The Saxifraga rivularis complex in Svalbard: Molecules, ploidy and morphology

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Flora 200 (2005) 207–221

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The Saxifraga rivularis complex in Svalbard:Molecules, ploidy and morphology

Ane Senstad Guldahla,1, Tove M. Gabrielsena, Anne-Cathrine Scheena, Liv Borgena,Snorre W. Steena, Sigmund Spjelkavikb, Christian Brochmanna,�

aNational Centre for Biosystematics, Natural History Museum, University of Oslo, P.O. Box 1172 Blindern,

NO-0318 Oslo, NorwaybThe University Centre in Svalbard, P.O. Box 156, NO-9171 Longyearbyen, Norway

Accepted 6 January 2005

Abstract

In many arctic plant groups, reticulate histories involving hybridization and polyploidization have resulted inproblems with species delimitation and phylogeny reconstruction. The Saxifraga rivularis complex is a typical example.Two taxa, S. rivularis and S. hyperborea, have been reported from the arctic archipelago of Svalbard (‘Spitsbergen’),but their delimitation, relationship, and taxonomic status are uncertain. We analyzed variation in chromosomenumber and DNA content (using flow cytometry), two types of molecular markers (RAPDs and isozymes), and 59morphological characters along transects in two large, mixed Svalbard populations. We also included presumedly‘pure’ reference populations from other sites to address whether the tentative taxa are distinct and whether theyhybridize in mixed populations. In the random amplified polymorphic DNA (RAPD) analysis, we identified two verydistinct groups of multilocus phenotypes, corresponding to the tentative species. These groups also differed in DNAcontent, chromosome number (S. hyperborea: 2n ¼ ca: 26; S. rivularis: 2n ¼ ca: 52; but endopolyploidy was observedin several plants), some morphological characters, and isozyme phenotypes (with a few exceptions). The cytosolic Gpi

locus was duplicated also at the 2n ¼ 26 level and fixed heterozygosity was observed in all populations, suggesting thatS. hyperborea and S. rivularis may be secondary allotetraploid and allooctoploid, respectively, rather than diploid andtetraploid as traditionally assumed. We conclude that the Svalbard populations represent two distinct species atdifferent ploidal levels and occurring in partly overlapping habitats, and that extensive hybridization presently doesnot occur between them even in mixed populations. Most molecular markers observed in S. hyperborea formed asubset of the S. rivularis markers, consistent with a hypothesis that S. rivularis is an allopolyploid lineage with the S.

hyperborea lineage as one of its progenitors.r 2005 Elsevier GmbH. All rights reserved.

Keywords: Arctic plants; Isozymes; Morphometry; Polyploidy; RAPDs; Taxonomy

e front matter r 2005 Elsevier GmbH. All rights reserved.

ra.2005.01.003

ing author. Tel.: +4722851611; fax: +4722851835.

ess: [email protected]

).

ress: Department of Natural History, Norwegian

ience and Technology, NO-7491 Trondheim, Norway.

Introduction

The arctic flora is taxonomically complex, probably asa result of recurrent hybridization and polyploidizationevents. The majority of the contemporary arctic plant

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ARTICLE IN PRESSA.S. Guldahl et al. / Flora 200 (2005) 207–221208

species appear to be of hybrid origin, stabilized throughallopolyploidization (Abbott and Brochmann, 2003;Brochmann et al., 2004). Recurrent hybridizationsbetween divergent genotypes have been facilitated byrepeated cycles of fragmentations, range expansions,and reunions of previously isolated populationsthroughout the large-scale climate changes of theQuaternary. These recurrent episodes of reticulateevolution have made it difficult to delimit taxonomicspecies and to unravel the evolutionary history of arcticspecies complexes.The arctic-alpine Saxifraga rivularis L. complex

(section Mesogyne Sternb.) is a typical example,presenting notoriously difficult delimitation of taxaand poorly known evolutionary relationships. In therecent Pan-Arctic Flora (PAF) Checklist, five specieswere tentatively accepted in this complex (Elven et al.,2003). Three of the species are Beringian, whereas thecircumpolar S. hyperborea R. Br. and the assumedlyamphi-Atlantic S. rivularis L. have been reported to co-occur in the arctic archipelago of Svalbard (formerlyoften named ‘Spitsbergen’). The taxonomic status ofthese two taxa and their distinction have been muchdebated. The opinions vary from treating them as asingle, variable species (S. rivularis; e.g. Hulten andFries, 1986) to treating them as two distinct taxa at thespecies level (Elven, 1994; Elven and Elvebakk, 1996;Elven et al., 2003; Rønning, 1996). A possible hybridbetween them has been reported from Svalbard (Borgenand Elven, 1983).The two taxa have been reported to have different

ploidal levels, referred to as diploid (2n ¼ 26) andtetraploid (2n ¼ 52), but it is unclear in many caseswhether the material has been identified to speciesbased on morphology or simply on chromosomenumbers. Saxifraga hyperborea has been reported with2n ¼ 26 throughout its circumpolar range (e.g. fromSvalbard by Flovik, 1940; Engelskjøn and Schweitzer,1970 (Bear Island); Borgen and Elven, 1983; fromGreenland by Holmen, 1952; Jørgensen et al., 1958;from N America by Love and Love, 1965, 1966;Johnson and Packer, 1968; and from northern Asia byZhukova et al., 1973). Saxifraga rivularis has beenreported with 2n ¼ 52 or ca. 52 throughout the North-Atlantic region, including Norway and Svalbard (Bor-gen and Elven, 1983; Engelskjøn and Knaben, 1971;Engelskjøn, 1979), Greenland (Bocher, 1938; Sørensenand Westergaard in Love and Love, 1948; Jørgensen etal., 1958), Iceland (Love and Love, 1956), and NorthAmerica (Love and Love, 1965, 1982). The plantsuggested to be a hybrid had 2n ¼ 39 (Borgen andElven, 1983).Although the variation in morphological characters

appears to be complex, differentiation in a fewcharacters has been suggested to be taxonomicallysignificant. Saxifraga rivularis has been described as

forming loose tufts or occasionally dense cushionswith subterranean runners (stolons), semicircular orkidney-shaped basal leaves shallowly divided intousually five lobes, unbranched stems up to 15 cm highwith more or less glandular hairs and a single ter-minal flower, a densely glandular-hairy hypanthium,and white to pink, rarely red, petals (Elven, 1994;Webb and Gornall, 1989). Saxifraga hyperborea hasbeen reported to differ from S. rivularis by its smallersize, more compact tufts, lack of runners, usuallythree-lobed basal leaves, and more reddish petals, leavesand stems.A previous molecular study of several arctic species

of section Mesogyne also included some material ofS. rivularis and S. hyperborea, but the main focusof that study was to address the origin of the endemicSvalbard polyploid S. svalbardensis D.O. Øvstedal(Brochmann et al., 1998). Saxifraga rivularis and S.

hyperborea had very similar sequences of the maternallyinherited chloroplast DNA (cpDNA) gene matK andwere more divergent for biparentally inherited nuclearmarkers (ITS sequences and random amplified poly-morphic DNAs – RAPDs). Whereas the RAPD datasuggested that S. svalbardensis originated as a hybridbetween S. rivularis and S. cernua L., the matK datawere consistent with a hypothesis that S. hyperborea

acted as a maternal progenitor of S. rivularis, which inturn acted as a maternal progenitor of S. svalbardensis

(Brochmann et al., 1998).Both S. hyperborea and S. rivularis are fully fertile

and set abundant seed (up to 100%) after sponta-neous selfing (Brochmann and H(apnes, 2001). Theflowers are small, only slightly protogynous, andautodeposit pollen at an early stage, consistent withtheir very low pollen/ovule ratios. The strongly auto-gamous mating system of the species must act as astrong barrier against hybridization between them.However, the existence of hybrid derivatives of S.

rivularis (S. svalbardensis and S. opdalensis A. Blytt;Brochmann et al., 1998; Steen et al., 2000) providedefinite evidence for occasional outcrossing, at least inthis species.Combination of detailed molecular and morphologi-

cal analyses can be a powerful tool for unravellingcomplex low-level taxonomy (e.g., Fjellheim et al., 2001;Guldahl et al., 2001; Hansen et al., 2000; Marhold et al.,2002; Scheen et al., 2002). In this paper, we analyzedvariation in chromosome number and DNA content(using flow cytometry), two types of molecular markers(RAPDs and isozymes), and 59 morphological char-acters along transects in two large, mixed Svalbardpopulations of tentative S. rivularis and S. hyperborea.We also included presumedly ‘pure’ reference popula-tions from other sites to address whether the tentativetaxa are distinct and whether they hybridize in the mixedpopulations.

ARTICLE IN PRESSA.S. Guldahl et al. / Flora 200 (2005) 207–221 209

Materials and methods

Plant materials

A total of 18 populations, 10 of putative S. rivularis

and 8 of putative S. hyperborea, were sampled onthe island of Spitsbergen in Svalbard (Table 1). Twosites close to Longyearbyen (Bjørndalen and Breinosa;Table 1) where both putative taxa occurred wereselected as the main study areas. At these sites, sampleswere taken every 50 cm (if present) along transectsto cover the observed variation in moisture andvegetation and thus to facilitate detection of po-tential hybrids. Plants tentatively belonging to S.

hyperborea occurred most frequently in the driestparts of the gradients, typically in late snowbeds thatonly were seasonally moist, whereas plants tenta-tively belonging to S. rivularis occurred most frequentlyin the wettest parts, typically in continuously wetmoss vegetation and along small brooks. The twotentative species appeared to occur in mixture in themiddle part of these gradients. In total, 60 plantswere sampled in Bjørndalen and 76 plants at Breinosa(referred to S. hyperborea (H) or S. rivularis (R) afterthe analyses were finished; i.e. populations 21H and 21Rin Bjørndalen and populations 24H and 24R atBreinosa; Table 1). Other, supposedly non-mixedsites in Svalbard were sampled in an attempt to obtain‘pure’ reference populations of each putative taxon(Table 1; at two of these sites, 29 and 30, subse-quent analyses showed that both taxa occurred). Theplants were brought to the laboratory at the UniversityCentre in Svalbard (UNIS). Because of the small size ofthe plants it was not in all cases possible to obtaincomplete data sets for each individual plant. A subset ofthe plants were first analyzed for morphometriccharacters, and fresh leaves from the same plants aswell as additional plants were immediately used forisozyme analysis in the UNIS laboratory. The remainderof the plants were either pressed as herbarium vouchers(deposited in O) or potted and transferred to thephytotron at the University of Oslo (cultivation condi-tions specified in Brochmann et al., 1992). Thecultivated plants were later used for RAPD analysis,flow cytometry analysis, and/or chromosome numberdeterminations.Because only a single cultivated Svalbard plant

was available of the short-lived S. hyperborea atthe time the flow cytometry analysis was carried out,we included some cultivated material of both spe-cies from other arctic areas for this analysis (Greenland,Iceland, Canada, and the island Prins Karls Forlandin Svalbard; Table 1). Many of these specimenswere taxonomically verified by inclusion in a cir-cumpolar study of the S. rivularis complex (Jørgensen,2004).

Chromosome number and DNA content

determination

Chromosome numbers were determined in ninecultivated plants. Root-tips were pretreated in 8-hydro-xychinoline for 2 h at room temperature followed by 2 hat 4 1C, fixed for 24 h in absolute ethanol:acetic acid(3:1), washed twice in 70% ethanol, and stored at�20 1C. The tips were stained in Feulgens (Schiffs)reagent. Procedures for pretreatment, staining andsquashing followed Jong (1997). For each plant, thechromosome number was determined in minimum threecells in mitotic metaphase.Ploidal levels were estimated in 65 cultivated plants by

flow cytometry conducted at the Plant CytometryServices, Schijndel, the Netherlands (Table 1). To isolatenuclei, fresh leaf material was chopped in an ice-coldneutral DNA buffer modified after Arumuganathan andEarle (1991) with 5mM Hepes, 10mM Magnesiumsulphate heptahydrate, 50mM Potassium chloride,0.2% Triton X-100, 1% DTE, and 2mg/l DAPI at pH7.0. This mixture was passed through a nylon filter of40 mm mesh size and sent through the flow cytometer(PAS II, Partec GmbH). Fluorescence of the DAPI-stained nuclei was measured by a photomultiplier andconverted into voltage pulses electronically processed toyield integral and peak signals further processed bycomputer. No internal standard was used, but S.

hyperborea was used as a calibration standard for theother measurements, and several of the chromosome-counted plants were also included in the flow cytometryanalysis (Table 1). The flow cytometer was adjusted sothat the median of the first peak of S. hyperborea

appeared at channel 50. The fluorescence of a leafsample is expected to vary proportionally to the DNAcontent (Husband and Schemske, 1998). The ploidallevels presented here (Table 1) were estimated from therelative fluorescence values.

RAPD analysis

DNA was extracted from silica-dried or fresh leafmaterial from the cultivated plants using the CTABmethod as specified in Gabrielsen and Brochmann(1998). The RAPD-polymerase chain reaction (PCR)protocol was modified after Williams et al. (1990) asdescribed by Gabrielsen et al. (1997). The PCR productswere separated on 1.4% agarose gels stained withethidium bromide, visualized under UV light, andcompared to a DNA ladder of known concentration(lDNA cut with EcoRI and HindIII) to estimatefragment lengths. Twenty-eight primers (from kits A,C, and D; Operon Technologies, Alameda, CA, USA)that had previously been used in similar studies inSaxifraga (Brochmann et al., 1998; Gabrielsen et al.,

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Table 1. Collection data, number of plants analyzed, and ploidal levels inferred for populations of Saxifraga rivularis and S. hyperborea

Taxon, pop.

no.

Geographic origin, sampling data No. of plants analyzed Gpi-2 phenotypes (no. of plants) Chromosome

number

Inferred main

ploidal level

Isozymes RAPDs Morphology Flow

cytometry

Chrom. no. ac bc abc

S. hyperborea

21H Svalbard, Nordenskiold Land, Bjørndalen,

NW of the road. 7811303000 N, 1511902900 E.

CB, TMG, SWS 1997.

15 2 15 1 15 2� (second. 4� ?)

24H Svalbard, Nordenskiold Land, NW of the

mountain Breinosa. 781902200 N, 161205200 E.

CB, TMG, SWS 1997.

47 25 47

26H Svalbard, Dickson land, Idodalen,

Kongressfjellet. 781330 N, 151220 E. RE 1997.

10 10 10

29H Svalbard, Sabine Land, Sassen, Gjelrabbane.

781150 N, 171 E. RE 1997.

10 10 10

30H Svalbard, Sabine Land, Sassen,

Coloradofjellet. 781200 N, 171100 E. RE 1997.

1 1 1

34H Svalbard, Nordenskiold Land, De Geer

dalen, S of Skjørlokstupet. 7714105400 N,

191202200 E. TMG 1997.

11 1 10 11

35H Svalbard, Nordenskiold Land,

Skjørlokstupet. 771405200 N, 191405300 E. SF,

ACS 1997.

2 2 2

41H Svalbard, Oscar II Land, NW of

Bohemanneset. 781240 N, 141350 E. RE 1997.

2 2 2 2 (23) 26 (38) 2� (second. 4� ?)

CB 99–27 Greenland, Liverpool Land, SE of

Scoresbysund , W of Mt. Inugsukajik. 701290

N, 211560 W. CB 1999.

2 2� (second. 4� ?)

CB 99–38 Greenland, Liverpool Land, NE of

Scoresbysund. 7012900 N, 211560 W. CB, JN,

PBE, SK 1999.

3 2� (second. 4� ?)

RE 2775 Canada, NW Territories, Queen Elizabeth

Isl., Banks Isl. NE. 7313703300 N, 11515103900

W. RE 1999.

1 2� (second. 4� ?)


Isl., Melville Isl., Beverly Inlet. 751605000 N,

10713804100 W. RE 1999.

1 2� (second. 4� ?)


Isl., Nunavut, Gabbro Penins. 781490280 N,

1031400490 W. RE 1999.

1 2� (second. 4� ?)


Isl., Devon Isl., Crocker Bay. 7413209200 N,

8214702500 W. RE 1999.

1 2� (second. 4� ?)

RE 3520 Canada, NW Territories, Baffin Isl., Cape

Hooper, Tanner Bay. 6812601800 N, 6614905000

W. RE 1999.

1 2� (second. 4� ?)

RE 3525 Canada, NW Territories, Baffin Isl., Cape

Hooper, Iqualuit. 681250000 N, 6614906000 W.

RE 1999.

1 2� (second. 4� ?)

A.S.Guldahletal./Flora200(2005)207–221

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SS. rivularis

21R Svalbard, Nordenskiold Land, Bjørndalen,

NW of the road. 7811303000 N, 1511902900 E.

CB, TMG, SWS 1997.

37 7 22 7 4 33 4� (second. 8� ?)

24R Svalbard, Nordenskiold Land, NW of the

mountain Breinosa. 781902200 N, 161205200 E.

CB, TMG, SWS 1997.

22 5 16 2 17 5 4� (second. 8� ?)

29R Svalbard, Sabine Land, Sassen, Gjelrabbane.

781150N, 171E. RE 1997.

1 1 1

30R Svalbard, Sabine Land, Sassen,

Coloradofjellet. 781200 N, 171100 E. RE 1997.

2 2 2

36R Svalbard, Oscar II Land, Alkhornet. 781130

N, 131500 E. TMG 1997.

1 1 1 1

37R Svalbard, Oscar II Land, Alkhornet, close to

36 R. 781130 N, 131500 E. TMG 1997.

21 10 10 10 3 8 13 (43) 52 (85) 4� (second. 8� ?)

38R Svalbard, Oscar II Land, Bohemanneset.

781230 N, 141400 E. RE 1997.

5 5 1 1 5 (47) 50–52 4� (second. 8� ?)

39R Svalbard, Oscar II Land, Bohemanneset.

781230 N, 141400 E. TMG 1997.

4 5 5 3 4 (26) 47, 52 (95) 4� (second. 8� ?)

40R Svalbard, Oscar II Land, NW of

Bohemanneset. 781240 N, 141350 E. RE 1997.

8 8 8

61R Svalbard, Nordenskiold Land, Adventdalen.

781120 N, 151400 E. SF, ACS 1997.

4 4 2 4 4� (second. 8� ?)

CB 93–133 Svalbard, Prins Karls Forland, Fuglehuken,

Fuglehukfjellet. 7815401400 N, 1012805100 E.

CB 1993.

1 4� (second. 8� ?)

CB 99–25 Greenland, Liverpool Land, E of Kap Tobin.

7012404800 N, 2115603800 W. CB, JN, PBE, SK

1999.

5 4� (second. 8� ?)

CB 99–39 Greenland, Liverpool Land, NE of

Scoresbysund. 7012900900 N, 2115605100 W.

CB, JN, PBE, SK 1999.

5 4� (second. 8� ?)

CB/IA 99–43 Greenland, Tunu, Kong Christian IX Land,

Tasiilaq E, Blomsterdalen. 6513703000 N,

371440 W. IGA, LL 1999.

2 4� (second. 8� ?)


Tasiilaq, E of Kuummiut. 6515201900 N,

3615703200 W. IGA, LL 1999.

4 4� (second. 8� ?)


Tasiilaq, Kulusuk—airport. 651510000 N,

3615804800 W. IGA, LL 1999.

5 4� (second. 8� ?)

CB/IA 99–49 Iceland, Vesturland, NE of Akranes,

Akrafjall, Berjadalur. 641203400 N, 211530 5900

W. IGA, LL 1999.

4 4� (second. 8� ?)

Total sample

size

203 47 128 65 9 106 22 75

All populations from Svalbard except CB 93–133 are from the island of Spitsbergen.

When several chromosome numbers were observed, rare extreme numbers are given in parentheses.

Vouchers for chromosome counts are (population number: individual plant number(s)) 41H: 1, 2; 37R: 9, 12, 15; 38R: 1; 39R: 3, 4, 7.

Abbreviation of collectors: IGA, Inger G. Alsos; CB, Christian Brochmann; PBE, Pernille B. Eidesen; RE, Reidar Elven; SF, Siri Fjellheim; TMG, Tove M. Gabrielsen; SK, Siri Kjølner; LL, Leidulf

Lund; JN, Jorun Nylehn; ACS, Anne-Cathrine Scheen; SWS, Snorre W. Steen.

A.S.Guldahletal./Flora200(2005)207–221

211


1997; Steen et al., 2000) were initially tested on eightplants. Six primers (A01, A04, A10, A11, C01, and C06)gave readily scorable, polymorphic, and reproduciblebands, and were used in the analysis of a set of 47 plants.Polymorphic bands were scored as present (1) or absent(0). Because of the high mortality of S. hyperborea incultivation, only eight plants were available of thisspecies when the RAPD analysis was carried out.However, because these eight plants represented fivedifferent populations, and because the two speciesturned out to be very distinct in the RAPD analysis,we considered this sample to be sufficient.

Isozyme analysis

Fresh leaves were used for starch gel electrophoresisfollowing Morden et al. (1987) as modified by Borgen(1997). Glucose-6-phosphate isomerase (GPI, E.C.5.3.1.9) was stained for gels run on a Tris-borate bufferwith pH 8.3. Phosphoglucomutase (PGM, E.C. 5.4.2.2),isocitric dehydrogenase (IDH, E.C. 1.1.1.42), anddihydrolipoamide dehydrogenase (DDH, E.C. 1.8.1.4 )were stained for gels run on a histidine-citrate bufferwith pH 6.5. A total of 203 plants were analyzed: 121plants from the two main sites and 1–21 plants fromeach of the other populations (Table 1).

Morphological analysis

The morphological analysis was performed on field-collected material only. A total of 128 plants from 11populations were analyzed for variation in 59 morpho-logical characters, 46 primary and 13 derived (Table 2).The characters were selected based on previous taxo-nomic treatments and floras (Elven, 1994; Elven andElvebakk, 1996; Engler and Irmscher, 1919; Øvstedal,1998; Rønning, 1996; Webb and Gornall, 1989). Thirty-five of the primary characters were scored as continuousvariables. When possible, three measurements weremade for each vegetative character for each plant andthe mean used for further analysis. Stem measurementswere made on the three tallest stems. Apical flowers ofthese stems were conserved in 70% ethanol and laterscored for floral characters. One measurement was madeper individual for each floral character.

Data analyses

Based on Jaccard similarity, the RAPD data weresubjected to principal coordinate analysis (PCO) andun-weighted pair-group method with arithmeticalaverages (UPGMA) using NTSYS-pc version 2.02 h(Rohlf, 1999). The morphological data were subjected toPCO analysis using the same program. All qualitativecharacters and one of the primary characters used in

each derived character were excluded from the morpho-logical data set prior to the PCO analysis. Because themeasurements made on the basal, middle and uppercauline leaves were found to correlate strongly, only thebasal leaf measurements were included in the analysis.The resulting data set contained 22 characters (Table 2).The data matrix was standardized by ranging and adistance matrix was computed based on Manhattandistance, given by (1=nÞ

Pkjxki2xkjj; where n is the

number of plants, k is the character, and i and j are apair of plants. Spearman’s rank correlation coefficients(r) were calculated between the morphological charac-ters using SPSS 11.0 (SPSS Inc., Chicago IL, USA). AMann–Whitney U-test for equality of medians for thetentative taxa was performed for the morphologicalcharacters using the same program.

Results

Chromosome numbers and DNA content

The chromosome numbers were somewhat variablewithin individual plants of both taxa, and aneuploidnumbers were frequent (Table 1). Many counts wereperformed in plants with variable numbers. Most of thecounts in the two plants examined of tentative S.

hyperborea gave 2n ¼ 26; but the number varied from2n ¼ 23 to 38. Most of the counts in each of the sevenplants examined of tentative S. rivularis gave 2n ¼ 52 orclose to this number. However, in this species some cellshad half that number (2n ¼ 26), and some cells had upto 2n ¼ 95:The 65 plants analyzed by flow cytometry formed two

distinct groups differing in their inferred relative DNAcontent values (Table 1). The plants of tentative S.

rivularis were inferred to have approximately twice asmuch DNA as the plants of tentative S. hyperborea.Thus, we found no evidence of hybrids, but we wereonly able to obtain flow cytometric data for a few plantsalong the transects (Table 1).

RAPD variation

We observed very low level of RAPD variation; most(27 of 39) of the amplified bands that could be reliablyscored were monomorphic. The 12 polymorphic bandsidentified 15 multilocus phenotypes among the 47 plantsanalyzed (Fig. 1). Eight markers showed none or onlylittle variation within the tentative species. Five markerswere exclusively observed in the high-ploid plants oftentative S. rivularis, three of them in all plantsanalyzed. Three markers were exclusively observed intentative S. hyperborea, but only one of them in allplants of this low-ploid species. In both multivariate

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Table 2. Characters used in the morphological analyses of Saxifraga rivularis and S. hyperborea in Svalbard

Character Type MA Note

1 Runners d Absent (0), present (1)

2 No. of flowering stems c *

3 No. of buds, flowers, and/or fruits per

flowering stem

c *

4 No. of cauline leaves per flowering stem c

5 Length of flowering stem to apical flower c *

6 No. of cauline leaves per flowering stem/

length of stem

r * Character 4/5

7 Color of flowering stem d Green (0), slightly anthocyanine-colored (1),

intensively anthocyanine-colored (2)

8 Color of hypanthium d Green (0), slightly anthocyanine-colored (1),


9 Color of sepals d Green (0), slightly anthocyanine-colored (1),


10 Color of ovary (upper part) d Green (0), slightly anthocyanine-colored (1),


11 Color of petals d White (0), white with less than half of nerve

red (1), white with more than half of nerve

red (2), light red (3)

Uppermost cauline leaf

12 Distinction of petiole d Distinct (0), not distinct (1)

13 Length of petiole c

14 Maximum length of leaf c Length from leaf base to apex of the longest

lobe

15 Maximum width of leaf c * Width including lobes

16 Length of petiole plus leaf c * Character 13+14

17 Leaf width/length r * Character 15/16

18 No. of lobes c *

19 Length of middle lobe c Length from base to apex of the middle lobe

20 Width of middle lobe c Maximum width of middle lobe

21 Middle lobe length/width r Character 20/19

Middle cauline leaf

22 Distinction of petiole d Distinct (0), not distinct (1)

23 Length of petiole c *

24 Maximum length of leaf c * Length from leaf base to apex of the longest

lobe

25 Maximum width of leaf c Width including lobes

26 Length of petiole plus leaf c *

27 Width of leaf/length of petiole plus leaf r * Character 25/26

28 No. of lobes c *

29 Length of middle lobe c Length from base to apex of the middle lobe

30 Width of middle lobe c * Maximum width of middle lobe

31 Middle lobe length/width r * Character 30/29

Basal leaves

32 Color d Green (0), slightly anthocyanine-colored (1),


33 Length of petiole c *

34 Maximum length of leaf c * Length from base to apex of the longest lobe

35 Maximum width of leaf c Width including lobes

36 Leaf width/length r * Character 35/34

37 No. of leaf lobes c *

38 Length of middle lobe c * Length from base to apex of the middle lobe

39 Width of middle lobe c * Maximum width of middle lobe

40 Middle lobe width/length r * Character 39/38

41 Middle lobe apex d * Rounded (0), obtuse (1), acute (2)

A.S. Guldahl et al. / Flora 200 (2005) 207–221 213

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Table 2. (continued )

Character Type MA Note

42 Middle lobe incision depth c

43 Distance from incision to lamina base c *

44 Relative incision depth r * Character 42/43

45 Relative incision depth II r Character 42/(42+43)

Hypanthium

46 Shape of base d U-shaped (0), V-shaped (1)

47 Length c *

48 Type of glandular hairs d Glandular hairs distinct (1) or not distinct (0)

49 Length of glandular hairs c *

Sepals

50 Length c *

51 Width c Maximum width

Petals

52 Length c Length from base to apex

53 Width c * Maximum width

Gynoecium

54 Length of style c * Length from point of style fusion to stigma

when stigma with receptive papillae

55 Length of hypanthium plus sepals c Character 47+50

56 Relative length of sepals r * Character 50/55

57 Petal length/sepal length r * Character 52/50

58 Sepal width/length r * Character 51/50

59 Sepal length/hypanthium length r * Character 50/47

Type of character is indicated as c (continuous), d (discrete), or r (ratio). Asterisks indicate characters used in mulivariate analysis (MA).

A.S. Guldahl et al. / Flora 200 (2005) 207–221214

analyses of the RAPD data, two very distinct groups ofmultilocus phenotypes were recognized, consistent withthe differences observed in chromosome number andrelative DNA content (UPGMA: Fig. 1; the PCOanalysis gave similar results and is not shown). Wefound no evidence of hybrids, but as with the flowcytometric analysis, we were only able to obtain RAPDdata for a few plants along the transects (Table 1).

Isozyme variation

We also observed very low level of isozyme variation.Four putative loci, Gpi-2, Pgm, Idh-2 and Ddh, wereinterpreted. All of the 203 plants analyzed werehomozygous for the same allele at Pgm and Ddh. Allplants were also homozygous at Idh-2, and with a singleexception (one plant of S. rivularis), they expressed thesame allele also at this locus.The cytosolic Gpi-2 locus was duplicated in all plants,

resulting in fixed-heterozygous three- or five-bandedphenotypes. Three different homomeric bands wereobserved at Gpi-2 (denoted a, b, and c), combininginto two different three-banded phenotypes (denoted ac

and bc) and one five-banded phenotype (denoted abc;

Table 1). All populations of the low-ploid S. hyperborea

were fixed for phenotype ac. In the high-ploid S.

rivularis, most (six) populations only expressed pheno-type abc, one population (a single plant only) ex-pressed bc, and four populations were polymorphic(expressing either abc and bc or abc and ac). Thus,the overall phenotype frequencies were 100% ac inS. hyperborea and 71% abc, 21% bc, and 8% ac in S.

rivularis, and whereas homomers a and c wereshared between the species, homomer b was only foundin S. rivularis.Large samples taken along the transects at the two

main mixed sites were included in the isozyme analysis.However, the variation pattern observed did not allowfor definite identification of hybrids, as hybridizationbetween the S. hyperborea phenotype (ac) at Gpi-2 andthe typical S. rivularis phenotypes (bc and abc) wouldresult in phenotype abc, indistinguishable from that ofS. rivularis.

Morphological variation

Many morphological characters showed more or lesscontinuous variation in the material analyzed. In the

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Runners: present absentHypanthium hair type: glandular non-glandularGpi-2 isozyme phenotype: abc bc acPloidal level: tetraploid (secondary octoploid?) diploid (secondary tetraploid?)

0.0 0.2 0.4 0.6 0.8 1.0

21R-62 21R-66 38R-1 21R-73 21R-76 21R-77 21R-68 36R-2 37R-12 37R-15 37R-2 37R-4 37R-5 37R-6 37R-7 37R-8 37R-9 39R-10 61R-2 61R-4 61R-6 61R-8 21R-72 24R-56 24R-57 24R-59 24R-60 30R-7 30R-9 38R-3 38R-4 38R-5 38R-8 39R-1 39R-7 39R-4 24R-58 39R-3 37R-1 21H-63 21H-71 34H-11 30H-3 35H-2 41H-1 35H-6 41H-2

Run

ners

Hyp

anth

ium

hair

type

Gpi

-2 is

ozym

eph

enot

ype

Plo

idal

leve

l

Fig. 1. UPGMA analysis of RAPD data (47 plants, 15 multilocus phenotypes) for Saxifraga rivularis and S. hyperborea in Svalbard.

Individual plants are identified by their population number (cf. Table 1), followed by their plant number (after hyphen). For each

plant, the isozyme multilocus phenotype, ploidal level inferred from chromosome number determination and/or flow cytometry

analysis, and two morphological characters distinguishing between the two groups of RAPD multilocus phenotypes, are given (dash

indicates missing data).


PCO analysis of the morphological data, no distinctgroupings were found (not shown). However, someindividual morphological characters discriminated be-tween the two distinct groups identified in the RAPDanalysis, corresponding to the two tentative species(Figs. 1 and 2; Table 3). Runners were almost invariablypresent in S. rivularis, whereas S. hyperborea alwayslacked runners. The hypanthium hairs were short (mean0.33mm) and invariably had distinct glandular tips in S.

rivularis, whereas S. hyperborea had longer hypanthiumhairs (mean 0.77mm) with indistinct glands. The stems,sepals, and hypanthia were typically more intensivelyanthocyanine-colored in S. hyperborea than in S.

rivularis (Table 3).Although we observed considerable overlap between

the two taxa in all quantitative morphological char-acters, they differed significantly in 30 of them (26 at thepo0:01 level; Table 3). Saxifraga hyperborea usually

had more and longer flowering stems with more flowersand cauline leaves than S. rivularis. The uppermost andbasal leaves were usually longest and broadest in S.

rivularis, which also usually had the longest, broadest,most incised, and least acute middle lobes on the basalleaves, the longest hypanthia and styles, and thebroadest sepals and petals. The two species only differedin very few shape (width/length) and other relative sizecharacters, for example that the sepals were broaderrelative to length in S. rivularis and that the sepals werelonger relative to the length of the hypanthium in S.

hyperborea (Table 3).Large samples taken along the transects at the two

main mixed sites were included in the morphologicalanalysis. However, because of the considerable overlapobserved in the morphological characters, potentialhybrids could not be identified with certainty based onmorphology alone.

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0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

Flowering stem length (mm)

Hyp

anth

ium

hai

r le

ng

th (

mm

)

Hypanthium hairs without distinct glandular tips

Hypanthium hairs with distinct glandular tips

(S. hyperborea)

(S. rivularis)

Fig. 2. Variation in flowering stem length, hypanthium hair length, and hypanthium hair type in Saxifraga rivularis and S.

hyperborea in Svalbard (N ¼ 128 plants).


Discussion

Two distinct species

The combined molecular, ploidal, and morphologicaldata suggest that there are two distinct species, S.

rivularis and S. hyperborea, in Svalbard, and we foundno definite evidence for hybridization between them.Although we only had limited material available of theshort-lived S. hyperborea at the time the RAPD analysiswas carried out, the results of this analysis were clear-cut; the plants were divided into two very distinct groupscharacterized by private markers and corresponding tothe two hypothesized species (Fig. 1). The flowcytometry analysis (Table 1; Fig. 1) also dividedthe material into two distinct groups; one (S. rivularis)characterized by approximately twice as much DNAas the other (S. hyperborea). Although we obser-ved considerable variation in mitotic chromosomenumber among different cells in individual plants,most counts in S. rivularis gave 2n ¼ ca: 52 andmost counts in S. hyperborea gave 2n ¼ 26: The iso-zyme analysis supported a similar grouping of thematerial, with S. hyperborea fixed for the single three-banded Gpi-2 phenotype with the homomers a and c andwith most plants of S. rivularis characterized by theadditional homomer b, combined into the three-bandedphenotype bc or the five-banded phenotype abc (Fig. 1;Table 1).

The two groups identified based on the molecular,DNA content and chromosome number data could alsobe readily distinguished based on three morphologicalcharacters; presence/absence of subterranean runners,and length and type of hypanthium hairs (Figs. 1 and 2;Table 3). Runners were never observed in S. hyperborea,neither in the field nor in the cultivated plants. In smallplants of S. rivularis, runners were occasionally lackingor poorly developed, but this character was generallyvery reliable in our material. This result is in agreementwith earlier treatments, in which S. hyperborea and S.

rivularis have been separated throughout their distribu-tions based on this single character (Webb and Gornall,1989; Elven, 1994). The type and length of hypanthiumhairs also separated the Svalbard material of S. rivularis

(short with distinct glandular tips) from that of S.

hyperborea (long without distinct glandular tips).In addition to these three characters, the flower stems,

hypanthium, and sepals were, on the average, moreintensively anthocyanine-colored in S. hyperborea thanin S. rivularis. Significant differences were also observedin many quantitative characters such as the lengthof the flowering stems and width and length ofthe leaves, but the ranges observed in the two taxaalways overlapped (Table 3). The absence of dis-tinct grouping by the multivariate analysis of thequantitative morphological data is probably caused bycomplex variation across taxa in several of thecharacters, concealing the information provided by the

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Table 3. Morphological variation in Saxifraga rivularis and S. hyperborea in Svalbard

Character M–W U-test S. hyperborea S. rivularis

N Mean SE Max. Min. N Mean SE Max. Min.

1 Runners 69 0 0 0 0 57 0.9 0.33 1 0

2 No. of flowering stems ** 59 2.0 1.38 6 1 56 1.2 0.53 3 0

3 No. of buds, flowers, and/or fruits per flowering stem ** 45 2.5 0.81 5 1 54 2.0 0.48 3 1

4 No. of cauline leaves per flowering stem ** 41 2.2 0.55 3 1 51 1.7 0.53 3 1

5 Length of flowering stem ** 69 27.4 10.86 58 4 56 18.0 12.28 51 3

6 No. of cauline leaves/length of flowering stem * 41 0.11 0.11 0.8 0.0 50 0.17 0.162 0.7 0.0

7 Color of flowering stem 69 1.5 0.51 2 0 57 0.3 0.49 2 0

8 Color of hypanthium 69 1.3 0.61 2 0 57 0.2 0.44 2 0

9 Color of sepals 69 1.7 0.55 2 0 57 0.6 0.55 2 0

10 Color of ovary (upper part) 66 1.9 0.40 2 0 49 0.8 0.77 2 0

11 Color of petals 68 2.0 0.27 2 0 52 1.6 0.82 2 0

Uppermost cauline leaf

12 Distinction of petiole 57 0.1 0.26 1 0 42 0.1 0.28 1 0

13 Length of petiole * 4 1.86 0.988 3.2 1.0 6 4.12 1.470 5.0 1.5

14 Length of leaf ** 69 4.39 1.416 12.0 2.5 57 5.77 1.707 9.5 2.1

15 Width of leaf ** 69 1.88 0.604 3.9 0.9 57 2.54 0.995 6.2 0.9

16 Length of petiole plus leaf ** 69 4.47 1.418 12.0 2.5 57 6.11 2.031 12.5 2.1

17 Leaf width/length 69 0.42 0.114 0.80 0.26 57 0.42 0.096 0.90 0.23

18 No. of lobes 69 0.2 0.60 3 0 57 0.0 0.26 2 0

19 Length of middle lobe 9 2.31 0.478 3.0 1.8 1 3.1 — 3.1 3.1

20 Width of middle lobe 9 2.00 0.412 2.8 1.3 1 2.8 — 2.8 2.8

21 Middle lobe length/width 9 0.88 0.152 1.10 0.67 1 0.90 — 0.90 0.90

Middle cauline leaf

22 Distinction of petiole 55 0.5 0.48 1 0 22 0.7 — 1 0

23 Length of petiole 36 4.7 2.40 15 2 41 5.9 3.34 19 1

24 Length of leaf * 64 5.00 1.546 10.1 2.6 41 5.72 1.814 11.6 3.0

25 Width of leaf 64 5.02 1.456 8.9 2.0 40 5.59 2.090 12.0 2.5

26 Length of petiole plus leaf * 64 7.12 3.272 23.2 3.1 40 8.82 4.057 25.0 3.2

27 Width of leaf/length of petiole+leaf 64 0.77 0.291 1.63 0.35 41 0.73 0.352 1.60 0.35

28 No. of lobes ** 64 2.8 0.82 5 0 30 2.1 1.43 5 0

29 Length of middle lobe 61 2.57 0.745 5.5 1.6 30 2.87 0.733 5.0 1.4

30 Width of middle lobe ** 61 1.97 0.499 3.1 1.1 30 2.43 0.727 4.3 1.1

31 Middle lobe length/width 61 0.798 0.188 1.40 0.22 58 0.84 0.133 1.13 0.52

Basal leaves

32 Color 68 0.6 0.53 2 0 58 0.6 0.50 1 0

33 Length of petiole 68 13.20 5.899 32.5 4.5 58 14.96 9.370 51.0 3.8

34 Length of leaf ** 68 4.54 0.956 7.1 3.0 58 5.50 1.535 12.5 2.4

35 Width of leaf ** 68 6.79 1.620 12.2 4.1 58 8.12 2.165 17.6 4.0

36 Leaf width/length 68 1.48 0.144 1.95 1.14 58 1.47 0.166 1.96 1.10

37 No. of leaf lobes 68 4.5 0.76 5 3 58 4.7 0.68 6 3

38 Length of middle lobe ** 68 2.42 0.504 3.8 1.7 58 2.95 0.738 6.0 1.2

39 Width of middle lobe ** 68 2.35 0.506 4.0 1.5 58 3.02 0.874 7.0 1.3

40 Middle lobe width/length 68 0.98 0.161 1.35 0.61 58 1.02 0.148 1.55 0.72

41 Middle lobe apex ** 67 1.1 0.31 2 0 58 0.8 0.38 1 0

42 Middle lobe incision depth ** 67 1.57 0.467 2.9 0.9 58 1.96 0.553 3.8 0.5

43 Distance from incision to lamina base 67 1.99 0.613 4.2 1.2 58 1.94 0.776 4.1 1.0

44 Relative incision depth ** 67 0.81 0.225 1.50 0.33 58 1.10 0.368 1.92 0.20

45 Relative incision depth II ** 67 0.44 0.068 0.60 0.25 58 0.51 0.089 0.66 0.17

Hypanthium

46 Shape of base 69 0.3 0.45 1 0 55 0.1 0.31 1 0

47 Length ** 69 1.11 0.400 3.0 0.5 55 1.33 0.371 2.3 0.6

48 Type of glandular hairs 68 0 0 0 0 55 1 0 1 1

49 Length of glandular hairs ** 69 0.74 0.302 1.6 0.1 55 0.33 0.155 0.9 0.1


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Table 3. (continued )

Character M–W U-test S. hyperborea S. rivularis

N Mean SE Max. Min. N Mean SE Max. Min.

Sepals

50 Length 69 2.07 0.391 3.2 0.9 55 2.14 0.451 3.0 0.8

51 Width ** 69 1.27 0.233 2.1 0.8 55 1.49 0.276 2.2 0.9

Petals

52 Length 69 3.85 0.732 5.5 1.6 49 4.00 0.601 5.3 3.0

53 Width ** 69 1.69 0.341 3.2 0.8 49 2.06 0.343 3.1 1.5

Gynoecium

54 Length of style ** 68 1.14 0.235 2.0 0.7 54 1.29 0.314 1.9 0.7

55 Length of hypanthium plus sepals ** 69 3.19 0.650 5.3 1.7 55 3.47 0.660 5.3 1.7

56 Relative length of sepals ** 69 0.66 0.730 0.81 0.43 55 0.62 0.072 0.75 0.39

57 Petal length/sepal length 69 1.92 0.516 4.17 0.71 49 1.94 0.524 4.40 1.35

58 Sepal width/length ** 69 0.62 0.120 1.06 0.39 55 0.73 0.297 2.67 0.40

59 Sepal length/hypanthium length ** 69 2.07 0.759 5.00 0.77 55 1.71 0.491 2.93 0.63

Measurements are given in mm. N, number of plants analyzed. Asterisks indicate significant differences between the taxa according to

Mann–Whitney U-tests (performed for quantitative characters only): ��po0:01; �po0:05:


taxonomically more significant ones (Fjellheim et al.,2001; Hansen et al., 2000).

Hybridization absent or infrequent

In spite of our quite extensive analysis alongenvironmental transects in the two large mixed popula-tions in Svalbard, we were unable to detect hybridsbetween the two species with certainty. In accordancewith our tentative field observations, S. hyperborea

occurred most frequently in the driest parts of thegradients, typically in late snowbeds that only wereseasonally moist, whereas S. rivularis occurred mostfrequently in the wettest parts, typically in continuouslywet moss vegetation and along small brooks. Our datacannot, however, exclude the possibility of occasionalhybridization between the species. We detected noobvious hybrids in the morphological analysis, butbecause many characters overlap between the species, itwould in any case be difficult to identify hybrids withcertainty based on morphology alone. The isozymeanalysis provided the most complete data set along thetransects, and potential hybridization between the S.

hyperborea phenotype (ac) at Gpi-2 and the typical S.

rivularis phenotypes (bc and abc) would result inphenotype abc, indistinguishable from that of S.

rivularis. The most efficient way to detect hybrids wouldhave been to obtain more complete RAPD and flowcytometry data sets along the transects, but this was notpossible because of the high mortality among thecultivated plants. Nevertheless, our data seem to excludethe possibility for extensive hybridization between thetwo species in Svalbard, consistent with the barrier

imposed by their strongly autogamous breeding systems(Brochmann and H(apnes, 2001). It is also possible thatthe previous report of hybridization between the speciesbased on a chromosome count of 2n ¼ 39 (Borgen andElven, 1983) in fact is based on endoploid variation in aplant with its main number at 2n ¼ 26 or 52 (cf.variation reported in Table 1), or on a plant resultingfrom fusion of one reduced and one unreduced gameteof S. hyperborea.

Endopolyploidy

We found variable chromosome numbers amongdifferent cells in plants of both species, especially in S.

rivularis (2n ¼ 26295; Table 1). Although mainlyreported with 2n ¼ 52; there are several earlier reportsof aneuploid or deviating numbers for this species(Bocher, 1938; Engelskjøn and Knaben, 1971; Engelsk-jøn, 1979). The chromosome number of S. hyperborea

seems to be more stable, as judged from earlier reports,but we nevertheless observed numbers in the range of2n ¼ 23238: It is possible that aneuploid and deviatingnumbers in these species of Saxifraga are underreportedin the literature. Multiple ploidal levels among cells inindividual plants are often referred to as endopolyploi-dy, resulting from endoreduplication. This is a naturallyoccurring disruption of the mitotic cell cycle, which inturn causes amplification of the genome (Nagl, 1978).Endopolyploidy typically occurs in highly specializedand large cells such as root hairs (Dosier and Riopel,1978). In a flow cytometric study of Brassica, Kudo andKimura (2001) suggest that endoreduplication couldpresent means for organisms to increase the number of


functional gene copies within each cell, thereby acting tomitigate any adverse effects of environmental influencessuch as ultraviolet radiation on transcription of thegenome. With respect to the arctic saxifrages, this is apossible explanation.

Low levels of molecular diversity

Very low levels of molecular variation were observedboth within and among the Svalbard populations of thetwo Saxifraga species, at RAPD loci as well as isozymeloci. The low level of within-population variation isconsistent with their strongly autogamous breedingsystem, partly also clonality via runners in S. rivularis,and it is similar to the levels found in many other speciesin Svalbard (Brochmann and Steen, 1999; Brochmann etal., 2004) as well as in S. rivularis in Britain (Hollings-worth et al., 1998). The low level of among-populationvariation in each species may reflect bottleneckingduring a single immigration event to this isolated,high-arctic archipelago, which was almost fully glaciatedat the Weichselian maximum (Brochmann et al., 2003),but it cannot be excluded that the two speciesimmigrated repeatedly from genetically depauperatesource populations.

Reticulate evolution

All plants examined of both species were fixed-heterozygous at the Gpi-2 locus, reflecting either asingle-gene duplication at the 2n ¼ 26 level or an oldgenome-wide duplication event involving a lower basicnumber than x ¼ 13: We favor the latter hypothesis.The basic number of x ¼ 8 also occurs in sectionMesogyne, and a wide range of other basic numbers isknown in Saxifraga, including 5 and 6 (Webb andGornall, 1989). These data suggest that aneuploidchromosome number evolution is frequent in the genus,and that x ¼ 13 may be a secondary basic number arisenvia aneuploid addition or removal of chromosomes aftera polyploidization event. Alternatively, x ¼ 13 may be asecondary number derived from hybridization andpolyploidization between taxa with different basicnumbers.The fixed heterozygosity observed at the Gpi-2 locus

may thus indicate that the species are geneticallyallopolyploid with disomic inheritance. Saxifraga hy-

perborea may be a secondary allotetraploid (withgenotype aacc) and S. rivularis may be a secondaryallooctoploid (with genotype bbbbcccc or with four-two-two dosage distribution for the abc phenotype).Our molecular data are consistent with the hypothesis

that the S. rivularis lineage has originated via hybridiza-tion and polyploidization with the S. hyperborea lineageas one of its progenitors (cf. Brochmann et al., 1998).

The isozyme markers and several RAPD markers foundin S. hyperborea formed a subset of those observed in S.

rivularis, which also contained additional markers thatmay have been derived from another progenitor lineage.Notably, evidence for the Beringian S. bracteata D. Don(2n ¼ 26) lineage as the second progenitor of S. rivularis

was recently obtained in a fully circumpolar analysis ofthe complex (Jørgensen, 2004). In a wide sense, S.

rivularis also comprises amphi-Beringian populationswith 2n ¼ 52 that have been referred to as S. arctolitor-

alis Jurtz. & V.V. Petrovsky. Saxifraga rivularis s. lat.was intermediate between S. bracteata and S. hyperbor-

ea in multivariate analyses of AFLP fingerprint data andmorphological data, and its DNA content was the sumof the DNA contents of the same two species. Thus, S.

rivularis s. lat. probably originated via polyploidizationin Beringia, followed by circumpolar expansion andslight morphological differentiation among Beringianand amphi-Atlantic populations (Jørgensen, 2004).

Acknowledgments

We thank the University Centre in Svalbard forproviding necessary facilities to set up an in situ isozymelaboratory and for logistic support to the field work. Weare indebted to Reidar Elven for advice and forproviding some of the material, including that fromCanada, which was obtained by participation in theTundra North-West (TNW) ‘99 expedition funded bythe Polar Research Secretariate at the Royal SwedishAcademy of Sciences. We also thank Inger G. Alsos,Pernille B. Eidesen, Siri Fjellheim, Siri Kjølner, LeidulfLund, and Jorun Nylehn for collection of material, andthe gardeners at the phytotron at the Department ofBiology, University of Oslo, for taking care of thecultivated plants. Marte Jørgensen is acknowledged forher very thorough and critical reading of an earlierversion of the paper. The study was supported by theUniversity Centre in Svalbard, the Natural HistoryMuseum at the University of Oslo, the NansenFoundation at the Norwegian Academy of Science andLetters, and Grant 146515/420 to CB and LB from theResearch Council of Norway.

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The Saxifraga rivularis complex in Svalbard: Molecules, ploidy and morphology

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