History, genetic differentiation and conservation strategies for disjunct

populations of Sibiraea species from Southeastern Europe and Asia

Dalibor Ballian1,�, Tine Grebenc2,�, Gregor Bozic2,*, Viktor Melnik3, Tone Wraber4 &Hojka Kraigher21Faculty of Forestry, University of Sarajevo, Zagrebacka 20, 71000, Sarajevo, Bosnia and Hercegovina;2Slovenian Forestry Institute, Vecna pot 2, SI, 1000, Ljubljana, Slovenia; 3National Botanical Garden,Academy of Sciences of Ukraine, Timiryazevska 1, 01014, Kiev, Ukraine; 4Department of Biology, Universityof Ljubljana, Vecna pot 111, 1000, Ljubljana, Slovenia (*Corresponding author: Phone: +386-1-200-7800;Fax: +386-1-2573589; E-mail: gregor.bozic@gozdis.si)

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 Croatianand 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 genomicDNA 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 digestedcpDNA 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 wasnoticed in two geographically distinct populations. No differences in the restriction pattern for the ITSregion in genomic rDNA indicates that all samples of sibirea belong to the same species since the ITS regionwas proven to be conserved within one taxonomic species. The minor differences that were obtained supportthe hypothesis that sibirea is an old tertiary relic that shows a minor variability, confirming previouspreliminary results from comparisons of the Croatian and Altaic sibireas at the morphological level. Ourdata 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 thatsurvived the glaciations, it deserves active conservation approaches through support of traditional use ofhigh-mountain pastures for reducing natural reforestation of sibirea ancient sites.

Introduction

Croatian sibirea (Sibiraea croatica Deg.),described by the Hungarian botanist Degen in1905 is a rare and endemic bushy species ofCroatian and Herzegovinian flora where it repre-sents a tertiary relic.

The taxonomy of sibirea has not been entirelyinvestigated yet. The genus name Sibiraea Maxim.derives from the Russian name for the region ofSiberia (‘‘Sibir’’ in Russian) and the ending of itsclosest genus Spiraea (Genaust 1996: 582). Sibi-raea croatica was described in the first part of the20th century as:• Sibiraea croatica Degen, Magyar. Bot. Lapok

4: 255, 1905�The first and second author provided an equal contribution tothe study.

Conservation Genetics (2006) 7:895–907 � Springer 2006DOI 10.1007/s10592-006-9131-z

• Sibiraea laevigata (L., 1771) Maxim., ActaHorti Petrop. 6: 213, 1879 subsp. croatica(Degen) Degen, Fl. Veleb. 2: 241, 1937

• Sibiraea altaiensis (Laxm., 1770)C.K.Schneidervar. 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 lateron Cabulja (Herzegovina) by O. Reiser, gainedconsiderable attention. Both discoveries wereevaluated by the Hungarian botanist A. Degen(1905), who estimated the morphological identityof both plants from the two European sites anddescribed them as Sibiraea croatica. He alsomentioned the high resemblance with the Asianplants and commented on the possibility that theEuropean plants were merely a subspecies of theAsian species, which he mentioned as S. altaiensis(=S. laevigata). He presented the same opinion inthe next publication in 1906, where the speciesname used was S. laevigata (Degen 1906). Only inthe final work, published after his death, was theEuropean plant decisively named S. laevigata (L.)Maxim. subsp. croatica (Degen) Degen (Degen1937).

A few preliminary comparative results from themorphological studies by Bosnian dendrologists,that were obtained by cultivation of the Croatianand Asian sibirea under comparable conditions inBosnia and Herzegovina, show that the differencesbetween them are minimal (Silic, personal com-munication). The taxonomical status of theEuropean 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 variationof the Asiatic populations is considered, it is notpossible to separate the European ones’’. In onecentury from its discovery, the knowledge of itsdistribution in the Velebit and Herzegovinian areahas widened, however its areal is still roughly thesame as it was when first discovered. Known ornew locations of its natural distribution have beenmentioned by Beck–Mannagetta (1927), Bosnjak(1935, 1936), Degen (1937), Forenbacher (1990),Fukarek (1962: map 6, 1965, 1975a: map 2, 1975b,1981: map 1), Kusan (1971: map 1, 1972), Maly(1930a, b), Silic (1973, 2002), Volaric–Mrsic(1994). According to these authors, Croatian sib-irea is scarce, growing in the mountainous regionof western Balkans. It grows mainly on barely

accessible rocky slopes of the Dinaric Alps, ataltitudes between 800 and 1600 m. In Croatia, itgrows in the region of northern and central partsof the mountain Velebit, in the plant association ofSeslerio-Ostryetum Horv. & H-ic., and at the edgesof the littoral common beech (Fagus sylvatica L.)forests. In Bosnia and Herzegovina, its area islimited to the mountains of Cvrsnica and Cabulja.There it grows on limestone substrates, at openrocky surfaces, screes and high mountain pasturesin a specific plant community. It represents anedge community between the Bosnian pine (Pinusheldreichii Christ) and common beech forests andthe anthropogenic mountain pastures communi-ties. It usually multiplies by its miniature seedswhich ripen at the end of July and the beginning ofAugust, or by branch-layering.

In its natural sites, sibirea has been protectedsince 1964; in 1997 it was included into the redlist of protected plant species in Croatia (Volaric–Mrsic 1994), but is missing from the last red listof vascular plants in Croatia (Nikolic and Topic2005). It is a protected endemic species in Bosniaand Herzegovina as well but no exact measuresfor protection have been proposed so far. Sepa-rate specimens of sibirea mostly originating fromthe Balkan region are present in dendrologicalcollections of many botanical gardens world-wide.

Sibirea also grows in Central Asia about5000 km away, in the area of the central and wes-tern Altai Mountains, at approximately 1300 maltitude, as well as in Western China in the Tian-shan 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 specificplant communities of high steppe and forest steppe,where it prevails and builds specific communities ofthe steppe vegetation. It usually grows in openspaces and at elevated parts of micro-relief withhigh humidity. The greatest obstacle to the normaldevelopment of sibirea lies in the decrease of theanthropogenic influence, above all in extensivesmall cattle farming (mainly sheep and goats)especially in areas with a small number of plantindividuals. An old plantation of unknown originof sibirea also grows in Southern Russia (theLipetsk area, approximately 1500 km eastwardsfrom the Balkan peninsula localities), where it wasused for protection of the soil against erosion.

896

The literature sources have reported thatthe genus Sibiraea contains five species growingin the European part of Southern Russia, Siberia,Western China and Southeastern Europe.S. angustata (Rehd.) Hand. Mazz. creates specificplant communities in the western part of theHimalayan plateau (in China) and is used for bio-mass production (Wu 1998). Cuizhi and Alexander(2003) reported three species for China, whileKrussmann (1978) reported two species, amongwhich S. laevigata (L.) Maxim. (=S. altaiensisSchneid.), with two varieties (var. angustata andvar. croatica (Deg.) Beck), and another speciesS. tomentosa Diels of a smaller habitus.

The name ‘‘Croatian sibirea’’ has been used inliterature for many years as S. croatica Deg.(Domac 1994) and its taxonomical classificationbased on morphological data has not yet beenprecisely established.

Based on the above information we questionedand discussed:– The genetic structure of the Croatian sibirea in

the western Balkan region, and its relationshipwith populations from Southern Russia andSouthern Siberia.

– The phylogenetic position of the genus Sibiraeawithin family Rosaceae as detected by analysisof internal transcribed spacers (ITS) 1 and ITS2in nuclear ribosomal DNA (rDNA) gene cluster.Due to the unique geographical and ecologicaldistribution of sibirea we consider it to be anexample for a discussion on protection of smalland fragmented populations by maintainingtraditional land use.

Material and methods

Branches with dormant buds in the initial stagewere collected in the natural populations of

Croatian sibirea in Bosnia and Herzegovina at themountain Cabulja and in Croatia at the mountainVelebit (with participation of colleagues from theForestry Faculty from Zagreb, Croatia), duringSeptember 2003, and at the end of winter period inFebruary 2004. The samples of Altaic sibirea wereobtained in its natural population from the area ofBarnaul in Southern Siberia (Altai area, Russia),and in a plantation from the area of Lipetsk inSouthern Russia (with generous assistance of ourcolleagues from the botanical garden in Kiev,Ukraine) (Table 1).

DNA extraction

DNA was extracted from dormant buds after theremoval of bud scales or from inner part of stemcore using Plant DNeasy Mini Kit (Qiagen). DNAwas re-suspended in pre-warmed, sterile milli-Qwater to the approximate final concentration of100 ng/ll. The success of extraction was checkedby electrophoresis in 1% agarose gel stained withethidium bromide.

PCR amplification

Several pairs of primers (Table 2) and amplifica-tion programmes were used since no data exists onthe use of polymerase chain reactions (PCR) withthe genus Sibiraea. We have tried to amplify ITSregions in genomic DNA and parts of chloroplastDNA (cpDNA). Primers were selected mainlyfrom references on amplification of DNA fromNicotiana tabacum. The amplification reactionswere done using two methods: (a) a standardprocedure as described for higher fungi (Whiteet al. 1990) in a total reaction volume of 40 ll withAmplyTaq polymerase (Perkin–Elmer) for ampli-fication of ITS region in genomic DNA or (b) amodified general method used for amplification ofSequence-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

Cabulja (Bosnia and Herzegovina) Rosne poljane 43�51¢42¢¢1 17�49¢60¢¢4 1343 33

43�54¢85¢¢3 17�54¢92¢¢7 1427

Velebit (Croatia) Mali Brizovac (Velinac) 44�56¢18¢¢2 15�07¢40¢¢7 992 33

44�56¢24¢¢7 15�07¢51¢¢4 1011

Lipecka oblast (plantation) (Russia) Stanovlianski raion 52�30¢ 39�32¢ 300 3

Altai (Russia) Barnaul 49�28¢ 85�37¢ Up to 1300 5

897

Bousguet 1998) in a final volume of 25 ll usingAmplyTaq Gold hot start polymerase (AppliedBiosystems).

We have performed amplification in a Perkin–Elmer 9700 DNA thermocycler with previouslypublished cycling parameters for amplificationwith ITS primers (Kraigher et al. 1995). Cyclingparameters for amplification of chloroplast DNA(cpDNA) comprised initial denaturation at 95 �Cfor 5 min, followed by 40 cycles of 1 min at 94 �C,annealing for 2 min at 55 �C (or 53–63 �C ifoptimisation of reaction was necessary), and anextension for 3 min at 72 �C, with the finalextension at 72 �C for 10 min and storage at 4 �C.Controls lacking DNA were run for each experi-ment to check the contamination of reagents.Positive controls were run only for ITS1 and ITS4pair of primers using fungal DNA.

The amplification product was separated byelectrophoresis in agarose gels containing 2% LEAgarose (SeaKem) run in 1� Tris–borate–EDTAbuffer for 1 h at 5 Vcm)1. The DNA was stainedwith ethidium bromide, visualised under ultravio-let light (302 nm) and recorded on Polaroid 667black and white film. The lengths of amplificationproducts were estimated by comparison to Gen-eRuler 100 bp DNA Ladder (Fermentas).

Restriction fragment length polymorphism analysisand data evaluation

Aliquots of amplified DNA from PCR yieldingonly a single fragment of expected length (as esti-mated from length of the product in Nicotianatabacum or basidiomycete – fungi) were digestedseparately with 1 unit of each restriction enzymeaccording to the manufacturers recommendationsin 10 ll total volume. The amplified ITS regions ingenomic DNA were cut by Hinf I (Promega), MboI (Promega) and Taq I (TaKaRa). PCR productsfrom cpDNA were cut by Hinf I (Promega),Hae III (Fermentas) and Alu I (Fermentas orTaKaRa). The enzymes were chosen on the basisof economic criteria (for cpDNA) and after pre-viously published sequences of the ITS region(Karen et al. 1997).

The restriction fragments were separated in a2% agarose gel in Tris–borate–EDTA buffer for3 h at 10 Vcm)1. The length of restriction frag-ments was estimated by comparison to GeneRuler100 bp DNA Ladder (Fermentas).

Gels were stained in ethidium bromide, ac-quired by GelDoc system (Biorad) and analysedwith Taxotron software (Pasteur Institute 1998,Paris, France) (Grimont 1998). The procedure is

Table 2. The list of primers (and reference for original publications) used for amplification of parts of nuclear or cpDNA with theirsequences and melting temperatures as given by authors

Primer code T am (�C) Reference Available at the internet site

ITS1 (nuclear) 64 White et al. (1990) http://www.plantbio.berkeley.edu/�bruns/primers.html

ITS4 (nuclear) 58 White et al. (1990) http://www.plantbio.berkeley.edu/�bruns/primers.html

ITS5 (nuclear) 59 Yulong et al. (1998) http://www.plantbio.berkeley.edu/�bruns/primers.html

ITS5-p (nuclear) 61 Moller and Cronk (1997) not available

atpB-1 (cpDNAb) 58 Chiang et al. (1998) http://www.ejournal.sinica.edu.tw/bbas/content/1998/4/bot94-10.html

rbcL-1 (cpDNA) 55 Chiang et al. (1998) http://www.ejournal.sinica.edu.tw/bbas/content/1998/4/bot94-10.html

ccmp10-R (cpDNA) 57 Weising and Gardner 1999 http://www.bfw.ac.at/200/2043.html

trnHM (cpDNA) 68 Heinze 2001 http://www.bfw.ac.at/200/2043.html

trnG-P (cpDNA) 65 Heinze 1998 http://www.bfw.ac.at/200/2043.html

trnMM (cpDNA) 62 Demesure et al. 1995 http://www.bfw.ac.at/200/2043.html

trnC-f (cpDNA) 60 Demesure et al. 1995 http://www.bfw.ac.at/200/2043.html

trnD-m (cpDNA) 56 Demesure et al. 1995 http://www.bfw.ac.at/200/2043.html

ycf9-P (cpDNA) 63 Heinze 2001 http://www.bfw.ac.at/200/2043.html

rps 14 (cpDNA) 60 Doyle et al. 1992 http://www.bfw.ac.at/200/2043.html

trnT (cpDNA) 56 Taberlet et al. 1991 http://www.bfw.ac.at/200/2043.html

trnF (cpDNA) 58 Taberlet et al. 1991 http://www.bfw.ac.at/200/2043.html

aTm – annealing temperature in �C.bcpDNA – chloroplast DNA.

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described in detail in Grebenc et al. (2000) andMartın and Karen (2000).

Sequencing

Prior to sequencing, the amplification productswere cleaned using the Wizard SV Gel and PCRClean-Up System (Promega). Both strands of theamplified DNA were sequenced separately usingprimers mentioned above. When weak or morethan one PCR product was obtained in PCR, theproducts of the desired length were cleaned fromthe agarose gel previously to sequencing themusing the same purification kit. DNA was se-quenced by commercially available sequencingservice (Sequiserve, Germany). Altogether, ninesamples were sequenced, three from each popula-tion in Croatia and Herzegovina, two from Altaiand one from the Lipetsk population. We havesequenced part of the genomic DNA using primersITS5-p and ITS4 and parts of cpDNA using ccmp10-R, trnHM, trnG-P and trnMM primers.

Sequence Navigator Software (Applied Bio-systems) was used to identify the consensussequence from the two strands of each isolate.Dialign 2 software was used for multiple alignmentof obtained sequences (Morgenstern et al. 1998)with minor manual corrections. The obtainedsequences have been lodged in The EuropeanMolecular Biology Laboratory database. Fromthe same database, sequences from different generafrom family Rosaceae were selected in a manner torepresent all tribes in the family. ‘‘SEQAPP’’software for multiple sequences was used to searchfor the best alignment. Where ambiguities in thealignment occurred, the alignment chosen was theone generating the fewest potentially informativecharacters. Alignment gaps were marked with ‘‘-’’,unresolved nucleotides and unknown sequenceswere indicated with ‘‘N’’.

The phylogenetic analysis

Using the programme PAUP* Version 3.11 forMacintosh (Swofford, 2002) heuristic search formost parsimonious trees and bootstrap analysiswas done for the full length of ITS1 and ITS2regions in rDNA. All characters were treated asunordered. Gaps were treated as characters in thisstudy because they may contain phylogeneticinformation and provide particularly clear

indications of relationship (Lloyd and Calder1991; Revera and Lake 1992; Baldwin 1993). Firsta maximum parsimony analysis (MP) under heu-ristic search was done; non-parametric bootstrapsupport (Felsenstein 1985) for each clade was tes-ted based on 10,000 replicates, using the fast-stepoption. The consistency index (CI; Kluge andFarris 1969), retention index (RI; Farris 1989), andre-scaled consistency index (RC; Farris 1989) wereobtained from PAUP*.

For all genera used in the phylogenetic com-parison with the newly obtained sequences ofSibiraea the sequences were retrieved from publicavailable databases with references therein: Agri-monia eupatoria L., U90798; Aremonia agrimono-ides (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; Fragariamoschata Duchesne, AF163505; Fragaria vesca L.,U90793; Fragaria vesca subsp. vesca forma alba(Ehrh.) Staudt., AF163516; Fragaria vesca L.subsp. vesca, AF163515; Fragaria � ananassaDuchesne, AF164494; Filipendula ulmaria (L.)Maxim., U90783; Filipendula vulgaris Moench,AJ416467; Geum montanum L., AJ302350; Geumrivale L., AJ302352; Malus sieversii (Ledeb.) M.Roem., AF186491; Malus � domestica Borkh.,U16195; Mespilus germanica L., U16196; Poten-tilla dickinsii Back., U90785; Potentilla fruticosaL., AF163478; Prunus armeniaca L., AF318756;Prunus avium L., AF318737; Prunus cerasus L.,AF318729; Prunus domestica L., AF318713; Pru-nus persica (L.) Batsch, AF318741; Pyrus pyrifolia(Burm. f.) Nakai., AF287240; Rosa canina L.,AB019495; Rosa persica Michx, AJ416468; Rubuscaucasicus Focke, AY083371; Rubus idaeus L.,AF055756; Rubus neomexicanus Gray, AY083358;Sanguisorba officinalis L., AY635040; Sorbus au-cuparia 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 primerpairs were used for molecular analysis of studiedpopulations. For three primer pairs, amplificationwas too weak or not successful at all while two

899

primer pairs yieldedmultiple bands. These productswere not used in further analysis. For PCR resultingin one clear DNA fragment within or betweenpopulation differences in the size of the amplifiedDNA product was not observed except for primerpair ccmp 10-R and trnHM with DNA fragment of280 bp in all samples from population Velebit andfor a few samples from population Altai comparedto all other samples with 274 bp (Table 3).

After the successful PCR resulting in a singleclear band, the DNA from all 74 samples was cut.The restriction analysis and separation in agarosegel showed no differences in length of the digestedcpDNA between or within populations. Sequenc-ing confirmed minor variability between popu-lations. The sequenced product using trnGPand trnMM primer pair showed no differenceswithin 246 bp long sequence (accession numbersAJ878754–AJ878762). Basic local alignmentsearch tools results (Altschul et al. 1997) for thesequence showed a close similarity to Prunus zip-peliana cpDNA trnG–trnM intergenic spacer re-gion (E=127, bit value=1e)26, accessionnumber=AB111589) indicating an amplificationof the correct part of the chloroplast genome. Se-quences obtained with ccmp 10-R and reverseprimer trnHM were 274 or 280 bp long with 6 bpduplication of GTATAT motif 215 bp down-

stream from 5¢ end (accession numbers AJ878763–AJ878770). Duplication was present in all samplesfrom Velebit and one sample from Altai. Bothsequences gave identical results after BLAST andwere similar to Vigna angularis chloroplast S10Boperon (E=92, bit value=7e)16, accession num-ber AF536225). The 6 bp long insertion was notdetectable on our agarose gels.

ITS regions and 5.8S in genomic rDNA for thesame samples were sequenced (accession numbersAJ876553–AJ876562). All sequences were 762 bplong using ITS5-p and ITS4 primers after deletingthe unreadable first part of the sequence. The se-quences showed inaccuracy or heterogeneity inbases 57, 630 and 666 downstream from 5¢ end ofITS1 independently of the origin (population) ofthe sample. From these nucleotide positions, thesequence could not be unequivocally read sincetwo bases gave approximately the same intensityof the signal in electrophoregram.

We have included only one sequence of the ITSregion of sibirea in phylogenetic analysis based onITS1 and ITS2 spacers in rDNA with selectedRosaceae species obtained from public availabledatabases (GenBank). We have constructed anunrooted bootstrap consensus phylogenetic treewith a Rohlf’s consistency index 0.741 (Figure 1left) 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 PCR quality

or length of

product [bp]

Restriction fragments of the PCR products [bp]

Hinf I Mbo I Taq I Alu I Hae III

ITS1, ITS4 weak

amplification

/a / / / /

ITS5, ITS4 multiple bands / / / / /

ITS5-p, ITS4 773 173, 157, 151,

128, 83, 67

582, 160 347, 260, 75, 60 / /

ccmp10-R,

trnHM

274 (280)b 140, 127,

7 (146,127,7)b274 (280)b

(no restriction)

/ 274 (280)b

(no restriction)

274 (280)b

(no restriction)

trnG-P, trnMM 240 240

(no restriction)

240

(no restriction)

/ 240

(no restriction)

240

(no restriction)

trnC-f, trnD-m multiple bands / / / / /

ycf9-P,

rps14Doyle

1150 / 880, 150, 70 / 850, 180, 120 1150

(no restriction)

atpB-1, rbcL-1 no amplification / / / / /

trnT, trnF weak

amplification

/ / / / /

We have used common names for primers.a/Reaction not performed.bFor samples from population Velebit and few samples from population Altai.

900

SIBSPICAN

SPIxVANFILVULFILULMWALGEO

GEURIVGEUMONRUBCAURUBIDE

RUBNEOPOTDICROSPER

ROSCANSANOFFFRAVESFRAVESALB

FRAVESVESFRAxANAFRAMOSPOTFRU

AGREUPAREAGRDRYOCTPRUARM

PRUDOMPRUPERPRUAVI

PRUCERCYDOBLSORTORPYRPYR

MALSIEMALxDOMSORAUCCRAMOL

MESGERCOTLAC

100

100

75

96

100

75

88

65

89

97

100

10096

9899

95

98

100

61

100

100

52100

100

100

62

50

100

98

BootstrapSIB

SPICAN

SPIxVANCYDOBL

SORTORPYRPYR

MALSIEMALxDOM

SORAUCCRAMOL

MESGERCOTLACPRUARM

PRUDOMPRUAVI

PRUCERPRUPER

DRYOCTFILVULFILULM

WALGEO

GEURIVGEUMON

RUBCAURUBIDE

RUBNEOPOTDIC

ROSPER

ROSCANSANOFF

AGREUPAREAGR

FRAVESFRAVESALBFRAVESVESFRAxANA

FRAMOSPOTFRU

Figure 1. Phylogenetic tree for selected genera from family Rosaceae. Left – consensus tree of 100 most maximum parsimony treeswith 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� ananassaDuchesne; 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, Rosacanina L.; ROSPER, Rosa persica Michx; RUBCAU, Rubus caucasicus Focke; RUBIDE, Rubus idaeus L.; RUBNEO, Rubus neo-mexicanus 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.

901

consistency index 0.618, a retention index 0.825and a re-scaled consistency index 0.548 (Figure 1right). Alignment and phylogenetic trees aredeposited in Treebase – http://www.treebase.org.We have performed the phylogenetic analysis onlyto determine the phylogenetic position of the genusSibiraea towards other genera in the family Rosa-ceae and not to infer the phylogenetic position anddistances of other genera (Alice and Campbell1999; Lee and Wen 2001; Eriksson et al. 2003).

Discussion

The origin of the disjunct populations of sibirea

During the Tertiary period, larger areas ofSoutheastern Europe, Southern Siberia and wes-tern China had been covered by steppe and steppeforests (Sitte et al. 1991; Adams 2002), and in theQuaternary period they achieved their maximumspan. In this period, the areas from the AdriaticSea to Eastern Siberia were relatively compact andcomprised of steppe and forest steppe with theircharacteristic floral elements, which can still befound in isolated areas of the Southern Siberia.Kuminova (1960) described these specific plantcommunities of the high steppe and the foreststeppe. Sibirea from the Altai Mountains is one ofthe most significant species of the steppe and theforest steppe plant communities, and as such itmust have been widely distributed during theTertiary period. After the climatic changes of thePleistocene the populations were separated intoisolated islands, i.e. into disjunct areas. Thus, theSoutheastern European sibirea became isolated atthe mountains of Velebit, Cabulja and Cvrsnicacomprising small subpopulations with relativelylow number of individuals. In Croatia, the sibireabushes appear in a plant community Seslerio-Os-tryetum Horv. & H-ic. (Volaric–Mrsic 1994), andin Bosnia and Herzegovina in separate plantcommunities in high mountain areas at brokenlimestone grounds, that have not been entirelyinvestigated yet, and that by their morphologypartially resemble the steppe forests. At least forSouthern Russia localities it is known where sibi-rea was artificially introduced for handcraft pur-poses and soil protection (Melnik and Janjicpersonal communication). Therefore, the presentdistribution in Southeastern Europe and Southern

Siberia most probably represent only the remainsof its initial large area from the Tertiary and theQuaternary period, supposedly being the Glacialrefugia that were created during the last glaciation.However, the paleontological analyses of the fossilpollen is not available to prove this assumption.

The close taxonomical affinity of the verydistant Croatian and South Siberian Sibiraea‘‘croatica’’ and S. altaiensis populations are notthe only case of such a geographical gap. Wellknown are the big disjunctions in the generaScopolia (1 species in Southeast Europe and 4 inEast Asia) and Pseudostellaria (1 species on thesouthern/southeastern foothills of the Alps and 10more in Middle/Eastern Asia) (Schaeftlein 1969).In the latter case, climate changes in Pleistoceneand post-Pleistocene period in the Near East, withthe subsequent areal drainage, is assumed(Schaeftlein 1969: 879). These and other similarcases are, of coarse, examples for mesophytic taxa,while Sibiraea has a more xerophytic character,although, comparable to Scopolia and Pseudo-stellaria, with a higher precipitations in theirdistribution areas, while as a taxon with thesteppe-character the genus Leontopodium can bediscussed. It has about 30–40 species in Eurasia,mostly in Inner-Asia, and only two of them growin Europe (Wagenitz 1965).

The migration of the European taxa from Asiais supposed to have happened in the early periodsof the Pleistocene, most presumably through thelowlands (because they are lacking in Caucasus andthe mountains of the Near East) (Wagenitz 1965).However, it is perhaps not necessary to search forthe explanation of the recent distribution of Sibi-raea ‘‘croatica’’ in the Pleistocene and the post-Pleistocene climate and vegetation history, sincethe distribution patterns of the genera Scopolia,Pseudostellaria, and Sibiraea, seem to be mucholder, going back to the Tertiary period. Addi-tionally to the previous cases, one could mentionthe genus Degenia, which as Sibiraea ‘‘croatica’’occurs in the Velebit Mountains. Its only species,Degenia velebitica, was first described as a speciesof 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 rela-tively slow rate of evolution and can be used for

902

differentiation between species and populations(Small et al. 1998). We have used six pairs ofprimers for amplification of parts of non-codingregions in cpDNA, general primers made for theamplification of atpB–rbcL genes spacer in mosses(Chiang et al. 1998) and primers made for tobacco(see Table 2). The selected primers were notdesigned specifically for amplification in sibireanor were they modified. Only the PCR was opti-mised for pairs with no or weak amplification. Theamplified products showed no differences betweenpopulation in restriction patterns (data gathered inTable 3) though the PCR in combination withrestriction fragment length polymorphism (PCR–RFLP) method is well known to separate mostspecies and some populations (White et al. 1990;Karen et al. 1997; Horton 2002). Only a minordifference of 6 bp duplication in DNA amplifiedwith ccmp 10-R and trnM primer pair was noticed.The duplication could be the result of a simpleinsertion in non-coding region and was observedin two geographically distinct populations. Anal-ysed regions in cpDNA represent only a small partof the genome. Polymorphic regions in cpDNAcan never be predicted, but since one polymorphicfragment was discovered further analysis of ge-netic diversity in sibirea should follow. For this, aselection of markers can be based on nuclearmarkers for Rosaceae since it has been demon-strated that molecular marker tools developed inone species (e.g. peach) are easily applied to otherspecies in the family (Dirlewanger et al. 2004; Junget al. 2004).

The lack of differences in the restriction patternfor the ITS region in genomic rDNA indicates thatall samples of sibirea belong to the same speciessince the ITS region was proven to be conservedwithin one taxonomic species. The observed sub-stitutions or inaccuracies in the sequence of thesame region gave a 0–2 bp difference betweendifferent sequences of ITS comparable to the dif-ferences observed within the Fragaria vesca com-plex with a 0–3 bp difference (Figure 1). Almostidentical sequences can indicate a very recentseparation and possible speciation of the studiedpopulations due to the recent colonisation event asobserved in species of Adenocarpus (Percy et al.2002). Vogler and DeSalle (1994) have discussedthat the main evolution force for ribosomal regionwas the concerted evolution, which should lead tohomogenisation of individual repeats and produce

mostly a uniform sequence in all repeats of a givenspecies. The concerted evolution might be aproblem if there are instances of allopolyploidybut we predicted Sibiraea spp. to be diploid. Be-sides this, conservation of ribosomal genes(including the ITS regions) was shown and usedtogether with maternally inherited cpDNA regionsfor phylogenetic analysis and species definition inmany plant groups (Alice and Campbell 1994; Leeand Wen 2001) as well as in other organisms suchas higher fungi, where we can observe some vari-ation within a morphological species (Agerer et al.1996; Karen et al. 1997; Horton 2002 and others).Within species, variability could explain inaccu-racies observed in some parts of the ITS se-quences. Point mutations could also occur andremain fixed within single species/sample resultingin heterozygocity of the ITS region (or at least insome copies of the rDNA cluster resulting in het-eroduplexes). The persistence of such mutations inthe populations can be explained by small, disjunctpopulations with high probability of self-crossing,self-pollination and vegetative reproduction bylayering. Similar polymorphic nucleotide sitesexplained as superposition of two or more distinctITS repeats within one or more ribosomal geneclusters were also observed and proven to persistin several taxa of Amelanchier showing commonasexual seed production. The authors proposefour possible reasons for persistence of poly-morphism: within individual polymorphism as aconsequence of transition stage in concertedevolution, interspecific hybridisation, evolution ofpseudogenes and disruption of concerted evolu-tion due to non-homologous position of rDNAloci (Campbell et al. 1997).

Sibirea’s closest relatives were the fewsequenced species from the genus Spiraea, sup-porting the position of the genus Sibiraea asdetermined after morphological characteristicswithin the tribe Spiraeae and next to the tribeMalonideae (Kubitzki 2004).

In studies of species from the genus Zelkovatwo endemic species, Zelkova abelicea (Lam.)Boisser and Zelkova sicula Di Pasquale, differonly in 1 bp in trnL intron and no within popu-lation variation was detected using differentchloroplast markers (Fineschi et al. 2004).According to the observed similarity of ITS1, 5.8Sand ITS2 regions in genomic rDNA clustersamplified from Croatian sibirea populations col-

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lected at Cabulja (Bosnia and Herzegovina) andVelebit (Croatia) and Altaic sibirea from bothlocations in Southern Russia we cannot confirmthe existence of Croatian sibirea (S. croatica) as aseparate species. However, it must be stressed thatthe lack of molecular evidence for consideringCroatian sibirea as a separate species should notreduce the importance of its conservation inthe regional flora in the Southeastern Europeanregion (Western Balkan mountains) where it is arare relic.

The process of evolution governing selectionin populations and mutation changes in smalldisjunct populations that would bring about thedifferentiation of populations is not well known(as reported by Savolainen and Kuittinen 2000).How the selection acts in a similar phenomenon ofsmall isolated populations can be exemplified inAbies nebrodensis (Lojac.) Mattei where the selec-tion favours heterozygotes (Vicario et al. 1995). Asimilar phenomenon was recorded for the popu-lations of Pinus leucodermis Ant. as reported byBoscherini et al. (1994) and in subpopulations ofPicea abies (L.) Karst. at extreme growing siteconditions (Bozic and Urbancic 2003). While insibirea the analysed gene pool was homogeneousas observed in genus Zelkova (Fineschi et al.2004), which is possibly due to the low number ofindividuals in subpopulations, to the specificity ofits androdioecious flowers or possibly to a bottle-neck effect after the separation of ancient popu-lation after the retrieval of glaciers.

Conservation measures for small disjunctpopulations in the Southeastern Europe

Many areas where sibirea is found naturally arealready under some kind of protection. The regionof Sichuan, Huanglong (Yellow-Dragon) in Chinais protected as a natural monument (Wenhua andXianying 1989) but sibirea is not included in thelist of species of interest for their rarity, endemismor ornamental and medical value (World HeritageConvention 1991) nor is it included in the WorldConservation Union (IUCN) Red list of threa-tened species (IUCN 1998). For the area of Vele-bit, some plant associations with several endemicspecies (including S. croatica) are included in theIUCN Biosphere reserve with strict protection of

the area of Paklenica National park and in theCroatian Act on Protection of Nature (2003). Nospecific measures for active protection of sibireaare given in any document although the specieswould demand an active protection.

There is no data about the evolution of sibirea,or about its populations from Tertiary or post-glacial periods. At least Southeastern Europeanpopulations were until recently maintainedby extensive small cattle farming or extreme,inaccessible rocky habitats thus providing suitableconditions for seed germination and reducingcompetitive species in areas suitable for sibirea,similar to steppe and forest steppe but with ahigher precipitation. Sibirea plants are in generalless or not of interest for small cattle, possibly dueto their thick leaves with dissuading secondarysubstances (Zhang et al. 1993). In many moun-tainous and subalpine rural areas in SoutheasternEurope resembling forest steppe, extensive smallcattle farming was abandoned in the last decadesfor economical and political reasons leading tostrong natural reforestation of many anthropo-genic-grasslands and steppe habitats. The maincompetitor of sibirea in its natural habitats inSoutheastern European localities are either Bos-nian (P. heldreichii, Silic 2002) or Austrian pine(P. nigra, Kusan 1971) and common beech causingserious reduction in growth of sibirea plants andfinally its disappearance if trees are not removed(usually by grazing).

Owing to its weak ecological competitiveness,the local sibirea subpopulations could becomeperiodically extinct due to the re-colonisation of itssites by neighbouring tree populations. Frag-mented populations are in a high risk of beingpermanently lost from small reserves.

Therefore, greater attention needs to be givento active management of autochthonous sibireagenetic resources in existing protected areas inorder to ensure the maintenance of specific habi-tats of the species. The solution for preservation ofnaturally occurring habitats of the species wouldbe either in the retention of traditional extensivesmall cattle farming in areas where sibirea occursnaturally thus protecting the specific anthropo-genic influence or through removal of competitivespecies which directly threaten the existing plants.None of the proposed measures have beenconsidered or officially undertaken so far.

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Acknowledgements

This research was part of the bilateral project BI-BA/04-05-010 between Bosnia-Herzegovina (Uni-versity of Sarajevo, Faculty of Forestry) and theRepublic of Slovenia (Slovenian Forestry Institute(SFI)), the research programme P4-0107 of theSFI, financed by the Ministry for Higher educa-tion, Science and Technology of the Republic ofSlovenia and its Young Researchers Scheme (TG).We would like to thank Dr. Monika Konnert,ASP Teisendorf, for advice on primers used in thisstudy and for suggestions and corrections of themanuscript, Dr. Josip Franjic, Faculty of Forestry,University of Zagreb, Croatia, for help in collec-tion of samples, colleagues from the NationalBotanical Garden in Kiev, Ukraine and to anon-ymous reviewers for very constructive suggestionsfor the improvement of the text.

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