Out of the Alps or Carpathians? Origin of Central European populations of Rosa
pendulina.
Z Alp nebo z Karpat? Původ středoevropských populací druhu Rosa pendulina.
Tomáš F é r1, Petr V a š á k2, Jaroslav V o j t a1  Karol M a r h o l d1,3
1
Department of Botany, Faculty of Science, Charles University, Benátská 2, CZ-128 01
Praha 2, Czech Republic, e-mail: tomas.fer@centrum.cz; 2Department of Dendrology and
Forest Tree Breeding, Faculty of Forestry and Environment, Czech University of Life
Sciences, Kamýcká 129, CZ-165 21 Praha 6, Czech Republic, e-mail: vasak.petr@post.cz;
3
Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, SK-845 23 Bratislava,
Slovakia, e-mail: karol.marhold@savba.sk
Running title: Phylogeography of Rosa pendulina
Keywords: phylogeography, trnL-trnF sequencing, cpDNA, postglacial migration, glacial
refugium
Abstract
Phylogeographical structure among 41 populations of temperate shrub Rosa pendulina in
Europe was studied using PCR-RFLP and sequencing of non-coding cpDNA regions. Our
study revealed a clear-cut geographic organization of cpDNA diversity, with three major
haplotypes that were geographically widespread and showed only little overlap in their
distribution areas. This picture suggests that postglacial expansion occurred from at least two
distinct glacial refugia, probably located in the edge of the Alp and in the Balkan Peninsula or
in the southern Carpathians. All populations in the Czech Republic and surrounding regions
are of the Carpathian origin. This finding disproves the speculation about Alpine origin of R.
pendulina populations in the Šumava Mts. (Czech Republic). The contact zone between
Carpathian and Alpine migration routes of R. pendulina is probably the Danube valley.
1
Introduction
Present distribution patterns of plants on the European subcontinent are to large extent
determined by the extensive climatic changes during the Quaternary, which caused repeated
cycles of species migrations throughout the continent (Hewitt 1999). Migration processes and
the origin of the present distribution patterns can be inferred only indirectly, on the basis of
palynological or, most recently, phylogeographical evidence. The latter deals with historical,
phylogenetic components of spatial distribution of gene lineages (Avise 2000).
The early phylogeographic studies of European taxa, dealing mainly with economically
important trees (e.g., Alnus, Quercus, Fagus, King & Ferris 1998, Dumolin-Lapègue et al.
1997, Demesure et al. 1996), were focused on the determination of patterns on the whole
continent scale. As a result, similar patterns of postglacial colonization routes were found for
several taxa, and three main refugia in southern Europe were postulated (Iberian and
Apennine Peninsula and the Balkans, see Taberlet et al. 1998). Cycles of area contraction and
expansion strongly affected also patterns of genetic diversity. Putative refugial populations
were found to harbour much higher diversity than populations in recolonized areas, which
experienced bottlenecks during the expansion after warming 13,000 years ago (Comes &
Kadereit 1998).
Previous large-scale studies revealed several hybridization- or suture-zones (a term
introduced by Remington 1968, for discussion see also Swenson  Howard 2004) in Europe,
where populations of the same or closely related taxa expanding from different refugia
(Balkan, Apennine and Iberian) came into contact (Hewitt 1988, 1999, Taberlet et al. 1998).
Although these suture-zones have not identical locations for all taxa, four main areas can be
identified: the Pyrenees, the Alps, central parts of the Scandinavian Peninsula, and central
Europe north of the Alps.
Willis et al. (2000) and Willis & van Andel (2004) based on palaeoecological evidence
concluded that there was no forestless landscape in Central Europe during the last glacial as
hypothesized before. Thermophilous trees (Carpinus betulus, Quercus, Corylus, Ulmus and
Tilia) must have survived in micro-environmentally favourable pockets in this area. Also
recently published evidence on Fagus sylvatica (Magri et al., 2006) suggests the presence of
open forests in Central Europe. This may considerably change the view on the origin of the
Central European flora and postglacial migrations established by early phylogeographic
studies.
2
When examining the present distribution patterns of taxa occurring in Central Europe
north of the Alps, both Alpine and Carpathian migration routes can be suggested, but
processes of postglacial migrations in this area are still not fully understood. The flora of the
area of the Czech Republic provides an amount of examples of taxa with different patterns of
postglacial immigration. In some cases it is possible to infer the postglacial origin of
populations in a given area unequivocally from the present distribution pattern. Among them,
a few examples of taxa with apparently Alpine origin are as follows: Soldanella montana (for
distribution map see Slavík 1990), Alnus viridis (indigenous distribution, Kučera 1966, Plzáková
1973), Cardamine amara subsp. austriaca (Marhold, unpubl.) and Willemetia stipitata
(Klášterský 1961, Kučera 1966), while Cardamine glanduligera (= Dentaria glandulosa; Slavík
1990, Lihová et al. 2007), Cardamine amara subsp. opicii (Hrouda & Marhold 1993), Centaurea
jacea subsp. oxylepis and Salix silesiaca (both Hendrych 1985) most likely immigrated to the
present area of the Czech Republic from the Carpathians. There is, however, an array of species
that could have immigrated from both the Alps and Carpathians (Hendrych 1985). Some
populations of these taxa can be unequivocally attributed to either the Alpine or Carpathian
origin, based on present distribution pattern, while the origin of others (or even most of them) is
uncertain. This phenomenon apparently reflects the existence of a suture-zone in the area of
Central Europe, including the Czech Republic.
One of the species for which both the Alpine and Carpathian postglacial immigration
into the area of the Czech Republic can be hypothesized is Rosa pendulina. It occurs
predominantly in mountains and hilly landscapes in the Alps, Carpathians, in the Balkan
Mountains, the Apennines and in the Pyrenees (Meusel et al. 1965, Kurtto et al. 2004; Fig. 1).
In the Czech Republic Rosa pendulina is common in mountains and hilly landscapes but it
does not occur in lowlands (Slavík et al. 1995: 38, Větvička 1995). At least two migration
routes into this area appear plausible, from the Carpathians in the east-west direction and from
the Alps throughout the Šumava Mts. in the south-north direction, but available distribution
data do not allow delimiting clear boundary between populations of these two migration
routes (Hendrych 1985).
In this study we analysed 41 samples of R. pendulina including populations from the
whole distribution area of the species. Using sequencing of non-coding trnL-trnF region of
cpDNA (widely used marker in phylogeographical studies; Taberlet et al. 1991) we searched
for intraspecific variability which enabled us to define haplotypes of samples from the Central
European region as well as the assumed refugia. The analysis allowed us to address following
3
questions: (1) Is there a phylogeographical structure in the distribution of cpDNA haplotypes?
(2) Can we trace the origin of Central European populations? Which of the population from
the potential refugia mainly contributed to the recolonization of Central Europe? (3) Are there
any suture zones in Central Europe and where? (4) Is the postglacial spread of R. pendulina
comparable to the pattern found in other species?
Methods
We analyzed individuals originated from 41 populations (one sample per population) from the
whole distribution area, including hypothetical refugia (Pyrenees, southern Italy, the Balkans;
Table 1). Young and healthy leaves were collected in the field and dried in silica gel. Voucher
specimens have been deposited in the herbarium PRC (Charles University, Prague, Czech
Republic).
DNA extraction, PCR-RFLP and sequencing
Total DNA was extracted using protocol of Doyle  Doyle (1987) and dissolved in 1 TE
buffer. DNA concentrations were measured photometrically and adjusted to 5 ng/l.
We used two methods to define chloroplast DNA (cpDNA) haplotype of all individuals.
First, we search for variability in three non-coding regions (psbC-trnS, trnK-trnK, trnC-trnD;
Demesure et al. 1995) using polymerase chain reaction – restriction fragment length
polymorphism (PCR-RFLP) approach. PCR reactions, containing 20 ng template DNA, 0.2
mM dNTP (Fermentas), 6 pmol of forward and reverse primer (Demesure et al. 1995), 2.0 μl
10× Buffer for RedTaq (Sigma), 0.5 U JumpStart REDTaq DNA Polymerase (Sigma), and
water added to final volume of 20 l, were placed in a Mastercycler ep gradient S thermal
cycler (Eppendorf). Reaction conditions were as follows: initial denaturation 1 min at 94 °C,
followed by 35 cycles of 45 s at 94 °C, 45 s at 53.5–57.2 °C, 2–4 min at 72 °C with a final
extension time 10 min at 72 °C (the exact annealing temperatures and elongation times
depend on the amplified region; see Table 2 for details). PCR products were cut separately
using 15 restriction enzymes as shown in Table 2. The restriction reactions contained 5.5 μl of
PCR product, 2.0 μl corresponding restriction buffer (Fermentas), 9.0 μl ddH2O, and 3 U of
restriction endonuclease (Fermentas). The digestions were performed for 4 h at 37 °C (HinfI,
Hin6I, MboI, Bsh1236RI, AluI, BsuRI, MspI, RsaI, PstI, BglI, HincII) or 65 °C (TaqI, TaiI,
4
TasI, Tru1I). About 1 μl of 6× loading dye (Fermentas) was added to each tube after the
restriction and the whole volumes of restriction products were loaded onto an 1.8% agarose
gel and electrophoresed for approx. 1 hour using HU13 vertical electrophoresis unit (SciePlas). Fragments were visualized under UV light after ethidium bromide staining using Gel
Logic 100 (Kodak) system and scored with the help of 1D Image Analysis Software (Kodak).
Altogether, we searched for variability in restriction profiles among 11 populations from
Romania (RP09), Ukraine (RP07), Slovakia (RP06), Austria (RP20), France (RP18), and
Czech Republic (RP01, RP05, RP13, RP14, RP15, and RP17).
Additionally, sequencing of trnL-trnF region of chloroplast DNA universal primers c
and f by Taberlet et al. (1991) were used for amplification of the trnL intron and spacer
between trnL and trnF regions in cpDNA. PCR amplification was carried out in a volume of
19 μl reaction using 5 ng of template DNA, 2 μl of 10 reaction buffer (Sigma), 0.4 μl of
dNTP mix (10 mM), 4 pmol of each primer, and 0.5 U of REDTaq DNA Polymerase (Sigma).
The reaction was optimized on a Mastercycler ep gradient S thermal cycler (Eppendorf) as
one cycle denaturation at 94 °C, 35 cycles of 45 s denaturation at 94 °C, 45 s annealing at
52.2 °C, and 2 min extension at 68 °C, followed by 10 min extension at 70 °C. Amplification
fragments were subsequently purified using JetQuick PCR Purification Kit (Genomed).
Sequencing reactions were performed using BigDye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems) according to the manufacturer’s instructions. For sequencing of both
strands the same primers were used. Purification of sequencing reactions followed the
ethanol/sodium acetate precipitation protocol provided with the sequencing kit. Products were
resolved on an ABI 3100 Avant automated sequencer (Applied Biosystems).
Data analysis
Sequences from both strands were manually assembled in one consensus sequence. All
sequences were automatically aligned using ClustalX v 1.83 (Thompson et al. 1997). The
resulted alignment was improved manually. Informative insertions/deletions were coded using
the “simple indel coding” approach following the rules by Simmons  Ochoterena (2000) as
implemented in SeqState (Müller 2005).
To reconstruct relationships among haplotypes, statistical parsimony network of
haplotypes (Templeton et al. 1992) have been constructed using TCS 1.21 (Clement et al.
2000). This software construct a haplotype network in that the required number of mutational
5
steps leading from one haplotype to another is minimized. Moreover, the position of a
haplotype in a network implies information regarding its age. Older haplotypes are thought to
have a greater likelihood of being located internally in the network (Posada & Crandall 2001).
The gaps were treated as missing data, but with additional indel coding (see above).
Results
We found no variability in cpDNA among 11 individuals from 11 populations using PCRRFLP approach. The combinations of cpDNA non-coding regions and restriction
endonucleases that were used for searching of polymorphism are in Table 2.
We sequenced the trnL-trnF region in 41 individuals from 41 populations (Table 1).
Total length of the whole aligned region was 832 bp from which eleven positions were
variable. We found nine point mutations and two length differences (one and four bp,
respectively). Based on this variability five unique haplotypes can be recognized (A –E; Table
3). The DNA sequences were submitted to the GenBank database (one locality per each
haplotype, accession numbers: haplotype A  XXX, B  XXX, C  XXX, D  XXX, E 
XXX). Three haplotypes (A, C, and D) are widely distributed, two additional haplotypes (B
and E) occurred only locally. The geographical distribution of haplotypes is shown on the Fig.
2. Haplotype A occurs in the Balkans, in the whole Carpathians and in all analysed
populations from Central Europe north of the Alps. Haplotype B was found only in one
population from the Jeseníky Mountains (Czech Republic). The distribution of haplotype C
includes Pyrenees, southern Italy and the western Balkans. All populations of R. pendulina in
the Alps and surrounding area have haplotype D. Haplotype E was found only in one
Bulgarian locality, namely in the Rila Mts (RP29).
Relationships among haplotypes were determined constructing statistical parsimony
network (Fig. 3). The input file includes the alignment and insertion/deletion were coded as
two additional characters using C for the insertion presence and A for the absence. The
haplotypes A and B found in the Carpathians and in Central Europe north of the Alps differ
only by one point mutation. Similarly, southern European haplotype C and the Alpine
haplotype D also differ by one point mutation. These two groups of haplotypes (A and B; C
and D) differ by two point mutations. Hence, a missing (unsampled or extinct) haplotype is
suggested among these groups. The haplotype E, found in the Balkans only, is much more
6
distinct from all other haplotypes. It differs by five point mutations and two indels from both
haplotypes A and C.
Discussion
Despite we analysed only 41 populations of R. pendulina (using sequencing of the trnL-trnF
region) our study revealed a clear-cut geographic organization of chloroplast DNA diversity,
with three major haplotypes that were geographically widespread and showed only little
overlap in their distribution areas. Nevertheless, no variability within three other non-coding
cpDNA regions have been found using PCR-RFLP approach.
Assumed refugia
Based on the results obtained, the present day distribution of cpDNA haplotypes in R.
pendulina suggests that postglacial expansion was from two major refugia. The first was
located in the Balkan Peninsula or somewhere in the Southern Carpathians. From here the
migration to the whole eastern and Central Europe occurred. This is one of the most common
scenarios for the colonization of the Central Europe as demonstrated for Fagus sylvatica
(Magri et al. 2006) or Carpinus betulus (Grivet  Petit 2003). The second refugium can be
located somewhere on the southern edge of the Alps, in northern Apennines or in the Dinaric
Alps. From here the species spread throughout the Alps and surrounding regions. Such
refugial area is postulated also for Alnus glutinosa (King  Ferris 1998), Fraxinus excelsior
(Heuertz et al. 2004) or Abies alba (Konnert & Bergmann 1995). Although no evidence of
common phylogeographic histories across Europe have been found among tens of species
compared (Taberlet et al. 1998) the postglacial history in R. pendulina is most similar to the
pattern found in Abies alba (Konnert & Bergmann 1995). Also in this species the
geographical distribution of genetic variation (allele frequencies at isozyme loci in this case)
indicates refugia in the Balkans and in Italy.
We found a common haplotype for all three hypothetical southern refugia indicating a
connection between Iberian, southern Italian and the Balkan populations of R. pendulina. This
haplotype (C) probably did not contribute to the present-day genetic pool of the Central
European populations. The distribution of haplotype C on both the western and eastern shore
of the Adriatic Sea illustrating the connection between refugia in the Italian and the Balkan
Peninsulas has been observed in common ash (Heuertz et al. 2004), oaks (Fineschi et al. 2002,
7
Petit et al. 2002) or ivy (Grivet  Petit 2002). Identical haplotype found in these areas
indicate some contact of populations from these regions during cold periods. Indeed, during
the last glacial maximum the sea level was considerable lower and there was a contact of the
Apennine and Balkan flora, especially in northern part of the Adriatic region (Frenzel et al.
1992, Adams 1997).
The most genetically distinct haplotype E (five point mutations and two indels apart
from both haplotypes A and C) was found in a single population in Bulgaria (RP29). A more
detailed study will be necessary to evaluate the haplotypic diversity in the Balkan Peninsula; a
potential taxonomic problem cannot be excluded as well. Many phylogeographical studies
revealed high diversity in this region (e.g. Fagus sylvatica – Magri et al. 2006; Alnus
glutinosa – King  Ferris 1998) but in most cases only a single lineage participated on the
postglacial colonization to the north.
Nevertheless, the hypothesis about survival of R. pendulina in Central Europe during the
last cold period in the light of results by Willis et al. (2000) and Willis & van Andel (2004)
may not be completely rejected on the basis of our data. More variable molecular marker (like
AFLPs; Vos et al. 1995) would be necessary to investigate present-day population structure
that can preserve some indication of past processes as bottleneck or founder effect (citace).
The suture-zone in the Central Europe
The suture zone between Carpathian and Alpine migration of R. pendulina is probably the
Danube valley. This fact can be related to the present day distribution gap between the
continuous Alpine area and the Danube river in Germany and Austria (Haeupler &
Schönfelder 1989, H. Niklfeld et al., unpublished) that might be associated with unsuitable
ecological conditions at low elevations. An alternative explanation of this phenomenon could
be the barrier of the Alps that prevented the northward expansion of populations isolated at
the southern edge of the Alps. Hence, the migration from the Alpine refugium was slowed
down compared to rapid expansion along the Carpathians, and the Šumava Mts. were
colonized from the Carpathian source. It is likely that first populations colonizing a region can
prevent the arrival of other ones because the space is already occupied (Taberlet et al. 1998).
In spite of the existence of this clear-cut border between Alpine and Carpathian colonization,
some migration over the Danube was detected. The Hochschwab (RP62) and Schneealpe
(RP49) populations in central eastern Austria have the Carpathian haplotype A. Since we
8
analysed only limited number of populations the more comprehensive study is necessary to
obtain detailed information about postglacial migration in this suture-zone and about the
extent of haplotype mixing.
Origin of populations in Central Europe north of the Alps
Based on the geographical pattern detected in variability of cpDNA, all populations in the
Czech Republic and surrounding regions are of the Carpathian origin and probably originated
from the postglacial migration from the Southern Carpathian or Balkan refugia. This finding
disproves the speculation about the Alpine origin of R. pendulina populations in the Šumava
Mts. (Hendrych 1985). The landscape with lower altitudes along the Danube was probably an
ecological barrier, which prevented the Alpine populations of R. pendulina from dispersal
further to the north.
One unique haplotype B was found in the Jeseníky Mountains (Czech Republic; RP01).
It is derived from the common Carpathian haplotype A from which it differs by a single point
mutation. This haplotype probably evolved recently or during postglacial recolonization of the
Central Europe from the Carpathian refugium. The terminal position of the haplotype in the
network is another indication of the recent haplotype age (Posada & Crandall 2001).
Acknowledgements
We thank to V. Machalová and E. Rejzková for the help with molecular work. We are
indebted to K. Boublík (Praha), H. Dvořáková (Praha), J. Fér (Kutná Hora), L. Férová
(Kolín), B. Frajman (Ljubljana), A. Kagalo (Lviv), M. Kolník (Bratislava), J. Kučera
(Rychnov n. Kněžnou), J. Lihová (Bratislava), D. Reich (Wien), E. Rejzková (Praha), P.
Schönswetter (Wien), M. Slovák (Bratislava), P. Stanimirova (Sofia), M. Stolařová (Praha),
A. Tribsch (Salzburg), V. Zabloudil (Žďár n. Sázavou), T. Wohlgemuth (Birmensdorf), and
other colleagues for the help with sampling of plant material and J. Lihová also for comments
on the manuscript. This work was supported by the Grant Agency of the Czech Republic no.
206/04/0770 and the MŠMT project no. 0021620828.
9
Souhrn
Použitím PCR-RFLP a sekvenování nekódujících úseků cpDNA byla studována
fylogeografická struktura mezi 41 populacemi růže převislé (Rosa pendulina) v Evropě. Naše
studie ukázala jednoznačné geografické uspořádání diversity chloroplastové DNA. Tři hlavní
haplotypy jsou geograficky široce rozšířené a vykazují jen velmi male přesahy v distribuci.
Tato situace svědčí o tom, že postglaciální kolonizace probíhala nejméně ze dvou odlišných
glaciálních refugií, která byla lokalizována na jižním okraji Alp a na Balkánském poloostrově
nebo v jižních Karpatech. Všechny populace v České republice a okolí jsou karpatského
původu. Toto zjištění vyvrací spekulace o možném alpském původu šumavských populací R.
pendulina. Kontaktní zóna mezi alpským a karpatským migračním proudem je
pravděpodobně údolí Dunaje.
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Figures
Fig. 1. – Distribution area of R. pendulina in Europe according to Kurtto et al. (2004)
(shaded) and location of 41 populations used in the study. For detailed descriptions of
the populations see Table 1.
Fig. 1. – Geographical distribution of five cpDNA haplotypes detected in R. pendulina.
The present day distribution is shaded (Kurtto et al. 2004).
Fig. 2. – Statistical parsimony network showing the relationships among five cpDNA
haplotypes detected in R. pendulina. The box sizes are proportional to the number of
localities where the haplotype was found.
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