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# LATE QUATERNARY HISTORY OF NORTH EURASIAN NORWAY SPRUCE (PICEA ABIES) AND SIBERIAN SPRUCE (PICEA OBOVATA) INFERRED FROM MACROFOSSILS, POLLEN AND CYTOPLASMIC DNA VARIATION

Mari Mette Tollefsrud<fnref n="1" />, Małgorzata Latałowa<fnref n="2" />, W. O. van der Knaap<fnref n="3" />, Christian Brochmann<fnref n="4" /> and Christoph Sperisen<fnref n="5" />

**Corresponding authors:**

Christoph Sperisen

Swiss Federal Research Institute for Forest, Snow and Landscape Research (WSL), Zürcherstrasse 111, CH-8903 Birmensdorf, Switzerland

E-mail: sperisen@wsl.ch

**Aim** We used combined palaeobotanical and genetic data to assess whether Norway spruce (Picea abies) and Siberian spruce (Picea obovata), two major components of the Eurasian boreal forests, occupied separate glacial refugia, and to test previous hypotheses on their distinction, geographical delimitation and introgression.

**Location** The range of Norway spruce in northern Europe and Siberian spruce in northern Asia.

**Methods** Pollen data and recently compiled macrofossil records were summarized for the Last Glacial Maximum (LGM), late glacial and Holocene. Genetic variation was assessed in 50 populations using one maternally (mitochondrial *nad1*) and one paternally (chloroplast *trnT-trnL*) inherited marker and analysed using spatial analyses of molecular variance (SAMOVA).

**Results** Macrofossils showed that spruce was present in both northern Europe and Siberia at the LGM. Congruent macrofossil and pollen data from the late glacial suggested widespread expansions of spruce in the East European Plain, West Siberian Plain, southern Siberian mountains and the Baikal region. Colonization was largely completed during the early Holocene, except in the formerly glaciated area of northern Europe. Both DNA markers distinguished two highly differentiated groups that correspond to Norway spruce and Siberian spruce and coincide spatially with separate LGM spruce occurrences. The division of the mtDNA variation was geographically well defined and occurred to the east of the Ural Mountains along the Ob River, whereas the cpDNA variation showed widespread admixture. Genetic diversity of both DNA markers was higher in western than in eastern populations.

**Main conclusions** North Eurasian Norway spruce and Siberian spruce are genetically distinct and occupied separate LGM refugia, Norway spruce on the East European Plain and Siberian spruce in southern Siberia, where they were already widespread during the late glacial. They came into contact in the basin of the Ob River and probably hybridized. The lower genetic diversity in the eastern populations may indicate that Siberian spruce suffered more from past climatic fluctuations than Norway spruce.

**Key words:** DNA markers, fossil records, glacial refugia, introgression, *Picea abies*, *Picea obovata*, post-glacial colonization.

Published in Journal of Biogeography (2015) 42: 1431-1442, with doi:10.1111/jbi.12484

Appendices S1, S2 and S3 and their respective references can be downloaded here:
http://onlinelibrary.wiley.com/wol1/doi/10.1111/jbi.12484/suppinfo

## Introduction

The boreal forest or taiga of Eurasia, with fir (*Abies*), larch (*Larix*), pine (*Pinus*) and spruce (*Picea*) as the most abundant components, is one of the largest continuous forest areas worldwide. Like most Northern Hemisphere biota, the taiga experienced repeated range contractions and expansions driven by the Quaternary climate fluctuations and the associated formation and decay of ice sheets and permafrost (Frenzel, 1968). However, little is known about the distribution of taiga taxa during glacial

maxima. Based on fossil pollen and macrofossils, it was assumed that the taiga was confined to small areas located south of its current limits during the Last Glacial Maximum (LGM) (Frenzel, 1968; Tarasov et al. 2000). This view has recently been challenged by new macrofossil data, demonstrating that several tree taxa, including birch (*Betula*), larch, pine and spruce, persisted locally at high latitudes during the LGM in areas south and east of the Scandinavian ice sheet (Binney et al. 2009). A more northerly and wider LGM distribution of taiga taxa is also supported by genetic data. For example, a detailed survey

<footnote n="1">¹ Norwegian Institute of Bioeconomy Research (NIBIO), NO-1431 Ås, Norway,</footnote>

<footnote n="2">² Laboratory of Palaeoecology and Archaeobotany, Department of Plant Ecology, University of Gdańsk, PL-80-308 Gdańsk, Poland,</footnote>

<footnote n="3">³ Institute of Plant Sciences and Oeschger Centre for Climate Change Research, University of Bern, CH-3013 Bern, Switzerland,</footnote>

<footnote n="4">⁴ National Centre for Biosystematics, Natural History Museum, University of Oslo, Blindern, NO-0318 Oslo, Norway,</footnote>

<footnote n="5">⁵ Swiss Federal Research Institute for Forest, Snow and Landscape Research (WSL), CH-8903 Birmensdorf, Switzerland.</footnote>
of molecular variation in Siberian larch (Larix sibirica) suggested the presence of several isolated refugia in the southern Siberian mountains, the foothills of the Sayan Mountains and northern Siberia (Semerikov et al. 2013). In Norway spruce [Picea abies (L.) H. Karst.], genetic data have corroborated the fossil-based inference that populations survived on the East European Plain (e.g. Tollefsrud et al. 2008) and suggested a refugium in Scandinavia (Parducci et al. 2012a).

In this study, we further examine the history of Norway spruce, and present data for it and the closely related Siberian spruce (Picea obovata Ledeb.), two major components of the taiga. Their combined range extends almost continuously from Norway in the west to the coast of the Sea of Okhotsk in the east (Fig. 1; Schmidt-Vogt, 1977). Despite their ecological importance, the delimitation and taxonomic rank of Norway spruce and Siberian spruce are not clear. Some authors recognize two separate species (Pravdin, 1975; Popov, 2003). Others consider them either as two closely related subspecies (Tutin et al. 1993) or geographical varieties (Schmidt-Vogt, 1974a), a suggestion which is supported by allozyme data: based on a limited number of populations, very little allozyme differentiation was detected between the two taxa, consistent with a rank of subspecies or variety (Krutovskii & Bergmann, 1995). Phylogenetic analyses using molecular markers have corroborated the close relationship (Ran et al. 2006) and indicated that the Norway spruce of northern Europe is more closely related to Siberian spruce than to the Norway spruce of central and south-eastern Europe (Lockwood et al. 2013).

At the morphological level, Norway spruce and Siberian spruce can only be distinguished by the shape of their

cone-scales (Schmidt-Vogt, 1974a). The cone-scales of Norway spruce are slightly pointed, whereas those of Siberian spruce are rounded, although a range of intermediate forms occur. Popov (2003) described a smooth longitudinal gradient of cone-scale morphology, with distinct Norway spruce populations found in the extreme west of northern Europe and distinct Siberian spruce populations east of the Ural Mountains (Fig. 1). It has been suggested that this morphological pattern is a result of introgression, when the two taxa came into contact after the last glaciation (Pravdin, 1975; Popov, 2003). Introgression is indeed supported by allozyme data, which revealed higher levels of genetic diversity in putative hybrid populations than in populations with 'pure' cone-scale shapes (Krutovskii & Bergmann, 1995). Schmidt-Vogt (1974a), on the other hand, argued that the morphological gradient reflects adaptation to cold climate rather than introgression, because the frequency of rounded forms also increases with latitude and elevation. The glacial and post-glacial history of Norway spruce has been intensively studied using fossil pollen (e.g. Giesecke & Bennett, 2004; Latałowa & van der Knaap, 2006), macrofossils (Terhürne-Berson, 2005), genetic data (e.g. Lagercrantz & Ryman, 1990; Vendramin et al. 2000; Sperisen et al. 2001; Collignon et al. 2002; Heuertz et al. 2006) and pollen and genetic data combined (Tollefsrud et al. 2008). These studies all agree that the Norway spruce of northern Europe and that of central and south-eastern Europe originate from separate glacial refugia. In the north, detailed pollen data (Giesecke & Bennett, 2004; Latałowa & van der Knaap, 2006), mitochondrial DNA (mtDNA) (Tollefsrud et al. 2008) and nuclear microsatellites (Tollefsrud et al. 2009) have consistently revealed the presence of a major refugium on the East European Plain. Inferences from macrofossils, stomata and pollen indicated that the refugium extended as far north as the Pechora region (Väliranta et al. 2011). The presence of a separate refugium in northern Scandinavia was inferred from ancient DNA and the current distribution of a mitotype that appears to be unique to Scandinavia (Parducci et al. 2012a,b). Population survival in northern Scandinavia was also suggested based on macrofossils (e.g. Kullman, 2008). The existence of refugial populations in Scandinavia has, however, been questioned (e.g. Birks et al. 2012; Vorren et al. 2013).

No comparable genetic and palaeobotanical data exist for spruce in Siberia. The available palaeobotanical data suggest that Siberian spruce survived in the lowlands of southern Siberia (Tarasov et al. 2000; Binney et al. 2009) and the Baikal region (Blyakharchuk et al. 2004; Bezrukova et al. 2005). These studies did not, however, consider cone-scale morphology, and it remains unclear whether Norway spruce and Siberian spruce also shared refugia, as was proposed based on allozyme data (Krutovskii & Bergmann, 1995).

<caption>FIGURE 1</caption>

Geographical distribution of Norway spruce (Picea abies) and Siberian spruce (Picea obovata) in northern Eurasia after Schmidt-Vogt (1974b; northern Europe and easternmost Siberia) and Bezrukova et al. (2005; Siberia). The current combined distribution of the two taxa is indicated by the green outline. The green dotted area marks the introgression zone suggested by Popov (2003, 2010). The shading illustrates topography: darker is higher altitude.

<figure><img src="image_82.png" /></figure>
Here, we summarize macrofossil and pollen data and assess cytoplasmic DNA variation in spruce populations sampled across boreal Eurasia to test whether Norway spruce and Siberian spruce occupied separate or shared glacial refugia and to clarify whether they are today genetically distinct and fully allopatric or connected by a zone of introgression. Cytoplasmic DNA variation was assessed in one mtDNA and one chloroplast DNA (cpDNA) marker. As in most conifers, mtDNA is maternally inherited in Picea (Sutton et al. 1991; Grivet et al. 1999) and thus dispersed by seeds only, whereas cpDNA is paternally inherited (Sutton et al. 1991) and dispersed by both pollen and seeds. Hence, their combined use provides opportunities for studying the geographical distribution of both maternal and paternal lineages. We hypothesize that Norway spruce and Siberian spruce underwent largely independent glacial histories. We therefore make the following predictions. (1) There are two major genetic groups, corresponding to the morphologically defined Norway spruce and Siberian spruce. (2) One genetic group coincides spatially with LGM spruce occurrences in northern Europe, and the other coincides with occurrences in Siberia. (3) The two genetic groups form a zone of contact along or in the vicinity of the Ural Mountains. (4) In the area of contact, the two genetic groups are admixed, reflecting introgression.

# Materials and methods

## Macrofossil and pollen data

Macrofossil and pollen data play complementary roles in palaeoecology. Macrofossils provide the most direct evidence for the past presence of a plant taxon in a particular area (Birks & Birks, 2000), but macrofossil sites are scarce in many regions. Owing to long-distance transport, pollen data alone have limited value in demonstrating the presence of a species, particularly when the pollen abundance of a given taxon is low. Pollen sites are, however, often numerous and may thus support other types of evidence. Pollen is also valuable for assessing the dynamics of expansion, because it can be quantified (e.g. Latałowa & van der Knaap, 2006). To obtain the best resolution, we used both types of records.

Macrofossil records were mainly derived from the Northern Eurasian Macrofossil Database (Binney et al. 2009). Although many records refer to either Norway spruce or Siberian spruce, the distinction between the taxa must be regarded as tentative because only very few records of cones exist in the database. Consequently, macrofossil records were used solely as evidence for the presence of spruce (Picea sp.). For records younger than 12 ¹⁴C kyr BP, calibrated years were taken from the table CALIBRATED_AGE BP in the database. Older radiocarbon dates were recalibrated using OXCAL 4.2, based on the IntCal13 and Marine13 calibration curves (Reimer et al. 2013). Additional macrofossil and stomata records were

included from recent studies or studies that are not integrated into the database. The compiled records are given in Appendix S1 of Supporting Information.

Pollen records were derived from the European Pollen Database (http://www.europeanpollendatabase.net/; see Appendix S2) and from the literature. A subset of pollen sites was taken from Latałowa & van der Knaap (2006). In the present study, we report ages when spruce pollen reached the 2% threshold for the first time, as we regard this as an acceptable marker for early population expansion. The possibilities and restrictions of this threshold are discussed in Latałowa & van der Knaap (2006).

Macrofossils and 2% pollen thresholds dated to 12–11 cal. kyr BP were carefully inspected to determine whether they belong to the late glacial or the Holocene. For plotting on maps, the palaeodata were divided into five time-slices: (1) > 27 cal. kyr BP, prior to the LGM; (2) 27 to > 18 cal. kyr BP, the local LGM when the Scandinavian and the Barents-Kara ice sheet were at their southern and south-western maximum, respectively (Clark et al. 2009); (3) 18 to > 11.7 cal. kyr BP, the late glacial, including the Younger Dryas, when the climate warmed and the ice retreated (Svendsen et al. 2004); the Holocene, divided into (4) 11.7 to > 6 cal. kyr BP and (5) 6 to 0 cal. kyr BP. In regions where sites are geographically clustered, only the oldest sites are shown on the maps. Ages are consistently expressed in calibrated years before present (cal. yr BP).

## Genetic analyses

The genetic analyses were based on 551 individuals from 50 populations distributed throughout the boreal range of Norway spruce and the range of Siberian spruce (Appendix S3). All the sampled populations represented putatively natural forests. DNA was extracted from frozen needles using the DNeasy 96 Plant Kit or the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). A subset of the Norway spruce DNAs (32 populations) and mtDNA data was taken from two previous studies (Tollefsrud et al. 2008, 2009). The mtDNA data of Siberian spruce and the cpDNA data are new to this study.

Variation in mtDNA was assessed using the second intron of the *nad1* gene, which includes two variable minisatellites of 32 and 34 bp (Sperisen et al. 2001). The polymorphic fragment was amplified, sized and scored according to Tollefsrud et al. (2008). Sequencing was performed for 15 samples collected to the west of the Ural Mountains, and 17 samples collected to the east. Additional sequences were taken from Tollefsrud et al. (2008) to obtain sequences for all the detected size variants. In a preliminary cpDNA screen, six intergenic spacer (IGS) regions were sequenced in eight samples collected on either side of the Ural Mountains. The IGS regions surveyed were trnH-psbA (for primer sequences, see Sang et al. 1997; Tate & Simpson, 2003), trnS-trnG (Hamilton, 1999),
FIGURE 2

Spruce macrofossils and pollen records from northern Eurasia in five time-slices (cal. kyr BP is calibrated thousand years before present): > 27 cal. kyr BP is the period prior to the LGM; 27 to > 18 cal. kyr BP represents the local LGM when the Scandinavian and the Barents-Kara ice sheet were at their southern and south-western maximum, respectively (Clark et al. 2009); 18 to > 11.7 cal. kyr BP is the late glacial including the Younger Dryas; 11.7 to > 6 cal. kyr BP is the first part of the Holocene; and 6 to 0 cal. kyr BP is the last part of the Holocene. Macrofossil sites: numbers given in millennia cal. kyr BP indicate the age of spruce

macrofossils. In the maps of the lateglacial and Holocene, only the oldest ages are shown. Pollen sites: numbers given in millennia cal. kyr BP indicate when spruce pollen reached the 2% threshold for the first time. Ice sheets are shown in blue with approximate extent according to time period: extent at 21 cal. kyr BP in the > 27 cal. kyr BP and 27 to > 18 cal. kyr BP maps; at 15 cal. kyr BP in the 18 to > 11.7 cal. kyr BP maps; and at 9 cal. kyr BP in the 11.7 to > 6 cal. kyr BP maps (Peltier, 1994). The current combined distribution of Norway spruce and Siberian spruce is indicated by the green outline.

<figure><img src="image_83.png" /></figure>
*trnS-trnfM* (Demesure *et al.* 1995) and *trnT-trnF* (Taberlet *et al.* 1991). Variation was detected in three regions, among which the most polymorphic (*trnT-trnF*) was genotyped in all samples. After amplification of the entire *trnT-trnF* region with primers 'a' and 'f' (Taberlet *et al.* 1991), a 593-bp fragment that included the *trnT-trnL* spacer and the *trnL* intron was sequenced with a newly designed forward primer (5'-GGAGGATAATAACATTGCAT-3') and the reverse primer 'd' (Taberlet *et al.* 1991). Mitochondrial and chloroplast sequences were assembled and refined manually with LASERGENE 7.7 (DNAStar, Madison, WI, USA) or with AUTOASSEMBLER (Applied Biosystems, Foster City, CA, USA). BIOEDIT (Hall, 1999) was used to align the sequences.

To establish the phylogenetic relationships among mitotypes and chlorotypes, a minimum spanning network was computed using ARLEQUIN 3.11 (Excoffier *et al.* 2005) and drawn in HAPSTAR (Teacher & Griffiths, 2011). In the alignment of the mitochondrial sequences, the two

minisatellites were coded as separate multistate ordered characters (0-2 for the 32-bp minisatellite and 0-9 for the 34-bp minisatellite).

We applied spatial analyses of molecular variance (SAMOVA; using SAMOVA 1.0) to investigate the spatial patterns of genetic subdivision across the ranges of the two taxa and across the two markers (Dupanloup *et al.* 2002). This approach uses an iterative procedure to delineate contiguous groups of populations that are maximally differentiated. In the mitochondrial marker, each repeat copy of the two minisatellites was coded as a single character (base for presence and gap for absence). The analysis was carried out for *k* groups, where *k* ranged from 2 to 10. The grouping was considered to be optimal when the differentiation among groups (*F*<sub>CT</sub>) reached a plateau and before single populations began to be delimited. Within-population gene diversity (*H*<sub>S</sub>), total gene diversity (*H*<sub>T</sub>) and genetic differentiation among populations (*G*<sub>ST</sub>) were estimated according to Pons & Petit (1995). Because

FIGURE 3

Variation in the mitochondrial *nad1* gene and the chloroplast *trnT-trnL* fragment in spruce populations from northern Eurasia. The current combined distribution of Norway spruce and Siberian spruce is indicated by the green outline. (a) Minimum spanning network of the 14 mitotypes identified in the *nad1* minisatellite region. The mitotypes are indicated according their size in base pairs. Characters separating the mitotypes are shown as black bars (single-base substitutions) and boxes (indels); black box, 9-bp indel;

blue boxes, indels of the 34-bp repeat; red boxes, indels of the 32-bp repeat. (b) Geographical distribution of the mitotypes. Their frequencies are represented in circle charts; chart size is proportional to the number of trees analysed, ranging from 7 to 16. (c) Minimum spanning network of the four chlorotypes identified in the *trnT-trnL* fragment. Chlorotypes are indicated with their polymorphic bases. Black bars indicate base substitutions. (d) Geographical distribution of chlorotypes.

<figure><img src="image_84.png" /></figure>
genetic differentiation depends on the level of variation, the standardized genetic differentiation was calculated as $G'_{ST} = G_{ST} (1 + H_S) / (1 - H_S)$ following Hedrick (2005), thus allowing a comparison between GST of the mitochondrial and chloroplast markers, which exhibited very different levels of variation. These indices were calculated over all populations, and for groups of populations delineated by SAMOVA.

# Results

## Macrofossil and pollen data

Macrofossils dated to the period before the LGM (> 27 cal. kyr BP) and to the LGM (27 to > 18 cal. kyr BP) were scarce, but indicated full-glacial presence of spruce in both northern Europe and Siberia (Fig. 2). Macrofossils dated to the LGM are reported at four sites, two on the East European Plain and two on the West Siberian Plain. The two sites on the West Siberian Plain were identical to the pre-LGM sites (Appendix S1), suggesting the long-term persistence of spruce in this area. Two additional occurrences, based on pollen dated to earlier than 18 cal. kyr BP, are described for the Baikal region.

Fossil records of the late glacial (18 to > 11.7 cal. kyr BP) were numerous and widespread, particularly the pollen records (Fig. 2). On the East European Plain, pollen records suggest that spruce established rapidly across large areas,

including the Baltic countries, the northern basin of the Volga River and the area west of the northern Ural Mountains. Most pollen sites reached the 2% threshold between 15 and 13 cal. kyr BP, including those situated close to the northern Ural Mountains. In southern Siberia, pollen and macrofossils consistently showed early spruce occurrences on the West Siberian Plain, Altai and Sayan mountains and the Baikal region. The records from these areas are generally older than those from the East European Plain, ranging from 18 to 13 cal. kyr BP.

In northern Europe, range expansion as recorded at the > 2% pollen threshold was very slow up to the millennium 9-8 cal. kyr BP. Northward expansion towards Finland was fast, particularly from 7 to 5 cal. kyr BP, and spruce reached its modern northern limits in Finland around 2 cal. kyr BP. Spruce expanded westwards towards the Baltic Sea and colonized Scandinavia from 5 cal. kyr BP to the present millennium. The timing of northward colonization in Siberia is less clear because of a scarcity of data, particularly for central Siberia. Fossil pollen records indicate that spruce had already reached its current northern limits in Siberia during the early Holocene (11-8 cal. kyr BP), and also revealed spruce occurrences north of these limits.

## Genetic data

Sizing and sequencing of the mitochondrial *nad1* marker revealed 14 mitotypes in the 50 populations surveyed (Fig. 3, Appendix S3). Three mitotypes were not previously reported. The size variation was mainly a result of copy-number variation in the two minisatellites. Two nucleotide substitutions and a 9-bp indel defined one western and one eastern lineage (Fig. 3a). The four mitotypes of the eastern lineage were restricted to Siberia and two populations in the northern Ural Mountains. The 10 mitotypes of the western lineage were mainly found in northern and north-eastern Europe. Mitotype 721 bp and the mitotypes 789, 857 and 891 bp were also detected in two separate populations situated in the western part of the West Siberian Plain (Fig. 3b). One of these populations and one population from the northern Ural Mountains included mitotypes of both lineages.

The chloroplast *trnT-trnL* marker included three polymorphic sites, together defining four chlorotypes (CAG, CCG, ACT and ACG). Only the first character was parsimony-informative (Fig. 3c). The rare chlorotype ACT was only found in Siberia and in one population from the northern Ural Mountains. The three other chlorotypes occurred across northern Eurasia, but their frequencies varied considerably between eastern and western regions (Fig. 3d). For example, chlorotype CAG was abundant in Siberia, but rare in most of northern Europe, whereas chlorotype CCG was rare in Siberia but abundant in northern Europe.

Spatial analysis of molecular variance (SAMOVA) delineated two highly differentiated groups in both markers (mtDNA, $F_{CT} = 0.69$; cpDNA, $F_{CT} = 0.44$), one

<caption>**FIGURE 4**</caption>

Population structure of northern Eurasian spruce populations as revealed by spatial analysis of molecular variance (SAMOVA). Populations delineated to a particular group are presented in the same colour: (a) mtDNA groups; (b) cpDNA groups.

<figure><img src="image_85.png" /></figure>
western and one eastern (Fig. 4). In the case of the mtDNA, a slightly higher $F_{CT}$ was obtained for $k = 3$ ($F_{CT} = 0.73$), but because the third group comprised two neighbouring populations in northernmost Fennoscandia, $k = 2$ was retained to capture the large-scale geographical structure across northern Eurasia. The two marker groups were largely congruent, with their division located to the east of the Ural Mountains. The division of the mtDNA variation was well defined and centred along the Ob River. This grouping roughly corresponds to the suggested ranges of Norway spruce and Siberian spruce, and coincides with LGM occurrences of spruce in northern Europe and Siberia. The western group also coincides with LGM populations on the West Siberian Plain.

On the northern Eurasian scale, population differentiation was higher for mtDNA ($G_{ST} = 0.569$) than for cpDNA ($G_{ST} = 0.194$), including when the level of variation was taken into account (Table 1). For assessing the genetic diversity within groups, we used the SAMOVA groups delineated by mtDNA, because these groups are geographically most structured. The within-population gene diversity was clearly higher in the western than in the eastern group, both in mtDNA (west, $H_S = 0.332$; east, $H_S = 0.086$) and cpDNA (west, $H_S = 0.580$; east, $H_S = 0.281$). In the western group, gene diversity was slightly higher when populations from formerly glaciated areas were excluded (Table 1).

# Discussion

## Genetic groups

Using cytoplasmic DNA markers, we identified two highly differentiated genetic groups, one western and one eastern, demonstrating for the first time that northern European Norway spruce and Siberian spruce should be considered two well-defined taxa. This finding resolves the long-term controversy on their distinction, which was primarily based on their high morphological similarity, the vast geographical cline in cone-scale shape (e.g. Schmidt-Vogt, 1974a; Popov, 2003) and their vague differentiation at allozyme loci (Krutovskii & Bergmann, 1995). The distinct east-west division of the mtDNA variation along the Ob River is consistent with the morphological data of Popov (2003). He described the most typical Siberian spruce cone-scales east of a line stretching from the Pechora River south-eastwards to the Ob River; in the area to the west, the scales gradually change to the shape of Norway spruce. Interestingly, the Ob River has also been found to constitute the border between Russian larch (*Larix sukaczewii*) and Siberian larch (Semerikov et al. 2007, 2013) as well as between several boreal animal species (Fedorov et al. 2008), suggesting that the same historical factors have been important to these taxa.

## Glacial refugia

The combined palaeobotanical and genetic data strongly suggest that Norway spruce and Siberian spruce occupied separate LGM refugia - Norway spruce on the East

European Plain and Siberian spruce in southern Siberia. Macrofossil records dated to the LGM and the widespread distribution of late glacial records on the East European Plain indicate that Norway spruce survived the LGM across vast areas, possibly including the area to the west of the northern Ural Mountains (cf. Väliranta et al. 2011). Consistent with a large refugium, current populations of the East European Plain were generally characterized by high mtDNA and cpDNA diversity, a pattern also reported for nuclear microsatellites (Tollefsrud et al. 2009). In contrast, populations in northern Scandinavia showed much lower diversity, both in mtDNA and microsatellites, reflecting the immigration history of spruce (Tollefsrud et al. 2009). The refugial population on the East European Plain may even have extended into the West Siberian Plain, where mitotypes of Norway spruce spatially coincide with LGM macrofossils (Figs 2 & 3).

Previous palaeobotanical studies, mainly based on pollen, have indicated that Siberian spruce persisted during the LGM in the southern part of the West Siberian Plain (e.g. Tarasov et al. 2000) and in the Baikal region, where large LGM populations were suggested (e.g. Bezruková et al. 2005). The data of the present study lend further support to these suggestions and indicate that, during the late glacial, Siberian spruce was present in large parts of southern Siberia, namely the West Siberian Plain, Altai and Sayan mountains and the Baikal region. Most records from these areas are situated in the basins of the rivers Irtysh, Ob and Yenisei, whereas many records from the Holocene are located on interfluves. River floodplains may have been locally sheltered and/or provided the moisture required for tree growth (cf. Binney et al. 2009).

## Post-glacial expansion

Based on pollen data, spruce started to expand on the East European Plain between 15 and 13 cal. kyr BP. No northward trend of decreasing age was detectable, suggesting that fossil pollen largely reflects local expansions. This supports the view of Väliranta et al. (2011) that expansion in the north-eastern region of European Russia is a result of local population expansion rather than migration from the south. In a former study, we hypothesized based on fossil cones with rounded scales that late-glacial expansions of spruce on the East European Plain involved Siberian spruce as the more cold-tolerant taxon, whereas Norway spruce was favoured during the Holocene expansion (Latałowa & van der Knaap, 2006). The genetic data of the present study do not support this hypothesis, but historical presence of Siberian spruce cannot be excluded. Following the argument of Schmidt-Vogt (1974a) that rounded cone-scales reflect adaptation to cold climate, the East European Plain may have harboured different Norway spruce ecotypes, some of them more cold-tolerant than others, and the cold-tolerant ecotypes may even have originated from introgression between Norway spruce and Siberian spruce during interglacials.
In Siberia, the pollen data show that the earliest expansions of spruce took place in the Baikal region and in the Altai and Sayan mountains (Fig. 2). Population expansion probably started in these areas concomitantly with the onset of climate warming after the LGM. The late-glacial expansion in westernmost Siberia possibly included both Norway spruce and Siberian spruce as suggested by mtDNA. The pollen data indicate that Siberian spruce reached the coast of the Kara and Laptev seas during the early Holocene. Many of these northernmost populations must, however, have declined again, because the northern limits of the modern Siberian spruce range are now further south (MacDonald *et al.* 2000; Binney *et al.* 2009). Whether northern populations expanded from southern or more northerly refugia cannot be inferred from our data. A survey of nuclear microsatellites and functional genes in six Siberian spruce populations located along the Yenisei River between 56° and 67° N latitude did not reveal any population structure, except for photoperiodic and circadian-clock genes which showed significant clinal variation and/or evidence of local selection (Chen *et al.* 2014). This pattern could indicate that Siberian spruce expanded from numerous source populations, including populations at high latitudes, but a wave-like front of population expansion from the south involving many individuals cannot be excluded.

## Contact and introgression between Norway spruce and Siberian spruce

Unlike the mitotypes, the chlorotypes showed widespread admixture. Three of the four chlorotypes were widely distributed and were shared between the two taxa. Haplotype sharing can be a result of introgression and/or retention of ancestral polymorphisms. Generally, introgression is predicted to be less common for genes that experience high rates of gene flow than for those that

experience little gene flow (Currat *et al.* 2008). The rationale is that high rates of gene flow help to dilute migrant alleles and thus to preserve the species' integrity. In our study, we observed a clear difference in overall population differentiation in the two markers (Table 1). As in other conifers (e.g. Du *et al.* 2009; Polezhaeva *et al.* 2010), population differentiation was lower in cpDNA than in mtDNA (Table 1), indicating greater gene flow in the former. Accordingly, it seems unlikely that the widespread distribution of the three chlorotypes solely reflects introgression. Rather, the retention of ancestral polymorphism, which is frequently observed between closely related *Picea* species (Li *et al.* 2010), seems to be the main cause for their widespread distribution. The rare chlorotype ACT, which was restricted to Siberian populations and one population in the northern Ural Mountains, may represent a more recent mutation.

In the north-western part of the West Siberian Plain, two populations exhibited mitotypes of both mitochondrial lineages, which strongly suggests that Norway spruce and Siberian spruce are indeed in contact today. The palaeobotanical data show that spruce was already present in this region during the late glacial, suggesting that the two taxa came into contact soon after the LGM. Based on morphological data, the north-western part of the West Siberian Plain is characterized by Siberian spruce (Popov, 2003). It is therefore not unlikely that mtDNA of Siberian spruce was replaced by that of Norway spruce as a consequence of recurrent hybridization. Replacement of mtDNA is also conceivable further west, in the Ural Mountains and the Pechora region (cf. Fig. 1). However, because the east-west division of the cpDNA variation was largely congruent with that of mtDNA, the zone of introgression may be narrower than suggested by morphological criteria (e.g. Pravdin, 1975; Popov, 2003).

<caption>TABLE 1</caption>

Genetic diversity within populations ($H_S$), total genetic diversity ($H_T$) and population differentiation estimated as $G_{ST}$ and standardized $G'_{ST}$ (Hedrick, 2005) for mtDNA and cpDNA of spruce populations of northern Eurasia, both globally and for the SAMOVA groups delineated by mtDNA. The western group consists of populations from the East European Plain, Fennoscandia,

the Ural Mountains and a population on the West Siberian Plain. Population-genetic parameters are also given for the western group, excluding populations from formerly glaciated areas. The eastern group consists of populations from Siberia. Standard errors are given in parentheses; n.c., not calculated because of low variation among populations.

<table><thead><tr><th rowspan="2">Grouping of populations</th><th colspan="4">Mitochondrial DNA</th><th colspan="4">Chloroplast DNA</th></tr><tr><th>H<sub>S</sub></th><th>H<sub>T</sub></th><th>G<sub>ST</sub></th><th>G'<sub>ST</sub></th><th>H<sub>S</sub></th><th>H<sub>T</sub></th><th>G<sub>ST</sub></th><th>G'<sub>ST</sub></th></tr></thead><tbody><tr><td>Northern Eurasia (n = 50)</td><td>0.278<br>(0.047)</td><td>0.644<br>(0.049)</td><td>0.569<br>(0.064)</td><td>1.001</td><td>0.514<br>(0.027)</td><td>0.638<br>(0.009)</td><td>0.194<br>(0.044)</td><td>0.605</td></tr><tr><td>Western group (n = 39)</td><td>0.332<br>(0.056)</td><td>0.492<br>(0.071)</td><td>0.326<br>(0.067)</td><td>0.649</td><td>0.580<br>(0.019)</td><td>0.606<br>(0.017)</td><td>0.043<br>(0.030)</td><td>0.163</td></tr><tr><td>Western group without glaciated areas (n = 14)</td><td>0.354<br>(0.094)</td><td>0.493<br>(0.110)</td><td>0.281<br>(0.073)</td><td>0.589</td><td>0.534<br>(0.044)</td><td>0.626<br>(0.026)</td><td>0.141<br>(0.052)</td><td>0.464</td></tr><tr><td>Eastern group (n = 11)</td><td>0.086<br>(0.058)</td><td>0.105<br>(0.067)</td><td>0.180<br>n.c.</td><td>0.214</td><td>0.281<br>(0.066)</td><td>0.347<br>(0.080)</td><td>0.192<br>n.c.</td><td>0.342</td></tr></tbody></table>
## Contrasting patterns of genetic diversity

The genetic diversity in both mtDNA and cpDNA was found to be clearly higher in western than in eastern populations, a pattern that also has been observed in allozymes, albeit less pronouncedly (Krutovskii & Bergmann, 1995). In the case of mtDNA, all but one of the populations of the eastern mitochondrial lineage were fixed for the 712-bp mitotype. The three other mitotypes, each containing several copies of the 34-bp minisatellite, were found in a single population located in the northern foothills of the southern Siberian mountains. Assuming stepwise copy-number changes in the minisatellite, it seems likely that ancestral populations carried more mitotypes.

The higher genetic diversity in Norway spruce than in Siberian spruce could have two causes – less pronounced fluctuations in population size of Norway spruce due to more stable and suitable climatic conditions during the Quaternary and/or gene flow from Siberian spruce. It seems unlikely that the clear difference in diversity is solely a result of introgression. Diversities of mtDNA and cpDNA are both quite high in the western part of the East European Plain, an area that has not been suggested to be influenced by introgression (cf. Fig. 1). Glaciological evidence indicates that the climate of the East European Plain was rather humid during glacial maxima, whereas that of Siberia was very dry, particularly during periods when the Scandinavian ice sheet was large, such as during the LGM, absorbing much of the moisture spread from the Atlantic Ocean (Stauch & Gualtieri, 2008; Krinner et al. 2011). A cold and extremely dry LGM climate in Siberia and much milder conditions on the East European Plain are also supported by palaeobotanical data (Hubberten et al. 2004) and palaeovegetation maps (Allen et al. 2010). Siberian spruce may thus have repeatedly experienced reductions in population size and consequently in genetic diversity, whereas Norway spruce was less affected. A similar pattern of reduced diversity into the continent was observed in *Abies* in the Russian Far East, where a more stable climate along the coast was suggested to have prevented major demographic fluctuations (Semerikova et al. 2011). It seems possible that glaciations also played a role, particularly during the Saalian stage, when glaciers in Siberia were more extensive (Stauch & Gualtieri, 2008). Indeed, climatic events pre-dating the LGM were suggested to have influenced the genetic structure of Siberian larch (Semerikov et al. 2013) and several Eurasian boreal animal species (Fedorov et al. 2008).

## Conclusions

The combined palaeobotanical and genetic data allowed us to draw the following major conclusions. (1) North Eurasian Norway spruce and Siberian spruce are genetically distinct and occupied separate LGM refugia. Norway spruce persisted during the LGM on the East

European Plain, where it was already widely distributed during the late glacial, suggesting widespread LGM tree occurrences. Siberian spruce persisted in southern Siberia, but may also have survived locally at high latitudes, facilitating rapid northward expansion. (2) The two taxa came into contact in the area of the Ob River, where they probably hybridized. (3) The higher cytoplasmic DNA diversity in Norway spruce than in Siberian spruce may be a consequence of more favourable past climatic conditions for Norway spruce and/or introgression from Siberian spruce.

Our survey of cytoplasmic DNA variation did not provide evidence for population subdivision either on the East European Plain or in Siberia. Multilocus surveys with nuclear markers will be necessary to provide further insights into the structure of refugial populations. Nuclear markers could also help to delineate the introgression zone and to elucidate patterns of past and current gene flow in this zone. Furthermore, variation in functional genes could be used to determine whether the gradient in cone-scale morphology is associated with introgression and/or climatic adaptation.

## Acknowledgements

We thank Paolo Cherubini, Thomas Geburek, Maxim Kapralov, Daniel Nievergelt, Andreas Rigling and Fritz Schweingruber for providing spruce samples from Russia. We are grateful to the staff at the ABI lab at the University of Oslo for producing excellent DNA sequences. We also thank Heather Binney for providing valuable information about the structure of the Eurasian Macrofossil Database and Arne Steffenrem, who assisted with extraction of data from the database. Marcelina Zimny is acknowledged for her help in preparing tables and references of palaeobotanical data, György Sipos for critically reading and commenting on the manuscript, Donatella Magri and an anonymous referee for their helpful comments. Finally, we thank the data contributors of the EMD and EPD. This study was supported by the Norwegian Institute of Bioeconomy Research, the Research Council of Norway (Strategic Institute Programme 501500: DNA markers for characterization of genetic variation in Norwegian Forest trees), and the EU network EVOLTREE; the work done by M.L. was based on statutory funds in the Faculty of Biology, University of Gdańsk (DS/530-L145-D486-14).

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EVOLTREE PARTNERS

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EHIATLANTIC, 2015

In green: core partners (i.e., partners who provide financial contribution to the network)

In blue: associate partners (i.e., partners who provide in-kind contribution to the network)

1. Austrian Institute of Technology GmbH (AIT)

2. Bundesforschungszentrum für Wald Institut für Waldgenetik (BWF), Austria

3. University of Sarejevo Faculty of Forestry (UNSA), Bosnia and Herzegovina

4. Croatian Forest Research Institute (CFRI)

5. Czech University of Life Sciences (CULS)

6. University of Copenhagen (UCPH), Denmark

7. Natural Resources Institute Finland (LUKE)

8. University of Oulu (UOULU), Finland

9. National Institute for Agricultural Research (INRA), France

10. Georg-August-University Göttingen (UGOE), Germany

11. Phillips University of Marburg (UNIMAR), Germany

12. Johann Heinrich von Thünen Institut, Germany

13. University of West Hungary Faculty of Forestry (UWH)

14. European Forest Institute (EFI), International

15. Institute of Plant Sciences Agriculture Research Organisation (ARO), Israel

16. National Research Council Plant Genetics Institute (CNR-IBBR), Italy

17. Institute for Applied Genomics (IGA-UNIUD), Italy

18. Wageningen UR (WUR), The Netherlands

19. Norwegian Institute of Bioeconomy Research (NIBIO)

20. University of Bydgoszcz (UKW), Poland

21. The Forest Research Institute (IBL), Poland

22. Universidade de Lisboa Instituto Superior de Agronomia (ISA), Portugal

23. Universitatea Transilvania Brasov (UNITBV), Romania

24. Russian Academy of Sciences Vavilov Institute of General Genetics (VIGG)

25. Institute of Lowland Forestry and Environment (ILFE), Serbia

26. Technical University of Zvolen (TUZVO), Slovakia

27. Slovenian Forestry Institute (SFI)

28. National Institute for Agriculture, Food Research and Technology (INIA), Spain

29. Uppsala University (UU), Norway

30. Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Switzerland

31. Centre for Ecology and Hydrology (NERC), UK

32. University of Southampton (SOTON), UK
AIT

AUSTRIAN INSTITUTE
OF TECHNOLOGY

BFW.

Bundesforschungszentrum für Wald

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CZECH
UNIVERSITY
OF LIFE SCIENCES PRAGUE

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UNIVERSITY OF
COPENHAGEN

Luke

NATURAL RESOURCES
INSTITUTE FINLAND

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UNIVERSITY
OF OULU

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INRA

SCIENCE & IMPACT

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GEORG-AUGUST-UNIVERSITÄT
GÖTTINGEN

Philippps

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Universität
Marburg

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THÜNEN

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E F I

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Consiglio
Nazionale delle
Ricerche

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IGA

ISTITUTO DI GENOMICA APPLICATA

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WABENINGEN UU

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NIBIO

NORWEGIAN INSTITUTE OF
BIOECONOMY RESEARCH

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INSTITUTO
SUPERIOR D
AGRONOMIA

Universidade de Lusópolis

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INSTITUTE OF LOWLAND FORESTRY
AND ENVIRONMENT (ILFE) SERBIA

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TECHNICKÁ UNIVERZITA VO ZVOLENE

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GOZDARSKI INSTITUT SLOVENJE
SLOVENIAN FORESTRY INSTITUTE

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INIA

Instituto Nacional de Investigación
y Tecnología Agraria y Alimentaria

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UPPSALA

UNIVERSITET

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SWITZERLAND

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Centre for
Ecology & Hydrology

NATURAL ENVIRONMENT RESEARCH COUNCIL

UNIVERSITY OF
Southampton

The EVOLTREE coordination team would like to thank all EVOLTREE participants, past and present, who have supported the network throughout the last 10 years by contributing to its successful work and activities. We are also grateful to the European Commission for funding during the period as Network of Excellence (2006-2010) under FP6 and to all institutional EVOLTREE members for self-funding after that period.
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EFI

THE EVOLTREE NETWORK
IS A EUROPEAN RESEARCH GROUP
FACILITATED BY THE EUROPEAN
FOREST INSTITUTE (EFI)

EVOLTREE/EFIATLANTIC

SITE DE RECHERCHE FORÊT-BOIS / 69 ROUTE D'ARCACHON / 33612 CESTAS / FRANCE