Conservation Genetics (2006) 7:895–907
DOI 10.1007/s10592-006-9131-z
Springer 2006
History, genetic differentiation and conservation strategies for disjunct
populations of Sibiraea species from Southeastern Europe and Asia
Dalibor Ballian1, , Tine Grebenc2, , Gregor Božič2,*, Viktor Melnik3, Tone Wraber4 &
Hojka Kraigher2
1
Faculty of Forestry, University of Sarajevo, Zagrebacˇka 20, 71000, Sarajevo, Bosnia and Hercegovina;
Slovenian Forestry Institute, Vecˇna pot 2, SI, 1000, Ljubljana, Slovenia; 3National Botanical Garden,
Academy of Sciences of Ukraine, Timiryazevska 1, 01014, Kiev, Ukraine; 4Department of Biology, University
of Ljubljana, Vecˇna pot 111, 1000, Ljubljana, Slovenia (*Corresponding author: Phone: +386-1-200-7800;
Fax: +386-1-2573589; E-mail: gregor.bozic@gozdis.si)
2
Received 15 July 2005; accepted 24 January 2006
Key words: conservation through active management, cpDNA, genomic rDNA spacers, Sibiraea altaiensis,
Sibiraea croatica
Abstract
The genetic structure of Croatian Sibirea (Sibiraea croatica), a rare and endemic tertiary relic of Croatian
and Herzegovinian flora, and its relationship with sibirea from Southern Russia and Southern Siberia
(Sibiraea altaiensis) was studied using amplification, restriction and sequencing of the ITS region in genomic
DNA and cpDNA and their comparisons with sequences of the Rosaceae species obtained from GenBank.
The restriction analysis and separation in agarose gel showed no differences in length of the digested
cpDNA between or within populations. Sequencing showed only minor variability between populations.
Only a minor difference of 6 bp duplication in DNA amplified with ccmp 10-R and trnM primer pair was
noticed in two geographically distinct populations. No differences in the restriction pattern for the ITS
region in genomic rDNA indicates that all samples of sibirea belong to the same species since the ITS region
was proven to be conserved within one taxonomic species. The minor differences that were obtained support
the hypothesis that sibirea is an old tertiary relic that shows a minor variability, confirming previous
preliminary results from comparisons of the Croatian and Altaic sibireas at the morphological level. Our
data suggests that Croatian sibirea from the Balkan is a disjunct population identical to the Altaic species.
Due to its disjunct occurrence in Southeastern Europe, the endemic status in the Dinarics, a relic that
survived the glaciations, it deserves active conservation approaches through support of traditional use of
high-mountain pastures for reducing natural reforestation of sibirea ancient sites.
Introduction
Croatian sibirea (Sibiraea croatica Deg.),
described by the Hungarian botanist Degen in
1905 is a rare and endemic bushy species of
Croatian and Herzegovinian flora where it represents a tertiary relic.
The first and second author provided an equal contribution to
the study.
The taxonomy of sibirea has not been entirely
investigated yet. The genus name Sibiraea Maxim.
derives from the Russian name for the region of
Siberia (‘‘Sibir’’ in Russian) and the ending of its
closest genus Spiraea (Genaust 1996: 582). Sibiraea croatica was described in the first part of the
20th century as:
• Sibiraea croatica Degen, Magyar. Bot. Lapok
4: 255, 1905
896
•
Sibiraea laevigata (L., 1771) Maxim., Acta
Horti Petrop. 6: 213, 1879 subsp. croatica
(Degen) Degen, Fl. Veleb. 2: 241, 1937
• Sibiraea altaiensis (Laxm., 1770) C. K. Schneider
var. croatica (Degen) G. Beck, Fl. Bosn. Herceg.
3: 4, 1927.
Its discovery in Europe in 1905, first by S.
Kocsis on Velebit (Croatia) and a few months later
on Čabulja (Herzegovina) by O. Reiser, gained
considerable attention. Both discoveries were
evaluated by the Hungarian botanist A. Degen
(1905), who estimated the morphological identity
of both plants from the two European sites and
described them as Sibiraea croatica. He also
mentioned the high resemblance with the Asian
plants and commented on the possibility that the
European plants were merely a subspecies of the
Asian species, which he mentioned as S. altaiensis
(=S. laevigata). He presented the same opinion in
the next publication in 1906, where the species
name used was S. laevigata (Degen 1906). Only in
the final work, published after his death, was the
European plant decisively named S. laevigata (L.)
Maxim. subsp. croatica (Degen) Degen (Degen
1937).
A few preliminary comparative results from the
morphological studies by Bosnian dendrologists,
that were obtained by cultivation of the Croatian
and Asian sibirea under comparable conditions in
Bosnia and Herzegovina, show that the differences
between them are minimal (Šilić, personal communication). The taxonomical status of the
European sibirea is still under consideration,
whether it is a species, subspecies or a variety. Ball
(1968) does not even consider it as a variety,
stating that (cit.) ‘‘when the range of the variation
of the Asiatic populations is considered, it is not
possible to separate the European ones’’. In one
century from its discovery, the knowledge of its
distribution in the Velebit and Herzegovinian area
has widened, however its areal is still roughly the
same as it was when first discovered. Known or
new locations of its natural distribution have been
mentioned by Beck–Mannagetta (1927), Bošnjak
(1935, 1936), Degen (1937), Forenbacher (1990),
Fukarek (1962: map 6, 1965, 1975a: map 2, 1975b,
1981: map 1), Kušan (1971: map 1, 1972), Malý
(1930a, b), Šilić (1973, 2002), Volarić–Mršić
(1994). According to these authors, Croatian sibirea is scarce, growing in the mountainous region
of western Balkans. It grows mainly on barely
accessible rocky slopes of the Dinaric Alps, at
altitudes between 800 and 1600 m. In Croatia, it
grows in the region of northern and central parts
of the mountain Velebit, in the plant association of
Seslerio-Ostryetum Horv. & H-ić., and at the edges
of the littoral common beech (Fagus sylvatica L.)
forests. In Bosnia and Herzegovina, its area is
limited to the mountains of Čvrsnica and Čabulja.
There it grows on limestone substrates, at open
rocky surfaces, screes and high mountain pastures
in a specific plant community. It represents an
edge community between the Bosnian pine (Pinus
heldreichii Christ) and common beech forests and
the anthropogenic mountain pastures communities. It usually multiplies by its miniature seeds
which ripen at the end of July and the beginning of
August, or by branch-layering.
In its natural sites, sibirea has been protected
since 1964; in 1997 it was included into the red
list of protected plant species in Croatia (Volarić–
Mršić 1994), but is missing from the last red list
of vascular plants in Croatia (Nikolić and Topić
2005). It is a protected endemic species in Bosnia
and Herzegovina as well but no exact measures
for protection have been proposed so far. Separate specimens of sibirea mostly originating from
the Balkan region are present in dendrological
collections of many botanical gardens worldwide.
Sibirea also grows in Central Asia about
5000 km away, in the area of the central and western Altai Mountains, at approximately 1300 m
altitude, as well as in Western China in the Tianshan massif. The latter species are S. altaiensis
(Laxm.) C. K. Schneider and S. tianschanica
(Krassn.) A. Pojark. According to Kuminova
(1960) sibirea at the Altai Mountains forms specific
plant communities of high steppe and forest steppe,
where it prevails and builds specific communities of
the steppe vegetation. It usually grows in open
spaces and at elevated parts of micro-relief with
high humidity. The greatest obstacle to the normal
development of sibirea lies in the decrease of the
anthropogenic influence, above all in extensive
small cattle farming (mainly sheep and goats)
especially in areas with a small number of plant
individuals. An old plantation of unknown origin
of sibirea also grows in Southern Russia (the
Lipetsk area, approximately 1500 km eastwards
from the Balkan peninsula localities), where it was
used for protection of the soil against erosion.
897
The literature sources have reported that
the genus Sibiraea contains five species growing
in the European part of Southern Russia, Siberia,
Western China and Southeastern Europe.
S. angustata (Rehd.) Hand. Mazz. creates specific
plant communities in the western part of the
Himalayan plateau (in China) and is used for biomass production (Wu 1998). Cuizhi and Alexander
(2003) reported three species for China, while
Krussmann (1978) reported two species, among
which S. laevigata (L.) Maxim. (=S. altaiensis
Schneid.), with two varieties (var. angustata and
var. croatica (Deg.) Beck), and another species
S. tomentosa Diels of a smaller habitus.
The name ‘‘Croatian sibirea’’ has been used in
literature for many years as S. croatica Deg.
(Domac 1994) and its taxonomical classification
based on morphological data has not yet been
precisely established.
Based on the above information we questioned
and discussed:
– The genetic structure of the Croatian sibirea in
the western Balkan region, and its relationship
with populations from Southern Russia and
Southern Siberia.
– The phylogenetic position of the genus Sibiraea
within family Rosaceae as detected by analysis
of internal transcribed spacers (ITS) 1 and ITS2
in nuclear ribosomal DNA (rDNA) gene cluster.
Due to the unique geographical and ecological
distribution of sibirea we consider it to be an
example for a discussion on protection of small
and fragmented populations by maintaining
traditional land use.
Material and methods
Branches with dormant buds in the initial stage
were collected in the natural populations of
Croatian sibirea in Bosnia and Herzegovina at the
mountain Čabulja and in Croatia at the mountain
Velebit (with participation of colleagues from the
Forestry Faculty from Zagreb, Croatia), during
September 2003, and at the end of winter period in
February 2004. The samples of Altaic sibirea were
obtained in its natural population from the area of
Barnaul in Southern Siberia (Altai area, Russia),
and in a plantation from the area of Lipetsk in
Southern Russia (with generous assistance of our
colleagues from the botanical garden in Kiev,
Ukraine) (Table 1).
DNA extraction
DNA was extracted from dormant buds after the
removal of bud scales or from inner part of stem
core using Plant DNeasy Mini Kit (Qiagen). DNA
was re-suspended in pre-warmed, sterile milli-Q
water to the approximate final concentration of
100 ng/ll. The success of extraction was checked
by electrophoresis in 1% agarose gel stained with
ethidium bromide.
PCR amplification
Several pairs of primers (Table 2) and amplification programmes were used since no data exists on
the use of polymerase chain reactions (PCR) with
the genus Sibiraea. We have tried to amplify ITS
regions in genomic DNA and parts of chloroplast
DNA (cpDNA). Primers were selected mainly
from references on amplification of DNA from
Nicotiana tabacum. The amplification reactions
were done using two methods: (a) a standard
procedure as described for higher fungi (White
et al. 1990) in a total reaction volume of 40 ll with
AmplyTaq polymerase (Perkin–Elmer) for amplification of ITS region in genomic DNA or (b) a
modified general method used for amplification of
Sequence-tagged-site (STS) in spruce (Perry and
Table 1. Sampling locations, their geographical co-ordinates, altitudinal span or average elevation and number of samples taken
Population
Location
Longitude
Latitude
Altitude (m)
Number of samples
Čabulja (Bosnia and Herzegovina)
Rosne poljane
Mali Brizovac (Velinac)
Lipecka oblast (plantation) (Russia)
Altai (Russia)
Stanovlianski raion
Barnaul
1749¢60¢¢4
1754¢92¢¢7
1507¢40¢¢7
1507¢51¢¢4
3932¢
8537¢
1343
1427
992
1011
300
Up to 1300
33
Velebit (Croatia)
4351¢42¢¢1
4354¢85¢¢3
4456¢18¢¢2
4456¢24¢¢7
5230¢
4928¢
33
3
5
898
Table 2. The list of primers (and reference for original publications) used for amplification of parts of nuclear or cpDNA with their
sequences and melting temperatures as given by authors
Primer code
ITS1 (nuclear)
ITS4 (nuclear)
ITS5 (nuclear)
ITS5-p (nuclear)
atpB-1 (cpDNAb)
rbcL-1 (cpDNA)
ccmp10-R (cpDNA)
trnHM (cpDNA)
trnG-P (cpDNA)
trnMM (cpDNA)
trnC-f (cpDNA)
trnD-m (cpDNA)
ycf9-P (cpDNA)
rps 14 (cpDNA)
trnT (cpDNA)
trnF (cpDNA)
a
Tma (C) Reference
64
58
59
61
58
55
57
68
65
62
60
56
63
60
56
58
White et al. (1990)
White et al. (1990)
Yulong et al. (1998)
Moller and Cronk (1997)
Chiang et al. (1998)
Chiang et al. (1998)
Weising and Gardner 1999
Heinze 2001
Heinze 1998
Demesure et al. 1995
Demesure et al. 1995
Demesure et al. 1995
Heinze 2001
Doyle et al. 1992
Taberlet et al. 1991
Taberlet et al. 1991
Available at the internet site
http://www.plantbio.berkeley.edu/bruns/primers.html
http://www.plantbio.berkeley.edu/bruns/primers.html
http://www.plantbio.berkeley.edu/bruns/primers.html
not available
http://www.ejournal.sinica.edu.tw/bbas/content/1998/4/bot94-10.html
http://www.ejournal.sinica.edu.tw/bbas/content/1998/4/bot94-10.html
http://www.bfw.ac.at/200/2043.html
http://www.bfw.ac.at/200/2043.html
http://www.bfw.ac.at/200/2043.html
http://www.bfw.ac.at/200/2043.html
http://www.bfw.ac.at/200/2043.html
http://www.bfw.ac.at/200/2043.html
http://www.bfw.ac.at/200/2043.html
http://www.bfw.ac.at/200/2043.html
http://www.bfw.ac.at/200/2043.html
http://www.bfw.ac.at/200/2043.html
Tm – annealing temperature in C.
cpDNA – chloroplast DNA.
b
Bousguet 1998) in a final volume of 25 ll using
AmplyTaq Gold hot start polymerase (Applied
Biosystems).
We have performed amplification in a Perkin–
Elmer 9700 DNA thermocycler with previously
published cycling parameters for amplification
with ITS primers (Kraigher et al. 1995). Cycling
parameters for amplification of chloroplast DNA
(cpDNA) comprised initial denaturation at 95 C
for 5 min, followed by 40 cycles of 1 min at 94 C,
annealing for 2 min at 55 C (or 53–63 C if
optimisation of reaction was necessary), and an
extension for 3 min at 72 C, with the final
extension at 72 C for 10 min and storage at 4 C.
Controls lacking DNA were run for each experiment to check the contamination of reagents.
Positive controls were run only for ITS1 and ITS4
pair of primers using fungal DNA.
The amplification product was separated by
electrophoresis in agarose gels containing 2% LE
Agarose (SeaKem) run in 1 Tris–borate–EDTA
buffer for 1 h at 5 Vcm)1. The DNA was stained
with ethidium bromide, visualised under ultraviolet light (302 nm) and recorded on Polaroid 667
black and white film. The lengths of amplification
products were estimated by comparison to GeneRuler 100 bp DNA Ladder (Fermentas).
Restriction fragment length polymorphism analysis
and data evaluation
Aliquots of amplified DNA from PCR yielding
only a single fragment of expected length (as estimated from length of the product in Nicotiana
tabacum or basidiomycete – fungi) were digested
separately with 1 unit of each restriction enzyme
according to the manufacturers recommendations
in 10 ll total volume. The amplified ITS regions in
genomic DNA were cut by Hinf I (Promega), Mbo
I (Promega) and Taq I (TaKaRa). PCR products
from cpDNA were cut by Hinf I (Promega),
Hae III (Fermentas) and Alu I (Fermentas or
TaKaRa). The enzymes were chosen on the basis
of economic criteria (for cpDNA) and after previously published sequences of the ITS region
(Kåren et al. 1997).
The restriction fragments were separated in a
2% agarose gel in Tris–borate–EDTA buffer for
3 h at 10 Vcm)1. The length of restriction fragments was estimated by comparison to GeneRuler
100 bp DNA Ladder (Fermentas).
Gels were stained in ethidium bromide, acquired by GelDoc system (Biorad) and analysed
with Taxotron software (Pasteur Institute 1998,
Paris, France) (Grimont 1998). The procedure is
899
described in detail in Grebenc et al. (2000) and
Martı́n and Kårén (2000).
Sequencing
Prior to sequencing, the amplification products
were cleaned using the Wizard SV Gel and PCR
Clean-Up System (Promega). Both strands of the
amplified DNA were sequenced separately using
primers mentioned above. When weak or more
than one PCR product was obtained in PCR, the
products of the desired length were cleaned from
the agarose gel previously to sequencing them
using the same purification kit. DNA was sequenced by commercially available sequencing
service (Sequiserve, Germany). Altogether, nine
samples were sequenced, three from each population in Croatia and Herzegovina, two from Altai
and one from the Lipetsk population. We have
sequenced part of the genomic DNA using primers
ITS5-p and ITS4 and parts of cpDNA using ccmp
10-R, trnHM, trnG-P and trnMM primers.
Sequence Navigator Software (Applied Biosystems) was used to identify the consensus
sequence from the two strands of each isolate.
Dialign 2 software was used for multiple alignment
of obtained sequences (Morgenstern et al. 1998)
with minor manual corrections. The obtained
sequences have been lodged in The European
Molecular Biology Laboratory database. From
the same database, sequences from different genera
from family Rosaceae were selected in a manner to
represent all tribes in the family. ‘‘SEQAPP’’
software for multiple sequences was used to search
for the best alignment. Where ambiguities in the
alignment occurred, the alignment chosen was the
one generating the fewest potentially informative
characters. Alignment gaps were marked with ‘‘-’’,
unresolved nucleotides and unknown sequences
were indicated with ‘‘N’’.
The phylogenetic analysis
Using the programme PAUP* Version 3.11 for
Macintosh (Swofford, 2002) heuristic search for
most parsimonious trees and bootstrap analysis
was done for the full length of ITS1 and ITS2
regions in rDNA. All characters were treated as
unordered. Gaps were treated as characters in this
study because they may contain phylogenetic
information and provide particularly clear
indications of relationship (Lloyd and Calder
1991; Revera and Lake 1992; Baldwin 1993). First
a maximum parsimony analysis (MP) under heuristic search was done; non-parametric bootstrap
support (Felsenstein 1985) for each clade was tested based on 10,000 replicates, using the fast-step
option. The consistency index (CI; Kluge and
Farris 1969), retention index (RI; Farris 1989), and
re-scaled consistency index (RC; Farris 1989) were
obtained from PAUP*.
For all genera used in the phylogenetic comparison with the newly obtained sequences of
Sibiraea the sequences were retrieved from public
available databases with references therein: Agrimonia eupatoria L., U90798; Aremonia agrimonoides (L.) D.C., U90799; Cotoneaster lacteus W.W.
Smith., U16188; Crataegus mollis (Torr. and A.
Gray) Scheele, U16190; Cydonia oblonga Mill.,
AF186531; Dryas octopetala L., U90804; Fragaria
moschata Duchesne, AF163505; Fragaria vesca L.,
U90793; Fragaria vesca subsp. vesca forma alba
(Ehrh.) Staudt., AF163516; Fragaria vesca L.
subsp. vesca, AF163515; Fragaria ananassa
Duchesne, AF164494; Filipendula ulmaria (L.)
Maxim., U90783; Filipendula vulgaris Moench,
AJ416467; Geum montanum L., AJ302350; Geum
rivale L., AJ302352; Malus sieversii (Ledeb.) M.
Roem., AF186491; Malus domestica Borkh.,
U16195; Mespilus germanica L., U16196; Potentilla dickinsii Back., U90785; Potentilla fruticosa
L., AF163478; Prunus armeniaca L., AF318756;
Prunus avium L., AF318737; Prunus cerasus L.,
AF318729; Prunus domestica L., AF318713; Prunus persica (L.) Batsch, AF318741; Pyrus pyrifolia
(Burm. f.) Nakai., AF287240; Rosa canina L.,
AB019495; Rosa persica Michx, AJ416468; Rubus
caucasicus Focke, AY083371; Rubus idaeus L.,
AF055756; Rubus neomexicanus Gray, AY083358;
Sanguisorba officinalis L., AY635040; Sorbus aucuparia L., U16204; Sorbus torminalis (L.) Crantz,
AF086533; Spiraea cantoniensis Lour, AF318722;
Spiraea vanhouttei (Briot) Zabel, U16205;
Waldsteinia geoides Willd., AJ302362.
Results
In total, 16 primers in nine combinations of primer
pairs were used for molecular analysis of studied
populations. For three primer pairs, amplification
was too weak or not successful at all while two
900
primer pairs yielded multiple bands. These products
were not used in further analysis. For PCR resulting
in one clear DNA fragment within or between
population differences in the size of the amplified
DNA product was not observed except for primer
pair ccmp 10-R and trnHM with DNA fragment of
280 bp in all samples from population Velebit and
for a few samples from population Altai compared
to all other samples with 274 bp (Table 3).
After the successful PCR resulting in a single
clear band, the DNA from all 74 samples was cut.
The restriction analysis and separation in agarose
gel showed no differences in length of the digested
cpDNA between or within populations. Sequencing confirmed minor variability between populations. The sequenced product using trnGP
and trnMM primer pair showed no differences
within 246 bp long sequence (accession numbers
AJ878754–AJ878762). Basic local alignment
search tools results (Altschul et al. 1997) for the
sequence showed a close similarity to Prunus zippeliana cpDNA trnG–trnM intergenic spacer region (E=127, bit value=1e)26, accession
number=AB111589) indicating an amplification
of the correct part of the chloroplast genome. Sequences obtained with ccmp 10-R and reverse
primer trnHM were 274 or 280 bp long with 6 bp
duplication of GTATAT motif 215 bp down-
stream from 5¢ end (accession numbers AJ878763–
AJ878770). Duplication was present in all samples
from Velebit and one sample from Altai. Both
sequences gave identical results after BLAST and
were similar to Vigna angularis chloroplast S10B
operon (E=92, bit value=7e)16, accession number AF536225). The 6 bp long insertion was not
detectable on our agarose gels.
ITS regions and 5.8S in genomic rDNA for the
same samples were sequenced (accession numbers
AJ876553–AJ876562). All sequences were 762 bp
long using ITS5-p and ITS4 primers after deleting
the unreadable first part of the sequence. The sequences showed inaccuracy or heterogeneity in
bases 57, 630 and 666 downstream from 5¢ end of
ITS1 independently of the origin (population) of
the sample. From these nucleotide positions, the
sequence could not be unequivocally read since
two bases gave approximately the same intensity
of the signal in electrophoregram.
We have included only one sequence of the ITS
region of sibirea in phylogenetic analysis based on
ITS1 and ITS2 spacers in rDNA with selected
Rosaceae species obtained from public available
databases (GenBank). We have constructed an
unrooted bootstrap consensus phylogenetic tree
with a Rohlf’s consistency index 0.741 (Figure 1
left) and an unrooted phylogenetic tree with a
Table 3. PCR amplification and length of restriction fragments [bp] for all positive reactions from different populations
Primer pair
ITS1, ITS4
ITS5, ITS4
ITS5-p, ITS4
ccmp10-R,
trnHM
trnG-P, trnMM
trnC-f, trnD-m
ycf9-P,
rps14Doyle
atpB-1, rbcL-1
trnT, trnF
PCR quality
or length of
product [bp]
Restriction fragments of the PCR products [bp]
Hinf I
Mbo I
Taq I
Alu I
Hae III
weak
amplification
multiple bands
773
/a
/
/
/
/
/
173, 157, 151,
128, 83, 67
140, 127,
7 (146,127,7)b
240
(no restriction)
/
/
/
582, 160
/
347, 260, 75, 60
/
/
/
/
274 (280)b
(no restriction)
240
(no restriction)
/
880, 150, 70
/
/
/
274 (280)b
(no restriction)
240
(no restriction)
/
850, 180, 120
/
/
/
/
/
/
/
/
274 (280)b
(no restriction)
240
(no restriction)
/
1150
(no restriction)
/
/
274 (280)b
240
multiple bands
1150
no amplification
weak
amplification
/
We have used common names for primers.
a
/Reaction not performed.
b
For samples from population Velebit and few samples from population Altai.
901
Bootstrap
100
100
97
100
96
89
99
65
98
88
75
95
61
100
100
98
96
100
75
100
52
100
100
100
50
100
62
100
98
SIB
SPICAN
SPIxVAN
FILVUL
FILULM
WALGEO
GEURIV
GEUMON
RUBCAU
RUBIDE
RUBNEO
POTDIC
ROSPER
ROSCAN
SANOFF
FRAVES
FRAVESALB
FRAVESVES
FRAxANA
FRAMOS
POTFRU
AGREUP
AREAGR
DRYOCT
PRUARM
PRUDOM
PRUPER
PRUAVI
PRUCER
CYDOBL
SORTOR
PYRPYR
MALSIE
MALxDOM
SORAUC
CRAMOL
MESGER
COTLAC
SIB
SPICAN
SPIxVAN
CYDOBL
SORTOR
PYRPYR
MALSIE
MALxDOM
SORAUC
CRAMOL
MESGER
COTLAC
PRUARM
PRUDOM
PRUAVI
PRUCER
PRUPER
DRYOCT
FILVUL
FILULM
WALGEO
GEURIV
GEUMON
RUBCAU
RUBIDE
RUBNEO
POTDIC
ROSPER
ROSCAN
SANOFF
AGREUP
AREAGR
FRAVES
FRAVESALB
FRAVESVES
FRAxANA
FRAMOS
POTFRU
Figure 1. Phylogenetic tree for selected genera from family Rosaceae. Left – consensus tree of 100 most maximum parsimony trees
with bootstrap values above branches. Right – maximum likelyhood tree (site variation with equal rates from all sites). Abbrevations:
AGREUP, Agrimonia eupatoria L.; AREAGR, Aremonia agrimonoides (L.) D.C.; COTLAC, Cotoneaster lacteus W.W. Smith.;
CRAMOL, Crataegus mollis (Torr. and A. Gray) Scheele; CYDOBL, Cydonia oblonga Mill.; DRAOCT, Dryas octopetala L.;
FRAMOS, Fragaria moschata Duchesne; FRAVES, Fragaria vesca L.; FRAVESALB, Fragaria vesca subsp. vesca forma alba (Ehrh.)
Staudt.; FRAVESVES, Fragaria vesca L. subsp. vesca; FRAxANA, Fragaria ananassa Duchesne; FILULM, Filipendula ulmaria (L.)
Maxim.; FILVUL, Filipendula vulgaris Moench; GEUMON, Geum montanum L.; GEURIV; Geum rivale L; MALSIE, Malus sieversii
(Ledeb.) M. Roem.; MALxDOM, Malus domestica Borkh.; MESGER, Mespilus germanica L.; POTDIC, Potentilla dickinsii Back.;
POTFRU, Potentilla fruticosa L.; PRUARM, Prunus armeniaca L.; PRUAVI, Prunus avium L.; PRUCER, Prunus cerasus L.;
PRUDOM, Prunus domestica L.; PRUPER, Prunus persica (L.) Batsch; PYRPYR, Pyrus pyrifolia (Burm. f.) Nakai.; ROSCAN, Rosa
canina L.; ROSPER, Rosa persica Michx; RUBCAU, Rubus caucasicus Focke; RUBIDE, Rubus idaeus L.; RUBNEO, Rubus neomexicanus Gray; SANOFF, Sanguisorba officinalis L.; SIB, Sibiraea altaiensis (Laxm.) C. K.Schneid. var. croatica (Degen) Beck;
SORAUC, Sorbus aucuparia L.; SORTOR, Sorbus torminalis (L.) Crantz; SPICAN, Spiraea cantoniense Lour; SPIxVAN, Spiraea
vanhouttei (Briot) Zabel; WALGEO, Waldsteinia geoides Willd.
902
consistency index 0.618, a retention index 0.825
and a re-scaled consistency index 0.548 (Figure 1
right). Alignment and phylogenetic trees are
deposited in Treebase – http://www.treebase.org.
We have performed the phylogenetic analysis only
to determine the phylogenetic position of the genus
Sibiraea towards other genera in the family Rosaceae and not to infer the phylogenetic position and
distances of other genera (Alice and Campbell
1999; Lee and Wen 2001; Eriksson et al. 2003).
Discussion
The origin of the disjunct populations of sibirea
During the Tertiary period, larger areas of
Southeastern Europe, Southern Siberia and western China had been covered by steppe and steppe
forests (Sitte et al. 1991; Adams 2002), and in the
Quaternary period they achieved their maximum
span. In this period, the areas from the Adriatic
Sea to Eastern Siberia were relatively compact and
comprised of steppe and forest steppe with their
characteristic floral elements, which can still be
found in isolated areas of the Southern Siberia.
Kuminova (1960) described these specific plant
communities of the high steppe and the forest
steppe. Sibirea from the Altai Mountains is one of
the most significant species of the steppe and the
forest steppe plant communities, and as such it
must have been widely distributed during the
Tertiary period. After the climatic changes of the
Pleistocene the populations were separated into
isolated islands, i.e. into disjunct areas. Thus, the
Southeastern European sibirea became isolated at
the mountains of Velebit, Čabulja and Čvrsnica
comprising small subpopulations with relatively
low number of individuals. In Croatia, the sibirea
bushes appear in a plant community Seslerio-Ostryetum Horv. & H-ić. (Volarić–Mršić 1994), and
in Bosnia and Herzegovina in separate plant
communities in high mountain areas at broken
limestone grounds, that have not been entirely
investigated yet, and that by their morphology
partially resemble the steppe forests. At least for
Southern Russia localities it is known where sibirea was artificially introduced for handcraft purposes and soil protection (Melnik and Janjić
personal communication). Therefore, the present
distribution in Southeastern Europe and Southern
Siberia most probably represent only the remains
of its initial large area from the Tertiary and the
Quaternary period, supposedly being the Glacial
refugia that were created during the last glaciation.
However, the paleontological analyses of the fossil
pollen is not available to prove this assumption.
The close taxonomical affinity of the very
distant Croatian and South Siberian Sibiraea
‘‘croatica’’ and S. altaiensis populations are not
the only case of such a geographical gap. Well
known are the big disjunctions in the genera
Scopolia (1 species in Southeast Europe and 4 in
East Asia) and Pseudostellaria (1 species on the
southern/southeastern foothills of the Alps and 10
more in Middle/Eastern Asia) (Schaeftlein 1969).
In the latter case, climate changes in Pleistocene
and post-Pleistocene period in the Near East, with
the subsequent areal drainage, is assumed
(Schaeftlein 1969: 879). These and other similar
cases are, of coarse, examples for mesophytic taxa,
while Sibiraea has a more xerophytic character,
although, comparable to Scopolia and Pseudostellaria, with a higher precipitations in their
distribution areas, while as a taxon with the
steppe-character the genus Leontopodium can be
discussed. It has about 30–40 species in Eurasia,
mostly in Inner-Asia, and only two of them grow
in Europe (Wagenitz 1965).
The migration of the European taxa from Asia
is supposed to have happened in the early periods
of the Pleistocene, most presumably through the
lowlands (because they are lacking in Caucasus and
the mountains of the Near East) (Wagenitz 1965).
However, it is perhaps not necessary to search for
the explanation of the recent distribution of Sibiraea ‘‘croatica’’ in the Pleistocene and the postPleistocene climate and vegetation history, since
the distribution patterns of the genera Scopolia,
Pseudostellaria, and Sibiraea, seem to be much
older, going back to the Tertiary period. Additionally to the previous cases, one could mention
the genus Degenia, which as Sibiraea ‘‘croatica’’
occurs in the Velebit Mountains. Its only species,
Degenia velebitica, was first described as a species
of the geographically very remote North-American
(!) genus Lesquerella (Degen 1909).
Little or no variation in cpDNA and ITS sequences
The non-coding regions in cpDNA show a relatively slow rate of evolution and can be used for
903
differentiation between species and populations
(Small et al. 1998). We have used six pairs of
primers for amplification of parts of non-coding
regions in cpDNA, general primers made for the
amplification of atpB–rbcL genes spacer in mosses
(Chiang et al. 1998) and primers made for tobacco
(see Table 2). The selected primers were not
designed specifically for amplification in sibirea
nor were they modified. Only the PCR was optimised for pairs with no or weak amplification. The
amplified products showed no differences between
population in restriction patterns (data gathered in
Table 3) though the PCR in combination with
restriction fragment length polymorphism (PCR–
RFLP) method is well known to separate most
species and some populations (White et al. 1990;
Kåren et al. 1997; Horton 2002). Only a minor
difference of 6 bp duplication in DNA amplified
with ccmp 10-R and trnM primer pair was noticed.
The duplication could be the result of a simple
insertion in non-coding region and was observed
in two geographically distinct populations. Analysed regions in cpDNA represent only a small part
of the genome. Polymorphic regions in cpDNA
can never be predicted, but since one polymorphic
fragment was discovered further analysis of genetic diversity in sibirea should follow. For this, a
selection of markers can be based on nuclear
markers for Rosaceae since it has been demonstrated that molecular marker tools developed in
one species (e.g. peach) are easily applied to other
species in the family (Dirlewanger et al. 2004; Jung
et al. 2004).
The lack of differences in the restriction pattern
for the ITS region in genomic rDNA indicates that
all samples of sibirea belong to the same species
since the ITS region was proven to be conserved
within one taxonomic species. The observed substitutions or inaccuracies in the sequence of the
same region gave a 0–2 bp difference between
different sequences of ITS comparable to the differences observed within the Fragaria vesca complex with a 0–3 bp difference (Figure 1). Almost
identical sequences can indicate a very recent
separation and possible speciation of the studied
populations due to the recent colonisation event as
observed in species of Adenocarpus (Percy et al.
2002). Vogler and DeSalle (1994) have discussed
that the main evolution force for ribosomal region
was the concerted evolution, which should lead to
homogenisation of individual repeats and produce
mostly a uniform sequence in all repeats of a given
species. The concerted evolution might be a
problem if there are instances of allopolyploidy
but we predicted Sibiraea spp. to be diploid. Besides this, conservation of ribosomal genes
(including the ITS regions) was shown and used
together with maternally inherited cpDNA regions
for phylogenetic analysis and species definition in
many plant groups (Alice and Campbell 1994; Lee
and Wen 2001) as well as in other organisms such
as higher fungi, where we can observe some variation within a morphological species (Agerer et al.
1996; Kåren et al. 1997; Horton 2002 and others).
Within species, variability could explain inaccuracies observed in some parts of the ITS sequences. Point mutations could also occur and
remain fixed within single species/sample resulting
in heterozygocity of the ITS region (or at least in
some copies of the rDNA cluster resulting in heteroduplexes). The persistence of such mutations in
the populations can be explained by small, disjunct
populations with high probability of self-crossing,
self-pollination and vegetative reproduction by
layering. Similar polymorphic nucleotide sites
explained as superposition of two or more distinct
ITS repeats within one or more ribosomal gene
clusters were also observed and proven to persist
in several taxa of Amelanchier showing common
asexual seed production. The authors propose
four possible reasons for persistence of polymorphism: within individual polymorphism as a
consequence of transition stage in concerted
evolution, interspecific hybridisation, evolution of
pseudogenes and disruption of concerted evolution due to non-homologous position of rDNA
loci (Campbell et al. 1997).
Sibirea’s closest relatives were the few
sequenced species from the genus Spiraea, supporting the position of the genus Sibiraea as
determined after morphological characteristics
within the tribe Spiraeae and next to the tribe
Malonideae (Kubitzki 2004).
In studies of species from the genus Zelkova
two endemic species, Zelkova abelicea (Lam.)
Boisser and Zelkova sicula Di Pasquale, differ
only in 1 bp in trnL intron and no within population variation was detected using different
chloroplast markers (Fineschi et al. 2004).
According to the observed similarity of ITS1, 5.8S
and ITS2 regions in genomic rDNA clusters
amplified from Croatian sibirea populations col-
904
lected at Čabulja (Bosnia and Herzegovina) and
Velebit (Croatia) and Altaic sibirea from both
locations in Southern Russia we cannot confirm
the existence of Croatian sibirea (S. croatica) as a
separate species. However, it must be stressed that
the lack of molecular evidence for considering
Croatian sibirea as a separate species should not
reduce the importance of its conservation in
the regional flora in the Southeastern European
region (Western Balkan mountains) where it is a
rare relic.
The process of evolution governing selection
in populations and mutation changes in small
disjunct populations that would bring about the
differentiation of populations is not well known
(as reported by Savolainen and Kuittinen 2000).
How the selection acts in a similar phenomenon of
small isolated populations can be exemplified in
Abies nebrodensis (Lojac.) Mattei where the selection favours heterozygotes (Vicario et al. 1995). A
similar phenomenon was recorded for the populations of Pinus leucodermis Ant. as reported by
Boscherini et al. (1994) and in subpopulations of
Picea abies (L.) Karst. at extreme growing site
conditions (Božič and Urbančič 2003). While in
sibirea the analysed gene pool was homogeneous
as observed in genus Zelkova (Fineschi et al.
2004), which is possibly due to the low number of
individuals in subpopulations, to the specificity of
its androdioecious flowers or possibly to a bottleneck effect after the separation of ancient population after the retrieval of glaciers.
Conservation measures for small disjunct
populations in the Southeastern Europe
Many areas where sibirea is found naturally are
already under some kind of protection. The region
of Sichuan, Huanglong (Yellow-Dragon) in China
is protected as a natural monument (Wenhua and
Xianying 1989) but sibirea is not included in the
list of species of interest for their rarity, endemism
or ornamental and medical value (World Heritage
Convention 1991) nor is it included in the World
Conservation Union (IUCN) Red list of threatened species (IUCN 1998). For the area of Velebit, some plant associations with several endemic
species (including S. croatica) are included in the
IUCN Biosphere reserve with strict protection of
the area of Paklenica National park and in the
Croatian Act on Protection of Nature (2003). No
specific measures for active protection of sibirea
are given in any document although the species
would demand an active protection.
There is no data about the evolution of sibirea,
or about its populations from Tertiary or postglacial periods. At least Southeastern European
populations were until recently maintained
by extensive small cattle farming or extreme,
inaccessible rocky habitats thus providing suitable
conditions for seed germination and reducing
competitive species in areas suitable for sibirea,
similar to steppe and forest steppe but with a
higher precipitation. Sibirea plants are in general
less or not of interest for small cattle, possibly due
to their thick leaves with dissuading secondary
substances (Zhang et al. 1993). In many mountainous and subalpine rural areas in Southeastern
Europe resembling forest steppe, extensive small
cattle farming was abandoned in the last decades
for economical and political reasons leading to
strong natural reforestation of many anthropogenic-grasslands and steppe habitats. The main
competitor of sibirea in its natural habitats in
Southeastern European localities are either Bosnian (P. heldreichii, Šilić 2002) or Austrian pine
(P. nigra, Kušan 1971) and common beech causing
serious reduction in growth of sibirea plants and
finally its disappearance if trees are not removed
(usually by grazing).
Owing to its weak ecological competitiveness,
the local sibirea subpopulations could become
periodically extinct due to the re-colonisation of its
sites by neighbouring tree populations. Fragmented populations are in a high risk of being
permanently lost from small reserves.
Therefore, greater attention needs to be given
to active management of autochthonous sibirea
genetic resources in existing protected areas in
order to ensure the maintenance of specific habitats of the species. The solution for preservation of
naturally occurring habitats of the species would
be either in the retention of traditional extensive
small cattle farming in areas where sibirea occurs
naturally thus protecting the specific anthropogenic influence or through removal of competitive
species which directly threaten the existing plants.
None of the proposed measures have been
considered or officially undertaken so far.
905
Acknowledgements
This research was part of the bilateral project BIBA/04-05-010 between Bosnia-Herzegovina (University of Sarajevo, Faculty of Forestry) and the
Republic of Slovenia (Slovenian Forestry Institute
(SFI)), the research programme P4-0107 of the
SFI, financed by the Ministry for Higher education, Science and Technology of the Republic of
Slovenia and its Young Researchers Scheme (TG).
We would like to thank Dr. Monika Konnert,
ASP Teisendorf, for advice on primers used in this
study and for suggestions and corrections of the
manuscript, Dr. Josip Franjić, Faculty of Forestry,
University of Zagreb, Croatia, for help in collection of samples, colleagues from the National
Botanical Garden in Kiev, Ukraine and to anonymous reviewers for very constructive suggestions
for the improvement of the text.
References
Act on Protection of Nature (2003) Regulation on collecting of
plants for purposes of remaking, trading and other kinds of
tradings. Narodne novine 162/30 (in Croatian).
Adams J (2002) Global land enviroments since the last interglacial, Europe during the last 150,000 years. Available
from: http://www.members.cox.net/quaternary/nercEUROPE.html (accessed June 2004).
Agerer R, Kraigher H, Javornik B (1996) Identification of ectomycorrhizae of Hydnum rufescens on Norway spruce and
the variability if the ITS region of H. rufescens and H. repandum (Basidiomycetes). Nova Hedwigia, 63(1–2), 183–194.
Alice LA, Campbell CS (1999) Phylogeny of Rubus (Rosaceae)
based on nuclear ribosomal DNA internal transcribed spacer
region sequences. Am. J. Bot., 86, 81–97.
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z,
Miller W, Lipman DJ (1997) Gapped BLAST and PSIBLAST: A new generation of protein database search programmes. Nucleic Acids Res., 25, 3389–3402.
Baldwin BG (1993) Molecular phylogenetics of Calycadenia
(Compositae) based on its sequences of nuclear ribosomal
DNA: Chromosomal and morphological evolution reexamined. Am. J. Bot., 80, 222–238.
Ball PW (1968) Sibiraea Maxim. In: Flora Europaea (eds. Tutin
TG et al.) Vol. 2. Cambridge University Press, Cambridge,
pp. 6.
Beck-Mannagetta G (1927) Flora Bosnae, Hercegovinae et
regionis Novipazar III. Choripetalae (finis). Posebna izdanja
SKA 63, prirodn. i matem. spisi (Beograd-Sarajevo), 15,
1–487.
Boscherini G, Morgante M, Rossi P, Vendramin GG (1994)
Allozyme and chloroplast DNA variation in Italian and
Greek population of Pinus leucodermis. Heredity, 73, 284–
290.
Bošnjak K (1935) Čabulja planina. Hrvat. planinar (Zagreb),
31, 76–82, 99–107.
Bošnjak K (1936) Iz hercegovačke flore. Glas. Hrvat. prir.
drusˇtva, 41–48, 17–70.
Božič G, Urbančič M (2003) The morphological and genetical
characterisation of native Norway spruce (Picea abies (L.)
Karst.) population in the area of Pokljuka mire. Acta Biol.
Slov., 46(1), 17–25.
Campbell SC, Wojciechowski MF, Baldwin BG, Alice LA,
Donoghue MJ (1997) Persistent nuclear ribosomal DNA
sequence polymorphism in the Amelanchier agamic complex
(Rosaceae). Mol. Biol. Evol., 14, 81–90.
Chiang TY, Schaal BA, Peng CI (1998) Universal primers for
amplification and sequencing a noncoding spacer between
the atpB and rbcL genes of chloroplast DNA. Bot. Bull.
Acad. Sinica, 39, 245–250.
Cuizhi G, Alexander C (2003) 2. SIBIRAEA Maximowicz,
Trudy Imp. Flora of China. S.-Peterburgsk-Bot. Sada 6:213.
1879, 9, 73–74.
Degen A (1905) Bemerkungen über einige orientalische
Pflanzenarten. XLV. Ueber das spontane Vorkommen eines
Vertreters der Gattung Sibiraea in Südkroatien und in der
Hercegovina. Magyar Bot. Lapok / Ungarische Botan.
Blätter, Budapest, 8/11, 245–259.
Degen A (1906) Remarques sur quelques Plantes rares. Extrait
du Bulletin de l’Association Pyrénéenne pour l’echange des
Plantes. Seizième année 1905 – 1906, p. 1–4.
Degen A (1909) Megjegyzések néhány keleti növényfajrol LIII.
A Lesquerella nemzetség egyik képviselöjének a Velebit hegységben törtent felfedezéséröl. Magyar Bot. Lapok, 8, 3–24.
Degen A (1937) Flora Velebitica, 2, 241–243. Verlag der Ungarischen Akademie der Wissenschaften, Budapest.
Demesure B, Sodzi N, Petit RJ (1995) A set of universal primers
for amplification of polymorphic non-coding regions of
mitochondrial and chloroplast DNA in plants. Mol. Ecol., 4,
129–131.
Dirlewnger E, Graziano E, Joobeur T, Garriga-Calderé F,
Cosson P, Howad W, Arús P (2004) Comparative mapping
and marker-assisted selection in Rosaceae fruit crop. PNAS,
101, 9891–9896.
Domac R (1994) Flora Hrvatske: Prirucˇnik za odred-ivanje bilja.,
Školska knjiga, Zagreb pp. 504.
Doyle JJ, Davis JI, Soreng RJ, Garvin D, Anderson MJ (1992)
Chloroplast DNA inversions and the origin of the grass
family (Poaceae). PNAS, 89, 7722–7726.
Eriksson T, Hibbs MS, Yoder AD, Delwiche CF, Donoghue
MJ (2003) The phylogeny of Rosoideae (Rosaceae) based on
sequence analysis of the internal transcribed spacers (ITS) of
nuclear ribosomal DNA and the trnL/F region of chloroplast DNA. Int. J. Plant Sci., 164(2), 197–211.
Farris JS (1989) The retention index and the rescaled consistency index. Cladistics, 5, 417–419.
Felsenstein J (1985) Confidence limits on phylogenies: An
approach using the bootstrap. Evolution, 39, 783–791.
Fineschi S, Cozzolino S, Migliaccio M, Vendramin GG (2004)
Genetics variation of relic tree species: The case Mediterranean Zelkova abelicea (Lam.) Boisser and Z. sicula Di Pasquale, Garfi and Quézel (Ulmaceae). For. Ecol. Manage.,
197(1–3), 273–278.
Forenbacher S (1990) Velebit i njegov biljni svijet XV, Školska
knjiga, Zagreb pp. 800.
906
Fukarek P (1962) Granice raširenja izrazitih flornih elemenata u
vegetaciji Hercegovine. Geografski pregled (Sarajevo), 6, 73–
96.
Fukarek P (1965) Rijetko šumsko drveče naše republike.
Narodni sˇumar (Sarajevo, 19, 125–134.
Fukarek P (1975a) Unterschiede in der Dendroflora der westlichen und östlichen Gebiete der Balkanhalbinsel. Problems
of Balkan flora and vegetation (Sofia), 146–161.
Fukarek P (1975b) Prilozi dendroflori Dalmacije, Hercegovine i
Crne Gore. Biosistematika (Beograd), 1(1), 61–67.
Fukarek P (1981) Endemne i rijetke vrste drveća i grmlja dinarskog područja i njihova introdukcija na područje Biokova. Acta biokovica (Makarska), 1, 169–188.
Genaust H (1996) Etymologisches Wörterbuch der botanischen
Pflanzennamen, 3rd edn. Birkhäuser Verlag, Basel, Boston,
Berlin.
Grebenc T, Piltaver A, Kraigher H (2000) Establishment of the
PCR–RFLP library for Basidiomycetes, Ascomycetes and
their ectomycorrhizae on Picea abies (L.) Karst. Phyton,
Ann. rei Bot., 40(4), 79–82.
Grimont PAD (1998) Taxotron User’s Manual, Institute Pasteur, Paris pp. 1–128.
Heinze B (1998) PCR-based chloroplast DNA assays for the
identification of native Populus nigra and introduced poplar
hybrids in Europe. For. Genet., 5(1), 31–38.
Heinze B (2001) A database for PCR primers in chloroplast
genome. Federal Forest Research Centre – Institute of
Forest Genetics, Vienna, Austria. Available from: http://
www.bfw.ac.at/200/2043.html (accessed February 2005.).
Horton TR (2002) Molecular approaches to ectomycorrhizal
diversity studies: Variation in ITS at a local scale. Plant Soil,
244, 29–39.
http://www.ejournal.sinica.edu.tw/bbas/content/1998/4/bot94–
10.html (Botanical Bulletin of Academia Sinica; accessed
December 2005.).
http://www.plantbio.berkeley.edu/bruns/primers.html (The
Bruns lab; accessed December 2005.).
IUCN (World Conservation Union) (1994) Guidelines for Protected Areas Management Categories, IUCN-WCPA and
WCMC, Gland, Switzerland.
IUCN (World Conservation Union) (1998) 1997 IUCN Red
List of Threatened Plants. In: Compiled by WCMC (ed.
Walter KS, Gillett HJ), IUCN, Publication Service Unit,
Cambridge.
Jung S, Jesudurai C, Staton M, Du Z, Ficklin S, Cho I, Abbott
A, Tomkins J, Main D (2004) GDR (Genome Database for
Rosaceae): Integrated web resources for Rosaceae genomics
and genetics research. BMC Bioinformatics, 5, 130.
Kåren O, Hogberg N, Dahlberg A, Jonsson L, Nylund JE
(1997) Inter- and intra-specific variation in the ITS region of
rDNA of ectomycorrhizal fungi in Fennoscandia as detected
by endonuclease analysis. New Phytol., 136, 313–325.
Kluge AG, Farris JS (1969) Quantitative phyletics and the
evolution of anurans. Syst. Zool., 18, 1–32.
Kraigher H, Agerer R, Javornik B (1995) Ectomycorrhizae of
Lactarius lignyotus on Norway spruce, characterised by
anatomical and molecular tools. Mycorrhizae, 5, 175–180.
Krussmann G (1978) Handbuch Der Laubgeholze, Band III, Pru
- Z, Verlag Paul Parey, Berlin Und Hamburg.
Kubitzki K(ed.) (2004) Flowering Plants. Dicotyledons Celastrales, Oxalidales, Rosales, Cornales, Ericales Series: The
Families and Genera of Vascular Plants, Vol. 6 Springer,
Köln.
Kuminova AV (1960) Rastiteljnii pokrov Altaia – Novosibirsk.
p. 450. Izd-bo SO AN SSSR.
Kušan F (1971) Novo nalazište svojte Sibiraea laevigata subsp.
croatica Degen na Velebitu. Acta Bot. Croat., 30, 131–134.
Kušan F (1972) Novo nalazište sibireje na Velebitu. Priroda
(Zagreb), 61, 97–98.
Lee C, Wen J (2001) A phylogenetic analysis of Prunus and the
Amygdaloideae (Rosaceae) using ITS sequences of nuclear
ribosomal DNA. Am. J. Bot., 88, 150–160.
Lloyd DG, Calder VL (1991) Multi-residue gaps, a class of
molecular characters with exceptional reliability for phylogenetic analyses. J. Evol. Biol., 4, 9–21.
Malý K (1930a) Znamenito drveće naše zemlje u riječi i slici.
Glasnik Zemaljskog muzeja u Bosni i Hercegovini, Sarajevo,
42(1), 115–132.
Malý K (1930b) Dendrologisches aus Illyrien. Mitt. Deutsh.
Dendro. Gesell., 42, 127–136.
Martı́n MP, Kårén O (2000) Taxotron and DNA databases to
identification of ectomycorrhizae. In: Protocols (ed. Martı́n
MP), pp. 45–47. Slovenian Forestry Institute, Ljubljana,
Slovenia.
Moller M., Cronk QCB (1997) Origin and relationships of
Saintpaulia (Gesneriaceae) based on ribosomal DNA internal
transcribed spacer (ITS) sequences. Am. J. Bot., 84, 956–965.
Morgenstern B, Frech K, Dress A, Werner T (1998) DIALIGN:
Finding local similarities by multiple sequence alignment.
Bioinformatics, 14, 290–294.
Nikolić T, Topić J (2005) Crvena knjiga vaskularne flore
Hrvatske = Red Book of Vascular Flora of Croatia, Min.
kulture, Državni zavod za zaštitut prirode, Zagreb, Republika Hrvatska.
Percy DM, Cronk QCB (2002) Different fates of island brooms:
Contrasting evolution in Adenocarpus, Genista, and Teline
(Genisteae, Fabaceae) in the Canary Island and Madeira.
Am. J. Bot., 89, 854–864.
Perry DJ, Bousguet J (1998) Sequence-tagged-site (STS)
markers of arbitrary genes: development, characterization
and analysis of linkage in Black Spruce. Genetics, 149, 1089–
1098.
Revera M, Lake JA (1992) Evidence that eukaryotes and eocyte
procaryotes are immediate relatives. Science, 257, 74–76.
Savolainen O, Kuittinen H (2000) Small Population Processes.
In: Forest Conservation Genetics – Principles and Practice
(ed. Young A, Boshier D, Boyle T), pp. 91–100. CABI –
Publishing, Wallingford.
Schaeftlein H (1969) Pseudostellaria Pax in Engler u. Prantl.
HEGI, Ill. Fl. Mitteleur. 2. Aufl., 3/2, Lief. 6, Carl Hanser
Verlag, München pp. 875–883.
Sitte P, Ziegler H, Ehrendorfer F, Bresinsky A (1991) Lehrbuch
der Botanik fur Hochschulen, Gustav Fischer Verlag, Stuttgart-Jena-New York pp. 1–1030.
Small RL, Ryburn JA, Cronn RC, Seelanan T, Wendel JF
(1998) The tortorse and the hare: Choosing between noncoding plastome and nuclear Adh sequences for phylogeny
reconstruction in a recent divergent plant group. Am. J. Bot.,
85, 1301–1315.
Swofford DL (2002) PAUP*. Phylogenetic Analysis Using
Parsimony (*and Other Methods). Version 4, Sinauer Associates, Sunderland Massachusetts.
907
Šilić Č (1973) Atlas šumskog drveća i grmlja. Svjetlost Sarajevo.
Šilić Č (2002) Endemične i rijedke biljke Parka prirode Blidinje,
Matica hrvatska, Ogranak čitluk pp. 280.
Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal
primers for amplification of three non-coding regions of
chloroplast DNA. Plant Mol. Biol., 17, 1105–1109.
Vicario F, Vendramin GG, Rossi P, Liò P, Giannini R (1995)
Allozyme, chloroplast DNA and RAPD markers for determining genetic relationships between Abies alba and the relic
population of Abies nebrodensis. Theor. Appl. Genet., 90(7–
8), 1012–1018.
Vogler AP, DeSalle R (1994) Evolution and phylogenetic
information content of the ITS-1 region in the tiger beetle
Cicindela dorsalis. Mol. Biol. Evol., 11, 393–405.
Volarić-Mršić I (1994) Sibiraea croatica Degen In: Crvena
knjiga biljnih vrsta Republike Hrvatske (ed. Šugar I), pp. 522–
. Ministarstvo graditeljstva i zaštite okoliša, Zavod, za zaštitu prirode Zagreb.
Weising K, Gardner R (1999) A set of conserved PCR primers
for the analysis of simple sequence repeat polymorphisms in
chloroplast genomes of dicotyledonous angiosperms. Genome, 42, 9–19.
Wenhua L, Xianying Z (1989) China‘s Nature Reserves, Foreign
Languages Press, Beijing pp. 1–19.
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and
direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols. A Guide to Methods and
Applications (ed. Innis MA, Gelfand DH, Sninsky JJ, White
TJ), pp. 315–322. Academic Press, San Diego.
World Heritage Convention (1991) World Heritage Convention. Natural Heritage: China. Huanglong Valley. In: Proposal for World heritage nomination, pp. 1–100. Ministry of
Construction, China.
Wu N (1998) The community types and biomass of Sibiraea angustata schrub and their relationship with environmental
factors in northwestern Sichuan. Acta Bot. Sin., 40(9), 860–
870.
Yulong S, Newbury HJ, Ford-Lloyd BV (1998) Identification
of taxa in the genus Beta using ITS1 sequence information.
Plant Mol. Biol. Rep., 16, 147–155.
Zhang CZ, Li C, Feng SL, Zhao CL, Wang WH (1993) Isolation and structure of sibirate from Sibiraea angustata. Yao
Xue Xue Bao, 28(10), 798–800 (in Chinese).
Gregor Bozic