Molecular Ecology (2007) 16, 3902– 3925
doi: 10.1111/j.1365-294X.2007.03425.x
Nuclear vs. plastid data: complex Pleistocene history of
a circumpolar key species
Blackwell Publishing Ltd
P . B . E I D E S E N ,* I . G . A L S O S ,* M . P O P P ,* Ø . S T E N S R U D ,* J . S U D A † and C . B R O C H M A N N *
*National Centre for Biosystematics, Natural History Museum, University of Oslo, PO Box 1172 Blindern, NO-0318 Oslo, Norway,
†Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, CZ-128 01 Prague, Czech Republic & Institute
of Botany, Academy of Sciences of the Czech Republic, Prùhonice 1, CZ-252 43, Czech Republic
Abstract
To fully understand the contemporary genetic structure of plants, both nuclear and plastid
markers are needed. Three chloroplast DNA (cpDNA) lineages, which probably diverged
before the major Pleistocene glaciations, have been identified in the circumpolar/circumboreal Vaccinium uliginosum. Here we investigate its nuclear DNA variation using nuclear
ribosomal internal transcribed spacer (ITS) sequences, DNA ploidy level measurements and
amplified fragment length polymorphisms (AFLPs). We also extend the cpDNA dataset.
Two ITS lineages, corresponding to diploids and tetraploids, respectively, were identified.
However, both main sequence types apparently occurred in most individual plants but
showed ploidy-biased homogenization and possibly reflect paralogy predating the origin
of V. uliginosum. The ploidy levels were largely consistent with the cpDNA lineages, suggesting that the initial cpDNA divergence followed early polyploidizations. Five main
AFLP groups were identified, consistent with recent glacial refugia in Beringia, western
Siberia, the southern European mountains and areas south/east of the Scandinavian and
Laurentide ice sheets. Except from the southern European mountains, there has been extensive
expansion from all refugia, resulting in several contact zones. Surprisingly, the presumably
older ploidy and cpDNA patterns were partly inconsistent with the main AFLP groups and
more consistent with AFLP subgroups. A likely major driver causing the inconsistencies is
recent nuclear gene flow via unreduced pollen from diploids to tetraploids. This may prevent
cytoplasmic introgression and result in overlayed patterns formed by processes dominating
at different time scales. The data also suggest more recent polyploidizations, as well as several
chloroplast capture events, further complicating this scenario. This study highlights the
importance of combining different marker systems to unravel intraspecific histories.
Keywords: AFLP, chloroplast DNA, heteroploid hybridization, ITS, phylogeography, Vaccinium
uliginosum
Received 4 November 2006; revision received 19 March 2007; accepted 16 May 2007
Introduction
Phylogeographers still pay too little attention to the criticism
of relying on single gene systems (Bermingham & Moritz
1998; Schaal et al. 1998; Hewitt 2001). The reason for this is
probably a lack of appropriate genetic markers; wellresolved intraspecific phylogenies from several independent
datasets are difficult and expensive to obtain. In phylogeographical analyses of plants, where no single molecular
Correspondence: Pernille Bronken Eidesen, Fax: +47 22 85 18 35;
E-mail: [email protected]
method stands out as the obvious first choice (Schaal et al.
1998), several more or less suboptimal marker systems
have frequently been used. There is typically a trade-off
between marker systems revealing sufficient levels of
variation vs. their suitability for inference of clear gene
genealogies. To meet the latter demand, marker systems
based on the haploid, usually nonrecombinant and
maternally inherited chloroplast DNA (cpDNA) have been
preferred (Comes & Kadereit 1998; Schaal et al. 1998), but
are often hampered by little variation (Schaal et al. 1998).
Inference of Pleistocene glacial-induced patterns has therefore typically been based on spatial arrangement of cpDNA
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P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3903
haplotypes rather than gene genealogies (Tremblay &
Schoen 1999; Abbott et al. 2000; Alsos et al. 2005; Koch et al.
2006). In addition, plants have high potential for interspecific gene flow, often leading to introgression of cpDNA
(Rieseberg & Soltis 1991; Soltis & Kuzoff 1995; King &
Ferris 2000; Petit et al. 2002; Chan & Levin 2005). Thus, a
cpDNA phylogeny may not be equivalent to the organism
phylogeny (Rieseberg & Soltis 1991).
Typically, markers revealing higher levels of variation
can reflect a more recent history (e.g. Magri et al. 2006;
Eidesen et al. 2007). In spite of possible problems caused by
recombination and heterozygosity, marker systems based
on nuclear DNA (nDNA) have shown to be useful for
revealing higher levels of variation. Especially fingerprinting techniques screening mainly nDNA, like amplified
fragment length polymorphism (AFLP), have recently
gained popularity (e.g. Stehlik et al. 2002; Kropf et al.
2003; Schönswetter et al. 2003; Skrede et al. 2006). However,
AFLP markers are anonymous and dominant, and comigrating bands are not necessarily homologous (Mueller &
Wolfenbarger 1999). Sequencing of the nuclear ribosomal
internal transcribed spacer (ITS) regions is another alternative
(e.g. Jeandroz et al. 1997; Hess et al. 2000; Koch et al. 2006),
but as part of the tandemly repeated 18S-5.8S-26S ribosomal
DNA region, ITS is subjected to the poorly understood
process of concerted evolution that confounds interpretation of sequence polymorphisms at the intraspecific level
(Zimmer et al. 1980; Wendel et al. 1995; Buckler et al. 1997).
Nevertheless, although appropriate marker systems for
inferring plant phylogeography are difficult to obtain, a
combination of suboptimal markers can provide a more
complete picture than a single (sometimes suboptimal)
marker. To account for events like introgression or concerted
evolution, and to better understand how the Pleistocene
glaciations shaped the contemporary genetic structure of
plants, comparative studies based on both cpDNA and
nuclear markers, reflecting both deep and shallow phylogeographical histories, are needed.
The circumpolar/circumboreal dwarf shrub Vaccinium
uliginosum L. s. lat. is a key component of northern ecosystems. It is a bird-dispersed, long-lived, insect-pollinated
and mainly outcrossing species complex containing considerable morphological and ploidy level variation (2n = 24,
48, 72; x = 12; Hultén 1970; Young 1970; Vander Kloet 1988;
Jacquemart 1996). On a circumpolar scale, it is suggested
that at least some of the variation in ploidy levels, morphological characters and geographical distribution is correlated (Löve & Boscaiu 1966; Young 1970; Hultén & Fries
1986). However, chromosome counts are scarce for most
areas and lacking from the southern European mountains.
V. uliginosum has earlier been investigated throughout
its vast distribution range for variation in cpDNA (Alsos
et al. 2005; Fig. 1) and morphology (Alsos 2003). Eighteen
cpDNA haplotypes were identified, grouping into three
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
major cpDNA lineages. The boreal Amphi-Atlantic cpDNA
lineage contained only two haplotypes. This lineage corresponded morphologically to the tetraploid V. uliginosum
ssp. uliginosum and probably expanded from refugia located
south of the ice in Europe and North America. The other
boreal cpDNA lineage was restricted to Beringia, a region
encompassing northwesternmost North America and
northeasternmost Asia. This Beringian cpDNA lineage
probably corresponds to several subspecies and ploidy
levels [V. uliginosum ssp. pedris (Harshb.) S. B. Young, V.
uliginosum ssp. occidentale var. occidentale (A. Gray) Hara
and V. uliginosum ssp. vulcanorum (Kom) Jurtsev]. The high
haplotype diversity and restricted distribution of this
lineage suggested longstanding survival in Beringia. The
circumpolar Arctic–Alpine cpDNA lineage showed some
morphological differentiation between arctic and alpine
populations; the arctic populations corresponded to the
mainly diploid V. uliginosum ssp. microphyllum (Lange)
Tolm., whereas the southern alpine populations were morphologically intermediate between ssp. microphyllum and
ssp. uliginosum. The three haplotypes found in southern
European alpine areas were not found elsewhere, suggesting some isolation. The high haplotype diversity as well as
the large range of single arctic haplotypes was interpreted
as a result of broad-fronted recolonization from the vast
periglacial tundras, as well as from more distant populations.
The relationships among the lineages were not resolved,
but they were estimated to have diverged 0.7–3.0 Ma ago,
suggesting that they could have been affected more or less
independently by the Pleistocene glaciations (Alsos 2003;
Alsos et al. 2005).
In this study, we investigate nDNA variation in V. uliginosum throughout its range using AFLPs, ITS sequences
and DNA ploidy level estimations. We also extend the
cpDNA dataset of Alsos et al. (2005). Our main aim is to use
nDNA variation in association with cpDNA and ploidy
level variation to infer the shallow (mid- to late Pleistocene) history of V. uliginosum in detail, including location
of refugia and migration patterns after the last glaciation.
We also aim to better understand the polyploidization
history in this complex.
Materials and methods
A total of 177 populations of Vaccinium uliginosum were
included in this study, of which 88 also were included in
Alsos et al. (2005) and 93 were sampled for this study
(Appendix I). For most of the new populations, 11 plants
each were collected 25 m apart along a 250 m transect. The
material for AFLP analysis was collected as fresh leaves
and stored on silica gel. Material for sequencing and DNA
ploidy level estimates was also taken from herbarium
specimens (Appendix I). Voucher specimens of all new
populations were deposited at the Botanical Museum in
3904 P . B . E I D E S E N E T A L .
Fig. 1 Various analyses of Vaccinium
uliginosum (a) Geographical origin of the 131
populations analyzed for AFLPs and their
grouping according to structure analyses.
Colours identify main groups; symbols
identify subgroups within main groups. The
geographical distribution of the species
(Hultén & Fries 1986) is outlined/shaded.
(b) Geographical distribution of ploidy levels,
including 19 chromosome counts adopted
from other sources (see Alsos (2003) and
references therein) and the three cpDNA
lineages found by Alsos et al. (2005): red:
Amphi-Atlantic lineage, blue: ArcticAlpine lineage, green: Beringian lineage.
(c) Intrapopulation diversity based on 105
AFLP markers. The maximum limits of the
late Weichselian/Wisconsian ice sheets are
redrawn from Abbott & Brochmann (2003)
and Brochmann et al. (2004). (d) The 95%
plausible set of haplotype networks inferred
for the 20 cpDNA haplotypes found by
Alsos et al. (2005; A–R) and in this study
(S and T). Lines represent the mutational
pathway interconnecting the haplotypes;
dots represent inferred intermediate haplotypes not observed in the samples. The size
of each circle is determined by the square
root of the sample size. Ploidy levels are
indicated (* denote unknown ploidy level).
Oslo (O; for populations with ID starting with ‘V’, see Alsos
(2003) and Alsos et al. (2005)). DNA was extracted using the
DNeasy™ Plant Mini Kit or DNeasy™ Plant 96 Kit (Qiagen)
following the manufacturer’s instructions. Sequences and
AFLPs were analysed using an ABI 3100 Genetic Analyser
(Applied Biosystems).
cpDNA
Thirty-three of the new populations were analysed for
cpDNA variation to extend the previous dataset (Alsos
et al. 2005). The trnL-trnF (Taberlet et al. 1991) and trnS-trnG
spacers (Hamilton 1999) were sequenced in 1–2 plants from
each population (Appendix I) following Alsos et al. (2005).
Parsimony analyses were performed on the extended cpDNA
dataset using TNT (Goloboff et al. 2003). Heuristic searches
were performed with 1000 random addition sequences,
saving 10 trees per replication. Collapsing rule was set to
minimum length = 0. Random seed was set to time. Jackknife
resampling with 36% deletion and GC frequencies as output
were performed with 10 000 replicates (10 random entry
orders and 10 trees saved each repetition) and collapsing
rule = TBR. Statistic parsimony analysis using the network
algorithm of Templeton et al. (1992) as implemented in the in
the tcs program (Clement et al. 2000) was also performed.
DNA ploidy level estimates
DNA ploidy levels (hereafter referred to as ploidy levels)
and 2C nuclear DNA values were estimated using a Partec
PA II flow cytometer (Partec GmbH) equipped with a
HBO-100 mercury arc lamp and an argon ion laser (488 nm).
One hundred and thirteen plants, which also were analysed
for cpDNA variation, were included in the analysis.
© 2007 The Authors
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P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3905
2C-values (= nuclear DNA content of holoploid chromosome
set) of fresh samples were determined according to Suda
et al. (2003), and the Lycopersicon esculentum Mill. cultivar
named Stupnické poln’ tyèkové rané (2C = 1.96 pg;
Dole6el et al. 1992) was used as an internal reference
standard. Estimation of ploidy levels in desiccated plant
material followed Suda & Trávnícek (2006). Herbarium
vouchers of V. uliginosum from population 147-V2, Colesdalen,
Svalbard were used as a primary reference standard. This
population corresponded to chromosome-counted material
by Flovik (1940; 2n = 24). Each plant was analysed at least
three times: (i) without internal standard to determine the
peak quality and to obtain a preliminary estimate of the
ploidy level (the above-mentioned diploid herbarium
voucher was used as an external standard and the position
of the diploid peak was always checked before the unknown
sample acquisition); (ii) with the above-mentioned diploid
herbarium voucher as an internal reference standard; and
(iii) with fresh leaves of chromosome-counted and flow
cytometry-confirmed hexaploid V. oxycoccos L. (2n = 6x = 72)
from southwestern Bohemia, the Novohut’ské mocály
peat bog (Suda & Lysák 2001) as an internal standard. We
obtained the same ploidy level estimate for each V. uliginosum
sample using all the above-mentioned modifications.
investigate intraindividual polymorphism (see below)
using a TOPO TA Cloning Kit (Invitrogen, San Diego, CA,
USA). All kits were used according to the manufacturer’s
recommendations.
ITS sequences were edited using staden version 1.6.0
(https://sourceforge.net/projects/staden). Polymorphic
sites were coded using IUPAC codes. Sequences obtained
from clones were usually free from polymorphism. The
sequences were manually aligned using Se-Al version 2.0a11
(Rambaut 1996). To reduce the impact of chimeric sequences
(Popp & Oxelman 2001; Cronn et al. 2002; Kanagawa 2003)
on the phylogenetic reconstruction, an initial maximum
parsimony analysis was performed including all cloned
sequences. The topology was inspected for obvious signs
of chimeric sequences, revealed as long-terminal branches
due to the increased homoplasy. After visual inspection,
the sequences suspected to be chimeras were excluded. To
minimize the effect of Taq errors in the clones (Hengen 1995;
Cline et al. 1996; Kanagawa 2003), substitutions not found
in at least one more clone or direct sequence (i.e. autapomorphic substitutions) were regarded as polymerase errors.
Ignoring these, ‘majority rule’ haplotype sequences were
constructed from otherwise identical clones. Parsimony
analyses were performed using TNT as described above.
ITS
AFLP
A representative subset of 53 plants from 51 populations
analysed for cpDNA variation (Alsos et al. 2005) and ploidy
level was selected for ITS analysis (Appendix I ). In addition
to sequences from Andromeda polifolia L., Zenobia pulverulenta
(Bartr ex Willd) Pollard, and Leucothoe fontanesiana (Steud.)
Sleumer used for outgroup rooting, 11 sequences from
closely related Vaccinium species (Kron et al. 2002) were
included in the ITS dataset.
The complete ITS region (ITS1, 5.8S and ITS2) was
amplified using the primers ITS5P (Möller & Cronk 1997)
and ITS4 (White et al. 1990). The reactions were performed
in 25 µL volumes, consisting of 2.5 µL Taq buffer (Applied
Biosystems), 2.5 µL MgCl2 (25 mm), 2.5 µL BSA (1 mg/mL),
2.5 µL TMACl (0.1 mm), 2.0 µL dNTPs solution (10 mm),
6.8 µL milliQ-H2O, 0.2 µL AmpliTaq (Applied Biosystems),
1.0 µL of each primer (10 µm) and 4 µL template. The
amplifications were run for 3 min at 94 °C followed by 35
cycles of 30 s at 94 °C, 30 s at 52 °C, and 60 s at 72 °C and a
final 10 min elongation step at 72 °C. Negative controls
(milliQ-H2O) were included in all amplifications.
Polymerase chain reaction (PCR) products and cyclesequencing products were cleaned with the Qiagen PCR
cleanup kit and an ethanol/sodium acetate precipitation
procedure (Applied Biosystems), respectively. Sequencing
reactions were performed using the BigDye Terminator
version 1.1 kit (Applied Biosystems) and the primers ITS5P
and ITS4. Six individuals (Appendix II) were cloned to
A total of 131 populations, containing up to 11 individuals
each (Appendix I), were analysed for AFLPs following
Gaudeul et al. (2000). For 44 populations, one randomly
chosen plant was duplicated as a control and repeatability
sample. Seventy-three primer combinations were tested
on four plants from different geographical areas. Primer
combinations producing polymorphic AFLP profiles with
clearly separated fragments were preferred. One HEXlabelled (EcoRI-ATC/Mse1-CAG) and two 6-FAM labelled
(EcoRI-ACT/Mse1-CTA, EcoRI-ATG/Mse1-CAG) primer
combinations were chosen and analysed for the complete
sample set. Each primer combination was analysed separately
using 2 µL labelled selective PCR product, 0.3 µL GeneScan
ROX 500 (Applied Biosystems) and 11.7 µL HiDi (formamide)
per run. The raw data were analysed in ABI prism GeneScan
Analysis Software version 3.7 (Applied Biosystems) and
imported into genographer version 1.6.0 (available at
http://hordeum.oscs.montana.edu/genographer/). Unambiguous fragments in the range 60–500 bp were scored as
present (1) or absent (0). The error rate was calculated
according to Bonin et al. (2004). The dataset was checked
for linkage among markers by making a neighbour joining
tree of an inverted matrix with the individual markers as
objects using treecon 1.3b (Van de Peer & De Wachter 1997).
Data format conversions and diversity/correlation estimations
for most analyses explained below were carried out with
the AFLPdat R-script (Ehrich 2006).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
3906 P . B . E I D E S E N E T A L .
Genetic groups were inferred using a Bayesian clustering
approach implemented in structure version. 2.1 (Pritchard
et al. 2000). As this program is developed for codominant
markers, the AFLP multilocus phenotypes were treated as
diploid multilocus genotypes with the unknown alleles
as missing values, and run under a no admixture model.
Iteration parameters were set to a burning-in period of
10 5 iterations followed by 10 6 iterations. The number
of clusters K was varied from 2 to 20 in 10 independent
runs. The structure analyses were run using the
www. bioportal.uio.no at the University of Oslo. Correlation between replicates was calculated according to
Rosenberg et al. (2002). The number of K in the dataset was
evaluated according to Pritchard et al. (2000) and Evanno
et al. (2005). After the number of K’s was decided, each
group was analysed separately using Structure under the
same settings as for the main analyses, except that maximum number of K was set from 1 to 10.
Intrapopulation diversity was calculated based on the
average proportion of pairwise differences between individuals for each population, corresponding to Nei’s gene
diversity calculated from marker frequencies (Nei 1987;
Kosman 2003).
Principal coordinate analyses (PCO) were used to
compare pairwise similarity among the AFLP multilocus
phenotypes. Analyses were executed in ntsys-pc (version
2.11T; Rohlf 2002) using Dice’s similarity coefficient.
The relationships between the AFLP multilocus phenotypes were also evaluated using different tree analyses.
Parsimony and jackknife analyses were performed in TNT
as described above. Neighbour-joining trees were calculated
using treecon 1.3b based on Nei & Li’s (1979) distance
measure. Branch support was estimated with 1000 bootstrap
replicates.
Differentiation between populations and groups was
quantified from analyses of molecular variance (amova;
Excoffier et al. 1992) based on pairwise distances (Simple
matching), using arlequin version. 2.000 (Schneider et al.
2000).
Multilocus assignment tests were performed using the
program aflpop version. 1.0 (Duchesne & Bernatchez 2002),
where differences in marker frequencies at polymorphic
loci were used to assign an individual to its most probable
source population based on its AFLP multilocus phenotype,
given a set of candidate populations. Marker frequencies
of zero were replaced by 0.001 and stringency level for
assignment was set to log-likelihood difference > 2.
Results
cpDNA and ploidy-level estimates
Three distinct ploidy levels were observed when relative
nuclei fluorescence of Vaccinium uliginosum samples was
measured with hexaploid V. oxycoccos as internal standard
(representing unit value). The sample-standard ratios
(mean ± SD) were 0.37 ± 0.1, 0.54 ± 0.3 and 0.78 ± 0.3,
corresponding to diploids (53 plants), triploids (5 plants)
and tetraploids (55 plants), respectively (Fig. 1b, Appendix
I). Most plants from the Arctic and the southern
European mountains were diploid whereas most plants
from northern Europe were tetraploid. Hexaploids, previously reported from Beringia (Fig. 1b), were not observed
in the material we successfully analysed with flow
cytometry (but see below). 2C-values for diploids and
tetraploids were 1.22 ± 0.01 pg and 2.67 ± 0.02 pg (mean ±
SE), respectively.
Two new combined (two-region) cpDNA haplotypes
were discovered (Table S1, Supplementary material), both
in areas with several geographically restricted haplotypes
and high diversity (cf. Alsos et al. 2005); haplotype S in
Beringia (found in two Alaskan tetraploids) and haplotype
T in the southern European mountains (found in one diploid
from Bulgaria; Appendix I). In total, 18 most parsimonious
trees with length 198 were retrieved from the heuristic
searches. The strict consensus tree indicated the same three
lineages with unresolved relationships as found by Alsos
et al. (2005), but there was no longer support for a single
Beringian cpDNA lineage (Fig. S1, Supplementary material).
The network was similar to that of Alsos et al. (2005;
Fig. 1d).
The distinct geographical pattern in ploidy levels was
mainly consistent with that of the three cpDNA lineages
(Fig. 1b). In total, we obtained both ploidy level and
cpDNA haplotype for 118 plants from 112 populations,
including cpDNA haplotypes for five herbarium vouchers
of previously chromosome-counted material; one diploid,
three tetraploids, and one hexaploid (Appendix I). Most
cpDNA haplotypes were associated with a single ploidy
level (Fig. 1d; Table S1, Supplementary material). It was
not possible to obtain ploidy levels for plants representing all Beringian cpDNA haplotypes. Most of those
obtained were tetraploid (two diploids, four triploids and
12 tetraploids). The Beringian haplotype Q was found in
two diploids as well as in one tetraploid (all from Alaska).
The two Amphi-Atlantic cpDNA haplotypes exclusively
belonged to tetraploids (39 plants). The Arctic-Alpine
cpDNA haplotypes mainly belonged to diploids (50
diploids, one triploid, six tetraploids and one hexaploid;
the latter taken from previously counted herbarium
material). Except for one tetraploid from Chukotka,
which had the unique Arctic-Alpine cpDNA haplotype
N; the remaining polyploids in this lineage contained
typically ‘diploid’ haplotypes (Fig. 1d; Table S1, Supplementary material). Notably, all polyploids found in the
Arctic-Alpine cpDNA lineage were sampled in or near
contact zones with one of the mainly ‘tetraploid’ cpDNA
lineages.
© 2007 The Authors
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P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3907
ITS
Forty-three plants from 41 populations were successfully
sequenced (Appendix I). Direct sequencing of the PCR
products resulted in unambiguous ITS sequences from
only 10 of the 43 plants, and 1–24 polymorphic nucleotide
sites were observed in the remaining plants (Appendix 2).
Sequencing of 45 clones from the six selected plants
resulted in 11 haplotypes (Appendices I and II).
Initial parsimony analyses including the directly sequenced fragments and the haplotypes constructed from
the clones separated the sequences of V. uliginosum into
two main groups. The groups were separated by six
substitutions in ITS1 and 10 in ITS2, but due to the polymorphisms in the directly sequenced fragments, the
actual number of parsimony informative group specific
differences was well below 16 for most pairwise comparisons of individual plants belonging to different
groups (Appendix II). Four sequences (147-V2a, 108-V43a,
176-V137 and 169-V152) were excluded from further
analyses due to too few unambiguous characters (= 2
of the 16 group specific characters). Two sequences
(173-V47 and 164-V81; Appendix II) appeared to be
mosaics of sequences from the two main groups and were
excluded.
The final ITS matrix including the outgroup consisted of
64 sequences and 665 aligned positions, of which 157 were
variable (including autapomorphies) and 71 parsimony
informative. For V. uliginosum, 63 variable and 23 parsimony
informative positions were found. The parsimony analysis
resulted in 14 most parsimonious trees with length 176 (Fig. 2).
Vaccinium uliginosum was resolved as nonmonophyletic.
Sequences obtained by direct sequencing from 13 of the 14
polyploids and two plants with unknown ploidy level
were supported with jackknife frequency (JK) 66% as
more closely related to a group (JK 77%) consisting of V.
arctostaphylos L. (southeastern Europe to western Asia), V.
padifolium (Hochst. ex Staud; Madeira) and V. cylindraceum
Sm. (the Azores) than to the diploid plants of V. uliginosum
(Fig. 2). All diploids and the tetraploid 177-V139 were
supported as monophyletic (JK 90%) and was resolved
as the sister to the former in the strict consensus tree
(JK < 50%). However, the high number of additive polymorphisms observed in sequences produced by direct
sequencing of both diploids and polyploids indicated that
ITS copies belonging to each of the two main groups were
present in varying numbers in most plants (Appendix II).
Haplotypes 1 and 2 (consisting of clones recovered from
the diploids 147-V2a, 147-V3a and 31-V30, and the tetraploids 66-V8 and 77-V17) were recovered in the ‘diploid’
clade (JK 90%), whereas haplotypes 3 –11 (consisting of
clones from the diploid 147-V2a and the tetraploids 66-V8,
77-V17a and 79-V18) were recovered with the tetraploids
(Fig. 2).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Fig. 2 Strict consensus of the 14 most parsimonious trees found in
the analyses of the ITS region in Vaccinium uliginosum. Numbers
associated with nodes indicate maximum parsimony jackknife
frequencies. Population numbers refer to Appendix 1. Haplo1 to
11 are haplotypes recovered from one or more specimens by
cloning (Appendix 2). Note that although the analyses divided V.
uliginosum into two groups largely corresponding to diploids and
polyploids, cloning as well as direct sequencing revealed that most
individuals contained sequences from both groups (Appendix 2).
Triploids are indicated by +, and unknown ploidy level by *.
3908 P . B . E I D E S E N E T A L .
Table 1 Pairwise hierarchical analysis of molecular variance (amova) of 949 plants of Vaccinium uliginosum between the main groups
inferred by the structure analyses. Values are percentage of variation between groups/percentage of variation among populations within
groups. P-values for all levels: < 0.001; N, number of populations; D, average population diversity
Siberian group
Beringian/north Canadian group
East Canadian/Greenlandic group
North European group
South European group
Siberian
group
Beringian/
north Canadian
group
East Canadian/
Greenlandic
group
North
European
group
South
European
group
N
D ± SD
—/16.7
9.3/21.3
13.8/15.5
24.2/12.9
27.9/17.4
—
—/27.5
9.4/21.1
22.6/17.1
24.9/23.0
—
—
—/18.8
25.4/13.4
28.7/16.7
—
—
—
—/17.2
17.5/17.8
—
—
—
—
—/39.8
15
36
25
29
12
0.154 ± 0.051
0.148 ± 0.037
0.158 ± 0.027
0.142 ± 0.027
0.096 ± 0.028
AFLP
A total of 958 plants from the 131 populations were
successfully analysed for AFLPs, and 105 unambiguous,
nonlinked, polymorphic markers were scored. For all
analyses except structure and PCO, the small and
putatively relict populations from Svalbard (cf. Alsos et al.
2002) were corrected for possible clones, reducing the total
number of individuals to 949. The error rate was 2.3%.
structure analyses (Pritchard et al. 2000) of the whole
dataset resulted in five main groups (the correlation
coefficient between replicates for K = 5 was 0.999; Fig. S2,
Supplementary material), here named (1) the Siberian
group, (2) the Beringian/north Canadian group (3), the east
Canadian/Greenlandic group (4), the north European group
and (5) the south European group (including Caucasus;
Fig. 1a). Separate analyses of each main group indicated three
subgroups within each of them except the Siberian, where
no substructure was detected (Fig. S3, Supplementary
material). The Beringian/north Canadian group contained
one widespread subgroup, here named the Arctic, and two
restricted Beringian groups (east and west). The east Canadian/
Greenlandic group was divided into one southern, one
central, and one eastern subgroup. The north European
group was divided into one southern, one western and one
eastern subgroup, and the south European group into one
western, one eastern, and one Caucasian subgroup (Fig. 1a).
The five main genetic groups were thus geographically
distinct, but intermixing was pronounced in several areas,
especially along east Greenland (between three groups)
and in east Siberia (between two groups; see cross hatched
symbols in Fig. 1a). For further analyses based on the
structure groups, all plants from the mixed populations
were included in the group to which the majority of the
population was assigned.
The five main groups inferred from the structure
analyses were also reflected in the PCO plot but with considerable intermingling (Fig. 3). The first axis (11.6% of the
variation) indicated that the main split in the dataset was
between the European samples and the others. Axis 3
separated the Siberian group somewhat from the Beringian
group, and the Caucasian subgroup from the rest (4.2%;
not shown). The parsimony and neighbour-joining analyses
showed weak structure except for clustering all European
populations together (no supported main groups; not shown).
The pairwise amovas of the five main AFLP groups confirmed the pattern found in the tree analyses and the PCO
plot; most variation was explained between the European
samples and the rest (Table 1). The south European and
north European groups were nevertheless well differentiated, and the subgroups and populations within the south
European group were more differentiated than the rest
(Table S2, Supplementary material). In spite of intermingling,
the main groups as well as the subgroups were significantly
differentiated. In amova analyses with three hierarchical
levels, the highest level of among-group variation was
explained among the 13 structure subgroups (23.8%),
and somewhat less among the five main structure groups
(20.2%) and between the European groups and the rest
(18.6%; Table 2). Grouping the subgroups according to
ploidy levels or cpDNA lineage only explained 7.2% and
11.7% of the total variation, respectively.
The aflpop analyses reflected the same genetic pattern
as the former analyses, and the intermingled nature of the
dataset was reflected in rather low allocation success. In
the reallocation analyses of the subgroups, most plants
were assigned to their own subgroup, although up to one
third of the plants were not assigned (results not shown).
When assignment of a plant to its group of origin was not
allowed, the allocated plants were mainly assigned to
another subgroup within their main group, except that
most plants from the south European subgroups were
assigned to north European subgroups (Table 3). The plants
from the Siberian AFLP group, which had no subgroups,
were assigned to the intermixed eastern east Canadian/
Greenlandic AFLP subgroup, and vice versa.
The average intrapopulation diversities in unglaciated
vs. glaciated areas (at last glacial maximum, LGM) were
almost identical [0.145 ± 0.037 vs. 0.144 ± 0.039, respectively
(mean values ± SD) P = 0.834, Student t-test]. The
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3909
Fig. 3 Principal coordinate analysis (PCO)
based on Dice’s similarity among AFLP
multilocus phenotypes of 958 plants from
131 populations of Vaccinium uliginosum.
Upper plot: symbols refer to different
geographical areas. Lower plot: symbols
and colours refer to different main Structure
groups (cf. Fig. 1).
intrapopulation diversity was typically high in populations that appeared as mixed in the structure analyses
(Fig. 1c, Appendix I). The average population diversity
was significantly lower in the south European group than
in the other main groups (P < 0.001, Student t-test; Table 1).
The southern north European subgroup also contained little diversity (Table 3). At the subgroup level, the highest
diversity was found in the eastern north European subgroup and in eastern east Canadian/Greenlandic subgroup, of which the latter contained several mixed
populations (Fig. 1a).
In two thirds of the populations analysed for AFLPs,
ploidy level and cpDNA data were available for at least
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
one individual (Appendix I). The five main AFLP groups
were partly inconsistent with the grouping based on ploidy
level and cpDNA data (Fig. 1, Table 2). The Beringian/north
Canadian and east Canadian/Greenlandic AFLP groups
corresponded to a mixture of ploidy levels and cpDNA lineages, but this incongruence was unexpectedly resolved at
the subgroup level (Table 4). Notably, all AFLP subgroups
were exclusively either diploid or polyploid. Except for
one diploid Alaskan plant that had the Beringian cpDNA
haplotype Q, all diploid AFLP subgroups belonged to the
Arctic-Alpine cpDNA lineage, and all cpDNA haplotypes
of the Beringian cpDNA lineage were restricted to tri- or
tetraploids of the western and eastern Beringian AFLP
3910 P . B . E I D E S E N E T A L .
Table 2 Analyses of molecular variance (amova) based on 105 AFLP markers for 117 populations of Vaccinium uliginosum (14 populations
represented by single individuals were excluded). P-values for all levels: < 0.001
All populations
Two groups — based on structure indicated in PCO plot: Siberian-, Beringian/
north Canadian-, and east Canadian/Greenlandic group vs. north Europeanand south European group
Five groups — based on main structure results. Siberian-, Beringian/N Canadian-,
east Canadian/Greenlandic-, northern European and south European group
Thirteen groups — based on substructure within the main structure groups
Two groups — structure subgroups only containing diploids vs. subgroups only
containing triploids or tetraploids
Three groups — structure subgroups assigned to the three main cpDNA lineages
subgroups. Furthermore, the southern east Canadian/
Greenlandic AFLP subgroup and all the north European
AFLP subgroups were tetraploid and mainly belonged to
the Amphi-Atlantic cpDNA lineage. However, contrary to
what should be expected based on the cpDNA data, these
nuclear-based subgroups showed no close relationship
across the Atlantic (e.g. Fig. 3). It is also noteworthy that
although three tetraploid plants (one from north Norway
and two from Ural) contained the widespread ‘diploid’
Arctic-Alpine cpDNA haplotype C, they grouped with the
other tetraploids in the north European AFLP group.
Discussion
Our results clearly show the importance of exploring
different marker systems. The various datasets were not
fully congruent, and if interpreted separately, they would
have indicated quite different histories. However, when
comparing the datasets and taking into account the most
likely time level each dataset reflects, a more complete
picture emerges. Our ploidy level data were largely consistent
with the three cpDNA lineages, which probably diverged
before the major glaciations (Alsos et al. 2005). On the other
hand, our wide sampling of the nuclear genome by AFLP
analysis revealed a distinct structuring into five main
groups, which can be interpreted nicely in terms of
different main refugial areas during the glaciations (see
below). However, the AFLP data show several pronounced
inconsistencies with the cpDNA and ploidy level structuring
(Fig. 1). Surprisingly, the presumably older cpDNA and
Source of
variation
d.f.
Percentage
of variation
Among pop.
Within pop.
Among groups
Among pop.
Within pop.
Among groups
Among pop.
Within pop.
Among groups
Among pop.
Within pop.
Among groups
Among pop.
Within pop.
Among groups
Among pop.
Within pop.
116
818
1
115
818
4
112
818
12
104
818
1
115
118
2
114
818
34.7
65.3
18.6
22.4
59.1
20.2
17.4
62.4
23.8
12.4
63.9
7.2
29.8
63.0
11.7
26.1
62.2
ploidy level patterns were more consistent with the
nuclear substructuring (AFLP subgroups), which should
be expected to show the most recent differentiation within
the species (Table 4). As discussed further below, it seems
that later processes have partly confounded the signatures
of the early polyploid formation and divergence. These
include occasional new polyploidizations and chloroplast
captures, but most importantly, nuclear gene flow across
ploidy levels in the absence of cytoplasmic introgression has
probably occurred to a large extent in Vaccinium uliginosum
in connection with cycles of refugial isolation and expansion.
This pattern is surprising and differs from what has been
found for the majority of plant species studied so far.
Usually, maternally inherited organelles introgress readily
both across ploidy levels and into other species (e.g.
Rieseberg & Soltis 1991; Renno et al. 2001; Petit et al. 2002)
Interestingly, we found two ITS lineages corresponding
to diploids and tetraploids in the phylogenetic analysis.
However, both main sequence types apparently occurred
in most individual plants but with ploidy-biased homogenization. The ITS data probably reflect both old (paralogy,
predating the origin of V. uliginosum) and ongoing processes. Thus, the ITS data could not convey unambiguous
information on the phylogeographical history of V. uliginosum
and are therefore discussed separately below.
Polyploidizations shaped the early history
In addition to inferring the glacial history, we aimed to
investigate the polyploidization history in V. uliginosum.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Table 3 Proportion of assignments of individuals of Vaccinium uliginosum to geographical groups inferred by structure analyses of AFLP data using aflpop (Duchesne & Bernatchez
2002). Assignment of a plant to its group of origin was not allowed. Stringency level for assignment was set to log-likelihood difference > 2. D, average population diversity within groups
(14 populations represented by single individuals were excluded). Abbreviations of main group names: Beringian/northern Canadian, Ber/N Can; east Canadian/Greenlandic, E Can/
Grl; north European, N Eur; south European, S Eur
Siberian
Ber/N Ber/N
Can
Can
(Arctic) (Ber W)
E Can/
Grl
(South)
E Can/
Grl
(East)
E Can/
Grl
N Eur
(Central) (West)
N Eur
(South)
N Eur
(East)
S Eur
(Caucasus)
S Eur
(East)
S Eur
(West)
Not
assigned
No. of
plants
D ± SD
—
—
0.03
0.12
0.02
0.31
—
—
—
—
—
—
—
0.02
—
0.10
0.03
—
—
—
—
—
0.01
—
—
—
0.02
0.77
—
0.16
0.03
—
0.05
—
—
—
0.18
—
—
—
—
—
0.01
—
0.09
0.29
—
—
—
—
—
—
0.34
—
0.01
—
—
—
0.03
—
—
—
—
—
—
—
—
0.04
—
0.52
0.04
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0.01
—
—
0.27
—
—
0.04
—
—
—
—
—
—
0.71
—
—
—
0.13
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0.21
—
—
—
—
—
—
—
—
—
—
—
0.08
—
0.58
0.18
0.76
0.67
0.42
0.56
0.63
0.29
0.88
0.25
0.55
0.79
0.61
126
39
113
73
60
75
76
168
43
89
11
48
28
0.154 ± 0.051
0.155 ± 0.063
0.143 ± 0.032
0.154 ± 0.021
0.160 ± 0.015
0.167 ± 0.040
0.150 ± 0.021
0.140 ± 0.018
0.100 ± 0.013
0.166 ± 0.021
0.055*
0.100 ± 0.034
0.101 ± 0.016
Assigned from
Siberian
Ber/N Can (Ber E)
Ber/N Can (Arctic)
Ber/N Can (Ber W)
E Can/Grl (South)
E Can/Grl (East)
E Can/Grl (Central)
N Eur (West)
N Eur (South)
N Eur (East)
S Eur (Caucasus)
S Eur (East)
S Eur (West)
*Only one population.
——
0.05
0.07
—
0.02
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0.12
0.74
—
—
0.18
P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3911
Assigned to
Ber/N
Can
(Ber E)
3912 P . B . E I D E S E N E T A L .
Table 4 Distribution of the cpDNA haplotypes and ploidy levels in Vaccinium uliginosum among the groups inferred from structure
analyses of AFLP data. N, number of individuals analysed for AFLPs, cpDNA and ploidy level variation. The southern north European
subgroup is not represented as none of the individuals screened for cpDNA and ploidy level variation belonged to this subgroup
cpDNA lineages
AmphiAtlantic
Arctic-Alpine
AFLP groups
K
A
N Eur (West)
N Eur (East)
E Can/Grl (South)
E Can/Grl (East)
E Can/Grl (Central)
S Eur (Caucasus)
S Eur (West)
S Eur (East)
Siberian
Ber/N Can (Arctic)
Ber/N Can (Ber W)
Ber/N Can (Ber E)
0.91
0.82
0.20
I
C*
Beringian
D
E
F
G
T
R
J*
Ploidy levels
Q*
S
2x
3x
0.09
0.18
0.80
0.33
0.50
0.33
0.33
0.33
0.17
0.25
0.33
0.81
0.69
0.33
0.19
0.08
0.15
0.40
0.80
0.20
N
1.00
1.00
1.00
11
11
5
3
6
1
4
3
7
13
5
5
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.75
0.33
4x
0.08
0.20
0.40
0.40
0.60
1.00
*Haplotype C and Q were found in both diploids and tetraploids, while haplotype J was found in both triploids and tetraploids.
N Eur, north European; E Can/Grl, east Canadian/Greenlandic; Ber/N Can, Beringian/north Canadian.
The three cpDNA lineages were largely consistent with
our ploidy level data (Fig. 1 and Table S1, Supplementary
material). This finding suggests that the initial cpDNA
divergence followed early polyploidizations, resulting in
one exclusively tetraploid boreal cpDNA lineage (the
Amphi-Atlantic) and one mainly tetraploid boreal lineage
(the Beringian), and leaving one mainly diploid, fully
circumpolar lineage (the Arctic-Alpine). At least for the
exclusively tetraploid Amphi-Atlantic cpDNA lineage, the
polyploidization event probably predates the divergence
of the main cpDNA lineages (0.7–3.0 Ma; Alsos et al. 2005).
Based on the AFLP data, it is tempting to localize this
polyploidization event to southern Europe. The exclusively
tetraploid north European AFLP group (dominated by
the Amphi-Atlantic cpDNA lineage) was most similar to
the exclusively diploid south European AFLP group
(belonging to the Arctic-Alpine cpDNA lineage). A southern
European origin of the Amphi-Atlantic cpDNA lineage is
consistent with morphology as the southern Alpine populations are intermediate between the northern European
and the arctic populations (Alsos 2003). However, as the
main AFLP groups seem to reflect a shallower pattern, the
similarity observed between tetraploids and diploids in
Europe at AFLP loci can alternatively be explained by later
nuclear interploidal gene flow (further discussed below),
as tetraploids and diploids in Europe may have experienced
periods of sympatry during the Plestocene glaciations.
Recent processes confound older patterns: new
polyploidizations, chloroplast capture, refugial isolation
and nuclear gene flow
Old polyploidization events have clearly initiated the
divergence within this species complex. However, later
independent polyploidization events have probably also
occurred in V. uliginosum, for instance leading to the
occasional occurrence of hexaploids. In addition, as also
suggested by Brochmann et al. (2004), the tetraploids with
typical ‘diploid’ cpDNA haplotypes may have originated
via independent polyploidization events. Alternatively,
they may have resulted from chloroplast capture. Among
the plants investigated for AFLPs, we observed four
tetraploids with cpDNA haplotypes otherwise associated
with diploids (haplotype C and Q; Table 4, Table S1 in
Supplementary material, Appendix I). These tetraploids
were not differentiated at AFLP loci from the other tetraploids in the same geographical area, and they occurred in
or near areas where diploids and tetraploids meet (Beringia
and northeast Europe). Thus, it is unlikely that these ‘aberrant’
tetraploids originated via independent polyploidizations;
rather, hybridization between diploids and tetraploids was
accompanied by chloroplast capture. The local occurrences
of triploids indicate that heteroploid hybridization occurs
in V. uliginosum (Fig. 1b), and chloroplast capture is common
between closely related species and within species
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3913
complexes (Rieseberg & Soltis 1991; Petit et al. 2002;
Palme et al. 2004). Still, independent polyploidizations and
chloroplast captures have probably not caused the major
discrepancies between our datasets (AFLPs vs. cpDNA
and ploidy level variation; see Fig. 1).
We rather suggest that much of the discrepancies can be
explained by major episodes of interploidal nuclear gene
flow. Polyploidy is traditionally considered as a way to
instantly cut off gene flow because of the chromosome
number difference between the progenitor diploid(s) and
the polyploid derivative. However, there is increasing
evidence that gene flow can continue to some degree across
a ploidy-level barrier (Brochmann et al. 2004; see Brochmann
et al. 1992 for an early example), rendering polyploidy
as way to decrease, rather than eliminate, gene flow. In
Vaccinium, unreduced gametes can result in unilateral
introgression from diploids to tetraploids (Lyrene et al. 2003).
Stress — temperature stress in particular — increases the production of unreduced pollen (Ramsey & Schemske 1998;
Levin 2000), which are quite frequent in Vaccinium (Ortiz
et al. 1992a, b). Thus, introgression from diploids to tetraploids in V. uliginosum may rather be mediated through
unreduced pollen than unreduced female gametes, in that
way keeping the cpDNA lineages associated with ploidy
level. The main AFLP groups are in conspicuous agreement with previously documented glacial refugia (cf.
Hewitt 2004): The Beringian/north Canadian group, a Beringian refugium; the Siberian group, a Siberian refugium
west of Beringia; the east Canadian/Greenlandic group, a
refugium southeast of the Laurentide ice sheet; the north
European group, a refugium south and/or east of the Scandinavian ice sheet; and the south European group, a collection
of peripheral refugia in the southern European mountains
and the Caucasus. The main contemporary AFLP structure
observed in V. uliginosum may thus reflect that different
lineages of diploids and tetraploids have shared recent
refugia and been connected via unilateral interploidal
nuclear gene flow. Because of this complex situation we
further consider the late Pleistocene history of the species
in relation to the subgroup structuring and ploidy levels.
Phylogeography of tetraploid AFLP subgroups
For the tetraploid AFLP subgroups, the cpDNA data are
consistent with one Beringian refugium and at least two
main Atlantic refugia, southeast of the Laurentide ice
sheet and south, and possibly east, of the Scandinavian ice
sheet. Whereas the tetraploids in Beringia show limited
expansion after the last glaciation, the Amphi-Atlantic
tetraploids expanded extensively northwards through the
previously glaciated areas on each side of the Atlantic
Ocean.
In the north European AFLP group, which was exclusively tetraploid and dominated by the Amphi-Atlantic
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
cpDNA lineage, we found a weak east–west substructure
and a somewhat more differentiated southern subgroup in
the Alps. This pattern may indicate several more or less
connected glacial refugia, in combination with further differentiation during colonization where the edge populations may have expanded southwards to higher altitudes,
and northwards on each side of the retracting ice. The low
cpDNA haplotype diversity of the tetraploid AmphiAtlantic lineage was explained by heavy bottlenecking
due to small glacial refugia by Alsos et al. (2005); however,
this lineage was not depauperate at AFLP loci. The higher
nuclear variation may have resulted from introgression
from diploids or larger effective population size (four
times higher in a nuclear tetraploid genome than a haploid
cpDNA genome).
Why the AFLP data do not reflect the Amphi-Atlantic
connection among the tetraploids seen in the cpDNA data
is probably a result of several factors. Firstly, bottlenecks
alone can create discordance between cytoplasmic and
nDNA (Fay & Wu 1999). Secondly, as discussed above,
occurrence of unilateral nuclear introgression from diploids to tetraploids may be frequent. In addition, during
colonization, pollen competition may further enhance unilateral and sex-biased hybridization; it has been hypothesized that pollen competition causes female flowers of
the less abundant species to hybridize disproportionately
(Rieseberg et al. 1996). Our haplotype network suggests
that the American cpDNA haplotype (I) was derived from
the European haplotype (K; Fig. 1d; Alsos et al. 2005). The
observed AFLP pattern can thus be explained by a strong
founder effect during initial westward colonization of
European tetraploids across the Atlantic in an earlier interglacial, followed by extensive heteroploid hybridization
through unreduced pollen from the American diploids
during colonization and/or during periods of increased
sympatry, e.g. in the rather restricted refugium southeast
of the Laurentide ice.
Phylogeography of diploid AFLP subgroups
Three refugia can be inferred for the arctic diploid AFLP
subgroups: Siberia, Beringia and southeast of the Laurentide
ice sheet. The arctic diploids apparently expanded east- and
westwards from Beringia, meeting the Siberian expanding
population in the Lena area, and the populations expanding
from the south through Canada and Greenland in central
Canada and northern Greenland. The occurrence of intermixed populations and the enhanced levels of diversity
indicate contact zones in these areas. The arctic diploids
expanding from Siberia did not proceed into Europe,
which was occupied by tetraploids expanding from the
south, but they dispersed to the arctic archipelago of
Svalbard and to northeast Greenland, where all three arctic
diploid groups meet.
3914 P . B . E I D E S E N E T A L .
We observed high AFLP diversity in arctic areas, and no
significant difference in diversity within populations from
glaciated vs. unglaciated areas, as also observed in the
cpDNA data (Alsos et al. 2005). In addition, the different
AFLP groups were rather intermingled. Thus, as concluded
on the basis of cpDNA data, there has probably been broadfronted colonization of arctic diploids from different
refugia. In contrast to what was inferred for the Beringian
tetraploids, Beringia appears to have been important as a
source for recolonization of arctic diploids after the last
glaciation. This may be explained ecologically; the arctic
diploids (ssp. microphyllum) are better adapted to the continental arctic environment than the Beringian tetraploids,
which appear to be restricted to more oceanic climates
(Alsos et al. 2005).
The diploids in the south European mountains formed
a distinct main AFLP group which also contained several
private cpDNA haplotypes and high haplotype diversity
(Alsos et al. 2005). They were also somewhat differentiated
from the arctic diploids in morphology (Alsos 2003),
suggesting isolation from the arctic diploids during
more than one glaciation. The south European AFLP
subgroups as well as the individual populations were
highly differentiated but contained low levels of diversity.
Thus, these populations probably survived the last glaciation in several smaller peripheral refugia surrounding the
alpine areas. Several studies have shown highly structured
genetic patterns in the southern Eurasian mountains,
suggesting several small refugia (Kropf et al. 2003;
Schönswetter et al. 2003; Stehlik 2003; Tribsch &
Schönswetter 2003; Schönswetter et al. 2005; Skrede et al.
2006). The alpine diploids were probably also blocked by
the expanding tetraploids, and did not expand northwards in Europe.
‘The ways of ITS are inscrutable’
Although the two main groups of ITS sequences resolved
in V. uliginosum largely corresponded to different ploidy
levels, the polymorphisms observed by direct sequencing
(Appendix II) show that both sequence types were present
in the majority of the diploid as well as the polyploid
plants, thus representing two paralogues. This was further
demonstrated by recovering sequences belonging to both
groups among the clones from individual plants (Fig. 2;
Appendix II). As a consequence, unless all ITS PCR products
are cloned, any phylogenetic signal useful for inferring
relatively recent phylogeographical patterns is effectively
concealed by the polymorphisms caused by the two
paralogous ITS repeats.
We therefore interpret the apparently deep split between
diploid and polyploid V. uliginosum in the ITS dataset to
result from directional bias in the homogenization process
of the ITS repeats. Such bias has been reported in a large
and increasing number of polyploids (Wendel et al. 1995;
Brochmann et al. 1996; Popp & Oxelman 2001; Kovarik
et al. 2005; Popp et al. 2005; Guggisberg et al. 2006). In V.
uliginosum, however, we have demonstrated strong ploidy
level correlated bidirectional homogenization bias of heterogenous ITS repeats found in both diploid and polyploid
individuals. Several scenarios may explain the origin of
such heterogeneity of intraindividual ITS repeats, including pseudogenization of some of the repeats, homoploid
hybridization between two lineages with different ITS
repeats, an ancient allopolyploidization (rendering today’s
diploid lineages palaeo-polyploids), incomplete lineage
sorting in a polymorphic ancestor followed by one or more
recent polyploidizations in the V. uliginosum lineage, and/or
occurrence of ITS repeats at two or more chromosomal
positions in each haploid genome. The almost complete lack
of variation in the 5.8S gene (haplo5 and haplo6 had a T
instead of a G in position 308, and population 135 ha an S
instead of a T; Appendix II) indicate that none of the sequences
represent a pseudogene. Furthermore, it is noteworthy
that although a close relationship between the polyploid V.
uliginosum group and the group consisting of V. arctostaphylos,
V. cylindraceum and V. padifolium could be demonstrated
by the ITS analysis, the same (or highly similar) ITS paralogues are found in a varying degree in most of the diploid
V. uliginosum (Appendix II). This may indicate that an
ancestor of the V. arctostaphylos, V. cylindraceum and V.
padifolium group was involved in a hybridization event,
or that the ITS lineages predate the lineage splits and that
concerted evolution has reached more or less completion
in these three species. To discriminate between these
hypotheses, one or more additional biparentally inherited
markers are needed.
In addition to the ploidy level correlated bidirectional
bias in the ITS dataset, we detected large individual
differences in the degree of homogenization, based on
the number of polymorphic nucleotide positions in the
sequences produced by direct sequencing of PCR products (Appendix II). For example, the tetraploid 169-V152
and the diploid 176-V137 showed very low levels of
homogenization, and the triploid 108-V43a appeared to be
unaffected by homogenization, thus completely contrasting the patterns seen in the diploid 28-V31b, the triploid
108-V43e and the tetraploid 163-V150 who all seemed to
be completely homogenized. Similar intraspecific variation in the degree of homogenization of ITS repeats has
been reported in taxa of hybrid origin in Tragopogon
(Kovarik et al. 2005). Although quantitative differences in
ITS copy number due to concerted evolution would result
in the observed pattern, PCR bias (Wagner et al. 1994)
cannot be ruled out as an explanation of some of the
individual differences in the level of inferred ITS homogenization. Further studies are needed to address the
nature of this pattern.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3915
Conclusion
The various datasets clearly reflect genetic differentiation
at different time levels, and that different processes have
contributed to this structuring. The ITS data could not
be used to infer phylogeographical history in Vaccinium
uliginosum but revealed an exceptional and interesting
pattern of strong ploidy level-correlated bidirectional homogenization bias of heterogenous ITS repeats found in both
diploid and polyploid individuals. The cpDNA data showed
strong congruence with ploidy levels, suggesting that the
tetraploid cpDNA lineages initially diverged because of a
few, very successful polyploidizations. Signals of the early
history of the species were preserved in the subgroup
structure of the AFLP dataset; the identified subgroups
were largely congruent with groupings based on ploidy
level and cpDNA. Surprisingly, the main structuring of the
AFLP data rather seem to reflect the most recent history of
the species and show several pronounced inconsistencies
with the cpDNA and ploidy level structuring. We suggest
that much of the discrepancies between our datasets can be
explained by major episodes of unidirectional nuclear gene
flow in shared refugia; from some diploid groups to some
tetraploid groups via unreduced pollen, which prevents
cytoplasmic introgression. This study provides a clear
example of a species for which both nuclear and plastid
data are needed to decipher intraspecific history. It also
highlights the importance of obtaining extensive data on
ploidy levels, to an extent that can only be achieved via
flow cytometric techniques, in systems where polyploidy
serves as an important evolutionary driver.
Acknowledgements
We thank all collectors (cf. Appendix 1) for providing plant material
for our study, Tor Carlsen and Reidar Elven for discussions and
advice and Siri Kjølner for technical assistance. We also thank the
reviewers for constructive criticism. Part of the material was obtained
via the Tundra Northwest (TNW) 1999 expedition funded by the Polar
Research Secretariat at the Royal Swedish Academy of Sciences.
The study was funded by grants 150322/720 and 146515/420 to
Christian Brochmann from the Research Council of Norway. Flow
cytometric analyses were supported by the Ministry of Education,
Youth and Sport of the Czech Republic (MSM 0021620828), and
the Academy of Sciences of the Czech Republic (AV0Z60050516).
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Pernille Bronken Eidesen is a PhD student at the National Centre
for Biosystematics (NCB) and works with comparative phylogeography as part of a larger project. She is supervised by Professor
Christian Brochmann, who is particularly interested in the
phylogeography and evolutionary history of arctic-alpine plants.
Inger Alsos worked as a postdoc on the main project and is
currently associate professor at the University Centre in Svalbard.
Magnus Popp has a postdoc position at the NCB. Øyvind
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Supplementary material
The following supplementary material is available for this article:
Fig. S1 Phylogenetic relationships among the 20 cpDNA haplotypes in Vaccinium uliginosum (strict consensus based on 18
equally most parsimonious trees). Numbers above branches indicate jackknife support values. Haplotypes associated with other
ploidy levels than the main ploidy level of the lineage are indicated in parentheses (* denote unknown ploidy level).
Fig. S2 Summary of structure analyses based on AFLP multilocus
phenotypes of 958 plants of Vaccinium uliginosum. Number of
clusters K was varied from 2 to 20 in 10 independent runs. (a)
Mean ln probability of the data for each K. Error bars indicate
standard deviation. (b) ∆K calculated according to Evanno et al.
(2005).
Fig. S3 Summary of separate structure analyses of each of the
five main Structure groups found in Vaccinium uliginosum. (A) The
3918 P . B . E I D E S E N E T A L .
north European group (B) the Beringian/north Canadian group
(C) the Siberian group (D) the east Canadian/Greenlandic group,
and (E) the south European group. Three subgroups were chosen
for group A, B, D and E (the fourth group indicated in E only
contained a couple of individuals). No subgroups were inferred
for group C, as the runs for K = 2 were not correlated and K > 4
contained empty groups. Number of clusters K was varied from 1
to 10 in 10 independent runs. Upper graph: Mean ln probability
of the data for each K. Error bars indicate standard deviation (SD).
Lower graph: ∆K calculated according to Evanno et al. (2005).
Table S2 Pairwise hierarchical analysis of molecular variance
(amova) of 949 plants of Vaccinium uliginosum between subgroups within the main groups inferred by structure analyses,
presented as percentage of variation between groups/percentage
of variation among populations within groups. P values for all
levels: < 0.001, except for the values obtained in comparisons with the
south European (Caucasus) subgroup, as this group only contained
one population (marked *). Abbreviations of main group names:
Beringian/north Canadian — Ber/N Can; east Canadian/Greenlandic
— E Can/Grl; north European — N Eur; south European—S Eur.
Table S1 Variable sites recorded in the trnL-trnF and trnS-trnG
cpDNA spacer region in Vaccinium uliginosum. Single-region and
combined (two-region) haplotypes are shown, and their relationships are indicated. Number of plants with known ploidy level
that also could be associated with a combined haplotype is listed.
Nucleotide positions refer to the number of bases from the first
position of the region. Outgroups and combined haplotypes A to
R are from Alsos et al. (2005).
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/abs/
10.1111/j.1365-294X.2007.03425.x
(This link will take you to the article abstract).
Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by
the authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Appendix I
Collection data and genetic diversity for populations of Vaccinium uliginosum analysed in this study. Letter after slash in population ID indicates type of material (f, fresh; s, silica-dried;
h, herbarium) and the number of the particular individual used for sequencing are given in parenthesis; Pl, Ploidy level (*, † and ‡ denote that chromosome numbers are found in the
literature: *Young (1965), ‡S. Vander Kloet (unpubublished) and ‡Zhukova (1966)); H, Combined (two-region) cpDNA haplotype (haplotypes assigned based on a single region and
somewhat ambiguous sequences are denoted as e.g. I′); h1/h2, trnL-trnF haplotype/trnS-trnG haplotype (Table S1, Supplementary material); I, Analysed for ITS (‘s’ successfully, ‘f’ failed);
A, number of plants successfully analysed for AFLPs; D, Intrapopulation diversity based on 105 AFLP markers. All sequences are deposited in GeneBank, accession nos cpDNA:
DQ073105-DQ073326 (population ID starting with ‘V’; Alsos et al. 2005) and EF502102-EF502167 (this study), accession nos ITS: AM491831-AM491884
Population:
§ID
Locality (longitude, latitude)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
AK3058/s
EE1-Vga/s
AK3051/s (4)
AK3233/s
SUP03-172/s
AK3238/s
SUP03-156/s
SUP03-359/s (1)
V147/s
V48/f (a)
V138/f
AK3201/s (1)
V52/s (a)
V50/s (a)
AK1141/s
V148/s
V49/s (a)
AK3004/s
SUP03-365/s
V149/s
AK3177/s (1)
V51/s (a)
AK3237/s
AK1268/s
V37/s (a)
V36/s (a)
V35/s (a)
AK808/s
V66/s (a)
V31/s (b)
V30/s (a)
V32/s (a)
AK4804/s (1)
Austria, Kärnten, Hohe Tauern, Goldberggr., Sadniggr. (46.952, 13.034)
Austria, Steiermark, Rax (47.697, 15.71)
Bulgaria, Vitosha Mt., beneath Goljam Rezen peak (42.5, 23.3)
Canada, BC, Atlin, Ruby Mtn. (59.703, –133.36)
Canada, BC, Cassiar Mts., Gnat Pass Summit on Cassiar hwy (58.281, –129.864)
Canada, BC, High Tuya (59.248, –130.522)
Canada, BC, Prince Rupert vic., Kaien I., Oliver Lake (54.279, –130.271)
Canada, BC, Skeena R. valley, Shames Mtn. ski area (54.484, –128.954)
Canada, Manitoba, Churchill, Northern Study Centre (58.733, –93.817)
Canada, Newfoundland, Bay du Nord Wilderness (Cult.) (48, –55)
Canada, Newfoundland, Holyrood, Hawke Hills (47.383, –53.133)
Canada, Newfoundland, Mt. Gros Morne (49.596, –57.791)
Canada, Nunavut, Ellesmere I., Grise Fiord (76.45, –82.667)
Canada, Nunavut, Melville I. (75, –110)
Canada, Nunavut, Pond Inlet (72.7, –77.967)
Canada, Nunavut, Rankin Inlet (62.8, –92.1)
Canada, Nunavut, Victoria I., Wollaston Pen. (69.333, –114.833)
Canada, NWT, Inuvik (68.317, –133.483)
Canada, NWT, Mackenzie Distr., Dempster hwy 174 km (67.721, –133.88)
Canada, NWT, Mackenzie R. Delta, Tuktoyaktut (69.433, –133.017)
Canada, Québec, Gaspésie, Mt. Jacques-Cartier (48.999, –65.943)
Canada, Québec, Ungava Pen. (62.367, –73.75)
Canada, Yukon Territory, Top of the world Hwy (64.194, –140.361)
Canada, Yukon, Dempster hwy, Tombstone Campsite (68.505, –138.221)
Finland, Kainuu, Kajaani, Koivukylä (64.017, 27.45)
Finland, Kainuu, Suomussalmi (64.95, 29.333)
Finland, Uusimaa, Sipoo, Pohjois-Paippinene (60.45, 25.2)
France, Isère, 21 km E of Grenoble, Lac de Crop (45.208, 5.975)
France, Isère, Belledonne, Chamrousse (45.117, 5.9)
France, Isère, Belledonne, Grandes Rousses (45.083, 6.067)
France, Provence-Alpes, Hautes-Alpes, Col du Lautaret (45.067, 6.383)
France, Pyrénées, Gabás, Vallée d’Ossau (42.833, –0.517)
Georgia, Borjomi region, Tskhratskaro pass (42.683, 43.508)
Pl
H
h1/h2
2x
T
ab/q
4x
4x
4x
4x†
4x
2x
2x
R′
I′
I
E′
I
G
E
g′/u′
c/—
c/l
—/p
c/l
b/r
b/p
2x
2x
G′
C
—/r
a/q
2x
2x
2x
C
E
E
a/q
b/p
b/p
4x
4x
4x
K
K
K
e/l
e/l
e/l
2x
2x
2x
2x
2x
A
D
A
A
C
a/n
b/n
a/n
a/n
a/q
I
s
s
s
s
s
s
s
s
s
s
A
D
11
11
6
11
5
11
5
5
2
0.083
0.113
0.162
0.159
0.124
0.172
0.114
0.046
0.124
11
5
5
11
5
5
11
5
5
11
4
11
11
1
0.152
0.13
0.08
0.159
0.179
0.118
0.152
0.131
0.143
0.15
0.076
0.148
0.131
1
10
4
5
5
0.124
0.097
0.107
0.097
11
0.055
P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3919
no.
Population:
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
no.
§ID
Locality (longitude, latitude)
Pl
H
h1/h2
I
A
D
34
35
36
37
38
39
40
41
42
43
44
45
46
V60/s (a)
V62/s (a)
V61/s (a)
AK113/s (1)
AK3027/s (1)
AK3002/s
AK1077/s
AK1116/s
V56/s (a)
V57/s (a)
V58/s (a)
V59/s (a)
V63/s (a)
V63/s (b)
AK368/s
AK348/s
AK202/s (1)
V64/s (a)
AK1078/s
AK279/s (1)
AK270/s
AK230/s
AK244/s (1)
V53/s (a)
AK1249/s
V54/s (a)
V55/s (a)
AK121/s
V65/s (a)
V4/s (a)
V11/s (a)
AK1152/s
AK858/s
V8/s (a)
AK888/s
V9/s (a)
V7/s (a)
AK853/s
V1/s (a)
V6/s (a)
AK852/s
Greenland, Anmagssalik, Kulusuk (65.567, –37.167)
Greenland, Anmagssalik, Kummiut (65.85, –36.983)
Greenland, Anmagssalik, Tasilak (65.617, –37.667)
Greenland, Ardencaple fjord (75, –20)
Greenland, Blosseville Kyst, Cape Dalton (69.456, –24.1)
Greenland, Clavering I., Eskimonaes (74.1, –21.275)
Greenland, Dove Bugt, Trekronerfjeldet (76.994, –20.15)
Greenland, Egedesminde (68.717, –52.867)
Greenland, Inglefield Ld, Firfinger Sø (78.983, –67.167)
Greenland, Inglefield Ld, Hiawatha (78.833, –67.3)
Greenland, Inglefield Ld, Marshall Bugt (78.833, –68.833)
Greenland, Inglefield Ld, Spejlsø (79.067, –66.4)
Greenland, Jameson Ld, Constable Point (70.733, –22.683)
Greenland, Jameson Ld, Constable Point (70.733, –22.683)
Greenland, Jameson Ld, Constable Point, Hareelven (70.712, –22.655)
Greenland, Jameson Ld, Constable Point, Primulaelven (70.747, –22.655)
Greenland, Kangerlussuaq (67, –51)
Greenland, Liverpool Ld, Scoresbysund (70.483, –21.933)
Greenland, Mestersvig (72.243, –23.898)
Greenland, Nanortalik (60.146, –45.227)
Greenland, Narsarsuaq, Kuusaqdalen, Blomsterdalen (61.202, –45.324)
Greenland, Nuuk, E of Nuussuaq (64.194, –51.699)
Greenland, Paamiut (62.004, –49.619)
Greenland, Qeqertarsuaq (Disko), Fortune Bay (69.25, –53.75)
Greenland, Qeqertarsuaq (Disko), near Arktisk Stasjon (69.25, –53.567)
Greenland, Upernavik, Langø (72.767, –56.083)
Greenland, Upernavik, Upernavik Ø (72.783, –56.117)
Greenland, Zackenberg (74.467, –20.55)
Greenland, Zackenberg (74.467, –20.55)
Iceland, Austurland, Egilsta3ir (65.35, –14.5)
Iceland, Austurland, Rey3arfjör3ur (65.017, –14.067)
Iceland, Austurland, Vopnafjör3ur (65.75, –14.83)
Iceland, Nor3urland Eystra, Akureyri, E of Eyjafjör3ur (65.69, –18.034)
Iceland, Nor3urland Eystra, Myvatn (65.567, –16.95)
Iceland, Nor3urland Eystra, Myvatn, near Hverfjall (65.611, –16.917)
Iceland, Nor3urland Eystra, Vesturhei3i (65.983, –17.867)
Iceland, Nor3urland Vestra, Kjölur (64.933, –19.5)
Iceland, Vestfir3i, Drangsnes/Holmavik, W of Laugarholl (65.766, –21.636)
Iceland, Vesturland, Akranes, Akrafjall (64.35, –21.9)
Iceland, Vesturland, Holtavör3uhei3i (64.867, –21.2)
Iceland, Vesturland, S of Hvalfjör3ur (64.353, –21.643)
2x
4x
2x
2x
2x
G
K
F
F
E
b/r
e/l
b/q
b/q
b/p
s
s
2x
2x
2x
2x
2x
3x
G
C
C
C
G
G
b/r
a/q
a/q
a/q
b/r
b/r
s
5
5
5
9
11
11
10
8
5
5
5
5
1
0.152
0.17
0.135
0.165
0.16
0.188
0.203
0.155
0.139
0.13
0.09
0.141
11
10
11
0.178
0.146
0.118
9
11
11
11
11
3
11
5
4
10
0.177
0.181
0.159
0.137
0.163
0.178
0.169
0.175
0.165
0.201
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
2x
2x
G
—/q′
b/r
4x
I′
—/l
4x
2x
I
F
c/l
b/q
2x
2x
C
C
a/q
a/q
2x
4x
4x
G
K
K
b/r
e/l
e/l
4x
K
e/l
4x
4x
K
K
e/l
e/l
4x
4x
K
K
e/l
e/l
s
s
s
s
s
1
1
11
11
1
8
1
1
11
1
1
11
0.146
0.148
0.136
0.161
0.142
3920 P . B . E I D E S E N E T A L .
Appendix I Continued
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Appendix I Continued
Population:
§ID
Locality (longitude, latitude)
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
ST-01–10/s
AK422/s
V71/h
V17/s (a)
AK706/s (1)
V18/s (a)
AK1347/s
V26/s (a)
AK414/s
AK515/s
AK495/s
AK750/s (1)
V14/s (a)
V23/s (a)
V21/s (a)
V24/s (a)
V22/s (a)
AK493/s
V25/s (a)
V27/s (a)
AK436/s
AK467/s
V13/s (a)
AK749/s
V15/s (a)
V16/s (a)
V20/s (a)
V19/s (a)
AK753 + V12/s (V12a)
AK3139/s (1)
AK873/s
AK4200/s
AK3081/s (1)
V98/h
V43/s (a)
V43/s (e)
01–05/s
09–04/s
V115/h
V44/s (a)
Italy, Piemonte, Torino, Cottische Alpen, around Pian di Re (44.7, 7.1)
Norway, Buskerud, Ål, N of Helsingset, Nystølane (60.738, 8.617)
Norway, Finnmark, Alta, Mattisdalen (69.85, 22.85)
Norway, Finnmark, Nordkapp, Duken (71.033, 25.8)
Norway, Finnmark, Nordkapp, Duken (71.075, 25.802)
Norway, Finnmark, Nordkapp, Dår’kacåk’ka (70.833, 25.783)
Norway, Finnmark, Vadsø, Ekkerøy (70.071, 30.111)
Norway, Hedmark, Folldal, Råtåsjøhøi (62.267, 9.8)
Norway, Hedmark, Varlisetra (61.353, 12.052)
Norway, Hordaland, Finse, towards Blåisen (60.587, 7.499)
Norway, Hordaland, Odda, Røldal, Røldalsfjell (59.835, 6.735)
Norway, Nordland, Andøy, Røyken (69.28, 16.007)
Norway, Nordland, Lofoten, Hadsel, Grunnførfjorden (68.417, 14.533)
Norway, Nordland, Vesterålen, Andøy, Måtind (69.25, 15.883)
Norway, Nordland, Vesterålen, Bø, Bufjellet (68.767, 14.467)
Norway, Nordland, Vesterålen, Hadsel, Storheia (68.533, 14.867)
Norway, Nordland, Vesterålen, Sortland, Holand (68.633, 15.25)
Norway, Nord-Trøndelag, Leka, Årdalsstrand (65.478, 12.219)
Norway, Oppland, Dovre, Geitryggen (62.2, 9.483)
Norway, Oppland, Dovre, Gråsida, Verkenssætri (62.05, 9.433)
Norway, Oppland, Lom, Bøverdalen, Borgasetra (61.682, 8.148)
Norway, Sør-Trøndlag, Oppdal, Vårstigen (62.343, 9.624)
Norway, Troms, Kvænangen, Kvænangsfjellet (69.9, 21.567)
Norway, Troms, Kåfjord, Kåfjordfjellet (69.401, 21.036)
Norway, Troms, Målselv, Skakteråsen (68.767, 19.667)
Norway, Troms, Målselv, Skakteråsen (68.767, 19.667)
Norway, Troms, Storfjord, Adjit (69.35, 20.367)
Norway, Troms, Storfjord, Adjit (69.367, 20.383)
Norway, Troms, Tromsø, Fløya (69.6, 19.017)
Poland, Krakow, Tatra Mts., Mt. Ornak (49.22, 19.833)
Romania, Gropile Pick, Rodnei Mts. (47.587, 24.637)
Romania, Southern Carpathians, Muntii Retezat, Lac Bucura (45.358, 22.877)
Romania, Southern Carpathians, Piatra Craiulu (45.525, 25.209)
Russia, Altai, Kurkure Range, Kuzulun R. (50, 85)
Russia, Chukotskiy AO, Anadyr Bay (64.617, 177.45)
Russia, Chukotskiy AO, Anadyr Bay (64.617, 177.45)
Russia, Chukotskiy AO, Anadyr Bay, Onemen Bay (64.783, 176.967)
Russia, Chukotskiy AO, Anadyr City, along Maly Dionisy R. (64.589, 177.212)
Russia, Chukotskiy AO, Bilibino, Besimyanny settlement (68, 166)
Russia, Chukotskiy AO, Lavrentia village (65.5, 171.033)
109
110
111
112
Pl
H
h1/h2
4x
4x
4x
4x
C
K
K
a/q
e/l
e/l
4x
K
e/l
4x
4x
4x
4x
4x
4x
K
K
K
K
K
K
e/l
e/l
e/l
e/l
e/l
e/l
4x
4x
K
K
e/l
e/l
4x
K
e/l
4x
4x
4x
4x
4x
2x
K
K
K
K
K
A
e/l
e/l
e/l
e/l
e/l
a/n
2x
C
F
J
J
a/q
b/q
d/m
d/m
3x
3x
4x‡
3x
N
J
a/w
d/m
I
s
A
D
5
11
0.112
0.143
5
11
0.137
0.157
11
0.148
11
11
11
11
0.134
0.144
0.129
0.13
11
1
1
11
11
0.144
0.154
0.149
11
0.179
9
10
5
11
11
0.145
0.067
0.09
0.083
0.085
4
0.133
11
5
0.165
0.181
5
0.158
s
s
s
s
f
s
P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3921
no.
Population:
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
no.
§ID
Locality (longitude, latitude)
Pl
H
h1/h2
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
AK4424/s (11)
AK4468/s
V40/s (a)
V39/s (a)
AK4442/s
AK4458/s
V38/s (a)
AK3706/s (1)
AK3712/s
AK3802/s (1)
AK3817/s
AK3886/s
AK3991/s (10)
AK3851/s (1)
V114/h
AK160/s (1)
AK3505/s
V99/h
V118/h
AK389/s
V41/s (a)
MW-01–08/s
AK161/s (1)
AK162/s (1)
V42/s (a)
AK3005/s
V29/s (a)
AK1324/s
V28/s (a)
AK1337/s
V33/s (a)
V34/s (a)
V67/s (a)
V5/s (a)
V10 +3 + 2/s (V10)
V10 +3 + 2/s (V3a)
V10 +3 + 2/s (V2a)
AK544/s
AK872/s
V93/s (a)
Russia, Komi Republic, Pechora, around airport (65.105, 57.156)
Russia, Komi Republic, upper Pechora valley, Seyda (67.053, 63.074)
Russia, Komi Republic, Vorkuta, Lyasmylk (67.467, 62.833)
Russia, Komi Republic, Vorkuta, Seyda (67.383, 62.917)
Russia, Komi Republic, Yugyd-Va Nat. Park, Balbanyu valley (65.341, 60.712)
Russia, Komi Republic, Yugyd-Va Nat. Park, Maldy Nyrd-range (65.354, 60.625)
Russia, Murmanskaya Obl, Kola Pen., Drozdovka (67.783, 40.533)
Russia, Nenetskiy AO, Naryan-Mar, Fakel (67.66, 53.114)
Russia, Nenetskiy AO, Nenetskaya Gryada, Korovinskaya Guba (68.336, 53.3)
Russia, Sakha Republic (Ya), 4–5 km WNW of Yakutsk (62.08, 129.682)
Russia, Sakha Republic (Ya), 7 km S of Zhigansk (66.707, 123.385)
Russia, Sakha Republic (Ya), Chekurovka (71.048, 127.523)
Russia, Sakha Republic (Ya), Kharaulakh Mts (71.925, 127.318)
Russia, Sakha Republic (Ya), Lena R. E bank, near Vartay I. (69.107, 124.195)
Russia, Sakha Republic (Ya), lower Indigirka drainage, Shandrin R. (62, 129)
Russia, Sverdlovskaya Obl, North Ural, Denezhkin Kamen Rock (60.433, 59.55)
Russia, Taimyrskiy AO, Ary-Mas nature r., NNW of Khatanga (72.464, 101.864)
Russia, Taimyrskiy AO, Khatanga settlement (71.95, 102.4)
Russia, Taimyrskiy AO, Taymyr Pen., Ragozinka R. mouth (75, 102)
Russia, Taimyrskiy AO, Taymyr, Dudinka (69.428, 86.245)
Russia, Vorkuta, Ileymusyur (67.133, 62.583)
Russia, Yamalo-Nenetskiy AO, Gydan Pen., Yamburg (68.35, 77.13)
Russia, Yamalo-Nenetskiy AO, Polar Ural, Slanzevaya Mtn. (66.917, 65.767)
Russia, Yamalo-Nenetskiy AO, S Yamal Pen., Ercuta-Yaha R. (68.2, 68.9)
Russia, Yamalo-Nenetskiy AO, Ural, Hadata (67.583, 66.317)
Russia, Yamalo-Nenetskiy AO, Yamburg (67.389, 77.929)
Scotland, Coire An T-Sneachda, Cairn Gorm (57.117, –3.667)
Scotland, Glencoe, Coire nan lochan valley (56.654, –5.012)
Scotland, Liathach, Wester Ross (57.55, –5.5)
Scotland, Lochnagar (56.958, –3.22)
Spain, Huesca, Bielsa, Valle del Trigoniero (42.7, 0.233)
Spain, Huesca, Canfranc, Somport Frontier (42.767, 0.533)
Spain, Pyrénées, Lérida, Barruera (42.517, 0.85)
Svalbard, Dickson Ld., Idodalen (78.583, 15.383)
Svalbard, Nordenskiöld Ld., Colesdalen (78.1, 15.133)
Svalbard, Nordenskiöld Ld., Colesdalen (78.1, 15.133)
Svalbard, Nordenskiöld Ld., Colesdalen (78.1, 15.133)
Svalbard, Nordenskjöld Ld, Rusanovodden (78.143, 15)
Sweden, Latujavagge, 250–500 m SE of Latnajaure (68.35, 18.48)
Switzerland, Appenzell Assuer-Rhoden, Schwägalp, Hungbuel (47.25, 9.333)
4x
K′
e/l′
2x
2x
C
H
a/q
b/s
148
149
150
4x
4x
K
C
2x
2x
2x
2x‡
4x
e/l
a/q
I
s
—/q
b/q
a/q
a/q
a/q
C′
H
—/q
b/s
f
f
2x
C
a/q
f
2x
2x
2x
C
C
C
a/q
a/q
a/q
4x
K
e/l
4x
K
e/l
2x
2x
2x
2x
2x
2x
2x
A
A
B
C
C
C
C
a/n
a/n
a/o
a/q
a/q
a/q
a/q
K
e/l
D
7
11
0.177
0.171
10
11
3
11
11
3
4
7
9
11
0.227
0.208
0.146
0.144
0.172
0.14
0.119
0.168
0.172
0.177
10
10
0.179
0.15
11
0.153
4
8
10
0.171
0.167
0.155
11
1
11
1
10
4
0.194
0.102
0.079
4
5
0.098
0.015
8
10
5
0.085
0.201
0.08
s
s
F
C
C
C
4x
A
s
s
s
s
f
0.103
s
s
s
3922 P . B . E I D E S E N E T A L .
Appendix I Continued
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Appendix I Continued
Population:
§ID
Locality (longitude, latitude)
Pl
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
V77/h
V87/h
V85/h
V86/h
SUP02-139/s (1)
SUP02-270/s (1)
AG03-2/s (2)
SUP02-160/s (1)
AK907/s (3)
V84/h
SUP03-112/s (1)
V46/s (a)
V150/s (a)
V81/h
AK575/s (1)
AK575/s (5)
V151/s
SUP02-206/s (1)
SUP02-165/s (1)
V152/s (a)
V152/s (c)
SUP02-187/s (1)
SUP02-195/s (1)
V78/h
V47/f (a)
V88/h
V142/f
V137/f
V139/f
USA, Alaska, Alaska Range, Rainbow Mtn. (63.3, –145.467)
USA, Alaska, Aleutian Is., Adak Quad, Adak I. (51.833, –176.583)
USA, Alaska, Aleutian Is., Atka Is. (52.067, –174.583)
USA, Alaska, Aleutian Is., Unalaska Quad, Umnak I. (53.383, –167.833)
USA, Alaska, Anchorage Area, Chugach Mts., Arctic Valley (61.247, –149.535)
USA, Alaska, Brooks Range, Endicott Mts., Chandler Lake (68.195, –152.744)
USA, Alaska, Kenai Pen., Kalifornsky Beach (60.433, –151.267)
USA, Alaska, Kenai Pen., S of Gilpatric, along Seward Hwy (60.567, –149.576)
USA, Alaska, Kenai Pen., W of Silver Lake (60.633, –150.819)
USA, Alaska, Ketchikan Quad, Ella Lake, NE shore (55.483, –131.067)
USA, Alaska, Mataniska-Glenallen, Mendeltna Creek (62.033, –146.533)
USA, Alaska, N of Brooks Range, along Sag R. (69.317, –148.717)
USA, Alaska, Noatak Quad, Sheshalik Split (67.017, –162.95)
USA, Alaska, Selawik Quad, Waring Mts. (66.967, –159.683)
USA, Alaska, Seward Pen., Cape Nome, along Council Road (64.444, –164.973)
USA, Alaska, Seward Pen., Cape Nome, along Council Road (64.444, –164.973)
USA, Alaska, Seward Pen., Cowpack Inlet (66.35, –164.95)
USA, Alaska, Seward Pen., Kigluaik Mts, Grand Singatook (64.907, –166.18)
USA, Alaska, Seward Pen., Skookum Pass (64.717, –164.05)
USA, Alaska, Seward Pen., Teller Road (64.683, –165.75)
USA, Alaska, Seward Pen., Teller Road (64.683, –165.75)
USA, Alaska, Seward Pen., Teller Road/Tisuk R. (64.97, –166.204)
USA, Alaska, Seward Pen., Teller, mire/shore E of settlement (65.253, –166.366)
USA, Alaska, Unalakleet Quad, Nulato Hills (63.8, –160.383)
USA, NE California, Sierra Nevada N, Adams Peak (Cult.) (40, –120)
USA, Nevada, Ormsby Co., Carson Range (39, –119)
USA, New Hampshire, Mt. Clinton, Rt302 (44, –71.5)
USA, New Hampshire, White Mts., Mt. Pierce (44, –71.5)
USA, New Hampshire, White Mts., Mt. Pierce (44, –71.5)
6x*
166
167
168
169
170
171
172
173
174
175
176
177
4x
2x
4x
4x
4x
2x
2x
4x
3x
4x
4x
4x
2x
4x
4x
2x
2x
4x
4x
2x
4x†
H
h1/h2
I
R
M
R
S
C
R
S′
J
R
C
C
O
J
J′
J
J
Q
Q
J
Q
C
J
P
L
L
I′
H
a/–
g/u
i/v
g/u
i/u
a/q
g/u
i/u′
d/m
g/u
a/q
a/q
j/x
d/m
d/m′
d/m
d/m
h/u
h/u
d/m
h/u
a/q
d/m
d/t
f/k
f/k
c/—
b/s
b/—
f
f
f
f
A
D
5
5
5
4
4
0.177
0.183
0.187
0.143
0.244
5
0.198
11
0.121
4
5
0.159
0.149
5
5
0.181
0.17
f
s
s
s
s
s
s
s
f
s
s
§Collectors: A. Gangardt, A. K. Brysting, A. Tribsch, A.-M. Gergö, B. J. Graae, B. Sabard, C. Pedersen, D. Ehrich, G. Bastholm, G. Bugge, G. H. Jacobsen, G. Schneeweiss, H. Meyer, H.
Solstad, I. G. Alsos, I. Skrede, J. J. Andersen, K. Albert, K. Hansen, K. Marr, K. Westergaard, L. Lund, M. Chiboshvili, M. H. Jørgensen, M. K. Holte, M. Kapralov, M. Puscas, M. Ronikier,
M. Tutkova van Loo, M. Wiedermann, O. Gilg, O. Paun, P. B. Eidesen, P. Kuss, P. Larsson, P. Schönswetter, R. Elven, R. Hebda, R. Nessa, S. Ertl, S. Kjølner, T. Carlsen, T. Englisch, T. M.
Gabrielsen, V. Razzhivin, and Ø. Stensrud. For populations with ID starting with ‘V’, collectors can be found in Alsos (2003) and Alsos et al. (2005).
P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3923
no.
3924 P . B . E I D E S E N E T A L .
Appendix II
Variable characters in the ITS matrix for Vaccinium uliginosum. Characters in bold are synapomorphies for the two main groups (Fig. 2).
Standard IUPAC ambiguity codes are used for polymorphic characters, dots represent the same character as in the Andromeda polifolia
sequence and question marks indicate lacking data. Positions 269–433 corresponds to the 5.8S gene. Plants containing identical ITS
haplotypes (recovered by cloning) are indicated in parentheses after each haplotype (haplo1–haplo11). Collection data and GenBank/
EMBL accession numbers for clones and sequences produced by direct sequencing of PCR fragments are given in Appendix 1
Species
V. uliginosum samples given with
pop. no. and ID (no — ID)
Andromeda polifolia
Zenobia pulverulenta
Leucothoe fontanesiana
V. crassifolium
V. crenatum
V. caespitosum
V. macrocarpon
V. vitis-idaea
V. myrtillus
V. acrobracteatum
V. scoparium
V. yatabei
V. arctostaphylos
V. cylindraceum 1
V. cylindraceum 2
V. padifolium
107-V98
172-V78
173-V47*
haplo1 (138-V2a, 31-V30, 77-V17†)
haplo2 (138-V2a, 138-V3a, 29,
66-V8, 77-V17†)
13-V52
14-V50
22-V51
31-V30
32-V32
29-V66
30-V31
34-V60
42-V56
46-V63
50-V64
56-V53
61-V65
137-V42
115-V40
116-V39
127-V114
143-V33
144-V34
147-V2a‡
147-V3a
162-V46
176-V137‡
haplo3 (66-V8, 77-V17, 138-V2a,
79-V18)
haplo4 (77-V17)
haplo5 (138-V2a)
haplo6 (79-V18)
Ploidy
level
?
?
?
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
1111111111111122222234444444445555555555566666666
123333556777890123445667788901234400344556680011124679911234445
350347688018749036272570415616991680849246804526748071638840190
CCATTAATGTCTCCGCCTAGTCAACACACCGGTGGTCCTCCCGCTCGTCCGACCAGGAACCAC
.T.......................................T..............-......
.....C..........G...........T............T......N......C...---.
....CC............G................................G..G......G.
....C..C...C......G................................G..G...G....
....CC.C...C...................................G...........T...
....CC.C...C.....CG............................G...G..G...G...G
....CC.C...C.....CN........--............N...N.N...N..N.......G
....CC.C...C...................................G...............
....CC.C...C.....C.................................G...........
....CC.C...C........................N..........G...............
....CC.C...C...................................G...............
....CC.C...C.....C.......................T..GT..........T......
....CC.C...C.....C.......................T..GT.......T..T..T...
....CC.C...C.....C.......................T..GT..........T..T...
....CC.C...C.....C.......................T..GT..........T..T...
?...CC.CA........C.......................T.TGT..........A...M.Y
??????????????...C..........Y.......Y.S..T.YKY.K......R.A.R....
.....Y.C...C.....C..Y.................A........G......G????????
.....C.....C.....C..........T.......T.A.-......G......G.T.G...T
.....C.....C.....C..........T.......T.A........G......G.T.G...T
...Y.C.....C.....C..........T.......T.A.......RG......G.TMR...T
.....C.....C.....C..........T.......T.A........G......G.T.G...T
.....C.....C.....C..........T.......T.A........G......G.T??????
....YC.Y...C.....C.........RT.......T.A.?......G......G.T.G..MT
.....C.....C.Y...C..........T.......T.A.-......G......G.T.G...T
....YC.Y...C.....C.........RT.......T.A........G......G.T.G...T
.....C.....C.....C..........T.......T.A........G......G.T.G...T
.....C....YC.....C..........T.......T.A........G......G.T.G...T
.....C....YC.....CS......R..T.......T.A........G......G.T.G...T
.....C....YC...Y.C..........T.......T.A........G.....YG.T.R...T
.....C....YC....MC......W...Y.......T.A........G.....YG.T.R...T
.....C....TC.....C..........T.......T.A........G.....TG.T.G...T
.....C....YC.....C..........T.......T.A........G.....YG.T.G...T
...Y.C.....C..R.MC..........Y.R.....T.A........G......R.T.R...T
...Y.C.....C.....C..........T.......T.A........G....Y.G.T.G...T
.....C.....C..R.MC..........T.......TMA........G......R.T.R...T
???........C.....C..........T..RY...T.AY.......G..K...G.T.G..??
.....C.....C.Y...C..........T.......T.A.-......G......G.T.R...T
.....C.....C.Y...C.W........T......ST.A.-......G......G.T.G...T
A...YC.YR..Y.....C......Y...Y.......Y.S.YYRYKYKY......R.A.R...Y
.....C.....C.....C..........Y.......T.A........G......G.T.R...T
.....C.....C.....C..........T.......T.A........G......G.T.G...T
.....C.YR..Y.....C........Y.Y....R..Y.S..Y.YKY.KY.....R.S.RY..Y
A...CC.CA........C.......................T.TGT..........A......
A...CC.CA........C.......................T.TGT..........A..T...
A...CC.CA........C......T.........T......T.TGT..........A......
A...CC.CA........C................T......T.TGT.....C....A......
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
P H Y L O G E O G R A P H Y O F V A C C I N I U M U L I G I N O S U M 3925
Appendix II Continued
Species
V. uliginosum samples given with
pop. no. and ID (no — ID)
haplo7 (138-V2a)
haplo8 (66-V8)
haplo9 (66-V8)
haplo10 (138-V2a, 79-V18)
haplo11 (66-V8)
108-V43(a)‡
108-V43(e)
112-V44
164-V81*
9-V147
10-V48
11-V138
35-V62
66-V8
77-V17
79-V18
119-V38
150-V93
163-V150
166-V151
169-V152‡
177-V139
Ploidy
level
1111111111111122222234444444445555555555566666666
123333556777890123445667788901234400344556680011124679911234445
350347688018749036272570415616991680849246804526748071638840190
3x
3x
3x
3x
4x
4x
4x
4x
4x
4x
4x
4x
4x
4x
4x
4x
4x
A...CC.CA........C......T................T.TGT..........A......
A...CC.CA........C...........T..C........T.TGT..........A......
A...CC.CA........C.......................T.TGT.C........A......
A...CC.CA........C......................TT.TGT..........A......
A...CC.CA........C...........T...........T.TGT..........A......
?.R.YCRYR..Y.....C.....R..Y.Y.R..R..Y.W..Y.YKY.KY.....R.W.RY..Y
A...CC.CA........C.......................T.TGT...A..???????????
?...YC.YR..Y.....C..........Y.......Y.S.YT.YGY.K......R.A.R...Y
??????.YR..Y.....C..........Y.......T.S....YK..G......R.A.R...T
A...CC.CA........C......................GT.TGT..........A...T..
M...YC.YA..Y.....C..........Y.......Y.W.GT.YGY.K......RKA.R.T..
?????????????....Y......Y................TRTGT........R.A......
MY..YC.YA..YY.R.MC...Y........R.......W..T.YGT..........A??????
?...YC.YR........C......W...YY...........T.TGT........R.A.R....
??????.CA........C......Y...Y...........STRTGT..........A....??
A...CC.CA........C....R..................T.TGT.....M....A......
A...CC.CA........C......................YT.TGT....?????????????
A...CC.CA........C.......................T.TGT..........A......
A...CC.CA........C.......................T.TGT..........A......
A...CC.CA........C.......................T.TGT..........A......
....YCRYRY.Y.....C......Y...Y.......Y.S..YRYKYRK......R.S.R...Y
??????....YY.....C..........Y.......T.A....Y...G.....YG.T.G...T
*Inferred recombinant and therefore excluded from the analyses. †Recombined clones contributing partially to haplo1 and partially to
haplo2. ‡Sequence excluded from the analyses due to too few (= 2) unambigous characters of the synapomorphies of the two main groups
in V. uliginosum.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd