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(American Journal of Botany. 2003;90:931-936.)
© 2003 Botanical Society of America, Inc.

Systematics

Phylogeography of the Arctic-Alpine Saxifraga oppositifolia (Saxifragaceae) and some related taxa based on cpDNA and ITS sequence variation1

Rolf Holderegger2 and Richard J. Abbott

Division of Environmental and Evolutionary Biology, Sir Harold Mitchell Building, University of St. Andrews, St. Andrews KY16 9TH, UK

Received for publication September 5, 2002. Accepted for publication January 10, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Saxifraga oppositifolia (Saxifragaceae) is an important model system for the evolution of Arctic-Alpine plant species. Sequences of the psbA–trnH intergenic spacer of chloroplast DNA and of the internal transcribed spacer region, ITS1–5.8S–ITS2, of the nuclear ribosomal DNA were used to investigate the intraspecific evolution and phylogeography of this species. Samples from nearly the species' entire circumpolar distribution were included in the analysis as well as samples from the closely related taxa S. smalliana from Alaska and S. blepharophylla and S. biflora from the Alps (S. aizoides served as outgroup). These latter taxa showed a low number of parsimony informative characters, in both cpDNA and ITS sequences, which separated them from S. oppositifolia. Two main cpDNA haplotypes were detected within S. oppositifolia, one with a Eurasian distribution and one with an East Asian-North American distribution. This confirmed the existence of two cpDNA lineages with different geographical distributions in this species, which had previously been reported based on a RFLP analysis. The ITS phylogeny was not useful with respect to the intraspecific evolution and phylogeography of S. oppositifolia, because it showed a largely unresolved topology with low statistical support. The cpDNA sequence analysis, however, also suggested a putative long-distance dispersal event. All investigated taxa had cpDNA haplotypes that were congruent with their geographical origin rather than their phylogeny. This could point to putative lineage sorting in S. oppositifolia and related taxa.

Key Words: contact zones • intraspecific evolution • ITS • lineage sorting • long-distance dispersal • phylogeography, psbA–trnH cpDNA • Saxifraga oppositifolia • Saxifragaceae


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Both the internal transcribed spacer (ITS) region of nuclear ribosomal (nr) DNA (Baldwin et al., 1995 ) and several genes or intergenic spacers of chloroplast (cp) DNA have been used to investigate phylogenetic relationships at the generic and higher taxonomic levels (e.g., Sang et al., 1995 , 1997 ; Soltis and Soltis, 1998 ). In the Saxifragaceae, ITS and cpDNA gene sequences have been used to infer evolutionary relationships within the whole family (Soltis and Soltis, 1997 ), within the genus Saxifraga (Soltis et al., 1996 ), or within sections of Saxifraga (Conti et al., 1999 ; Vargas, 2000 ). However, the usefulness of sequence data at lower taxonomic levels, i.e., within a group of closely related species or within species, is still debated because of the few universal primers available (Bachmann, 2001 ) and the chronically low amount of polymorphism detected. For the latter reason, Soltis and Soltis (1998) doubted the applicability of ITS sequences to intraspecific phylogenies. However, sequence variation of genes or intergenic spacers of cpDNA has helped to resolve relationships within or between several closely related species (e.g., Vijverberg et al., 1999 ; Widmer and Baltisberger, 1999 ; Utelli et al., 2000 ). Moreover, in the genus Saxifraga, Brochmann et al. (1998) found substantial levels of ITS variation in four geographically close populations of S. cernua in Svalbard, while Vargas et al. (1999) resolved an evolutionary split between populations of S. globulifera from the north and south of the Strait of Gibraltar in an ITS phylogeny.

The circumpolar cushion plant S. oppositifolia has been an important model species for the analysis of the Quaternary phylogeography and evolution of Arctic-Alpine species and also for examining the relevance of intraspecific polymorphisms in adaptation to environmental heterogeneity at small spatial scales and to climate change (e.g., Crawford et al., 1993). In particular, the species has been the subject of several molecular genetic studies examining restriction fragment length polymorphisms (RFLPs) of cpDNA and random amplified polymorphic DNA (RAPDs; Abbott et al., 1995 , 2000 ; Gabrielsen et al., 1997 ; Gugerli et al., 1999 ; Holderegger et al., 2002 ). Based on fossil evidence and a study of cpDNA phylogeography, Abbott et al. (2000) suggested that S. oppositifolia was relict to the Arctic in western Beringia (northeastern and north-central Siberia) during the late Tertiary, before it migrated eastwards and westwards to obtain a circumpolar distribution. There is also reliable macrofossil and molecular evidence showing that the species survived the Pleistocene glaciations in several major refugial regions of the Alaskan and Canadian high Arctic, of northern Siberia, and of Eurasia to the south and east of the main ice sheets (Abbott and Brochmann, 2003 ). The present study evaluates whether these phylogeographical patterns are supported by cpDNA and nrDNA sequences. Specifically, we asked whether glacial survival in different major glacial refugia resulted in the evolution of distinct intraspecific lineages in S. oppositifolia, as could be expected because of genetic drift or differential adaptive selection during long-term isolation. We analyzed the psbA–trnH spacer of the chloroplast genome (Sang et al., 1997 ) and the ITS region of nrDNA of samples of S. oppositifolia from almost the species' entire distribution. For comparison, some closely related taxa (species or subspecies) occurring within the distribution area of S. oppositifolia were included.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Species and sampling
The purple saxifrages (section Porphyrion; Saxifragaceae) comprise several entities of doubtful taxonomic status. The latter are sometimes designated as varieties, subspecies, or species (Webb and Gornall, 1989 ). For simplicity, we treat them as species. The most widespread of these taxa, S. oppositifolia L. (including S. oppositifolia subsp. oppositifolia and S. oppositifolia subsp. glandulisepala, which are difficult to tell apart; Webb and Gornall, 1989 ) occurs throughout the Arctic and in many southern mountain ranges of Asia, Europe, and North America. We investigated 19 geographically widespread samples of S. oppositifolia (Table 1, Fig. 1) from the Arctic and the Alps, representing major distributional regions. Samples from the same locations, though not necessarily the same individuals, had previously been included in RFLP analyses of cpDNA (Abbott et al., 2000 ; Holderegger et al., 2002 ; Table 1).


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  		Table 1. Numbering and localities of 19 samples of Saxifraga oppositifolia and of one sample each of S. biflora, S. blepharophylla, S. smalliana, and S. aizoides included in the analyses of psbA–trnH (cpDNA) and ITS (nrDNA) sequences

 


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  		Fig. 1. Localities and distribution of psbA–trnH cpDNA haplotypes of 19 samples of Saxifraga oppositifolia (circles) and of one sample each of S. biflora (star), S. blepharophylla (triangle), S. smalliana (square), and S. aizoides (hexagon). Filled symbols, haplotypes with indel 1; open symbols, haplotypes without indel 1 (Fig. 2 ). For population numbering see Table 1

 
In addition, we studied one sample each of S. blepharophylla Kerner ex. Hayek (S. oppositifolia subsp. blepharophylla according to Webb and Gornall, 1989 ) from the Austrian Alps, which is characterized by columnar leafy shoots, of S. smalliana (Engler & Irmscher) Hultén (S. oppositifolia subsp. smalliana according to Webb and Gornall, 1989 ) from Alaska with four rows of densely packed leaves (Table 1, Fig. 1), and of S. biflora All. endemic to the Alps and characterized by two or more flowers per stem (Kaplan, 1995 ; Table 1, Fig. 1). As in Abbott et al. (2000) , one sample of S. aizoides L. (section Xanthizoon) collected in the Alps (Table 1, Fig. 1) served as outgroup in the phylogenetic analyses. Leaf tissue of all samples was collected in the field and dried with silica gel.

DNA extraction
Genomic DNA was extracted from 80 mg of dried leaf material using the hexadectyltrimethylammoniumbromide (CTAB) protocol of Whittemore and Schaal (1991) with the modifications and cleaning steps given in Milne et al. (1999) . Total DNA was additionally cleaned with 7.5 mol/L ammonium acetate and a Wizard DNA cleanup kit (Promega, Madison, Wisconsin, USA).

The psbA–trnH sequencing
Amplifications of the psbA–trnH intergenic spacer of cpDNA using the primers psbAF and trnHR (Operon, Alameda, California, USA) from Sang et al. (1997) were performed in 50 µL volumes containing 24.1 µL of ddH2O, 10 µL of Q-solution (enhancing the quality of the PCR product; Qiagen, Valencia, California, USA), 4 µL of 25 mmol/L MgCl2, 2.5 µL of a 2 mmol/L dNTP solution in equimolar ratio, 1 µL of each primer at 10 pmol/µL, 0.4 µL of Taq DNA-polymerase A (5 units/µL; Promega, Madison, Wisconsin, USA), 5 µL of the provided 10x enzyme buffer, and 2 µL of genomic DNA at 20 ng/µL. Polymerase chain reaction (PCR) was performed with a PTC-100 thermocycler (MJ Research, Waltham, Massachusetts, USA) set at an initial 1 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C with a final extension of 10 min at 72°C.

The PCR products were cleaned with a Wizard PCR prep kit (Promega, Madison, Wisconsin, USA). Sequence analysis in forward and reverse directions was performed on an ABI PRISM 377 automated sequencer (Applied Biosystems, Foster City, California, USA) according to the manufacturer's instructions (4.75% Long Ranger gels) using the Dye Deoxy Terminator sequencing kit. For cycle sequencing (35 cycles of 10 s at 96°C, 5 s at 50°C, and 90 s at 60°C), 0.5 µg of template DNA, and 5 pmol of the same primers as given above were used. Sequences were edited on SEQUENCE NAVIGATOR 1.0 (Applied Biosystems, Foster City, California, USA) and manually aligned with the psbA–trnH sequence of Saxifraga spathularis (GenBank accession number AF374742) using SEQUENCHER 3.0 (Applied Biosystems, Foster City, California, USA). For each sample, the PCR reaction and the sequence determination were repeated at least twice. No statistical treatment was applied, because we found only two phylogenetically informative haplotypes.

GenBank accession numbers for psbA–trnH are: S. oppositifolia 217-base pair (bp) sequence "with indel 1" (sample 9), AF502090; S. oppositifolia 201-bp sequence "without indel 1" (sample 22), AF502091; S. biflora (sample 2), AF504550; S. blepharophylla (sample 3), AF504548; S. smalliana (sample 4), AF504549; S. aizoides (sample 1), AF504551 (Table 1, Fig. 2).



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  		Fig. 2. The psbA–trnH cpDNA sequences of 19 samples of Saxifraga oppositifolia and of one sample each of S. biflora, S. blepharophylla, S. smalliana, and S. aizoides. 1S. oppositifolia consensus sequence without indel 1; 2S. oppositifolia consensus sequence with indel 1

 
The ITS sequencing
Sequencing of the entire ITS1–5.8S–ITS2 region of nrDNA (Baldwin et al., 1995 ) was performed with three different primer combinations for each of the 23 samples: ITS5/ITS4, ITS1/ITS2, and ITS3/ITS4 (White et al., 1990 ). Amplifications were performed as described above, except that the thermocycler was set at an initial 3 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, and 1.5 min at 72°C with a final extension of 4 min at 72°C. Cycle sequencing, using the same primer combinations as given above, and sequence editing followed the same procedure as described for the psbA–trnH sequencing. The sequences obtained were manually aligned to ITS1 (GenBank accession number: AF087592) and ITS2 (AF087622) sequences of S. oppositifolia.

For parsimony analysis of ITS sequences, we used PAUP 4.0 (Sinauer, Sutherland, Massachusetts, USA) taking into account both nucleotide substitution and indel information (coded as present or absent irrespective of indel length) with heuristic search, Fitch algorithm, steepest descent, ACCTRAN, tree bisection reconnection (TBR) swamping, and MULPARS in effect. Saxifraga aizoides served as outgroup, and bootstrap estimates were based on 1000 replicates. As an alternative analysis, we additionally generated a neighbor-joining clustering based on uncorrected DNA-DNA distances with PAUP 4.0, again using 1000 bootstrap replicates for significance testing.

GenBank accession numbers for ITS1–5.8S–ITS2 are as follows: S. oppositifolia (sample 22), AF502089; S. biflora (sample 2), AF504546; S. blepharophylla (sample 3), AF504545; S. smalliana (sample 4), AF504544; S. aizoides (sample 1), AF504547 (Table 1).

The complete data set on the psbA–trnH and ITS sequences is available from the first author upon request.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The psbA–trnH sequences
The 19 psbA–trnH sequences of S. oppositifolia were either 201 bp (12 samples "without indel 1" in the following) or 217 bp (seven samples "with indel 1") in length due to an indel of 16 bp (Figs. 1 and 2). Apart from this major and, with respect to parsimony, informative polymorphism, only four noninformative nucleotide substitutions were found: two substitutions occurred in sample 18 and one in sample 17, both from northwestern Canada, and one in sample 5 from north-central Russia. The geographical distributions of the two main cpDNA haplotypes found in S. oppositifolia were, in general, distinct (Fig. 1): samples having a psbA–trnH sequence with indel 1 occurred from the Taymyr Peninsula in north-central Siberia westwards to Franz Joseph Land, Svalbard, and the Alps in Europe and to central parts of east and west Greenland. Samples with sequences without indel 1 ranged from the Taymyr region eastwards through northern central Siberia, Alaska, the Rocky Mountains, and Arctic Canada to northern Greenland (Fig. 1). However, the haplotype without indel 1 was also found disjunctly in sample 12 from the North Cape in Scandinavia (Fig. 1).

Saxifraga blepharophylla (217 bp), S. biflora (217 bp), and S. aizoides (207 bp) all showed psbA–trnH sequences with indel 1 (Fig. 2) in accordance with their geographical origin (Fig. 1). The latter also held for S. smalliana from Alaska with a 201-bp sequence without indel 1 (Figs. 1 and 2). Otherwise, S. blepharophylla showed none, while S. biflora and S. smalliana each had one additional nucleotide substitution not found within the S. oppositifolia samples. These substitutions were noninformative with respect to parsimony. Saxifraga aizoides exhibited two additional indels (indel 2, 1 bp; indel 3, 9 bp) and a single additional nucleotide substitution (Fig. 2).

The ITS sequences
The consensus ITS1–5.8S–ITS2 sequence of the 19 S. oppositifolia samples had a length of 676 bp. Within S. oppositifolia, ITS1 exhibited four parsimony informative and 12 noninformative nucleotide substitutions as well as two informative and four noninformative indels. ITS2 showed less variation: one informative and one noninformative nucleotide substitution and a single informative indel. Even the 5.8S region exhibited one informative and one noninformative nucleotide substitution. Hence, nine phylogenetically informative sites were found within the entire ITS data set of S. oppositifolia. Pairwise sequence differences among samples ranged between zero and 10 (26 in total).

In S. biflora (sequence length: 675 bp), S. smalliana (676 bp), and S. blepharophylla (675 bp), only the latter showed a single nucleotide substitution otherwise not found within the ITS variation of the 19 S. oppositifolia samples. In contrast, S. aizoides (675 bp) exhibited 31 additional nucleotide substitutions compared with the other four taxa in the analysis.

All ITS trees generated exhibited largely unresolved topologies with low bootstrap support irrespective of the methods (parsimony or neighbor-joining) and the settings used. As an example, we present the 50% majority-rule consensus tree of the 25 most parsimonious trees found (Fig. 3). It also had low phylogenetic resolution with a consistency index (CI) of only 0.192. Only two aspects were recurring in the different analyses of the ITS sequences: (1) samples of S. oppositifolia with a cpDNA haplotype without indel 1 (see above; Fig. 2) were mainly grouped in a clade including S. smalliana (top clade in Fig. 3), and (2) S. biflora, S. blepharophylla, and S. smalliana were positioned amongst the S. oppositifolia samples.



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  		Fig. 3. Fifty percent majority-rule consensus tree (consistency index = 0.192) of the ITS1–5.8S–ITS2 nrDNA region of 19 samples of Saxifraga oppositifolia and of one sample each of S. biflora, S. blepharophylla, S. smalliana, and S. aizoides (outgroup). Bootstrap support values are given above branches; symbols represent psbA–trnH cpDNA haplotypes according to Fig. 1 .

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Intraspecific evolution of Saxifraga oppositifolia
Abbott et al. (2000) hypothesized that S. oppositifolia occurred in the Arctic in western Beringia during the late Tertiary before migrating east and west to gain a circumpolar distribution. This suggestion was based on the results of an RFLP analysis of the cpDNA of S. oppositifolia and on fossil evidence. This RFLP analysis identified 14 different cpDNA haplotypes in S. oppositifolia, which were resolved into two major clades. The basal haplotypes of each clade occurred only in western Beringia, although a subsequent survey (Holderegger et al., 2002 ) indicated that one of these haplotypes may also occur rarely in the Alps. In the present study, two main cpDNA haplotypes, characterized by a 16-bp indel, were found based on sequences for the psbA–trnH intergenic spacer. The geographical distributions of these two haplotypes (Fig. 1) were consistent with those of the two major clades of cpDNA-RFLP haplotypes detected by Abbott et al. (2000) , i.e., a Eurasian and an East Asian-North American clade. Hence, our data support the occurrence of two major evolutionary lineages of S. oppositifolia, which most likely have been geographically isolated from each other during the Pleistocene (Abbott et al., 2000 ).

The phylogenetic analysis of ITS sequences (Fig. 3) resulted in a poorly resolved topology with low bootstrap support. Hence, the two main cpDNA lineages detected in S. oppositifolia were not precisely reflected in the species' nrDNA sequence variation, although samples of S. oppositifolia with a cpDNA haplotype without indel 1 were mainly grouped together in an ITS clade that included S. smalliana. In view of this poor resolution, a determination of divergence time between the two cpDNA lineages of S. oppositifolia must be left to future studies.

The two cpDNA lineages remained separate throughout their distributions in the study of Abbott et al. (2000) , except in northern Greenland and north-central Siberia where secondary contact zones appeared to exist. In the present study, we detected one exception to this pattern in that sample 12 of S. oppositifolia from the North Cape in Scandinavia possessed the cpDNA sequence without indel 1, which was not concordant with its geographical location (Fig. 1). A possible explanation for this particular pattern could be rare long-distance dispersal through seed from a population of S. oppositifolia either from northern Greenland or from the Siberian Taymyr Peninsula (Fig. 1). Such long-distance migration is now considered to be rather common in many Arctic plant species (Hagen et al., 2001 ; Abbott and Brochmann, 2003 ). However, without having a comparative and reliable nuclear gene phylogeny at hand, the alternative explanation of an ancient polymorphism cannot be rejected.

In regions where migrants from different glacial refugia have come into contact, higher levels of genetic diversity can be expected (Comes and Kadereit, 1998 ; Stehlik, 2002 ). Such postglacial sink regions between the East Asian-North American and the Eurasian lineages of S. oppositifolia are likely to occur in north-central Siberia and northern Greenland/northeastern Arctic Canada, where the two cpDNA lineages co-occur (Abbott et al., 2000 ; Fig. 1). In contrast to maternally inherited cpDNA, biparentally inherited nrDNA would be expected to mix easily in such contact zones through sexual reproduction in the mainly outbred S. oppositifolia (Gugerli et al., 1999 ). Unfortunately, the unresolved ITS-phylogeny prevented a corresponding phylogeographical analysis. As there are no comparative data available on intraspecific sequence variation in other Arctic plant species, it is not yet possible to infer whether contact zones are general biogeographical features of circumpolar taxa.

The usefulness of ITS for intraspecific phylogenetic analyses
The limited variation in the ITS sequences of S. oppositifolia (i.e., nine phylogenetically informative sites) resulted in a poor resolution of this species' intraspecific evolution and phylogeography. The samples investigated exhibited between zero and 10 pairwise site differences. This ITS variation of individuals covering almost the species' entire distribution area was low compared with the ITS variation found in other Saxifraga species sampled at far smaller spatial scales. Brochmann et al. (1998) detected 2–21 divergent sites in pairwise sequence comparisons of four populations of S. cernua collected within only 140 km on the Arctic Svalbard, while values for seven populations of S. globulifera from both sides of the Strait of Gibraltar ranged between zero and 24 differences (Vargas et al., 1999 ). Therefore, the applicability of ITS sequences to intraspecific phylogenies (Soltis and Soltis, 1998 ) varies within comparatively closely related species and for different geographical scales. This sheds doubt on the accuracy of estimates of divergence times at lower taxonomic levels (Ayala, 1999 ) in cases where sequence variation within a studied taxon or a group of taxa is not adequately covered.

Sequence differences between Saxifraga oppositifolia and closely related taxa
The S. smalliana sample from Alaska had a cpDNA haplotype without indel 1 and differed in only one nucleotide substitution from samples of S. oppositifolia from the same region (Figs. 1 and 2). Moreover, its ITS sequence was congruent with the consensus sequence of all S. oppositifolia samples. Saxifraga blepharophylla from the European Alps, often treated as a subspecies of S. oppositifolia (Webb and Gornall, 1989 ), exhibited a psbA–trnH haplotype identical to the one found in the Alpine samples of S. oppositifolia (i.e., with indel 1; Figs. 1 and 2), and its ITS sequence had only one nucleotide substitution not found in S. oppositifolia. It may be concluded from this that S. smalliana and S. blepharophylla are very closely related to S. oppositifolia.

Based on the present molecular data, taxonomic conclusions on S. smalliana and S. blepharophylla cannot be drawn. For such an aim, many more samples of these two taxa had to be included in morphological, biosystematic (e.g., common garden transplants or cross pollinations), and molecular genetic analyses. It was nevertheless striking that even the morphologically distinct, but closely related Alpine species S. biflora (Kaplan, 1995 ) showed an ITS sequence encompassed by the variation found in S. oppositifolia. In addition, this species' cpDNA sequence (with indel 1) had only one nucleotide substitution differing from the sequences of the other samples investigated (Figs. 1 and 3). Alpine samples of S. biflora and S. oppositifolia were also not distinguishable based on a preliminary survey of cpDNA RFLPs using the Southern blotting technique (R. Holderegger and R. J. Abbott, unpublished data). In natural populations, these two species hybridize, and introgression is observed (Gugerli, 1997 ). This may account for the observed lack of differentiation between these two taxa.

All investigated samples, including S. aizoides, either contained or lacked the psbA–trnH indel 1 according to their geographical origin. A corresponding pattern, where individuals do not possess taxon-specific but region-specific cpDNA haplotypes, was found in the European white oaks (Petit et al., 2002 ), which was attributed to lineage sorting. Lineage sorting due to the presence of ancestral polymorphisms coupled with differential survival of the variants results in gene phylogenies not matching organismal phylogenies (Page and Holmes, 2000 ). It has also been invoked in the evolution of Mediterranean Senecio (Comes and Abbott, 2001 ) and Alpine Draba species (Widmer and Baltisberger, 1999 ). Whether the geographical pattern of the cpDNA sequences found in several Saxifraga taxa in the present study is due to lineage sorting cannot be answered based on the present data set; there are several alternative explanations. (1) It is possible that indel 1 represents an ancestral state in S. oppositifolia and other Saxifraga species, which the East Asian-North American lineage of S. oppositifolia has lost. (2) Saxifraga smalliana and S. blepharophylla may simply represent subspecies or even varieties of S. oppositifolia of regional origin. Thus, it would not be surprising that they exhibit the main cpDNA haplotype typical for the corresponding regions. (3) Only one sample each of S. biflora and S. aizoides, both from the European Alps, were included in the analysis. While the former species is an Alpine endemic, the latter has a wide circumpolar distribution. It would thus be interesting to investigate whether samples of S. aizoides from other regions, e.g., from North America, also show region-specific cpDNA haplotypes. (4) Ancient hybridization events as found in the Boykinia and Heuchera groups of Saxifragaceae (Soltis and Kuzoff, 1995 ; Soltis et al., 1996 ) could be involved and might cause similar phylogeographic patterns.

Conclusions
The two main cpDNA haplotypes based on psbA–trnH sequences found in S. oppositifolia were consistent with the existence of two intraspecific evolutionary lineages (East Asian-North American and Eurasian) in this species. In contrast, an analysis of intraspecific ITS variation resulted in low phylogenetic resolution. A possible long-distance dispersal event in S. oppositifolia and the occurrence of putative lineage sorting within a group of taxa of the genus Saxifraga were also indicated by the molecular analyses conducted.


   FOOTNOTES
 
1 The authors thank Felix Gugerli, Peter Schönswetter, and Robert M. M. Crawford for providing plant material of S. biflora, S. blepharophylla, and S. smalliana, Anette Becher, Frank Blattner, Hans Peter Comes, Elena Conti, Jenny Gleason, and Michael Möller for technical advice, and Felix Gugerli, Susan Hoebee, Ivana Stehlik, and two anonymous reviewers for critical comments on the manuscript. Grants from the Swiss National Science Foundation, the Novartis Fonds, and the Julius Klaus Foundation for Genetic Research to R.H. are acknowledged. Back

2 Author for reprint requests, present address: Division of Ecological Genetics, WSL Swiss Federal Research Institute, Zürcherstrasse 111, CH-8903 Birmensdorf, Switzerland (phone: +41 1 739 25 27; FAX: +41 1 739 22 15; rolf.holderegger{at}wsl.ch ) Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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