 T E C H N I C A L A D V A N C E S

but nevertheless provides high taxonomic resolution

compared to other regions.

The first analyses of permafrost sediments presented

here illustrate the potential of the P6 loop for palaeo-

ecological reconstruction based on degraded DNA. A

high proportion of the sequences retrieved could be

identified to species, species groups and genera. We

could also assess the relative abundances of the differ-

ent haplotypes from the 454 sequencing, although it is

still not clear to what degree these reflect the actual rel-

ative abundances of the different taxa in the past vege-

tation. The species composition of the older sample we

analysed, which appears to be dominated by Bistorta

vivipara and Equisetum sp. (most likely E. arvense in this

case), indicates a species-rich, mesic meadow ⁄ moist

heath community. The species composition of the youn-

ger sample, which appears to be dominated by Trollius

sp., Valeriana sp. and grasses, indicates a nutrient-rich

tall-forb meadow. This also might be expected to be

species-rich, and it is probable that analysis of more

samples from the same locality would reveal additional

species. Thus, the first test results presented here sug-

gest that full analysis of sites in permafrost regions may

allow for detailed reconstructions of past plant commu-

nities and give new insights into their change over

time.

The main constraint of our approach is clearly the lim-

ited resolution at the species level, but this apparently

must be accepted as unavoidable when working with

degraded DNA, and particularly so in the young arctic

ecosystem, which formed only 2–3 million years ago

(Brochmann & Brysting 2008). The low sequence varia-

tion observed in many arctic plant groups can be

explained by recent (Pleistocene) speciation, for example

in Papaver, Salix, Draba and Cardamine (reviewed in

Brochmann & Brysting 2008). Further complicating the

analysis of conservative molecular markers such as chlo-

roplast DNA in recently originated arctic species is

potential incomplete lineage sorting of old polymor-

phisms, which can be shared among closely related spe-

cies. The arctic flora is also known for high levels of

interspecific hybridization, which may lead to transfer of

chloroplasts between species (Brochmann & Brysting

2008). For the taxonomic reference database constructed

here, we sequenced two geographically widely separated

specimens of each species to minimize this problem, but

in some cases, such processes may admittedly lead to

erroneous discrimination among species based on our

current database. Nevertheless, despite the inherent limi-

tations associated with the evolutionary history of arctic

plants, the Arctic provides unique possibilities for

long-term DNA preservation in the permafrost and thus

ideal conditions for reconstructing historical species

assemblages.

It is possible that the taxonomic resolution of our

approach can be improved by using a tiered approach,

with the P6 loop as the first tier and subsequently design

family ⁄ genus-specific primers around short regions in

the chloroplast or nuclear genome with higher power for

species separation (Newmaster et al. 2006). The current

resolution should nevertheless be sufficient for quite

detailed large-scale reconstruction of past plant commu-

nities. Inferences of past climates based on the ecological

requirements of the identified species, inferred from

niche modelling of current species distributions, do not

require identification of all plant species in past commu-

nities.

Two important possibilities need to be explored in fur-

ther studies. First, it should be possible to apply the same

approach also to other groups of organisms, by designing

appropriate short barcode systems and constructing

taxonomic reference databases. Second, the possibility of

quantitative reconstruction of past communities should

be explored by analysis of DNA from modern soils

relative to present composition of the vegetation. The

accuracy of quantitative reconstruction can possibly be

improved by carrying out emulsion PCR that better pre-

serve the initial concentrations (Williams et al. 2006) and

by increasing the number of sequences from each sample

in the 454 analysis.

To summarize, we have shown that use of the P6

loop is promising for improved palaeoecological recon-

struction. Our approach can also be useful in other

kinds of ecological studies depending on a short bar

coding region, for example in studies of species com-

position in faeces or other animal remains (Valentini

et al. 2009). Besides enabling amplification from

degraded DNA, short DNA regions can also be easily

retrieved via ultra-high-throughput sequencing, as

demonstrated for the permafrost soil samples analysed

here. With this technology, a high number of DNA

fragments can be sequenced in parallel without the

need for traditional cloning. We conclude that such

short DNA barcode systems, together with the parallel

pyrosequencing technology, open new avenues for

DNA-based biodiversity assessment using environmen-

tal samples.

Acknowledgements

This study was funded by the European Commission, via the

ECOCHANGE project (GOCE-2006-036866). We thank Professor

Mark Ungerer and three anonymous reviewers for valuable

comments on improving the manuscript. We also thank Corinne

Cruaud and Patrick Wincker for the 454 sequencing at Geno-

scope, France and Virginia Mirre for technical assistance in the

Oslo laboratory. The late A.V. Sher identified the Quaternary

sequence at Mayn River and established the stratigraphic context

for sample collection.

� 2010 Blackwell Publishing Ltd

T E C H N I C A L A D V A N C E S 9

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Supporting Information

Additional Supporting Information may be found in the

online version of this article.

Appendix S1. List of species included in the Arctic plant

database, with P6 haplotypes (each number refers to a

unique haplotype) and trnL haplotypes and notes on

intraspecific variation in parentheses (number of substi-

tutions, number of indels).

Appendix S2. Variability in g and h primer sites (Taberlet

et al. 2007), with variable characters given.

Appendix S3. Variability in DNA barcoding markers

from Chase et al. (2007) compared with the trnL intron

and the P6 loop.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting information

supplied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.

� 2010 Blackwell Publishing Ltd

10 T E C H N I C A L A D V A N C E S