www.sciencemag.org/cgi/content/full/335/6072/1083/DC1

Supporting Online Material for

Glacial Survival of Boreal Trees in Northern Scandinavia

Laura Parducci, Tina Jørgensen, Mari Mette Tollefsrud, Ellen Elverland, Torbjørn Alm, Sonia L. Fontana, K. D. Bennett, James Haile, Irina Matetovici, Yoshihisa Suyama, Mary

E. Edwards, Kenneth Andersen, Morten Rasmussen, Sanne Boessenkool, Eric Coissac, Christian Brochmann, Pierre Taberlet, Michael Houmark-Nielsen, Nicolaj Krog Larsen,

Ludovic Orlando, M. Thomas P. Gilbert, Kurt H. Kjær, Inger Greve Alsos, Eske Willerslev*

*To whom correspondence should be addressed. E-mail: ewillerslev@snm.ku.dk

Published 2 March 2012, Science 335, 1083 (2012)

DOI: 10.1126/science.1216043

This PDF file includes:

Materials and Methods SOM Text Figs. S1 to S5 Tables S1 to S5 References (40–92)

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Materials and Methods

Material In 2009 we conducted a genetic survey of needles from more than 100 forest stands of

Norway spruce Picea abies, the only native spruce in Scandinavia) sampled across the north European species range (Fig. 1b; Table S1). The majority of samples came from (20). For the present study we collected additional samples from seven populations in central Scandinavia (populations 56, 57 and 98-102 in Table S1).

To ensure that we collected spruce material from natural stands, when possible we gathered local information on stand history. Accordingly, we did not sample at Andøya, where the only extant spruce was planted within the last 200 years (Fylkesmannen i Nordland, Pers. comm. 2010). To ensure that sampled stands were not planted or dispersed provenances from the central European species range, we included 15 spruce populations from Germany, Switzerland, Serbia, Italy, and Austria as controls (Table S1). For information on collection see also (20, 21). For populations from central Scandinavia collected primarily for this study, we collected needles from an average of 16 trees. Sampled trees were between 20 and 50 m apart. As reference species, we analyzed trees from the two closely related spruce taxa, Siberian spruce (Picea obovata) and white spruce (Picea glauca), and from the more distantly related Scots pine (Pinus sylvestris). For Siberian spruce, we sampled seeds from six individuals originating from three populations located in different localities along the Ural Mountains. White spruce is widely distributed across Canada and Alaska, and we therefore analyzed 11 seeds from a seed orchard with mixed individuals originating from Québec (Canada). For Scots pine, we sampled needles from 10 trees originating from one population from central Sweden. The initial primer screening on a subset of 228 Norway spruce trees selected from populations 57, 39 and 98-102 (Table S1) was performed at Uppsala University in a building physically separated from molecular laboratory where analysis on ancient DNA (aDNA) was performed. The rest of the populations were analyzed at the Norwegian Forest and Landscape Institute in Ås, Norway.

Andøya The island of Andøya (69° N, 16° E) in Northern Norway is a key locality for studying lateglacial and Holocene paleoenvironments in Scandinavia as the long, continuous lacustrine sedimentary records and the early deglaciation date (~ 26,000 calibrated years before present (cal. yr BP) are unique in northern Europe. Basal sediments from several lakes yield ages between 22,000 and 26,000 cal. yr BP (29-31). Offshore, the continental shelf is narrower than anywhere else along the Norwegian coast (~10 km), thus limiting the maximum potential extension of a grounded ice sheet. Several studies focusing on palaeoenvironments of Andøya and Andfjord fiord have been conducted; onshore studies have provided data on late-glacial to Holocene terrestrial palaeovegetation (29-32, 40, 41) and fauna (42-45) and offshore studies have provided information on palaeoclimate (38). Between ~ 26,000 and 11,000 cal. yr BP, seven main glacial events occurred in the Andfjord - Vågsfjord area. Atlantic water penetrated the area at ~ 16, 000 cal. yr BP, and an atmospheric warming started between 14,400 and 14,200 cal. yr BP During the Allerød interstadial, the glaciers retreated to the fjord heads or even farther inland (38). Three major climatic warming events are recorded, at ~ 16,000, 12,800 and 12,000 14C yr BP (~ 19,000, 15,000 and 14,000 cal. yr BP), and two minor ones at ~ 19,500 and 18,500 14C yr BP (~ 23,000 and 22,000 cal. yr BP); and two cold periods with High Arctic climate occurred ~ 17,900 to 16,000 and 13,700 to 12,800 14C yr BP (~ 21,000-19,000 and 17,000-15,000 cal. yr

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BP) (30). A time-scale correlation diagram was constructed by Vorren (30), extended and revised by Alm (29) comparing the local pollen assemblage zones for Endletvatn and two nearby lakes (29-31). For the period 22,000 to 10,000 BP, they constructed July mean temperature climate curves. A more thermophilous vegetation indicated a July mean temperature of about 10 °C during the warming events (29). The curves were principally based on bryophytes and pollen data, however pine and spruce pollen data was excluded and not used for climate reconstruction, as they were assumed to be from long distance origin.

In 2002, a lake sediment core was recovered from the south-west end of lake Endletvatn (69° 15' N Lat., 16°05’ E Long.) by means of a 100 mm Geonor clay sampler. The core was 14C dated and macrofossil analysis was performed on one core half. The remaining half was stored at + 4 °C, and in 2010 additional samples were taken at 10 cm intervals for sedimentary ancient DNA (sedaDNA) analysis.

As macrofossil analysis and DNA analysis were performed on different core halves and DNA analysis was performed after storage for several years, there is a risk that the remaining core half may have shrunk or cracked, rendering centimeter measurements inaccurate. For this reason and as the number of macrofossils for each vertical centimeter is generally low, the macrofossil investigation included material from three vertical cm of sediment, whereas the DNA analysis only included material from one vertical cm placed within the three cm interval.

Trøndelag

The region of Trøndelag in Central Norway is an area of particular paleoecological interest due to megafossil finds of boreal species in the central part of the Scandes Mountains between central Sweden and Norway (10). Many of these finds are radiocarbon-dated to the early Holocene. The two earliest spruce fossil remains of Norway spruce were dated 11,020 cal. yr BP and 10,250 cal. yr BP, respectively, which led to speculations about the survival of this species off the Norwegian coast during the Late Glacial. At first, pollen investigations from this area yielded limited evidence in support of the survival hypothesis for spruce (14, 46, 47) giving rise to debate on the issue (11, 12, 48). However, detailed pollen records from several sites in southern, central and northern Norway, especially at the west, have lately revealed the presence of spruce pollen well before the time conventionally recognized for the immigration of spruce in the Scandinavian Peninsula [~ 3000 cal. yr BP; (19) and references therein].

In winter 2008 a sediment core was recovered from lake Rundtjørna in North Trøndelag (63° 22' 32'' N Lat., 11°49’44” E Long). The core was 14C dated (see below) and stored at +4 °C and in 2009 samples were taken for sedaDNA and pollen analysis.

For pollen analysis, 43 sub-samples of 0.35 to 0.5 cm3 were taken at intervals of ca. 7 cm along the core, using sterilized plastic syringes of 0.5 cm diameter. For sedaDNA analysis, we took four samples with a sterile scalpel at 145-147, 182.5-185, 217-220.5, 257-259 cm depth. To minimize contamination risk we removed the outer 1–2 cm of sediment and approximately 10 g from the inner core were placed in sterile falcon tubes to minimize contamination. We collected a fifth sample at 174.5-175.5 cm depth where the palynological analysis identified the first presence of Norway spruce pollen corresponding to an interpolated age of 6,200 cal. yr BP Methods Chronological analysis

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The lake sediment core from Andøya was dated at the Laboratory for Radiological Dating in Trondheim, Norway and the Svedberg Laboratory in Uppsala, Sweden. The Trøndelag sediments were dated at the Tandem Laboratory, Uppsala University, Sweden.

The age models are based on radiocarbon dates of macrofossils and bulk dates that have been converted into calendar years using Oxcal 4.1 with IntCal09 (49, 50) and are quoted as cal. yr BP (Table S2). In the age model for the lake Endletvatn (Fig. S1) calibrated radiocarbon dates from three cores [the present core and two cores from (31)] have been used and based on eleven bulk dates and two macrofossil dates and constructed using the depositional model in OxCal 4.1 with a k value of 10 yielding Amodel=79%. The deglaciation age of the area is assumed to be 26,000 cal. yr BP (29) with a re-advance at 23,000 to 21, 000 cal. yr BP (31) and this younger deglaciation event has been used as the lower marker in the age model. Further, a lower boundary at 1210 cm (upward transition from sand to gyttja) was chosen to take into account a possible change in accumulation rate. The age model for the Rundtjørna lake is based on seven macrofossil and two bulk dates and constructed using the depositional model in OxCal 4.1 with a k value of 100 yielding Amodel=81% (Fig. S2). Ice-sheet model

The map of extent and retreat of the Scandinavian Ice Sheet (SIS) (Fig. 1A) is compiled from multiple datasets. The mapping of end moraines combined with radiocarbon and luminescence-dating of inter till-sediments deposited during ice free episodes or in extra-marginal settings permits the establishment of time-distance models for the ice sheet. When the time-distance models along major ice flow paths within the whole ice sheet are put together the spatial distribution of the SIS during several time slots within the Weichselian and Early Holocene can be outlined. The diverse data set underlying these reconstructions includes new and old data of different quality that may be in conflict with each other, and the quality of areal coverage may vary largely from region to region. Cosmogenic exposure ages indicates, that the plateaux of NW-Andøya remained ice-free during the LGM (51), thus the > 250 m high plateaus immediately above the examined sites could have hosted potential refugia that for a short while during the Late Weichselian were nunataks surrounded by glaciers flowing along the fringes of the Scandinavian Ice SIS. This leaves reconstructions open to interpretation; however, ice sheet development in key areas along the Atlantic and Barents Sea coast is based on a densely distributed set of stratigraphic and geo-chronological data (52-62). Weather the margin of the SIS was at the shelf break or somewhere across the Andøya island itself (58), it does not affect the potential refugial areas, due to the low gradient of the ice sheet (51).

Analyses on modern spruce populations

DNA extraction, PCR amplifications and sequencing: At Uppsala, we extracted DNA from needles from seven populations (56, 57 and 98-102 in Table S1) using DNeasy Plant Mini Kit (Qiagen). The rest of the DNA samples came from (20, 21) and were extracted in the DNA lab at the Norwegian Forest and Landscape Institute in Ås, Norway. We chose variable non-coding parts of the mitochondrial DNA (mtDNA) and tested 11 primer pairs amplifying the following mtDNA regions of spruce: mh05, mh35, mh02, mh33, mh38, mh44 (63), nad5 (64), nad7/1-2 (65), mt15D02, mt23D02, and mtH01(66). PCRs were performed in a 15 µl final volume containing 5.3 mM Tris-HCl, 2 mM MgCl2, 100 µM each of dNTPs, 0.2 µM of primers, 25 ng of genomic DNA template and 0.5 units of High-Fidelity DNA polymerase (Phusion, F-530) with the following program: 4 min at 94°C, 35 cycles of 45 s at 94°C, 45 s at 50-60°C

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(depending on the primer pairs), 45s at 72°C, and 72°C for 10 min. Primers used and observed fragment length for each region are presented in Table S3. PCR products were checked on a 2% agarose gel. To identify the origin of the only polymorphism detected in one of the regions (mh05), and to detect possible additional variation not visible on gel we sequenced 32 individuals from populations 56, 57 and 98-102 (Table S1). As the only polymorphism in the mh05 was due to an insertion/deletion of 21 base pairs (bp) easily detectable on agarose gel variation in the rest of the populations was also screened on gel. DNA sequencing was performed by Macrogen DNA Sequencing service (Korea) and we used BioEdit to trim, edit and combine DNA sequences. Andøya

DNA extraction, PCR amplification and high-throughput sequencing: Subsampling for sedaDNA was carried out at Tromsø University Museum in a sterile flow hood bench where ca. 10 g of sediment from the inner core was sampled into sterile falcon tubes. DNA extraction and amplifications from sedimentary samples followed previously demonstrated protocols for sedaDNA analysis, and were carried out in dedicated ancient DNA facilities at the University of Copenhagen using established precautions to monitor for, and prevent, contamination, including extraction and PCR blank controls (67): Approximately 10 g of sediment was placed in PowerMaxTM Soil PowerBead tubes (Cambio UK Ltd), and dissolved in 24 ml lysis buffer (68). The tubes were then agitated vigorously for 1 min and left to incubate overnight at 65oC under gentle agitation. Following extraction, the DNA was purified using the PowerMaxTM Soil DNA Isolation Kit protocol according to manufacturer’s instructions, where all samples were eluted in the same volume of 1.5ml. The PCR amplifications were performed with the chloroplast trnL g-h primers (33). The trnL g-h primers contained a 46 bp flanking sequence for the GS FLX sequencer, and an 8 bp ‘barcoding’ tag both on forward and reverse primer to enable differentiation of the samples after sequencing. PCRs were performed in a 25 µl final volume, using 1 µl of DNA as template in an amplification mixture containing 1 U Platinum® Taq High Fidelity DNA Polymerase (Invitrogen, Carlsbad, CA), with 1X HiFi buffer, 5 mM MgSO4, 2 mM mixed dNTPs, 1 mg/ml bovine serum albumin (BSA) and 0.4 mM of each primer. The DNA was subjected to 55-60 cycles of PCR (4 min initial denaturation at 97oC, 45 s at 94oC, 45 s at 55oC, 45 s at 68oC and a final cycle at 72oC for 10 min). PCR products were checked on 2% agarose gels, and for each sample, successful PCR replicates (between 2-4 per sample) were purified using the E.Z.N.A.® Gel Extraction kit (Omega) and pooled before sequencing. Amplification products were sequenced on the Roche GS FLX DNA sequencing platform following the manufacturer’s guide for amplicon sequencing, using five 8th of a Pico-Titer-Plate.

We also attempted amplifying a shorter internal region of the mtDNA fragment mh05 (mh05int; Table S3) from the DNA extracts following the same amplification methods as described below for Trøndelag.

Macrofossil and pollen analysis: Macrofossils were washed using sieves with mesh sizes 0.2 and 1 mm, identified and counted under a stereomicroscope; they are displayed as presence only in Fig. S3. The macrofossils were identified according to seed identification keys (69-72) and the seed/fruit collection available at Tromsø Museum (University of Tromsø). The macrofossil/sedaDNA diagram was produced using TILIA 2.0.b4 (73) and TGView 2.02 (74) and the layout modified by use of CorelDRAW12. To evaluate if pollen could be the source for sedaDNA for spruce and pine, a direct pollen analysis was performed on 1 cm3 of material from

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the three samples that yielded aDNA attributed to these species. The pollen preparations followed (75).

Taxonomic assignment of sedaDNA: For the taxonomic assignments of the trnL g-h region, raw sequences were assigned to sample according to the DNA tag used (zero mismatches permitted). Only sequences containing PCR primers (2 mismatches allowed) were retained (76, 77). All marker sequences shorter than 20 bp were filtered out, resulting in between 202 and 10 669 sequences per sample. Manipulation of sequences and taxonomic assignment was performed using a series of scripts freely available from the OBITools package (http://www.grenoble.prabi.fr/trac/OBITools). Analyses were performed using the computing facilities provided by the Norwegian Metacenter for Computational Science (Notur; http://www.notur.no/). To account for sequencing or PCR errors, all sequences represented by less than five reads were removed from the dataset, resulting in 94 682 usable reads. The taxa were assigned using an exact global alignment algorithm (78) as implemented in the program ecoTag [database created 01-02-2008; (33) (79)). Identity between the query sequence and database sequences were estimated through the length of the longest common subsequence (LCS). Identity percent is computed by dividing the LCS length by the length of the longest sequence involved in the alignment. If two or more taxa have the same sequence, we assigned this sequence to the lowest common taxonomic level. Following this method, sequences were compared with a database of 842 species representing all widespread and/or ecologically important taxa of the arctic flora [further referred to as the Arctic database, GenBank accession number GQ244527 to GQ245667 (80), where only sequences with a 100% match were considered as true taxa; Fig. S3]. This highly conservative approach resulted in a total of 12% of the sequences assigned to taxa (11,343 reads), ranging between 0 and 91% of the sequences per sample, where the younger sediments had a larger proportion of sequences passing the sorting criteria and assigned to arctic plant taxa (Fig. S3). Between one and four taxa were identified per sample. Samples from which taxa were identified to the Arctic database with less than 100% certainty are shown as a column in fig. S3. To investigate the identity of sequences not assignable to the arctic database, sequences were compared against a database created by in silico amplification of the g-h primers against EMBL using the ecoPCR software (79). However with the assignment against EMBL, occasionally sequences were recovered that resemble known food plants: soya, banana and rice. This is unavoidabe when using generic primers as such DNA is found in minor quantities throughout laboratory reagents (25, 81).

Identification of nettle (Urtica dioica): An unidentified plant fragment found in the macrofossil analysis was bleached (5%) to remove any outer modern contamination and powdered in dry condition in a mixer mill (Merck Retsch) and extracted with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the manufacture’s protocol. PCR amplifications were done following the protocol described for the tagged primers, but with primers without the tags. The amplified PCR products were cloned and sequenced using Sanger sequencing on ABI chemistry by the commercial Macrogen facility (Macrogen, Amsterdam, Netherlands). Blasting (word size, 11; Match-mismatch scores, 2,-3; Gap cost, Existence 5, extension 2) the sequences against NCBI resulted in 100 % match to Urtica dioica (e-value 3e-07) .

Sediment description To assess the integrity of sedimentation and to ensure that downward particle transport was not an alternative explanation for the presence of tree DNA into the oldest sediment, the lithography of the Andøya core was described and XRF scanning was performed on the older sediments (Fig. S4). We used the Rubidium test and an ITRAX Core scanning procedure, which involves a combined analytical instrument for simultaneous micro-XRF (X-ray

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Fluorescence) and digital x-ray micro radiography of sediment samples. Combined with a Bartington (MS2) magnetic susceptibility sensor, the instrument provides a state-of-the-art core scanning for sediment characteristics based on analysis of the split cores. The instrument non-destructively collected optical and X-radiographic images, and provided high-resolution

elemental profiles. Most of the elements recorded, from Mg and heavier are covered, with important elements like Al, Si, K, Ca Ti and most of the heavier metals, including Mn, Fe, Cu, Zn, Sr, Ba, Pb. The detection limits range from bulk and down to the low parts per million (PPM) level for most metals. In this study, we scanned with a lateral resolution of 500 microns and repeated measurements show high reproducibility between repeated scans of the same core. Trøndelag

DNA extraction, PCR amplification, cloning and Sanger sequencing: DNA extraction from sedimentary samples were carried out in dedicated ancient DNA facilities at the University of Copenhagen following the same protocols for sedaDNA analysis and precautions used for the Andøya material (67). PCR set up was prepared in two replicates in DNA-free rooms in two separate buildings at Uppsala University (EBC and GEO) physically separated from the laboratory where analyses on modern DNA are performed. We also attempted to extract DNA from the oldest pollen grains of spruce found in the Rundtjørna core at 174.5-175.5 cm depth with an interpolated age of 6,300 cal. yr BP We dissolved ca. 0.5 g of sediment sample in sterile distilled water and sieved through a 120µm filter cloth to collect spruce pollen. The solution was kept at 5°C for no more than 2 days. Drops of the solution were then moved on a glass slide and under a light microscope (10 to 40 X magnification) we selected pollen grains using a standard micropipette (0.5-10 µl-capacity). The grains were then washed 30-50 times in sterile distilled water drops and transferred in four separated sterilized PCR tubes (ca. five grains/tube) containing 2 µl of extraction buffer with 0.01% M sodium dodecyl sulfate (SDS), 0.1 g/L proteinase K, 0.01M Tris Hydrochloride (Tris HCl) pH 7.8, and 0.01 ethylenediaminetetraacetic acid (EDTA). The tubes were incubated at 56°C for 60 min for DNA extraction and at 95°C for 10 min for inactivation of proteinase.

For amplification from sedaDNA and pollen ancient DNA we used mh05int primers (Table S3) and the Qiagen Multiplex PCR kit, following the protocol from the manufacturer and increasing the number of cycles to 38. The volume of the reaction mixtures was 20 µl, including 2 µl of DNA template, 1x Multiplex PCR Master Mix (Qiagen), 0.2 µM of each primer, and water for adjusting the final volume. Five µL of PCR products were screened on 2% agarose, purified using ExoSAP-IT (Affymetrix, Inc.) and used for cloning with the CloneJet PCR Cloning Kit (Fermentas) following manufacturer’s instructions. All clones with inserts of expected sizes were sequenced using Macrogen DNA Sequencing service (Korea). We used BioEdit to trim, edit and combine all DNA sequences and to identify mismatch due to base call errors, post-damage lesions, and true polymorphism.

Pollen analysis: Pollen preparation followed Bennett and Willis (82). A minimum of 500 pollen grains and spores of terrestrial vascular plants was counted in each sample. Pollen of aquatic plants and Sphagnum spores were excluded from the total pollen sum. Pollen taxonomy follows Bennett (83), modified for Sweden using the checklist provided by (84).

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SOM Text Analyses on modern spruce populations

The initial screening of the mtDNA revealed size polymorphism for the mh05 region only.

Sequencing analysis showed that the polymorphism was due to a deletion of 21 bp yielding two variants of 241 and 262 bp (haplotype A and B, Table S4). No other polymorphism was detected in the regions flanking this deletion. The frequencies of the two haplotypes in all populations analysed are presented in Table S1.

Ancient spruce polymorphism restricted to Scandinavia: The hypothesis of an early Holocene origin of haplotype A in the east with an expansion towards west across the distribution of haplotype B in north-central Europe is very unlikely, since haplotype A was not detected outside Scandinavia (except in one individual on Åland island in the Baltic Sea). An important assumption for this interpretation is that the age of haplotype A predates the last glaciation. Assuming that white spruce diverged from Norway spruce around 5 Ma 6 (85) and, because the haplotype A was detected in sediments dated 10,300 cal. yr BP in Trøndelag, haplotype A is at most 5 Myr old and at least 10,300 yr old. Assuming a constant demography in a population of effective size N, it takes about 2Ne generations within a haploid population for a neutral mutation to reach fixation. Therefore, with a generation time of approximately 40 yr for spruce, and an effective population size varying between 2,500 and 200,000, it should take between 200,000 and 16 million years for haplotype A to spread to a whole population – if the mutation has no selective advantage. With a smaller constant population size (Ne =100) the time for spread would be 8 000 years (2Ne generations) but in this case, the successive founder events of colonization would create a decreasing level of genetic diversity, with increasing genetic distance from the glacial populations. This gradient would be accentuated by the fast spread of spruce seeds on iced surfaces (86), as such rapid expansions increase the rate of loss of genetic diversity (87, 88). Current populations from Scandinavia however, do not show such gradient of decreasing nuclear diversity from east to west (21). This agrees with the hypothesis of an older origin of A likely surviving the LGM in microenvironmentally favorable pockets in western Norway and a bidirectional mixing of two separate lineages (A and B) in Scandinavia after climate warming.

Chronological analysis

The radiocarbon dated macrofossils and bulk samples show an increasing age trend with increasing depth (Figs. S1 and S2). Bulk dates integrate carbon from various sources and for that reason may yield ages too old. However, bulk dates from Andøya in northern Norway have previously been successfully used (31) Because of this, and given the good agreement between the bulk dates and the plant macrofossil dates, the bulk samples have been included in the constructed age model for both lakes. These dates have been anchored to dates from the main sediment core used for DNA analysis using biostratigraphic correlation and loss on ignition data. Analyses of ancient material

The attempt to amplify mtDNA from the oldest sediments at Andøya yielded only a few visible amplification products among multiple replicates. Such results are typical if only minor amount of target DNA is present and may result from sporadic contamination; therefore, these

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results were omitted. On the other hand, we recovered mtDNA from younger sedaDNA samples in Trøndelag and from pollen dating to 6,300 cal yr BP (Fig. 2; Table S5A). No PCR products were detected on gel after amplification from the blank samples.

Alignment of ancient and modern sequences revealed mismatches at ten nucleotide positions in the region flanking the deletion. Seven transition substitutions (four C → T and three A → G) were interpreted as nucleotide miss-incorporations during PCR, due to post-mortem lesions in the degraded DNA molecules, a common problem when amplifications are performed from ancient DNA molecules (89-91). The nucleotide miss-incorporation pattern is compatible with post-mortem C deamination in DNA templates, suggesting that PCR reaction primed/started on ancient and not modern templates. The remaining three transversion substitutions yielded three new haplotypes (C, D and E in Table S4) unique to the ancient material. However, as it is difficult to prove the authenticity of point mutations in ancient sequences, this polymorphism was not considered further. In Table S5 we present the percentage of cloned sequences with the 21-bp deletion. Between 26 and 42 % of the ancient sequences at Rundtjørna carried the short haplotype (A or C). More than 40% of current haplotypes at Rundtjørna showed also the 21-bp deletion (population 56 in Table S1). As the deletion was absent from all sister spruce species and from pine (Table S1), it is considered specific to Norway spruce, and excludes the possibility that A originated from pine DNA also likely present in the Trøndelag sediments. The sequence similarity observed between pine and spruce sequences in absence of deletion implies also that the frequencies of A may be underestimated in our calculations. All DNA sequences from ancient and modern material are available upon request.

At Andøya, the overall reconstruction of vegetation based on plant taxa detected both with sedaDNA and macrofossils (Fig. S3), indicates a change in vegetation from a polar desert or open pioneer vegetation with Poaceae, Papaver and Brassicaceae as Draba to a higher biodiversity and more herbs such as Saxifraga, Sagina, Cerastium and Oxyria digyna towards the Holocene from ca 15,000 cal. yr BP. These results correspond well with the overall trend of deglacial warming, but they do not detect the smaller changes described from earlier pollen analyses (29-31).

The pollen analysis conducted at Trøndelag showed an unexpectedly early presence of spruce in the core in a sample dated 6,300 cal. yr BP (Fig. S5). Here pollen is present in the samples with an abundance of up to 2.5 % of total terrestrial pollen over a section corresponding to about 200-300 years, indicating the occurrence of a small population in the catchment area of the lake. Thereafter, no spruce pollen is encountered in younger samples until a continuous pollen curve starts at 4,300 cal. yr BP. The long tail of the curve, with regular and continuous occurrence of spruce pollen for about 2,500 years before a sustained rise to high abundance at 1,500 cal. yr BP (10% of total terrestrial pollen), suggests that small isolated populations may have occurred in the Trøndelag region during this period. After 1,500 cal. yr BP, spruce populations around the site expanded rapidly. At 1,200 cal. yr BP, spruce became abundant and slowly declined thereafter to its present abundance.

At Andøya, no spruce or pine pollen was observed in the samples in which spruce and pine were detected with sedaDNA, and the pollen spectra otherwise conformed with previous studies, containing low concentrations of pollen of Poacaeae, Papaver and other taxa likely to have grown in an arctic environment. Lithostratigraphy and XRF scanning associated with X-ray detection indicate clearly laminated sediments (Fig. S4), thus the fine-grained sediments are undisturbed. This is further supported by Rb (Rubidium), which has low environmental mobility due to rapid absorption-adsorption to clay minerals, and is thus a robust indicator for vertical

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particle transport in the Andøya lake sediments. Rb shows high-frequency variation at a low amplitude on a multiple-laminae scale.

In Trøndelag, mtDNA of spruce was recovered from sediment in which no pollen was detected (7,650 and 10,300 cal. yr BP). Leaching of DNA from younger sediments cannot explain the presence of A in older sediments as this would require the DNA molecules to have migrated more than 1 m downward without leaving detectable traces (i.e., in the 5,300-yr BP layer; Fig. 2 and Table S5B).

Other possible sources for DNA at Andøya: driftwood and resedimentation: Macrofossils of a marine macro alga (Desmarestia cf. aculeata) dated to ~ 20,500 cal. yr BP indicate a marine transgression at Andøya. It is legitimate therefore to question whether driftwood could be a possible explanation of pine and spruce DNA at 19,000 and 17,500 cal. BP at this site. However, no marine incursion is recorded at ~ 22,000 cal. BP, where pine is detected the first time. More importantly, the likelihood that a few pine or spruce logs washing up on the shore of the island would contribute to aDNA in lake sediments some hundred meters inland is extremely low.

Early Weichselian sediments containing resedimented Eemian spruce pollen were previously reported further north in North Norway at Tromsø ca. 120 km in North Norway (92), and pollen-bearing Jurassic and Cretaceous rocks are present in the Andøya area, but due to their age it is extremely unlikely they contribute detectable DNA of any kind, and none would probably be picked up by the extraction techniques used in this study. Author contributions LP, TJ, MMT, EE, IGA and EW jointly conceived and designed the work; LP, TJ, MMT, EE, TA, JH, SLF, YS, MEE, TA, IGA, EW, TG, MR, and LO contributed to the molecular and paleoecological data and interpretation of the results; KDB and YS contributed to the fieldwork in Trøndelag and interpretation of the results; KA contributed to investigation of sedaDNA samples from Andøya and IM contributed to investigation of sedaDNA samples from Trøndelag; SB, EC, PT and CB contributed to the assignment analysis of sedaDNA results from Andøya; MHN compiled data on the distribution of the ice sheet and MHN, NKL and KHK contributed to analyses and interpretation of sediments; LP, TJ, MEE, IGA and EW wrote the paper with contributions from other authors. References

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16

Table S1. Sample information for Norway spruce and distribution of the haplotypes A and B found at the mitochondrial locus mh05. S, source of the sample: a, (20); b, (21); c, this study; d, (85); e, (37); N, sample size; (*), Norway spruce (P. abies); (†), Siberian spruce (P. obovata); (‡), White spruce (P. glauca) (seeds originating from orchard with individuals originating from multiple populations); (§), Scots pine (P. sylvestris).

Country S Lat. N Long. N Hap A Hap B

Freq. A 1 Austria* a 46.50 14.60 E 15 0 15 0..00 2 Austria* a 46.90 10.60 E 15 0 15 0.00 3 Austria* a 47.00 10.40 E 5 0 5 0.00 4 Austria* a 47.00 12.50 E 13 0 13 0.00 5 Bosnia Herz.* a 43.92 19.41 E 13 0 13 0.00 6 Belarus* a 53.70 24.80 E 7 0 7 0.00 7 Belarus* a 54.10 26.50 E 16 0 16 0.00 8 Belarus* a 55.10 30.20 E 9 0 9 0.00 9 Estonia* a 58.09 27.45 E 16 0 16 0.00 10 Estonia* a 58.50 22.30 E 12 0 12 0.00 11 Estonia* a 59.00 26.50 E 16 0 16 0.00 12 Estonia* a 58.10 25.30 E 10 0 10 0.00 13 Estonia* a 58.90 24.40 E 12 0 12 0.00 14 Finland* a 60.22 20.05 E 15 1 14 0.07 15 Finland* a 60.35 25.07 E 16 0 16 0.00 16 Finland* a 60.37 23.68 E 16 0 16 0.00 17 Finland* a 60.82 21.87 E 16 0 16 0.00 18 Finland* b 62.00 24.65 E 32 0 32 0.00 19 Finland* a 62.28 26.25 E 16 0 16 0.00 20 Finland* a 62.65 27.37 E 16 0 16 0.00 21 Finland* a 63.10 29.81 E 16 0 16 0.00 22 Finland* a 63.22 22.15 E 16 0 16 0.00 23 Finland* a 63.93 27.37 E 16 0 16 0.00 24 Finland* a 64.58 24.43 E 16 0 16 0.00 25 Finland* a 65.63 26.73 E 15 0 15 0.00 26 Finland* a 65.89 27.80 E 16 0 16 0.00 27 Finland* a 67.44 29.72 E 16 0 16 0.00 28 Finland* a 67.53 24.93 E 15 0 15 0.00 29 Finland* a 68.40 27.42 E 7 0 7 0.00 30 Germany* a 48.00 11.70 E 15 0 15 0.00 31 Germany* a 49.50 12.30 E 14 0 14 0.00 32 Germany* a 49.60 11.50 E 7 0 7 0.00 33 Italy* a 46.60 12.70 E 11 0 11 0.00 34 Latvia* a 56.40 24.30 E 8 0 8 0.00 35 Lithuania* b 54.51 24.05 E 32 0 32 0.00 36 Lithuania* a 54.95 24.07 E 16 0 16 0.00 37 Lithuania* a 55.10 22.30 E 16 0 16 0.00 38 Lithuania* a 55.48 26.18 E 15 0 15 0.00 39 Lithuania* a 55.83 21.35 E 16 0 16 0.00 40 Lithuania* a 55.95 23.10 E 16 0 16 0.00 41 Lithuania* a 56.12 27.58 E 16 0 16 0.00 42 Norway* a/b 58.78 8.30 E 32 29 3 0.91 43 Norway* a 59.30 7.42 E 16 4 12 0.25 44 Norway* a 59.45 6.34 E 16 0 16 0.00 45 Norway* c 59.74 8.14 E 24 20 4 0.83 46 Norway* a 60.09 10.52 E 16 10 6 0.63 47 Norway* a 60.15 8.40 E 16 14 2 0.88 48 Norway* a/b 60.60 6.54 E 32 0 32 0.00 49 Norway* a/b 60.82 5.76 E 32 30 2 0.94 50 Norway* a/b 61.19 9.88 E 32 15 17 0.47 51 Norway* c 61.20 8.73 E 15 9 6 0.60 52 Norway* a/b 61.26 7.34 E 32 0 32 0.00 53 Norway* a 61.27 12.43 E 5 4 1 0.80 54 Norway* a 61.90 11.05 E 16 7 9 0.44

17

55 Norway* a 63.05 11.63 E 16 0 16 0.00 56 Norway* c 63.37 11.82 E 52 21 31 0.40 57 Norway* c 63.78 10.96 E 30 9 21 0.30 58 Norway* a 65.33 13.35 E 16 11 5 0.69 59 Norway* a 66.38 14.68 E 16 16 0 1.00 60 Norway* c 67.00 14.57 E 3 3 0 1.00 61 Norway* a 69.25 29.08 E 16 0 16 0.00 62 Norway* a/b 69.45 30.07 E 32 0 32 0.00 63 Poland* a 50.80 20.00 E 8 0 8 0.00 64 Russia* a 53.30 33.70 E 12 0 12 0.00 65 Russia* a 53.50 31.25 E 11 0 11 0.00 66 Russia* a 54.50 32.33 E 12 0 12 0.00 67 Russia† d 54.64 99.91 E 2 0 2 0.00 68 Russia* a 55.50 35.30 E 13 0 13 0.00 69 Russia† d 55.88 98.12 E 2 0 2 0.00 70 Russia* b 56.80 60.60 E 32 0 32 0.00 71 Russia* a 57.58 41.00 E 11 0 11 0.00 72 Russia* a 57.80 27.00 E 8 0 8 0.00 73 Russia* a 57.80 36.50 E 8 0 8 0.00 74 Russia* a 57.83 30.00 E 11 0 11 0.00 75 Russia* a 58.00 33.20 E 13 0 13 0.00 76 Russia* a 58.00 39.00 E 7 0 7 0.00 77 Russia* a 60.18 30.03 E 16 0 16 0.00 78 Russia* a 62.25 36.72 E 11 0 11 0.00 79 Russia* a 62.88 34.42 E 11 0 11 0.00 80 Russia* a 64.58 40.00 E 11 0 11 0.00 81 Russia† d 67.46 86.56 E 2 0 2 0.00 82 Russia* a 55.70 37.50 E 10 0 10 0.00 83 Serbia* a 43.25 20.83 E 16 0 16 0.00 84 Serbia* a 43.29 20.32 E 15 0 15 0.00 85 Serbia* a 43.57 20.85 E 15 0 15 0.00 86 Sweden* c 56.63 15.57 E 16 0 16 0.00 87 Sweden* a 57.02 13.67 E 16 0 16 0.00 88 Sweden* b 57.76 15.60 E 32 6 26 0.19 89 Sweden* a 59.00 14.60 E 14 3 11 0.21 90 Sweden* a 59.10 16.40 E 16 2 14 0.13 91 Sweden* a 59.20 17.90 E 5 4 1 0.80 92 Sweden* a 59.94 17.74 E 16 0 16 0.00 93 Sweden* a 60.17 12.83 E 16 10 6 0.63 94 Sweden* a/b 60.33 15.20 E 32 24 8 0.75 95 Sweden* a 61.40 13.20 E 15 13 2 0.87 96 Sweden* a 62.50 15.20 E 9 0 9 0.00 97 Sweden* a 63.25 12.40 E 24 2 22 0.08 98 Sweden* c 63.24 12.43 E 30 5 25 0.17 99 Sweden* c 63.29 12.38 E 30 4 26 0.13 100 Sweden* c 63.44 12.73 E 26 4 22 0.15 101 Sweden* c 63.49 12.49 E 30 5 25 0.17 102 Sweden* c 63.57 12.28 E 30 0 30 0.00 103 Sweden* a 63.78 20.37 E 16 0 16 0.00 104 Sweden* a 64.20 19.70 E 4 0 4 0.00 105 Sweden* a/b 64.67 15.47 E 32 8 24 0.25 106 Sweden* a 65.56 16.13 E 16 6 10 0.38 107 Sweden* a/b 66.00 17.33 E 32 0 32 0.00 108 Sweden* a 66.05 20.62 E 16 0 16 0.00 109 Sweden* a/b 66.70 19.92 E 11 0 11 0.00 110 Sweden* a 67.39 22.08 E 15 0 15 0.00 111 Sweden* a 67.67 20.90 E 11 0 11 0.00 112 Switzerland* a 46.18 7.61 E 10 0 10 0.00 113 Switzerland* a 46.23 8.56 E 16 0 16 0.00 114 Canada‡ d 45. 13 - 48. 83 70. 66-79..50 W 11 0 11 0.00 115 Sweden§ e 60. 39 15. 56 E 10 0 10 0.00

18

Table S2. Radiocarbon dates from Endletvatn Lake (Andøya) and Rundtjørna Lake (Trøndelag), calibrated for the age models. Ref. 1, this study; ref. 2 corresponds to ref. (31).

Lab. no. Locality Depth (cm) Material Method 14C age BP

Model age (cal. yr

BP) Ref

Ua-­‐39895   Rundtjørna   13-­‐14   Macro   AMS   1369±32   1290±26   1  

Ua-­‐39896   Rundtjørna   46-­‐47   Macro   AMS   2197±31   2229±55   1  Ua-­‐39897   Rundtjørna   76-­‐77   Bulk   AMS   3151±30   3378±34   1  

Ua-­‐39898   Rundtjørna   105-­‐106   Macro   AMS   3624±34   3943±55   1  

Ua-­‐39899   Rundtjørna   134-­‐135   Macro   AMS   4412±31   4990±86   1  Ua-­‐39900   Rundtjørna   165-­‐166   Macro   AMS   5226±38   5992±65   1  

Ua-­‐39901   Rundtjørna   193-­‐194   Macro   AMS   5930±41   6757±54   1  Ua-­‐39902   Rundtjørna   222-­‐223   Macro   AMS   6930±52   7769±60   1  

Ua-­‐39903   Rundtjørna  256-­‐257.5   Bulk   AMS   9233±52   10387±79   1  

               TRa-­‐814A   Endletvatn   850   Bulk   AMS   10605±55     12575±56   1  

T-­‐1887   Endletvatn   930   Bulk   Conventional   12920±110     15449±239   2  

T-­‐963   Endletvatn   940   Bulk   Conventional   12710±200   15781±264   2  TRa-­‐790A   Endletvatn   965   Bulk   AMS   13910±75   16857±76   1  

TUa-­‐4894   Endletvatn   1035   Macro   AMS   14240±130   17706±126   1  T-­‐2152A   Endletvatn   1070   Bulk   Conventional   14990±130   18404±120   2  

T-­‐2152B   Endletvatn   1070   Bulk   Conventional   15300±290   18399±121   2  TRa-­‐800A   Endletvatn   1080   Bulk   AMS   15375±95   18631±73   1  

TRa-­‐804A   Endletvatn   1110   Bulk   AMS   15990±135   19291±113   1  

TUa-­‐4925   Endletvatn   1150   Macro   AMS   17725±190   21284±229   1  T-­‐1512   Endletvatn   1160   Bulk   Conventional   18710±40   22116±218   2  

T-­‐17775A   Endletvatn   1180   Bulk   Conventional   18100±800     22425±147   2  T-­‐1775B   Endletvatn   1180   Bulk   Conventional   19100±270   22421±147   2  

19

Table S3. Targeted regions, sequences and approximate product length (bp) of PCR for primer pairs used to amplify 11 mtDNA regions of Norway spruce. Pa; poor amplification.

Region Sequence Length

mh05int 5' CCCCTAAGTAAGTAAACCTCTA-3' 5' TCAGAGCCAGAAGCAGATTCAC-3' 120-141

mh05 5'-GGGAGTCAGCGAAAGAAGTAAG-3' 5'-AGTCTCAGAGCCAGAAGCAG-3' 241-262

mh35 5'-CGATGACATCTCTTAGCTTCC-3' 5'-TGGGGAATAGGATTCGGGTAAG-3' 1000

mh02 5'-TTTTAGGGCCATTTGCCTGC-3' 5'-TCTATGGACAAGAGCCCGACCT-3' 950

mh33' 5'-CGAAGGAAGGAATGAAGGTG-3' 5'-GCTCTTAAGTGCTGGTTGATG-3' 850

mh38 5'-CCGTCCCCTATCCATCAAAC-3' 5'-CCCTGAGCGAGATTGAATTAG-3' 1000

nad5 5'-AGTCCAATAGGGACAGCAC-3' 5'-ACCCGACGATAACTAGCTTC-3' Pa

nad7/1-2 5'-ACCTCAACATCCTGCTGCTC-3' 5'-CGATCAGAATAAGGTAAAGC-3' Pa

mh44 5'-ATGACTGGAAGAATTGCTCAC-3' 5'-TTCACTTGATACTCACCCCC-3' 157

mt15-D02 5'-TATCTGACTTGCCTTATC-3' 5'-ATCCGAATACATACACC-3' 750

mt23D02 5'-CACCCTTGGGTAGACTGG-3' 5'-GGTTCACGCAGTGCTTCT-3' Pa

mt1H01 5'-AAGATGGATCGCCCTTACGC-3' 5'-GAGGAGGAGGCTTCGTCGTC-3' 700

20

Table S4. Polymorphism detected at mh05 in Rundtjørna samples (Trøndelag) with nomenclature used for haplotypes. The polymorphic internal segment of the mh05 region obtained with primers mh05int is shown (Table S3) from position 121 to 262 on the full length of the mh05 region.

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Table S5. Summary of the haplotypes (A-E) observed after amplification and cloning of the short internal fragment mh05int (Table S3): A) DNA extracted from ancient pollen and B) DNA extracted from sediments at Rundtjørna Lake (Trøndelag). Between 26% and 42% of the ancient sequences carried the short haplotype (A or C). A 5%-95% confidence interval (CI) is provided for each frequency estimate using the cumulative binomial probability function. A PCR np c A B C A+C A+C/Total CI, 5% CI, 95% 6.2RD (EBC) 5 13 11 1 1 12 6.2RD (EBC) 5 0 0 0 0 0 6.2RD (EBC) 5 0 0 0 0 0 6.2RD (EBC) 5 16 0 16 0 0 Total EBC 29 11 17 1 12 41.4% 29.1% 58.3% 6.2RD (GEO) 5 12 6 6 0 6 6.2RD (GEO) 5 2 0 2 0 0 6.2RD (GEO) 5 0 0 0 0 0 6.2RD (GEO) 5 0 0 0 0 0 Total GEO 14 6 8 0 6 42.9% 26.5% 67.5%

Total 43 17 25 1 18 41.9% 31.3% 55.6%

B PCR c A B C D E A+C A+C/Total CI, 5% CI, 95% 5.3RD (EBC) 0 0 0 0 0 0 0 5.3RD (GEO) 0 0 0 0 0 0 0 6.5RD (EBC) 0 0 0 0 0 0 0 6.5RD (GEO) 4 1 3 0 0 0 1 Total 6.5RD 4 1 3 0 0 0 1 25.0% 9.9% 75.2% 7.6RD (EBC) 2 0 2 0 0 0 0 7.6RD (GEO) 1 0 1 0 0 0 0 Total 7.6RD 3 0 3 0 0 0 0 0.0% 1.8% 63.3% 10.3RD (EBC) 6 2 4 0 0 0 2 10.3RD (EBC) 10 3 5 0 1 1 3 Total 10.3RD 16 5 9 0 1 1 5 31.3% 17.9% 54.9%

Total 23 6 15 0 1 1 6 26.1% 15.4% 45.1%

Amplifications were independently performed in two separate buildings (EBC and GEO) at Uppsala University following identical procedures. np, number of pollen grains; c, total number of clones.

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Fig. S1.

&

Depthinmetres

Modelled age years BP

Age model for Endletvatn, Andøya. 14C-ages are calibrated using OxCal v 4.1.7 (48). Atmospheric data from (49). Red data set from: Core 1972 (31), Blue dataset: Core 1974 (31), Black-grey: Present study.

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Fig. S2.

Age model for Rundtjørna, Trøndelag. 14C-ages are calibrated using OxCal v 4.1.7 (49). Atmospheric data from (50).

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Fig. S3.

Diagram of all taxa detected with macrofossils (red), sedaDNA (light blue), and DNA from macrofossil remains (green) in the Andøya core. Left-hand four columns indicate age (14C and calendar dates), lithology and loss on ignition.

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Fig. S4.

XRF scan from 10-11 and 11-12m of the Andøya core.

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Fig. S5.

Norway spruce pollen percentage curve from Rundtjørna (Trøndelag) with the outline curve representing an exaggeration of 10 ×. Ages in calendar years are based on nine radiocarbon dates (asterisks) on terrestrial plant remains and bulk organic matter obtained by accelerator mass spectrometry. Rectangles indicate the position of the sediment samples used for sedaDNA (filled) and pollaDNA analysis (open).