Am. J. Bot.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koch, M.
Right arrow Articles by Bernhardt, K.-G.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Koch, M.
Right arrow Articles by Bernhardt, K.-G.
Agricola
Right arrow Articles by Koch, M.
Right arrow Articles by Bernhardt, K.-G.
(American Journal of Botany. 2004;91:115-124.)
© 2004 Botanical Society of America, Inc.

Systematics

Comparative biogeography of the cytotypes of annual Microthlaspi perfoliatum (Brassicaceae) in Europe using isozymes and cpDNA data: refugia, diversity centers, and postglacial colonization1

Marcus Koch2,4 and Karl-Georg Bernhardt3

2Institute for Plant Sciences, Biodiversity and Plant Systematics, University of Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany; 3Institute of Botany, University of Agricultural Sciences Vienna, Gregor-Mendel-Str. 33, A-1180 Vienna, Austria

Received for publication April 1, 2003. Accepted for publication July 18, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
In the last few years, the biogeography of many European plant species has been analyzed using molecular markers, and some consistent patterns of Pleistocenic differentiation and range fluctuations have been established. These studies mostly focused on perennial herbs or woody species, rarely considering annual taxa. This study focused on the annual Microthlaspi perfoliatum, which is distributed all over Europe and comprises three cytotypes. Morphologically, these cytotypes are hard to distinguish, although, based on molecular markers, they should be treated as two different species. Diploid and polyploid cytotypes had a different biogeographical history, with distinct glacial refugia. For the polyploids, a well-known distribution pattern of relict areas was confirmed, with Iberia, Italy, and the Balkans serving as primary Pleistocene refugia. Secondary refuge areas have been detected in southeastern France and neighboring Switzerland, with closer affinity to the Iberian refugium than to any other region based on allozyme and cpDNA haplotype data. For the diploids, two refugia have been characterized, one of which is congruent to the secondary refugium of the polyploids in France and Switzerland. The second refuge of diploid populations is located in unglaciated lowland areas of East Austria and Croatia. Isozyme and cpDNA haplotype data favor a postglacial colonization of diploid populations into Germany from Austrian lowland areas along the Danube River as well as from Switzerland. This scenario is also true for polyploids in Germany, Belgium, and Sweden.

Key Words: biogeography • Brassicaceae • chloroplast DNA • Europe • isozymes • Microthlaspi perfoliatum • Pleistocene • refugia • Thlaspi


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Since the term "phylogeography" was defined (Avise et al., 1987 ), interest has been continuously increasing in the distribution of organismal diversity in space and time. This is especially true for Pleistocenic range fluctuations forced by major climate changes, which were caused by glaciation and deglaciation cycles and have fascinated biologists for decades (Dahl, 1946 ; Hewitt, 2000 ). The rapid development of new molecular tools and approaches for studying genetic differentiation enables scientists to elucidate phylogenetic relationships as well as genetic relatedness of populations and individuals (Bachmann, 1997 ; Koch et al., 2003a). Comparative phylogeographic approaches are based on the assumption that species with similar habitats and a comparable distribution might share a similar biogeographic history (Gugerli and Holderegger, 2001 ). For high alpine taxa on silicious or calcareous rocks, this approach has been successfully used to determine Pleistocene refugia on a local scale (Schonswetter et al., 2002 , 2003 ; Tribsch et al., 2002 ; Tribsch and Schönswetter, 2003 ). Comparisons of the existing data of phylogeographic scenarios revealed congruent biogeographies. These patterns indicated several Pleistocene glacial refugia in Europe (Konnert and Bergmann, 1995 ; Hewitt, 1996 , 1999 ; Comes and Kadereit, 1998 ; Taberlet et al., 1998 ) as well as several postglacial colonization routes (Koch et al., 1998b ; Taberlet et al., 1998 ; Sharbel et al., 2000 ). On a broader comparative scale, three main refugia from the southern areas can be distinguished (Iberia, Italy, the Balkans). However, for some taxa or populations other refugia might have been more important for periglacial survial, which were located either outside Europe, in the Southeast of the British Isles, along the Norwegian coast (an area for which ice free zones have been also proposed [Forsström and Punkari, 1997 ]), or for example in lowland (Koch et al., 2003b) or mountanous regions (Tremetsberger et al., 2002 ) in East Austria and adjacent areas. Other taxa such as common beech (Demesure et al., 1996 ), black alder (King and Ferris, 1998 ), or Scots pine (Sinclair et al., 1999 ) survived the Pleistocene close to the periglacial frontline. More distinctive refugia patterns are obvious in studies with an extremely dense sampling all over Europe, such as for the fern Asplenium ceterach (Trewick et al., 2002 ), or with a fine scale sampling, such as for the high alpine Saponaria pumila (Tribsch et al., 2002 ) or Eritrichum nanum (Stehlik et al., 2001 ).

However, among plants, these studies mostly focused on perennial herbs and woody species, and in Europe annuals have been only rarely tested for geographic structuring of genetic variation, which led to phylogeographic hypotheses. Some of these few examples came from Arabidopsis (Sharbel et al., 2000 ), Cochlearia danica (Koch et al., 1998a), or several Senecio species (Comes and Abbott, 2000 , 2001 ).

The annual Microthlaspi perfoliatum is a representative of the genus Microthlaspi, which according to Meyer (1973 , 1979 ) consists of four annual species, namely M. perfoliatum (L.) F. K. Meyer, M. natolicum (Boiss.) F. K. Meyer, M. granatense (Boiss. & Reut.) F. K. Meyer, and M. umbellatum (Steven ex DC.) F. K. Meyer. However, this classification is questionable, and on the basis of molecular data only M. perfoliatum and M. natolicum represent a monophyletic species assemblage (Koch and Mummenhoff, 2001 ). Both M. granatense from the orophile vegetation from the Sierra Nevada in southern Spain to the Algerian-Moroccan Atlas Mountains (Galland and Favarger, 1990 ) and M. umbellatum from the Gilyan Province in North Iran (Bush, 1985 ) appear as paraphyletic taxa (Koch and Mummenhoff, 2001 ). Taxonomical difficulties are obvious, and even for Iberian M. granatense mixed populations with polyploid M. perfoliatum have been reported (Galland and Favarger, 1990 ). For this reason, the occurrence of M. granatense on Sicily remain unexplained until independent molecular analysis have shown its genetic relatedness to the Iberian-Algerian-Moroccan populations. Microthlaspi natolicum comprises four subspecies, two of which are found in Anatolia (subsp. natolicum and subsp. longistylum), one in Lebanon (subsp. gaillardotii), and one in Cyprus and the southern Sporades Islands (subsp. sporadium). Microthlaspi granatense is diploid, and dipoids as well as hexaploids of M. natolicum have been reported (Polatschek, 1983 ; Koch and Hurka, 1999 ). Microthlaspi perfoliatum is distributed all over Europe and consists of three cytotypes of which the polyploids (2n = 4x = 28 and 2n = 6x = 42) are widely distributed. Polyploid M. perfoliatum occurs also in all Mediterranean countries except Egypt and is extending to the Middle East and Central Asia (Jalas et al., 1996 ). It also has been reported as introduced species from North America (Hill, 1982 ), and a detailed overview of chromosome counts within Microthlaspi is provided in Jalas et al. (1996) and Koch et al. (1998b) . This is in sharp contrast to the local distribution of diploid populations in Central Europe (Koch, 1997 ; Koch et al., 1998b ; Koch and Hurka, 1999 ). However, in several populations the sympatric occurrence of different cytotypes has been reported (Koch and Hurka, 1999 ). This finding raises the question whether these cytotypes are separated genetically and whether they can be distinguished morphologically. The application of molecular markers revealed some interesting findings. Polyploid M. perfoliatum has been constituted via hybridization and subsequent polyploidization between diploid M. natolicum and M. perfoliatum accessions, most likely before Pleistocenic glaciation processes started (Mummenhoff et al., 1997 ). Accordingly, diploid and polyploid cytotypes are distinguishable by different chloroplast DNA haplotypes classes (Koch et al., 1998b ), and no ongoing geneflow between cytotypes has been detected (Koch et al., 1998b ; Koch and Hurka, 1999 ). However, comparisons of different molecular marker sets revealed that gene flow involving different ploidy levels has occurred in the past after constitution of the different cytotypes, e.g., an Iberian hexaploid population has a diploid-related chloroplast DNA haplotype and a diploid-related nuclear ribosomal DNA operon (Koch et al., 1998b ), contrary to a set of allozymes typically found in other Iberian hexaploid populations.

The morphology of M. perfoliatum has been analyzed first by Jordan (1852 , 1864 ), who described Thlaspi erraticum Jordan and T. improperum Jordan, respectively. Thlaspi improperum is identical to the taxon described first by Linné (1753) as T. perfoliatum L. (Schultze-Motel, 1986 ). Interestingly, Markgraf (1958) proposed a more southern distribution of T. improperum than for T. erraticum, a pattern that also was found for chloroplast DNA haplotypes variation among diploids and polyploids (Koch et al., 1998b ). Nonetheless, Hess et al. (1977) and Galland and Favarger (1990) concluded for a larger European sampling that no clear separation between the cytotypes was possible based on morphological characters. In contrast, on a more local scale in Germany, a significant morphological separation between diplods and polyploids occurred (Koch, 1997 ). The corresponding shematic fruit types are shown in Fig. 1a, b. The reason for this is simple. There are only quantitative characters, and the variances of this morphological character variation are much larger in the polyploids than in the diploid, and on a European scale, the diploids represent only one extreme of character variation also found in some polyploids.



View larger version (141K):
[in this window]
[in a new window]
  		Fig. 1. Characteristic phenotypes of (a) polyploid and (b) diploid Microthlaspi perfoliatum

 
The overlapping distribution patterns of the three cytotypes in M. perfoliatum, mixed populations, and any missing obvious ecological differentiation between cytotypes raised the question of whether the cytotypes have a similar biogeographical history since the polyploids have arisen via hybridization and polyploidization. The rapid, annual life cycle, a selfing mating system, and an ecological niche as pioneer in open habitats or in vegetation gaps enforces processes such as founder effects, genetic drift, or bottleneck events, processes that might blur geographical structuring of genetic variation. In Arabidopsis thaliana, which shared several characteristics with M. perfoliatum, significant structuring of genetic variation was detected on a broad geographical scale (Sharbel et al., 2000 ). However, in A. thaliana multilocus linkage disequilibrium has been detected indicating historical recombination at such a high degree that tree-like analysis failed to recognize inter-regional differentiation.

Therefore we have undertaken a study to investigate the genetic variation across an enlarged set of M. perfoliatum populations using isozyme analysis. Our aim was to answer or test the following questions and hypotheses. (1) Are there any centers of diversity or Pleistocene refugia shared by the different cytotypes or in agreement with other phylogeographic studies? (2) Is it possible to perform a comparative biogeographic analysis of the different cytotypes? And how are these scenarios embedded in and influenced by the hypothesized evolutionary history of polyploid M. perfoliatum? (3) Colonization routes should be formulated with a focus on postglacial recolonization of lowland regions in Central Europe.

In the past decade, amplified fragment length polymorphisms (AFLPs) (Vos et al., 1995 ) have been introduced as a marker of choice to obtain nuclear-encoded polymorphisms, which have proven to be reliable and efficient for obtaining a high number of informative characters (Mueller and Wolfenbarger, 1999 ). This marker system has been successfully applied in infraspecific phylogeographic studies in Europe focusing on Quarternary differentiation processes (Stehlik et al., 2001 ; Schönswetter et al., 2002 , 2003 ; Tribsch et al., 2002 ; Koch et al., 2003b ). However, with the AFLP marker system, anonymous DNA fragments are analyzed without any information about fragment characteristics (intron, repetitive DNA, microsatellites, etc.) or loci and alleles. In contrast, isozymes provide a genetically well-defined codominant marker system solely based on coding DNA, but which might suffer from a lower number of informative characters. Therefore, we also address the question of whether isozymes could be analyzed in a similar way to that for AFLPs to obtain significant phylogeographical information.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material
Previous molecular studies on the genus Microthlaspi focused primarily on the evolutionary history of the genus as a whole (Koch et al., 1998b ; Koch and Hurka, 1999 ), and a broad sampling of Microthlaspi populations (accession numbers 1–117) has been achieved. A consecutive enumeration of accessions is followed herein using population numbers 123–176 for 54 newly investigated populations. Population numbers 118–122 have been used for outgroup taxa (Koch et al., 1998b ). In this study we investigated 54 populations of Microthlaspi perfoliatum (Table 1) with 10 individuals in each population. The individuals were raised from seeds that were collected from different mother plants in the wild. From each of the 540 individuals cultivated, 0.5 g fresh leaf material was harvested and stored immediately at –80°C for subsequent protein extract preparation. From each of three individuals per population, 1 g fresh leaf material was harvested for subsequent isolation of total DNA to investigate cpDNA haplotype variation.


View this table:
[in this window]
[in a new window]
  		Table 1. Accession data, chromosome number, cpDNA haplotype, and isozyme haplotype for European populations of Microthlaspi perfoliatum. Isozyme haplotypes from this study and previous published data (Koch and Hurka, 1999) are shown in the Appendix (see Supplementary Data accompanying the online version of this article). D = Germany, AUS = Austria, SWE = Sweden, CZ = Czech Republic, BG = Bulgaria, CRO = Croatia, m.a.s.l. = meters above sea level, S = southern, N = northern

 
In addition, seeds were collected from all offspring to analyze isozyme segregation patterns in the F2. Because all populations revealed uniform within-population genotypes, we had to test only one individual per population for segregation of allozymes, analyzing five individuals from the corresponding F2.

Chromosome counts
Chromosome counts were performed using young root tips with a Giemsa staining procedure (Koch and Hurka, 1999 ). However, diploid and tetraploid/hexaploid were easily distinguished by their isozyme patterns. Therefore, chromosome counts were only necessary to distinguish between the two polyploid cytotypes.

Isozyme analysis
The following isozyme systems have been studied: aspartate aminotransferase E.C. 2.6.1.1 (AAT), phospho glucomutase E.C. 2.7.5.1 (PGM), leucine aminopeptidase E.C. 3.4.11.1(IAP), and phospho glucoisomerase E.C. 5.3.1.9 (PGI). Detailed protocols are given in Koch and Hurka (1999) , with the corresponding diagrams of allozyme distribution among the several loci of the different Microthlaspi taxa. However, in the polyploids, it was not possible to distinguish between duplicated loci.

Variation in cpDNA haplotypes
An extensive survey of cpDNA haplotypes based on restriction fragment length polymorphism (RFLP) data covered a broad range of polyploid Microthlaspi perfoliatum (Koch et al., 1998b ). However, diploid Microthlaspi perfoliatum from an assumed relict area in East Austria was represented by only one accession. To draw biological inferences, it was necessary to fill this data gap. Therefore, all populations from this study were scored subsequently for their cpDNA haplotypes. The experimental work followed Koch et al. (1998b) . For the haplotype screening, it was not necessary to use the total set of 18 restriction endonucleases (Koch et al., 1998b ), but nine enzymes (ScaI, XhoI, PvuII, SmaI, PstI, SstI, HindIII, EcoRI, NcoI) were sufficient to detect the diagnostic mutations resulting in the different RFLP patterns of M. perfoliatum cpDNA haplotypes.

Data analysis
The allozyme patterns were analyzed based on relative migration distances, using an isozyme system elaborated previously (Koch and Hurka, 1999 ). The occurrence of each allele was scored as an absent or present binary character, resulting in a 0/1 matrix comprising 45 different alleles from nine loci. Because each population consisted of totally uniform multilocus genotypes only (herein referred to as "haplotypes"), the Jaccard distance was used to measure interpopulational differentiation (implemented in SYNTAX 5.0, Podani, 1993). The corresponding distance matrix was used to perform a principal coordinate analysis (PCoA) using SYNTAX 5.0 (Podani, 1993). The results were visualized using SPSS version 10.07 (Chicago, Illinois). Based on haplotype data interpopulational differentiation and geographic structuring of the genetic variation was analyzed with an analysis of molecular variance (AMOVA) using ARLEQUIN 2.0 (Schneider et al., 2000 ). The AMOVA estimates genetic structure indices using information on the occurrence of alleles, as well as their frequencies (Excoiffier et al., 1992 ).

The number of rare alleles (fr) with a frequency less than 10% (considering all Microthlaspi perfoliatum populations [Koch and Hurka 1999 ] and populations analyzed herein) and the number of private (or unique) alleles (fu) were counted for different regions after combining the corresponding populations. As an independent measure for regional interactions, the numbers of shared rare alleles between regions (fsr) were also counted.

To detect and visualize similar or even identical haplotypes among polyploid populations, a neighbor-joining tree of all haplotypes was constructed with TREECON version 1.3b (Van de Peer and De Wachter, 1994 ) based on distances according to Nei and Li (1979) . The bootstrap option was used with 1000 replicates to infer statistical support of branching patterns.

Mantel tests were conducted in two different ways. (1) The matrix of genetic interindividual Jaccard distances was compared with a matrix of geographic distances in kilometers (Mantel, 1967 ), and (ii) the genetic distance matrix was compared to a model matrix of 15 geographical distance classes (Gabrielsen et al., 1997 ; Stehlik et al., 2001 ) to test the goodness-of-fit of the genetic distance matrix with a model matrix of distance classes. The distance classes were classified as follow: 1 = 0–50 km, 2 = 51–100 km, 3 = 101–150 km, 4 = 151–200 km, 5 = 251–300 km, 6 = 351–400 km, 7 = 451–550 km, 8 = 551–650 km, 9 = 651–750 km, 10 = 751–1001 km, 11 = 1000–1250 km, 12 = 1251–1500 km, 13 = 1501–1750 km, 14 = 1751–2000 km, 15 = 2001–3300 km. All Mantel RM-values were calculated and Bonferroni-corrected using R Package 4.0 (Casgrain and Legendre, 2001 ).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Chromosome numbers
Among the different populations, all ploidy levels (2n = 2x = 14, 2n = 4x = 28, 2n = 6x = 42) reported previously for Microthlaspi perfoliatum were detected. The data are summarized in Table 1. Some additional studies of rare diploids have been carried in populations from Austria (populations 133, 134, 154, 159) and from Croatia/Istria (population 171). However, the majority of the material is represented by polyploid cytotypes (Table 1). Interestingly, despite a low overall frequency of tetraploids (<5%), there was a high number of tetraploid populations in Austria (hexaploids vs. tetraploids = 28 populations vs. 6 populations), and in Croatia/Istria, all investigated polyploid populations turned out to be tetraploid.

Isozyme analysis
No within-population variation has been detected, and also the F2 individuals had the same multilocus allozyme pattern as the original F1 individuals. This indicates that all individuals were homozygous for all loci tested. Especially the polyploids with duplicated (tetraploids) or triplicated (hexaploids) loci exhibited total homozygosity. Accordingly, the allozyme patterns were coded as binary 0/1 matrix with each allele representing a homozygous locus. Because of the missing intrapopulational differentiation, each population is characterized by one single allozyme phenotype or "haplotype." Considering all data (Koch and Hurka [1999 ] and populations analyzed herein), 51 haplotypes were distinguished. Two new alleles were identified with Lap2–4 and Aat3–3 from several polyploid Austrian populations and from population 168 (Croatia), respectively. The corresponding 0/1 data matrix is provided in the Appendix (see Supplementary Data accompanying the online version of this article) and isozyme haplotypes are indicated in Table 1.

The PCoA analysis of the distribution of the genetic diversity demonstrates that most of the variation within the data set is explained along the first three axes (axis 1, 53.9%; axis 2, 8.6%; axis 3, 6.5%). The two-dimensional plot representing the first two axes had a significant separation of diploid vs. polyploid populations. However, differentiation between tetraploids and hexaploids is not obvious (Fig. 2a). Similarly, there is hardly any clear signal within the polyploids to group them into different regions. In Fig. 2b several regions and the corresponding polyploids have been indicated.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 2. Principal coordinate analysis based on pairwise Jaccard distances indicating (a) cytological and (b) geographical differentiation of Microthlaspi perfoliatum

 
Analysis of molecular variance (AMOVA) was performed for several combinations of regions considering only the polyploids. In contrast to the PCoA analysis, some significant findings are notable. The most significant regional combinations are listed in Table 2. Here, Spain and Italy have been characterized as regions with most of the diversity distributed between and not within regions. A more detailed overview was obtained, when alleles with a total frequency less than 10% were scored. The number of rare alleles (fr) and the number of unique rare alleles (fu) within a defined region nicely correlates with the results from the AMOVA identifying Spain and Italy as areas with higher numbers of rare alleles (Table 3). Moreover, the number of shared rare alleles between regions (fsr) showed some interesting patterns, with the highest affinity between the Iberian and the France/Switzerland region (Table 3). The findings are summarized separately for the polyploids (Fig. 3) and the diploids (Fig. 4).


View this table:
[in this window]
[in a new window]
  		Table 2. Analysis of molecular variance for allozyme phenotypes in polyploid Microthlaspi perfoliatum from European populations

 

View this table:
[in this window]
[in a new window]
  		Table 3. Distribution of rare alleles of polyploid Microthlaspi perfoliatum in geographic regions. Rare alleles are defined by a total frequency <10%. The single numbers in the lower triangle show successively the number of shared rare alleles (fsr) between regions. The triplets of numbers on the diagonal represent the number of unique alleles (fu) and rare alleles (fr) within a region and the total number of alleles. The mean number of alleles per population in all regions ranges between 14 and 15

 


View larger version (110K):
[in this window]
[in a new window]
  		Fig. 3. Phylogeography of polyploid Microthlaspi perfoliatum. Chloroplast haplotypes are indicated as S1–S7. The numbers of unique (fu), rare (fr), and shared rare (fsr) alleles (black arrows) are indicated. The black areas mark the assumed primary refugia. The greyish areas mark regions of late Pleistocenic lowland survival and recent postglacial immigration (hatched areas), with migration direction indicated by arrows

 


View larger version (95K):
[in this window]
[in a new window]
  		Fig. 4. Phylogeography of diploid Microthlaspi perfoliatum. Chloroplast haplotypes are indicated as N1–N5. Allozyme haplotypes are indicated as types A–C. Postglacial migration is indicated by arrows. See Fig. 3 for explanation of greyish areas

 
The diploid populations exhibited only limited variation with three multilocus genotypes considering all populations investigated. From these three genotypes, one (type B) was found exclusively in Switzerland, a second (type C) was exclusively documented for eastern Austria and Croatia, and the third genotype (type B) occurred in both regions as well as in the German distribution range (Fig. 4). Interestingly, all alleles in the diploids are common and do have a total frequency >10%. The only exception is allele Aat2–4 from the Austrian populations, which is also a rare allele in the polyploids. In total 13 alleles are found in Austrian and Croatian diploids, 10 alleles in population from France and Switzerland, and nine alleles in populations from Germany.

The overall mantel RM value calculated with genetic and geographical distance matrix for polyploid individuals only is 0.39 (P < 0.001), demonstrating a significant correlation between geographical and genetic distances, i.e., "isolation by distance."

The Mantel test for polyploids with distance classes (Fig. 5) reveals a significant positive correlation among populations separated by up to 1250 km. In most cases, the correlation is significantly negative from 1250 to 3300 km. The highest RM value was found in distance class 10 (751–1000 km) (RM = 0.13, P = 0.001), the lowest in distance class 14 (1751–2000 km) (RM = –0.40, P = 0.001).



View larger version (68K):
[in this window]
[in a new window]
  		Fig. 5. Correlogram of Mantel RM per distance class. Filled circles indicate Bonferroni-corrected RM values significantly different from 0 at P ≤ 0.05

 
Variation in cpDNA haplotypes
The cpDNA haplotypes have been characterized according to Koch et al. (1998b) , and they are summarized in Table 1. For the diploid German populations, the occurrence of cpDNA haplotypes N1 and N2 was confirmed (N = northern). Among the five diploid populations from Austria and Croatia, the existence of type N2 in Austria was confirmed, but haplotype N3 (population 154, Table 1), an extremely rare cpDNA type in Germany (Koch et al., 1998b ) was also found. Among the polyploids, the widely distributed cpDNA haplotypes S2 and S3 were identified.

A compilation of all cpDNA haplotypes detected so far is provided in Figs. 3 and 4 for polyploid and diploid Microthlaspi perfoliatum, respectively. Regional structuring was drawn in accordance with the isozyme data.

In summary, the distribution of cpDNA types among regions is in congruence with the isozyme data: e.g., the highest number of different S (S = southern) cpDNA haplotypes were documented for polyploids from Spain, a region for which also the highest allozyme diversity was scored. The remaining regions had less significant differentiation.

The cpDNA haplotype data of the diploid populations revealed that the type N3, which is rarely found in Germany and absent from France/Switzerland, is also present in eastern Austria, as is the more common N2 type. It should be mentioned that the cpDNA haplotype N4 from Germany (Koch et al., 1998b ) is also extremely rare, and it might be a matter of sampling to detect this haplotype in Austria. However, it must be considered that diploids in Austria are also rare, which reduces the probability of finding the N4 haplotype. The cpDNA haplotype N5, which is exclusively distributed in a diploid population from France, represents an intermediate haplotype (between S types of the polyploids and N types of the diploids). Interestingly, this type is most similar to plastome type S1 from polyploid populations from Spain (Koch et al., 1998b ), a finding that supports the interrelationship between these two regions and was also demonstrated by the isozyme data.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Centers of diversity, refugia, and suture zones
Within the polyploids, no separation between hexaploids and tetraploids is possible based on either morphological or molecular data. Therefore, these cytotypes were regarded as a single taxonomic unit. However, it is obvious that the relative occurrence of tetraploids increased from eastern Austria and Croatia to Turkey (Koch and Hurka, 1999 ; and accessions cited in Table 1). It can be hypothesized that these findings might reflect ancient speciation processes (hybridization and reticulation) between diploid M. perfoliatum and diploid M. natolicum. In the Late Tertiary, a larger hybrid and suture zone between these diploids might have existed, and this zone migrated from the eastern Mediterranean along lowland coastal sites northwestward (Koch et al., 1998b ) with subsequent extinction of most of the original diploids. All tetraploids analyzed from these regions are represented by isozyme haplotypes not found in the hexaploids. This is different for tetraploids in Germany or France with isozyme haplotypes also found in adjacent hexaploids, which might demonstrate a secondary chromosome number reduction. However, it was not possible to detect any recent gene flow over ploidy levels by comparing biparentally inherited allozymes and strictly maternally transmissed cpDNA types (Harris and Ingram, 1991 ; Reboud and Zeyl, 1994 ). Compared to the diploids, the polyploids had significantly higher levels of genetic diversity. This is true for isozymes as well as for plastid types and correlates with increased morphological variation in the polyploids and a largely extended distribution range.

All diploids carry a characteristic N cpDNA haplotype, and all polyploids can be identified by a corresponding S cpDNA haplotype (Fig. 6). The same strict separation is seen for allozyme haplotypes (Fig. 2a). However, potential geographical suture zones exist in the diploid refuge areas in France/Switzerland and Austria. The rare sympatric occurrence of different cytotypes within smaller areas or even within the same population have been described from France and Germany (Koch and Hurka, 1999 ). However, genetic barriers are upheld by the selfing mating system, which does not propagate outbreeding or hybridization. Presently, this is clearly seen on the homoploid level with totally fixed heterozygotes for diploids as well as for polyploids. Consequently, it must be assumed that presently gene flow over ploidy levels presently plays no significant role, in accordance with the notion that no gene flow over ploidy levels was detected. Nonetheless, it is remarkable that especially between the Iberian refuge and the France/Switzerland region, close relationships based on shared rare alleles were observed (Table 3). Accordingly, the plastid type S1 from a polyploid population from central Spain represents an "intermediate" type such as N5 from diploids in France (Fig. 6). This finding indicates that hybrid zones had existed with low levels of gene flow over ploidy levels, which became extinct during the Pleistocene.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 6. Chloroplast DNA phylogeny of the genus Microthlaspi based on cpDNA restriction site polymorphisms (strict consensus tree redrawn from a parsimony analysis; Koch et al., 1998b )

 
Isozyme data revealed different patterns of diversity centers and assumed Pleistocene refugia when diploid and polyploid populations are compared. Polyploid M. perfoliatum had a "classical" pattern of Pleistocene refugia: Iberia, Italy, and the Balkans. A fourth center of diversity is found in southeast France and parts of Switzerland, a range with major portions of unglaciated areas. These refugia patterns are supported by isozyme as well as cpDNA haplotype data (Fig. 3). The Mantel tests also confirm the existence of several diversity centers. The overall Mantel analysis showed significant genetic isolation by distance. Moreover, the Mantel test using distance classes had significant positive RM values up to 1250 km. From 1250 km on, RM values were significantly negative (Fig. 5), the approximate distance among the three main refugia (Iberia, Italy, and the Balkans).

The diploids are restricted to three areas, two of which, France/Switzerland and Austria/Croatia, can be regarded as Pleistocene refugia. The third area in Germany represents a postglacially colonized distribution range. Therefore, it can be concluded that diploid M. perfoliatum was forced into similar refugia such as polyploid M. perfoliatum, but the diploid populations did not occur in the three "main" refugia, Iberia, Italy, and the Balkans (on a larger scale).

Phylogenetic evidence and glacial refugia
The hybrid origin of polyploid M. perfoliatum has been demonstrated recently (Mummenhoff et al., 1997 ). In this analysis, plastome types of the different cytotypes of M. perfoliatum resemble a monophyletic clade, and the plastome type of M. natolicum is basal to this clade (Fig. 6), indicating close relationships of the different cytotypes of M. perfoliatum regarding the maternally inherited chloroplast genome. However, sequence comparisons of the internal transcribed spacer region of the nuclear ribosomal DNA revealed that the sequence of polyploid M. perfoliatum resembles the original sequences of diploid M. natolicum and M. perfoliatum in a mosaic-like fashion (Mummenhoff et al., 1997 ). A closer look at this mosaic indicates that polyploid M. perfoliatum shares 13 nucleotide positions with diploid M. perfoliatum and another 13 nucleotide positions with diploid M. natolicum. More importantly, all three taxa have also nine (diploid M. perfoliatum) or 10 (polyploid M. perfoliatum and diploid M. natolicum) unique characters not found in the other species or cytotypes. This finding led to two conclusions. First, all three taxa or cytotypes evolved mostly independently from each other after the hybridization had occurred, and second, the mutation rate is consistent for all three taxa. Taking a molecular clock into account, the accumulation of approximately 10 mutations along the 650-bp DNA fragment in each taxon resulted in a sequence divergence between the different ITS sequences of 3.1%. In recent studies of cruciferous plants, 1% ITS sequence divergence corresponded to approximately 0.5 to 1.0 million years (Koch and Al-Shehbaz, 2000 , 2002 ), which indicates that the hybridization event dates back to the late Tertiary, therefore, prior to the Pleistocene glaciation and deglaciation cycles. This age estimate might be important to understand and explain the distribution of the refugia across Europe. It might be assumed that if a taxon had occupied all major periglacial refugia in Iberia, Italy, and the Balkans, this taxon should be as old as major Pleistocene glaciation and deglaciation cycles, which forced the species into these refugia. Unfortunately, there are not many time estimates of the evolutionary history of species analyzed for their Pleistocene refugia. Arabidopsis thaliana (Sharbel et al., 2000 ), which was also distributed within these major refuge areas, diverged from its closest relatives approximately 5.8 million years ago (Koch et al., 2000 , 2001 ), long before Pleistocene glaciation processes started. Similarly, Anthyllis montana diverged from its closest relatives at the Tertiary-Quaternary boundary (Kropf, 2002 ; Kropf et al., 2002 , but intraspecific molecular differentiation might have occurred only in the late Pleistocene 0.25–0.66 million years ago (Kropf, 2002 ).

In the case of polyploid M. perfoliatum, the long species history enabled the plant to expand through most parts of southern and southeastern Europe. Eventually, this colonization process was enforced by Pleistocene climate changes. However, during this period of time, the same climatic factors led to the extinction of most of the genetic variation in diploid M. perfoliatum. By the end of Pleistocene glaciation and deglaciation cycles, this cytotype was forced into two refugia, of which the eastern Austrian-Croatian refugium correlates to some extent with the Balkan refugium, which was found in many other plant species (e.g., Trewick et al., 2002 ; Palmé et al., 2003 ). The second refugium in southeast France has been also described for other species such as silver birch (Palmé et al., 2003 ) and white oaks (Petit et al., 2002 ). However, for the situation in white oak, the recolonization of France from Iberia is superimposed on the present day diversity in France (Palmé et al., 2003 ). This fragmentation and extinction model for diploid populations of M. perfoliatum in general is also supported by the lack of molecular variation within the regions, which is also true for its morphological variation.

Postglacial colonization of Central Europe
Isozyme and cpDNA haplotype data indicate that diploid M. perfoliatum postglacially migrated to Germany and adjacent regions from both refugia, Switzerland/France and Austria. The same is true for the polyploids.

The neighbor-joining distance tree of isozyme haplotypes from polyploid populations in Germany, the Czech Republic, Belgium, and Sweden revealed five different types distributed among 12 populations analyzed (distance tree not shown). Nearly all of them are most closely related or even identical to haplotypes from Austria or Hungary with one exception, population 75 near Pettstadt, Germany, with a haplotype close to population 103 from France (Messigny, Côte d'Or). This finding indicates that most polyploids invaded from Austria. It is unknown if this colonization is recent and caused by human settlement or if colonization occurred naturally in postglacial times in lowland regions along the Danube River, which might have provided appropriate base-rich, open pioneer habitats. It is noteworthy that in Germany, Great Britain, or Sweden diploid as well as polyploid populations of M. perfoliatum are restricted to limestone areas with corresponding habitat types such as calcareous grassland or disturbed road sites or other edges at agricultural fields or under woods. However, in most floras, M. perfoliatum is treated as native (Lid, 1989 ; Stace, 1991 ). Most of these habitats have been created or maintained through human activity, and we can at least conclude that M. perfoliatum did not have a high invasion capacity into the northern territories.


   FOOTNOTES
 
1 The authors thank Peter Schönswetter and Andreas Tribsch for support with applying Mantel tests and Michael Ancev, Nicole Galland, Monika Kriechbaum, Ingrid Möllenkamp, Peter Poschlod, and Isabelle Waser for collecting seed material. Special thanks go to Bernhard Haubold for critical reading of the manuscript. Back

4 Author for correspondence and reprint request (marcus.koch{at}urz.uni-heidelberg.de ) Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Avise J. C. J. Arnold R. M. Ball E. Bermingham T. Lamb J. E. Neigel C. A. Reeb N. C. Saunders 1987 Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics. Annual Reviews in Ecology and Systematics 18: 489-522

Bachmann K. 1997 Nuclear DNA markers in plant biosystematic research. Opera Botanica 132: 137-148

Bush N. A. 1985 Thlaspi. In V. L. Komarov and N. A. Bush [eds.], Flora of the U.S.S.R., vol. 8. Leningrad, Moskow, U.S.S.R

Casgrain P. P. Legendre 2001 The R package for multivariate and spatial analysis, version 4.0 d3: user's manual. Départment de sciences biologiques, Université de Montréal, Montréal, Quebec, Canada. Available at http://www.fas.umontreal.ca/BIOL/legendre/

Comes H. P. R. J. Abbott 2000 Random amplified polymorphic DNA (RAPD) and quantitative trait analyses across a major phylogeographical break in the Mediterranean ragwort Senecio gallicus Vill. (Asteraceae). Molecular Ecology 9: 61-76[CrossRef][Medline]

Comes H. P. R. J. Abbott 2001 Molecular phylogeography, reticulation, and lineage sorting in Mediterranean Senecio sect. Senecio (Asteraceae). Evolution 55: 1943-1962[ISI][Medline]

Comes H. P. J. W. Kadereit 1998 The effects of Quaternary climatic changes on plant distribution and evolution. Trends in Plant Science 3: 432-438[CrossRef][ISI]

Dahl E. 1946 On different types of unglaciated areas during ice ages and their significance to phytogeography. New Phytologist 45: 225-242[CrossRef][ISI]

Demesure B. B. Comps R. Petit 1996 Chloroplast DNA phylogeography of the common beech (Fagus sylvatica) in Europe. Evolution 50: 2515-2520[CrossRef][ISI]

Excoiffier L. P. Smouse J. Quattro 1992 Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: 479-491[Abstract]

Forsström L. M. Punkari 1997 Initiation of the last glaciation in Northern Europe. Quaternary Science Review 16: 1197-1215[CrossRef]

Gabrielsen T. M. K. Bachmann K. S. Jacobsen C. Brochmann 1997 Glacial survival does not matter. RAPD phylogeography of Nordic Saxifraga oppositifolia. Molecular Ecology 6: 831-842[CrossRef]

Galland N. C. Favarger 1990 Thlaspi tineoi Nyman et T. granatense Boiss. & Reut.: position systématique et valeur biogéographique de deux taxons ouest-médeterranéens au sein du complexe polyploide T. perfoliatum L. (Brassicaceae). Ecologia Mediterranea 16: 41-49

Gugerli F. R. Holderegger 2001 Nunatak survival, tabula rasa and the influence of the pleistocene ice-ages on plant evolution in mountain areas. Trends in Plant Science 6: 397-398[CrossRef][Medline]

Harris S. A. R. Ingram 1991 Chloroplast DNA and biosystematics: the effect of intraspecific diversity and plastid transmission. Taxon 40: 393-412

Hess H. E. E. Landolt R. Hirzel 1977 Flora der Schweiz und angrenzender Gebiete, vol. II., 2nd ed., 142. Birkhäuser, Basel, Stuttgart, Germany

Hewitt G. M. 1996 Some genetic consequences of ice ages, and their role in divergence and speciation. Biological Journal of the Linnean Society 58: 247-276[CrossRef]

Hewitt G. M. 1999 Post-glacial re-colonization of European biota. Biological Journal of the Linnean Society 68: 87-112[CrossRef][ISI]

Hewitt G. M. 2000 The genetic legacy of the Quaternary ice ages. Nature 405: 907-913

Hill L. M. 1982 IOPB chromosome number reports LXXIV. Taxon 31: 119-128

Jalas J. J. Suominen R. Lampinen 1996 Atlas florae Europaea. Distribution of vascular plants in Europe. Helsinki University Printing House, Helsinki, Finland

Jordan A. 1852 Pugillus plantarum novarum: 12

Jordan A. 1864 Diagnoses d'Especes Nouveau 1: 250

King R. A. C. Ferris 1998 Chloroplast DNA phylogeography of Alnus glutinosa (L.) Gaertn. Molecular Ecology 7: 1151-1161[CrossRef]

Koch M. 1997 Zur Morphologie, Systematik und Verbreitung des Polyploidkomplexes Thlaspi perfoliatum L. [Microthlaspi perfoliatum (L.) F. K. Meyer] in Deutschland. Osnabrücker Naturwissenschaftliche Mitteilungen 23: 157-168

Koch M. I. A. Al-Shehbaz 2000 Systematics of Chinese Genera Yinshania, Hilliella, and Cochleariella (Brassicaceae): evidence from plastid trnL intron and nuclear ITS DNA sequence data. Annals of the Missouri Botanical Garden 87: 246-272[CrossRef][ISI]

Koch M. I. A. Al-Shehbaz 2002 Molecular data indicate complex intra- and intercontinental differentiation of American Draba (Brassicaceae). Annals of the Missouri Botanical Garden 89: 88-109[CrossRef][ISI]

Koch M. I. A. Al-Shehbaz K. Mummenhoff 2003a Molecular systematics, evolution, and population biology in the mustard family (Brassicaceae). Annals of the Missouri Botanical Garden 90: 151-171[CrossRef][ISI]

Koch M. C. Dobes K.-G. Bernhardt J. Kochjarová 2003b Cochlearia macrorrhiza (Brassicaceae): a bridging species between Cochlearia taxa from the Eastern Alps and the Carpathians?. Plant Systematics and Evolution, in press

Koch M. B. Haubold T. Mitchell-Olds 2000 Comparative evolutionary analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis, Arabis and related genera. Molecular Biology and Evolution 17: 1483-1498[Abstract/Free Full Text]

Koch M. B. Haubold T. Mitchell-Olds 2001 Molecular systematics of the cruciferae: evidence from coding plastome matK and nuclear CHS sequences. American Journal of Botany 88: 534-544[Abstract/Free Full Text]

Koch M. H. Hurka 1999 Isozyme analysis in the polyploid complex Microthlaspi perfoliatum (L.) F.K. Meyer: morphology, biogeography and evolutionary history. Flora 194: 33-48

Koch M. M. Huthmann H. Hurka 1998a Isozymes, speciation and evolution in the polyploid complex Cochlearia L. (Brassicaceae). Botanica Acta 111: 451-466

Koch M. K. Mummenhoff 2001 Thlaspi s.str. (Brassicaceae) versus Thlaspi s.l.: morphological and anatomical characters in the light of molecular data. Plant Systematics and Evolution 227: 209-225[CrossRef][ISI]

Koch M. K. Mummenhoff H. Hurka 1998b Molecular biogeography and evolution of the Microthlaspi perfoliatum s.l. polyploid complex (Brassicaceae): chloroplast DNA and nuclear ribosomal DNA restriction site variation. Canadian Journal of Botany 76: 382-396[CrossRef][ISI]

Konnert M. F. Bergmann 1995 The geographical distribution of genetic variation of silver fir (Abies alba, Pinaceae) in relation to its migration history. Plant Systematics and Evolution 196: 19-30[CrossRef][ISI]

Kropf M. 2002 Vergleichende Biogeographie europäischer Gebirgspflanzen: Molekulare und morphometrische Untersuchungen an der montan-subalpinen Anthyllis montana L. (Fabaceae) und der hochalpinen Pritzelago alpina (L.) O. Kuntze (Brassicaceae). Cuvillier Verlag, Göttingen, Germany

Kropf M. J. W. Kadereit H. P. Comes 2002 Late Quaternary distributional stasis in the submediterranean mountain plant Anthyllis montana L. (Fabaceae) inferred from ITS sequences and amplified fragment length polymorphism markers. Molecular Ecology 11: 447-463[CrossRef][Medline]

Lid J. 1989 Norsk, Svensk, Finsk Flora, 174–175. Det Norske Samlaget, Stockholm, Sweden

Linné C. 1753 Species plantarum: 641. Paris, France

Mantel N. 1967 The detection of disease clustering and a generalized regression approach. Cancer Research 27: 209-220[ISI][Medline]

Markgraf F. 1958 Cruciferae. In G. Hegi [ed.], Illustrierte Flora von Mitteleuropa, vol. IV, 1. Parey, Berlin, Germany

Meyer F. K. 1973 Conspectus der "Thlaspi"—Arten Europas, Afrikas und Vorderasiens. Feddes Repertorium 84: 449-470

Meyer F. K. 1979 Kritische Revision der "Thlaspi"—Arten Europas, Afrikas und Vorderasiens. I. Geschichte, Morphologie und Chorologie. Feddes Repertorium 90: 129-154

Mueller U. G. L. Wolfenbarger 1999 AFLP genotyping and fingerprinting. Trends in Ecology and Evolution 14: 389-394

Mummenhoff K. A. Franzke M. Koch 1997 Molecular phylogenetics of Thlaspi s.l. (Brassicaceae) based on chloroplast DNA restriction site variation and sequences of the internal transcribed spacer of nuclear ribosomal DNA. Canadian Journal of Botany 75: 129-154[ISI]

Nei M. W.-H. Li 1979 Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences, USA 186: 5269-5273

Palmé E. Q. Su A. Rautenberg F. Manni M. Lascoux 2003 Postglacial recolonization and cpDNA variation of silver birch, Betula pendula. Molecular Ecology 12: 201-212[CrossRef][Medline]

Petit R. J. et al 2002 Chloroplast DNA variation in European white oaks phylogeography and patterns of diversity based on data from over 2600 populations. Forest Ecology and Management 156: 5-26[CrossRef][ISI]

Podani J. 1993 Computer porgrams for multivariate data analysis in ecology and systematics, version 5.0 User's guide. Scientia Publishing, Budapest, Hungary .

Polatschek A. 1983 Chromosomenzahlen und Hinweise auf Systematik und Verbreitung von Brassicaceae-Arten aus Europa, Nordafrika, Asien und Australien. Phyton (Austria) 28: 127-139

Reboud X. C. Zeyl 1994 Organelle inheritance in plants. Heredity 72: 132-140[ISI]

Schneider S. D. Roessli L. Excoiffier 2000 Arlequin vers. 2.000: a software for population genetics data analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland

Schönswetter P. A. Tribsch M. Barfuss H. Niklfeld 2002 Several Pleistocene refugia detected in the high alpine plant Phyteuma globulariifolium Sternb. & Hoppe (Campanulaceae) in the European Alps. Molecular Ecology 11: 2637-2647[CrossRef][Medline]

Schönswetter P. A. Tribsch G. M. Schneeweiss H. Niklfeld 2003 Disjunctions in relict alpine plants: phylogeography of Androsace brevis and A. wulfeniana (Primulaceae). Botanical Journal of the Linnean Society 141: 437-446[CrossRef]

Schultze-Motel W. 1986 Thlaspi. In W. Schultze-Motel [ed.], Illustrierte Flora von Mitteleuropa, vol. IV, 3rd ed. Parey, Berlin, Germany

Sharbel T. F. B. Haubold T. Mitchell-Olds 2000 Genetic isolation by distance in Arabidopsis thaliana: biogeography and post-glacial colonization of Europe. Molecular Ecology 9: 2109-2118[CrossRef][Medline]

Sinclair W. T. J. D. Mormon R. A. Enos 1999 The postglacial history of Scots pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial DNA variation. Molecular Ecology 8: 83-88

Stace C. 1991 New Flora of the British Isles. Cambridge University Press, Cambridge, UK

Stehlik I. J. J. Schneller K. Bachmann 2001 Resistence or emigration: response of the high-alpine plant Eritrichum namum (L.) Gaudin to the ice age within the Central Alps. Molecular Ecology 10: 357-370[CrossRef][Medline]

Taberlet P. L. Fumagalli A. G. Wustsaucy J. F. Cosson 1998 Comparative phylogeography and postglacial colonization routes in Europe. Molecular Ecology 7: 453-464[CrossRef][Medline]

Tremetsberger K. C. König R. Samuel W. Pinsker T. F. Stuessy 2002 Infraspecific genetic variation in Biscutella laevigata (Brassiaceae): new focus on Irene Manton's hypothesis. Plant Systematics and Evolution 233: 163-181[CrossRef][ISI]

Trewick S. A. M. Morgan-Richards S. J. Russell S. Henderson F. J. Rumsey I. Pintér J. A. Barrett M. Gibby J. C. Vogel 2002 Polyploidy, phylogeography and Pleistocene refugia of the rockfern Asplenium ceterach: evidence from chloroplast DNA. Molecular Ecology 11: 2003-2012[CrossRef][Medline]

Tribsch A. P. Schönswetter 2003 Patterns of endemism and comparative phylogeography confirm palaeo-environmental evidence in the Eastern Alps. Taxon 52: 477-498[CrossRef][ISI]

Tribsch A. P. Schönswetter T. Stuessy 2002 Saponaria pumila (Caryophyllaceae) and the ice age in the European Alps. American Journal of Botany 89: 2024-2033[Abstract/Free Full Text]

Van de Peer Y. R. De Wachter 1994 TEECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Computer Application for Bioscience 10: 569-570

Vos P. R. Hogers M. Bleeker M. Reijans T. Van De Lee M. Hornes A. Frijters J. Pot J. Peleman M. Kuiper M. Zabeau 1995 AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23: 4407-4414[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Am. J. Bot.Home page
J. Lihova, K. Marhold, H. Kudoh, and M. A. Koch
Worldwide phylogeny and biogeography of Cardamine flexuosa (Brassicaceae) and its relatives
Am. J. Botany, August 1, 2006; 93(8): 1206 - 1221.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science