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(American Journal of Botany. 2001;88:103-112.)
© 2001 Botanical Society of America, Inc.

Trans-Atlantic dispersal and phylogeography of Cerastium arcticum (Caryophyllaceae) inferred from RAPD and SCAR markers1

Aslaug R. Hagen2, Henriette Giese3 and Christian Brochmann4,2

2 Botanical Garden and Museum, University of Oslo, Sarsgate 1, N-0562 Oslo, Norway; and 3 Department of Ecology and Molecular Biology, Section of Genetics and Microbiology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark

Received for publication June 29, 1999. Accepted for publication March 10, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Cerastium arcticum is an autogamous pioneer species with a distribution limited to the North Atlantic region. It has been suggested that such species must have survived in ice-free refugia on both sides of the Atlantic throughout the last, or even several, of the Pleistocene glaciations, because they lack special adaptations for long-distance dispersal. To address the possibility for recent trans-Atlantic dispersal of C. arcticum, we analyzed random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) differentiation among 26 populations of this high-polyploid species. Three SCAR markers were obtained that verified the main patterns identified in the RAPD analysis. Eighty-four multilocus RAPD phenotypes were observed in the 126 plants analyzed, based on 35 polymorphic markers. Multivariate analyses and analyses of molecular variance (AMOVAs) identified two highly divergent groups of populations: one arctic group (western and eastern Greenland, and the archipelagos of Svalbard and Franz Josef Land) and one nonarctic group (southern and northern Norway, and Iceland), indicating that C. arcticum is composed of two lineages with different evolutionary histories. However, there was little geographic structuring within each lineage, in spite of the fact that both lineages are disjunctly distributed across the Atlantic. Occurrence of very similar, in some cases even identical RAPD multilocus phenotypes on both sides of the Atlantic in this autogamous allopolyploid is most probably caused by postglacial dispersal. The present geographic distribution of C. arcticum may thus have been established after trans-Atlantic expansion from two Weichselian refugia, one for each evolutionary lineage. Unexpectedly, the level of intrapopulational variation increased towards the north. This may reflect that interpopulational migration is most extensive in the treeless arctic environment, where the species has a more continuous distribution than in the more southerly areas.

Key Words: Cerastium arcticum • long-distance dispersal • phylogeography • RAPD • SCAR


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Long-distance dispersal of arctic-alpine plants has been considered to be extremely rare because most of them lack special adaptations such as hairs or wings on the diaspores (e.g., Dahl, 1963, 1987 ). The North Atlantic Ocean has therefore been regarded as a virtually impenetrable barrier to dispersal of arctic-alpine plants, and it has been suggested that arctic floras are of ancient origin. The present similarities between the floras on different sides of the North Atlantic have been ascribed to migration across a Tertiary landbridge and subsequent survival during the Quaternary glaciations (Dahl, 1963, 1987 ; Löve, 1963 ). It is possible, however, that specialized dispersal mechanisms are of much less importance for occasional long-distance dispersal than for regular short-distance dispersal (Berg, 1983 ). There are, for example, only minor floristic differences between the isolated, volcanic island of Jan Mayen and northern Europe (Lid, 1964 ). This island has never been connected by a land bridge (Bennike and Hedenäs, 1995 ), but it is nevertheless populated by many species that lack special adaptations to long-distance dispersal. It is possible that long-distance dispersal by migrating birds and strong winds over sea-ice or other ice- and snow-covered surfaces may be particularly important in the treeless arctic environment (Savile, 1972 ; Nordal, 1987 ). The North Atlantic Ocean was ice-covered in winter southwards to France during the late Weichselian, when many coastal areas of northern Europe already were ice-free (Andersen and Borns, 1994 ). This sea-ice may have given ample opportunities for trans-Atlantic dispersal.

Reconstruction of Late Weichselian floras has shown that their composition has changed rapidly in response to climate changes. Plant fossil records from Andøya in northern Norway show, for example, that there have been repeated cycles of immigration and expansion during climatic ameliorations, resulting in decline and extinction of populations during colder phases (Alm and Birks, 1991 ). Recent paleoclimatic reconstructions have demonstrated that the climate changed very rapidly (e.g., Alley et al., 1993 ; Taylor et al., 1993 ; Lowe and NASP Members, 1995 ). In central Greenland, conditions probably switched from glacial to near-interglacial in periods of 10–20 yr (Dansgaard et al., 1993 ; Taylor et al., 1993 ), probably inducing severe biotic responses.

Geographic patterns of intraspecific genetic variation are determined by migration history. Molecular genetics have provided plant geographers with new, powerful tools for studying intraspecific variation. Earlier inferences on migration history based on present geographic distributions can now be re-examined by studying the geographic structuring of the genetic variation. Phylogeographic variation has recently been related to the postglacial migration history of some arctic-alpine plant species, e.g., Vahlodea atropurpurea (Haraldsen, Ødegaard, and Nordal, 1991 ), Draba spp. (Brochmann, Soltis, and Soltis, 1992 ), Lychnis alpina (Haraldsen and Wesenberg, 1993 ), Saxifraga oppositifolia (Gabrielsen et al., 1997 ), S. cespitosa (Tollefsrud et al., 1998 ), Dryas integrifolia (Tremblay and Schoen, 1999 ), and Phippsia spp. (Aares, Nurminiemi, and Brochmann, 2000 ). Several of these studies indicate that there has been recent gene flow among North Atlantic regions (e.g., Greenland, the arctic archipelago of Svalbard, Iceland, and/or Scandinavia).

Among the amphi-Atlantic disjuncts in the arctic-alpine flora, the so-called "West-arctic" species have attracted particular interest in Nordic phytogeography. These species (~30) occur in alpine Scandinavia and/or Svalbard as well as on the opposite side of the Atlantic (Greenland and/or northeastern continental North America), but they are absent from the Central European mountains and most of the remaining Arctic and Sub-Arctic (e.g., Dahl, 1963, 1987 ). The current eastern Atlantic distribution area of these species (Hultén and Fries, 1986 ) is situated within the Pleistocene area of glaciation (Andersen and Borns, 1994 ). The present occurrence of these West-arctic species in Fennoscandia and/or Svalbard has provided the strongest argument in favor of survival in local ice-free refugia within these geographic areas throughout one or several of the Pleistocene glaciations ("the nunatak hypothesis"; e.g., Dahl, 1963, 1987 ; Rønning, 1963 ; Gjærevoll, 1992 ).

In this paper, we report the first phylogeographic analysis of one of the West-arctic species, Cerastium arcticum Lange (Caryophyllaceae). This autogamous pioneer species (Ekstam, 1898 ; Asplund, 1918 ; Warming, 1920 ; A. R. Hagen, University of Oslo, unpublished data) is found in the northern part of the British Isles, disjunct in the Scandinavian mountain range, the Faeroe Islands, Iceland, the northwestern arctic Russian islands, Svalbard, Greenland, and the eastern Canadian Arctic (Hultén and Fries, 1986 ). Cerastium arcticum belongs to the mature C. alpinum–C. arcticum polyploid complex, in which extensive and complex morphological variation has led to the recognition of several species, subspecies, and varieties (e.g., Tolmachev, 1930 ; Hultén, 1956 ; Böcher, 1977 ). Cerastium arcticum is high-polyploid, usually with 2n = 12x = 108 (e.g., Brett, 1955 ; Löve and Löve, 1956, 1975 ; Jørgensen, Sørensen, and Westergaard, 1958 ; Engelskjøn, 1979 ), and it is highly polymorphic in morphology and occurs in a variety of habitats (Elven and Elvebakk, 1996 ). The present study is part of a larger effort at the University of Oslo to unravel the evolutionary history, taxonomy, and phylogeography of the C. alpinum–C. arcticum complex (cf. Hagen and Sæther, 1993 ; Hagen et al., 1995 ; Schjøll, 1995 ; Brochmann et al., 1996 ; Brysting and Hagen, 1999 ; Brysting and Borgen, 2000 ; Brysting and Elven, 2000 ).

In this paper, we use RAPD analysis (Random Amplified Polymorphic DNA; Welsh and McClelland, 1990; Williams et al., 1990 ) and SCAR analysis (Sequence Characterized Amplified Regions; Paran and Michelmore, 1993 ) to address the possibility of recent trans-Atlantic dispersal of C. arcticum. Populations from most of the distribution area were examined with special reference to the partitioning of the genetic variation within and among geographic regions. RAPD analysis is simple and has been successfully used in several phylogeographic studies (e.g., Gabrielsen et al., 1997 ; Tollefsrud et al., 1998 ), but the method has several well-known problems and limitations. One problem is reproducibility, which may be affected by the quality and concentration of the DNA, the specific polymerase used, and the assay conditions, and it is also possible that some of the comigrating, anonymous fragments are nonhomologous (e.g., Ellsworth, Rittenhouse, and Honeycutt, 1993 ; Giese et al., 1994 ). The SCAR technique depends on sequence information in order to design longer primer pairs, but results in a more robust marker system (Paran and Michelmore, 1993 ). Herein we report on the development of SCAR markers designed on the basis of polymorphic RAPD fragments identified in this study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant materials
Material was sampled from 26 populations of Cerastium arcticum in seven geographic regions (Fig. 1; Table 1): Iceland (four populations), southern Norway (two populations), northern Norway (five populations), eastern Greenland (two populations), western Greenland (three populations), Svalbard (nine populations), and the arctic Russian archipelago of Franz Josef Land (one population). We were not able to obtain fresh material from the eastern Canadian Arctic, but western Greenland is situated close to the American mainland, and the western Greenlandic material was therefore considered sufficient for our purpose.



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  		Fig. 1. Map showing the sampling sites for the 26 populations analyzed of Cerastium arcticum. Populations in the seven main geograpic regions are indicated with different symbols (cf. Table 1 ; Figs. 2–5 )

 

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  		Table 1. Collection data, number of RAPD phenotypes observed, and percentage of polymorphic markers observed in the populations of Cerastium arcticum investigated. Percentage of polymorphic markers is calculated based on the total number of the 35 markers scored that were polymorphic within each population. Five plants were analysed from each population except for population IC-1 (four plants) and WG-1 (two plants). Collectors: AB (A. K. Brysting), AH (A. R. Hagen), AO (A. Often), BS (B. Strandberg), HP (H. Pauli), JB (J. B{ö}cher), MG (M. Grønbech), MT (M. M. Tollefsrud), MS (M. Strand), OS (O. Schjøll), TG (T. M. Gabrielsen), and TS (T. Sæther)

 
When analyzing geographic structuring of genetic variation, the most efficient sampling procedure is sampling of many populations covering the species distribution area, rather than sampling many individuals per population (Templeton, Routman, and Phillips, 1995 ). As Cerastium arcticum is a predominantly autogamous allopolyploid with high levels of fixed heterozygosity at isozyme loci (Hagen and Sæther, 1993 ; Brysting and Borgen, 2000 ), and our primary aim was to investigate among-populational variation, a sample size of five plants per population was considered sufficient for the RAPD analysis. The plants were collected with an interplant distance of at least 10 m to increase the possibility of detecting potential among-individual variation. Most plants were cultivated in the phytotrone at the University of Oslo with cycles of 3 mo of summer (18-h day at 12°C) and 3 mo of vernalization (10-h day at 5–6°C). Voucher specimens from all populations are deposited at the Botanical Museum, University of Oslo, Norway.

RAPD analysis
Total DNA was extracted from ~100 mg fresh leaf tissue from the cultivated plants or ~30 mg silica-dried leaf tissue from the field-collected plants using a modification of the cTAB (cetyltrimethylammonium bromide) protocol of Saghai-Maroof et al. (1984) , differing primarily in the use of a 2x extraction buffer and RNase treatment. Leaf material was ground in liquid nitrogen, mixed with 700 µL extraction buffer of 65°C (1.4 mol/L NaCl, 100 mmol/L Tris-HCl (pH 8.0), 20 mmol/L EDTA, 2% cTAB, and 1% 2-mercaptoethanol), and incubated at 65°C for 45 min with shaking every 10 min. Proteins were extracted twice with 500 µL chloroform-isoamyl alcohol (24:1) for 10 min and then centrifuged at 12 000 rpm for 5 min. RNase (10 µg/mL) was added to the resulting supernatants for 30 min at 37°C, and the DNA was precipitated with ice-cold 2-propanol (two-thirds of the supernatant volume) for at least 1 h and centrifuged at 12 000 rpm for 10 min. The pellet was washed twice in 70% ethanol, vacuum-dried, and resuspended in 150 µL TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0). Approximate DNA concentrations were estimated by visual inspection of 0.7% agarose gels run in TBE buffer (89 mmol/L Tris base, 89 mmol/L boric acid, 2 mmol/L EDTA, pH 8.3) and stained with ethidium bromide, by comparing staining intensity of the samples to a {lambda} EcoRI/HindIII marker.

PCRs (polymerase chain reaction) were modified after Williams et al. (1990) . Reaction volumes were 20 µL with 2.5 ng genomic DNA, 0.2 µmol/L primer (Operon Technologies, Alameda, California, USA), 100 µmol/L of each dNTP (Promega, Madison, Wisconsin, USA), 1x PCR buffer [10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.01% gelatin, and 0.1% Triton X-100; HT Biotechnology Ltd., Cambridge, UK], and 0.25 U Super Taq polymerase (HT Biotechnology Ltd.). The reaction mixtures were overlaid with mineral oil prior to PCR to prevent evaporation. Amplifications were performed in a PTC-100 thermocycler (MJ Research Inc., Watertown, Massachusetts, USA) using microtiter plates, except for amplifications with primers of the F kit, for which a Omnigene thermocycler (Hybaid Ltd., Middlesex, UK) was used. An initial denaturing step for 2 min at 94°C was followed by 45 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C. A final extension step at 72°C for 10 min was included for extra elongation. A negative control was run for each primer. Amplification products were separated on 1.4% agarose gels run in 1x TBE buffer, detected by ethidium bromide staining, and photographed under UV light. {lambda} EcoRI/HindIII was used as a molecular size marker.

Eighty random 10-mer primers (Operon kits A, C, D, and F) were initially tested using one plant from each of four geographic regions. Twelve primers that produced strongly amplified polymorphic bands with these test templates were selected for full analysis (Table 2). The gels were scored conservatively, i.e., only the most reliable and distinct bands were scored, as 1, present, or 0, absent. The reproducibility of all initially scored bands was rechecked by comparing the profiles of a number of individual plants that each was run several times (in the first primer test, in the main analysis, and/or in final reruns) to verify marker alignment across gels.


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  		Table 2. Sequences of RAPD and SCAR primers used in RAPD- and SCAR-PCR analyses of Cerastium arcticum. The part of the SCAR sequences that was obtained from sequencing of RAPD markers is in italics

 
SCAR analysis
Five RAPD markers that showed among-region variation were pipetted out of the agarose gels and dissolved in 300 µL warm TE buffer (pH 7.6). Two microlitres of each solution was used in five re-amplifications using the original RAPD primer. Replicas of each sample were excised from the gel, pooled, and purified using QIAEX II Agarose Gel Extraction (QIAGEN Inc., Chatsworth, California, USA). The DNA fragments were blunt-end ligated into a precut pTAg plasmid vector included in the LigATor PCR cloning kit (R&D Systems Ltd., Minneapolis, USA). The plasmids were transformed into the competent cells provided in the kit and spread on LB agar plates containing ampicillin and tetracycline (the latter to prevent loss of F' plasmids). Colony PCR was performed as a rapid screening for presence or absence of inserts using the primers pTAg SEQ 3' and 5'. Positive clones were transferred to SOC media containing ampicillin and tetracycline and incubated overnight. Minipreps of the cultures were purified using QIAGEN-tip 20 (QIAGEN Inc.) and quantified on 0.8% agarose gels. Twenty to fifty nanograms of the purified fragments were sequenced on an ABI 373 automated sequencer using the ABI PRISM TaqFS Dye Terminator kit (Perkin-Elmer Corp., Foster City, California, USA), and 3 pmol of each of the sequencing primers pTag SEQ 3' and pTag SEQ 5'.

For each cloned RAPD marker (Table 2), two oligonucleotides were designed as SCAR primers, each consisting of the original ten bases of the RAPD primer plus the next 14 internal bases (cf. Paran and Michelmore, 1993 ). The primers were obtained from Pharmacia Biotech (Uppsala, Sweden) and DNA technology ApS (Aarhus, Denmark). Amplification of genomic DNA using the SCAR primers was carried out under standard PCR conditions at high annealing temperatures using 20 ng template and 1 U Taq polymerase per reaction. The PTC-100 thermocycler was programmed for 5 min at 94°C followed by 30 cycles of 1 min at 94°C, 1 min at 60°C to 70°C, and 2 min at 72°C, followed by a final extension step at 72°C for 10 min. Amplification products were separated on 2.0% agarose gels run in 1x TBE buffer and detected by ethidium bromide staining.

Data analyses
The RAPD phenotype of each plant was expressed as a vector of zeros and ones corresponding to the scores of the RAPD bands, and the resulting matrix was analyzed using NTSYS-pc (Rohlf, 1998 ). Similarities between all pairs of phenotypes were calculated using the Dice, Simple Matching, and Jaccard coefficients. The three similarity matrices were highly correlated (r = 0.99 for all combinations, P = 0.002; the normalized Mantel statistics z). The similarity matrices were subjected to UPGMA (Unweighted Pair Group Method with Arithmetic Averages), PCO (Principal Coordinate Analysis), and Minimum Spanning Tree analysis (MST), and similar results were obtained for all three similarity coefficients. Only the analyses based on Dice's coefficient are therefore presented.

Analyses of Molecular Variance (AMOVA; Excoffier, Smouse, and Quattro, 1992 ) were used to partition the RAPD variation among main geographic regions, among populations within geographic regions, and among individuals within populations, using the program WINAMOVA 1.55 provided from L. Excoffier (http://anthropologie.unige.ch/ftp/comp; see also Gabrielsen et al., 1997 ).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
RAPD analysis
Thirty-seven polymorphic RAPD markers were scored for the 12 primers used in the full analysis (Table 2). Two of these RAPD markers (OPF-06700 and OPF-13775) were excluded based on the subsequent SCAR analysis (see below), resulting in a final RAPD data set consisting of 35 markers. One to seven markers (mean 2.9), ranging in size from 300 bp to 1400 bp, were scored for each primer. Eighty-four multilocus RAPD phenotypes were identified among the 126 plants analyzed. Seven markers showed no intrapopulational variation. Most populations contained several RAPD phenotypes (mean 3.2); only three populations contained a single phenotype (Table 1). Six phenotypes were observed in more than one population (cf. Fig. 2). One of these phenotypes was particularly widespread; it was observed in five populations from Iceland, northern Norway, and southern Norway. The two most distant populations containing this phenotype were separated by >1800 km.



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  		Fig. 2. UPGMA analysis of 84 multilocus RAPD phenotypes observed in 126 plants from 26 populations of Cerastium arcticum (based on Dice's similarity index). Presence of each SCAR marker is indicated. Individual plants are identified by their population number (Table 1 ) followed by the plant number

 
The UPGMA analysis of the RAPD multilocus phenotypes resulted in two very distinct clusters separated at a high level (Fig. 2), one consisting of the arctic populations (western Greenland, eastern Greenland, and the archipelagos of Svalbard and Franz Josef Land) and one consisting of the nonarctic populations (Iceland, southern Norway, and northern Norway). One RAPD marker was fixed in the arctic group and absent from the nonarctic group, four markers were fixed in the arctic group and rare in the nonarctic group, and five markers were rare in the arctic group and fixed in the nonarctic group. The "nonarctic" cluster in the UPGMA analysis consisted of two rather distinct subclusters. However, both of them contained Icelandic as well as Norwegian populations, and there was no distinct geographic structure within the subclusters. The "arctic" cluster was also poorly structured with respect to geographic origin of the populations.

In a PCO analysis of all RAPD phenotypes, the arctic and nonarctic population groups were clearly separated along the first axis (65.7% of the variation; Fig. 3). Because of the high proportion of the total variation extracted by the first axis in this analysis, PCO analyses were also performed separately for the arctic and the nonarctic populations to clarify the relationships within these groups. In the analysis of the nonarctic group (Fig. 4), no distinct geographic structure was identified even though the first three axes extracted a large proportion (70.4%) of the total variation. The first axis (48.5%) separated the same two groups as the UPGMA analysis (Fig. 2), both of them including Icelandic as well as Norwegian plants. In the PCO analysis of the arctic group (Fig. 5), the population from Franz Josef Land was somewhat separated from the other populations along the first axis (19.8%), whereas the populations from western Greenland, eastern Greenland, and Svalbard were highly intermingled. The minimum spanning trees superimposed on the three PCO analyses consisted of many trans-Atlantic connections (Figs. 3–5).



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  		Fig. 3. PCO analysis with a minimum spanning tree superimposed of 84 multilocus RAPD phenotypes observed in 126 plants from 26 populations of Cerastium arcticum (based on Dice's similarity index). For simplicity, only the longest branches of the minimum spanning tree are shown

 


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  		Fig. 4. PCO analysis with a minimum spanning tree superimposed of 27 multilocus RAPD phenotypes observed in the 54 plants analyzed of the 11 nonarctic populations of Cerastium arcticum (based on Dice's similarity index)

 


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  		Fig. 5. PCO analysis with a minimum spanning tree superimposed of 57 multilocus RAPD phenotypes observed in the 72 plants analyzed of the 15 arctic populations of Cerastium arcticum (based on Dice's similarity index)

 
In a nested AMOVA analysis based on the two main groups (the arctic and the nonarctic) identified in the UPGMA and PCO analyses, 73.3% of the total variation was found between the groups (Table 3). In a nested analysis of the nonarctic group, the variance component among the three geographic regions (Iceland, southern Norway, and northern Norway) was negative (-5.6%; but insignificant), possibly indicating that RAPD phenotypes from different geographic regions were more similar than those from the same geographic region. Also in nested analyses of the arctic group, most of the variation was found among and within populations from the same geographic region; much less variation was found among the geographic regions. In an AMOVA analysis of all arctic populations, disregarding geographic region, more than half of the variation (51.2%) was found within populations. In contrast, the variation within the nonarctic populations was only 22.4%. In AMOVAs performed separately for each geographic region, the within-population variation increased consistently from the nonarctic regions (15.8–27.0%) to the arctic regions (58.0–67.7%).


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  		Table 3. Analyses of molecular variance (AMOVAs) based on 84 RAPD phenotypes observed in 126 plants from 26 populations of Cerastium arcticum. All P values obtained were <0.001, except for one negative variance component. Significance levels were not obtained for the intrapopulational level of variation when each region was analysed separately. Abbreviations: df, degrees of freedom; SSD, sum of squared deviations; MSD, mean squared deviations

 
SCAR analysis
Five RAPD markers were selected for the development of SCARs (Table 2). Each RAPD fragment was cloned from two PCR reactions carried out on DNA from plants from two different geographic regions (cf. Table 1). The sequences of the RAPD markers OPF-06700 and OPF-13775 differed between the two geographic regions. These RAPD markers were thus excluded, although the result did not necessarily indicate nonhomology (the sequences may rather have been derived from two different comigrating RAPD bands from each plant, but this possibility was not further investigated; see Paran and Michelmore, 1993 ). Twenty-four-mer SCAR primer pairs were designed for the three remaining RAPD markers. Genomic DNA of all of the 126 plants used in the RAPD analysis were used as templates in the SCAR-PCRs, which verified the results of the RAPD analysis (Fig. 2).

Two of the SCARs (OPF-06900 and OPC-191000) were dominant markers resulting in patterns identical to those in the RAPD analysis, whereas a subsequent restriction fragment analysis of the third SCAR (OPC-15550) revealed that this was a codominant marker. This marker was obtained with similar intensity for all plants in the SCAR-PCR. To search for polymorphisms corresponding to the original RAPD pattern, the 550-bp SCAR fragments obtained from one plant from each of 17 populations were purified using Qiaquick gel extraction and digested with HaeIII and HhaI, respectively. The digestions revealed fragment length polymorphisms that corresponded exactly to the presence/absence pattern of the original RAPD marker. Included in this restriction fragment analysis were seven arctic populations and the nonarctic populations IC-1, NN-17, and NN-50, all of which possessed the 550-bp RAPD marker, and seven other nonarctic populations, which did not possess the 550-bp RAPD marker (cf. Fig. 2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The RAPD and SCAR analyses revealed two very divergent groups of populations within C. arcticum. One group consists of the arctic populations (western Greenland, eastern Greenland, and the archipelagos of Svalbard and Franz Josef Land), and the other group consists of the nonarctic populations (Iceland, southern Norway, and northern Norway). Most of the genetic variation (73.3%) was ascribed to differentiation between the arctic and the nonarctic group (AMOVA; Table 3). This result suggests that the high-polyploid C. arcticum comprises two distinct evolutionary lineages, that each of these lineages occurs on both sides of the Atlantic, and that they have independent migration histories.

In spite of their trans-Atlantic disjunctions, no distinct geographic structuring was observed in the molecular variation within the two lineages of C. arcticum, except that the arctic Franz Josef Land population was somewhat separated from the remaining arctic populations. Rather, many similar and even some identical multilocus phenotypes were observed across the Atlantic; one phenotype occurred in two populations separated by >1800 km. Populations from different sides of the Atlantic were to a large extent intermingled in the UPGMA and PCO analyses of the genetic data, in agreement with the AMOVA analyses, which suggest that the populations from different Atlantic regions are poorly differentiated (Figs. 2–5; Table 3). Within the nonarctic lineage, for example, there are two somewhat differentiated groups of populations, but both groups consist of Icelandic as well as Norwegian populations.

RAPD markers supporting the main division into an arctic and a nonarctic group of populations were converted into locus-specific SCAR markers so that the homology and identity of the crucial bands could be ascertained. While RAPD markers can be obtained inexpensively and quickly, the reliability of each individual polymorphic band as a marker is limited by its anonymity and the lack of information on the type, frequency, and uniqueness of the underlying mutation. As shown above for the five bands tested by us, there is considerable variation among RAPD bands for the details of the molecular polymorphism. The three SCAR markers developed from crucial bands not only support our interpretation, they are also tools for further, larger surveys. This applies especially to the codominant SCAR marker for which allele frequencies can be determined. We are at present trying to isolate the intact 550-bp fragment from herbarium specimens in order to check for allelic polymorphisms between geographic regions.

Thus, the results of this first phylogeographic analysis of one of the so-called "West-arctic" species, whose occurrence on both sides of the Atlantic has provided the strongest argument in favor of the "glacial survival hypothesis" for Scandinavia and Svalbard (e.g., Dahl, 1963, 1987 ; Rønning, 1963 ; Gjærevoll, 1992 ), are contradictory to the expectations under this hypothesis. If the contemporary populations of C. arcticum (within each of the lineages identified herein) on different sides of the Atlantic Ocean have been isolated from each other since the late Tertiary (i.e., at least 2–3 million years) and survived throughout the Quaternary glaciations in refugia situated on both sides of the Atlantic (cf. Dahl, 1963 ), these populations should have shown considerable genetic divergence. The occurrence of identical, or very similar, complex RAPD multilocus phenotypes on both sides of the Atlantic in a high-polyploid, alloploid, and autogamous species such as C. arcticum is also improbable under a less extreme version of the glacial survival hypothesis, i.e., isolation and survival in different refugia throughout the last glaciation (cf. Nordal, 1987 ). Notably, these multilocus phenotypes are based on a high number (35) of polymorphic, putative loci representing widely different parts of the genome (cf. Williams et al., 1990, 1993 ). The most reasonable interpretation of these data is that a very short time has been available for genetic divergence and that the present geographic distribution of each evolutionary lineage therefore results from post-Weichselian expansion across the Atlantic from one refugium for each lineage. The most probable areas for refugial survival are situated on the western side of the Atlantic (Greenland or Northeast Canada) for the arctic lineage of C. arcticum, and on the North Sea Continent or south of the North European ice sheet for the nonarctic lineage of the species (cf. Funder, 1979 ; Pielou, 1991 ; Andersen and Borns, 1994 ; Landvik et al., 1998 ; Steig, Wolfe, and Miller, 1998 ).

The lack of genetic divergence within the lineages of C. arcticum is clearly most consistent with a scenario involving long-distance, trans-Atlantic dispersal after the last glaciation. Independent survival of genetically variable populations in refugia on both sides of the Atlantic throughout the last glaciation is unlikely, because genetic drift and probably new mutations in different glacial survivor populations would have resulted in more distinct divergence at RAPD loci. Divergence caused by drift would have been expected because of the autogamous reproductive system of C. arcticum and because refugial population sizes must have been limited by the extensive glaciation. In particular, if populations survived in Svalbard and Scandinavia, they must have been small because these areas were extensively glaciated (Andersen and Borns, 1994 ).

The general lack of geographic structuring of the molecular variation within the two evolutionary lineages of this West-arctic species is consistent with the results of other studies of arctic-alpine plants in the North Atlantic region (Haraldsen, Ødegaard, and Nordal, 1991 ; Brochmann, Soltis, and Soltis, 1992 ; Haraldsen and Wesenberg, 1993 ; Gabrielsen et al., 1997 ; Tollefsrud et al., 1998 ; Aares, Nurminiemi, and Brochmann, 2000 ). This lack of geographic structure suggests that arctic-alpine plants may migrate long distances, even across sea barriers, despite their lack of classic adaptations to long-distance dispersal. The conditions for long-distance seed dispersal may, however, have been more favorable in the late glacial and early postglacial period than today, because of the considerable extension of the winter sea ice in the Atlantic at that time (Andersen and Borns, 1994 ). A continuous surface of frozen sea may represent a less severe barrier to wind dispersal than open sea (cf. Savile, 1972 ).

We found no particular "hot spots" with high molecular diversity in C. arcticum that could be taken as evidence for long-term isolation and differentiation in glacial refugia, although the species showed high total levels of molecular variation. Neither were distinct hot spots observed in RAPD studies of Saxifraga oppositifolia and S. cespitosa in the North Atlantic region (Gabrielsen et al., 1997 ; Tollefsrud et al., 1998 ). However, absence of such hot spots does not necessarily indicate absence of refugia. Arctic refugial populations may have been bottlenecked because marginal environmental conditions and small ice-free areas limited population size, in contrast to more southern refugial populations of temperate species, which usually have been examined in phylogeographic studies so far (cf. Ferris et al., 1995; Konnert and Bergmann, 1995 ; Demesure, Comps, and Petit, 1996 ; Hewitt, 1996 ; Soltis et al., 1997 ; Comes and Kadereit, 1998 ; Schaal et al., 1998 ; Taberlet, 1998 ; Taberlet et al., 1998 ).

An unexpected result of this study was that the level of intrapopulational molecular variation in the autogamous C. arcticum increased towards the north (from 15.8 to 67.7%; Table 3). In contrast, the intrapopulational proportion of the RAPD variation observed in another autogamous species in the North Atlantic region, Saxifraga cespitosa (Tollefsrud et al., 1998 ), as well as that in the allogamous S. oppositifolia (Gabrielsen et al., 1997 ), decreased towards the north (from southern Norway to Svalbard). More inbreeding towards the north is usually expected because of decreasing pollinator activity. Seed set experiments do not indicate that there are increasing levels of outcrossing in C. arcticum towards the north (A. R. Hagen, University of Oslo, unpublished data), which otherwise could have explained the results for this species. One possible explanation for the different levels of intrapopulational variation is that migration among populations is most frequent in the treeless arctic environment, where C. arcticum often is continuously distributed over large areas. The species has much more scattered occurrences in the Scandinavian mountains, and the populations in this area are isolated by forested valleys.

The existence of two highly divergent lineages within C. arcticum demonstrated in this study may indicate that this high-polyploid species has originated recurrently via independent polyploidizations, a hypothesis first proposed by Böcher (1977) . What traditionally has been named C. arcticum may actually consist of two distinct, 12-ploid taxa that have different evolutionary origins and migration histories. The divergence in RAPD markers observed herein is paralleled by differentiation in morphology and isozymes (Hagen et al., 1995 ; Brysting and Borgen, 2000 ; Brysting and Elven, 2000 ). The arctic plants are more pubescent and have smaller seeds and narrower capsules than the nonarctic plants, and they have bracts with distinct hyaline margins, in contrast to the nonarctic ones. The correct name for the arctic taxon is C. arcticum sensu stricto (cf. Hultén, 1956 ), and the southern taxon should be named C. nigrescens (H. C. Watson) Edmondston ex H. C. Watson (cf. Brummit et al., 1987 ; Brysting and Elven, 2000 ). These two lineages of C. arcticum sensu lato are isozymically as different from each other as they are from the octoploid C. alpinum, with which both share several, but different, enzyme bands (Hagen and Sæther, 1993 ; Hagen et al., 1995 ; Brysting and Borgen, 2000 ). It is thus possible that C. alpinum has been involved in the formation of both lineages of C. arcticum s. l. A second parent of the arctic lineage may have been restricted to the Beringian area, where several species of Cerastium occur today (Hultén, 1956, 1968 ; Murray, 1995 ). A second parent of the nonarctic lineage is possibly the tetraploid C. uniflorum Clairv. from the Alps (Söllner, 1953 ; Böcher, 1977 ; Boscaiu, 1996 ; Brysting and Borgen, 2000 ; Brysting and Elven, 2000 ).


   FOOTNOTES
 
1 The authors thank the collectors of plant material (cf. Table 1 ); the Norwegian Polar Institute for logistic support on MS Lance during the Svalbard expedition; Tove M. Gabrielsen, Marit Grønbech, Anders Often, Morten Strand, and Mari Mette Tollefsrud for field assistance; Margit Elm Andersen, Ann-Charlotte Heiberg, Inge Merete Larsen, Camilla Lothe Nesbø, and Rikke Sillesen for laboratory assistance; Kjetill S. Jakobsen and Anders Often for fruitful discussions; Anne K. Brysting, Camilla Lothe Nesbø, Pierre Taberlet, and two anonymous reviewers for comments on the manuscript; and Liv Borgen for her encouragement and support. The laboratory work was carried out in the DNA laboratory for biosystematics and ecology at the Department of Biology, University of Oslo, and at Risø National Laboratory, Denmark. This project was supported by grants to A. R. Hagen from the Nansen Foundation (grant 101/95), the Norwegian Polar Institute (grant 4/94), and the University of Oslo ("Polar Research grant"). Back

4 Author for reprint requests (e-mail: christian.brochmann{at}toyen.uio.no.) Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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