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Geobotanisches Institut ETH, Zollikerstr. 107, CH-8008 Zürich, Switzerland
Received for publication August 17, 1998. Accepted for publication February 1, 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Brassicaceae • cpDNA • hybridization • ITS nucleotide sequences • polyploid evolution • speciation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Masterson, 1994
) and because polyploidization may be a significant mode of speciation (Leitch and Bennett, 1998
). Within the last decade, the application of molecular markers has changed our concepts about polyploids (i.e., lack of genetic variation or single origin of polyploids) and has provided significant new insights into the evolutionary dynamics of polyploid speciation in plants. Polyploid species are often polyphyletic with two or more independent origins (Soltis, Doyle, and Soltis, 1992
), especially in polyploids with wide geographic distribution. In many cases their diploid relatives have large geographical distributions as well (Ehrendorfer, 1980
), suggesting that polyploids are typically polyphyletic when their parental species co-occur on a large geographic scale.
The study of polyploid evolution has been facilitated by the development of several molecular techniques that help to identify progenitor species of allopolyploid taxa and assess genetic variation within both progenitors and polyploids. Nuclear genetic variation, which is often assessed through allozyme electrophoresis, Randomly Amplified Polymorphic DNA analysis (RAPD), or sequencing, may identify parental species because plants of polyploid or hybrid origin typically exhibit additivity of parental genomes (Soltis, Doyle, and Soltis, 1992
). In comparison to allozyme and RAPD data, sequences from nuclear genes, such as the nuclear ribosomal Internal Transcribed Spacer (ITS) not only allow the identification of parents, but may provide information about the age of the hybrid species.
The application of ITS sequences to the study of hybrid speciation, however, may be hindered by the particular mode of ITS sequence evolution (Wendel, Schnabel, and Seelanan, 1995
). ITS sequences are part of the ribosomal DNA (rDNA), which in higher plants is grouped into arrays that contain hundreds or even thousands of repeats. Evolutionary processes, usually referred to as concerted evolution, are thought to homogenize these repeats such that only a single copy is present (Zimmer et al., 1980
). In polyploids of hybrid origin, additivity of parental rDNA sequences is often observed (Soltis, Doyle, and Soltis, 1992
). However, interlocus concerted evolution may homogenize repeats from different arrays, effectively removing one parental rDNA type from the hybrid genome (Wendel, Schnabel, and Seelanan, 1995
). Conclusions based on the presence of a single rDNA type, i.e., that species of presumed hybrid origin are in fact autopolyploids, may therefore be premature. How fast one of the parental rDNA repeat types may be removed from the hybrid genome and by what evolutionary forces, however, is currently unresolved.
Maternal inheritance of the chloroplast genome predominates in angiosperms (Harris and Ingram, 1991
; but see Testolin and Cipriani, 1997
) making chloroplast DNA (cpDNA) sequences an ideal marker for the identification of maternal species in studies of polyploid speciation. The large variation in the average rate of evolution between different genes and between coding and noncoding regions of the chloroplast genome allow the choice of suitable regions for study. Rapidly evolving sequences, such as the coding matK gene (Brochmann, Nilsson, and Gabrielsen 1996
), and noncoding cpDNA sequences, such as the trnL (UAA) intron and the intergenic spacer between the trnL (UAA) 3' exon and the trnF (GAA) gene (Taberlet et al., 1991
), are considered ideal markers for the study of phylogenetic relationships among closely related taxa, due to their high average rate of evolution. They have been used intensively for studying relationships at lower taxonomic levels (Kim and Jansen, 1994
; Mes and ‘T Hart, 1994
; Kita, Ueda, and Kadota, 1995
; Gielly and Taberlet, 1996
; Kim, ‘T Hart, and Mes, 1996
; Mes, Vanbrederode, and ‘T Hart, 1996
; Mes, Wijers, and ‘T Hart, 1997
) and are considered an extremely valuable tool for the differentiation of closely related taxa (Gielly and Taberlet, 1994
). Despite the presumably conservative mode of cpDNA evolution, several cases of intraspecific variation have been reported (Rajora and Dancik, 1995
; Demesure, Comps, and Petit, 1996
; Jordan, Courtney, and Neigel, 1996;
Soltis et al., 1996
). Such intraspecific variation offers great possibilities for the study of allopolyploid evolution because it may indicate repeated origin of hybrid species (Brochmann et al., 1998
).
The tetraploid Draba ladina is a narrow endemic to the Swiss Alps and was first described in 1919 [ Braun-Blanquet, 1919 (1920)
]. It is restricted to few (<12) mountains of the Ofenpassgruppe in the Lower Engadin (Unterengadin) east of Zernez where it grows in dolomite crevices and in scree on limestone at altitudes between 2600 and 3000 m. Schulz (1927)
suggested that D. ladina is a hybrid between D. hoppeana and D. tomentosa but gave no indication how this conclusion was reached. Markgraf (1958)
refused this hypothesis, stating that it was based solely on the yellowish flower color, which was intermediate between the yellow flowers of D. hoppeana and the white flowers of D. tomentosa. However, D. hoppeana does not occur in the area [Markgraf, 1958;
and personal observations, but see Braun-Blanquet, 1919 (1920)
]. Buttler (1967) rejected a recent hybrid origin of D. ladina, stating that such hybrids were always sterile, and considered an ancient allopolyploid origin unlikely. He preferred the hypothesis that D. ladina represents a western outpost of an asian Draba species group, and further proposed that D. ladina survived the last glaciation period in the Alps and later failed to expand its geographic range after the retreat of the glaciers (Buttler, 1967
). In 1969, Buttler suggested an allopolyploid origin for D. ladina based on morphological and caryological data and identified the diploid D. aizoides and D. tomentosa as potential parental species. Draba ladina occurs in sympatry with its proposed diploid progenitors, D. aizoides, D. tomentosa, and another white-flowered species, D. dubia [ Braun-Blanquet, 1919 (1920)
; Markgraf, 1958;
and personal observations]. These species share the same chromosome number (2n = 2x = 16; Hess, Landolt, and Hirzel, 1967)
and are typical members of the alpine flora where they often co-occur (Welten and Sutter, 1982
).
In this study, we used a molecular approach involving sequencing of chloroplast and nuclear rDNA to reconstruct the evolutionary history of D. ladina, to verify its polyploid origin (auto- or allopolyploid), to identify the parental species in the case of an allopolyploid origin, to assess whether it evolved once or repeatedly, and to estimate the age of the species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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were used to amplify the trnL (UAA) intron, the trnL (UAA) 3' exon, and the intergenic spacer between the trnL (UAA) 3' exon and trnF (GAA). PCRs were carried out in a total reaction volume of 50 µL, containing 1x reaction buffer (Promega, Catalys AG, Wallisellen, Switzerland),1.5–2.5 mmol/L MgCl2, 0.3 µmol/L of each primer (Microsynth GmbH, Balgach, Switzerland), 100 µmol/L of each dNTP (Boehringer Mannheim, Rotkreuz, Switzerland), and 2.5 Units of Taq Polymerase (Promega, Catalys AG, Wallisellen, Switzerland). The thermocycling profile consisted of an initial denaturation step at 94°C for 2 min, followed by 25 cycles with 30 s at 94°C, 30 s at 55°C, and 2 min at 72°C. PCRs were performed either on a PTC-100 temperature cycler (MJ Research Inc., Watertown, Massachusetts) or on a GeneAmp PCR System 2400 (PE Biosystems, Foster City, CA). PCR fragments were purified with a QIAquick PCR purification kit (Qiagen, Basel, Switzerland) to remove unincorporated primers and dNTPs. Primers c, d, e, and f of Taberlet et al. (1991)
were used to sequence both strands of the PCR fragment to unambiguously identify all sites. Sequencing was done by the dideoxy method (Sanger, Nicklen, and Coulson, 1977
) using AmpliTaq DNA Polymerase FS and fluorescent labelled dNTP's (PE Biosystems, Foster City, CA). Sequencing reactions were set up according to the supplier's recommendations (Perkin-Elmer, 1995
) and purified using ethanol precipitation at room temperature. Automatic DNA sequencing was performed on an ABI PRISMTM310 Genetic Analyzer (PE Biosystems, Foster City, CA). Both strands were sequenced at least once for each species and DNA fragment, to unambiguously identify all sites. CpDNA sequences were obtained from ten individuals of D. dubia, 20 of D. ladina, and ten of D. tomentosa. Sequences of nine D. aizoides haplotypes (DA1-DA9) found among 53 individuals (unpublished data) were included in the analysis.
ITS amplification, cloning, and sequencing
Nuclear rDNA-gene spacers ITS1 and ITS2 were amplified by polymerase chain reaction (PCR) using the primers "ITS 2," "ITS 3," "ITS 4," and "ITS 5" (White et al., 1990
). 5–25 ng of DNA were submitted to amplification in a total volume of 50 µL, containing 1x reaction buffer, 1.5–2 mmol/L MgCl2, 0.4 µmol/L of each primer, 200 µmol/L of each dNTP and 0.5 U of Pfu DNA polymerase (Stratagene AG, Basel, Switzerland) or 1.2 U of AmpliTaq Gold (Perkin Elmer Europe B.V., Rotkreuz, Switzerland). The typical thermocycling profile consisted of 25 cycles with 30 s at 94°C, 30 s at 55°C, and 2 min at 72°C. With AmpliTaq Gold Polymerase, a preheating step of 8 min at 94°C was necessary. All PCR reactions were performed on a PTC-100 temperature cycler.
To test for intra-individual ITS sequence variation, corresponding PCR products (primers "ITS4" and "ITS5") of three D. ladina were cloned. PCR products were purified with the QIAquick PCR purification kit and subsequently cloned into the Sma1 site of Bluescript KS+, using E. coli strain DH5
as host (Stratagene AG, Basel, Switzerland). The plasmid preparation was performed based on the alkaline lysis method of Birnboim and Doyle (1979)
. The universal primers SK and KS and primer "ITS3" were used for sequencing three clones each of three D. ladina. PCR-amplified ITS sequences of other taxa were sequenced as described above. ITS sequences other than from D. ladina were obtained from 20 individuals of D. aizoides (including at least one individual per population and per chloroplast DNA haplotype), eight of D. dubia, and ten of D. tomentosa.
Sequence analyses
Sequence files obtained from the ABI PRISMTM310 Genetic Analyzer (PE Biosystems, Foster City, CA) were imported separately for each individual into Sequence Navigator version 1.0.1 (PE Biosystems, Foster City, CA) and complementary strands were aligned using the Clustal V option as provided in the program. Ambiguous sites were checked manually and corrected by comparing the electropherograms from both strands. Consensus sequences were obtained for each individual. Multiple alignments based on consensus sequences were carried out in Sequence Navigator, using Clustal V. Minor adjustments were made manually to minimize the number of inferred insertions/deletions (indels).
PAUP 4.0d64 written by D. L. Swofford was used for phylogenetic analyses, using maximum parsimony. One-base pair (bp) indels were treated as binary presence/absence characters. Longer indels were considered as one independent evolutionary event each and subsequently treated as one polymorphic position each. The presence and frequency of indels suggest that only a parsimony approach should be used for phylogenetic reconstructions, since no reliable genetic distances can be estimated from both substitutions and indels (Gielly and Taberlet, 1994
). All characters were specified as unordered and weighted equally. The presence of phylogenetic signal in the sequence matrices was tested using the g1 statistic test based on the skewness of tree-length distributions (Hillis and Huelsenbeck, 1992). The g1 was estimated by generating 105 trees from the complete data set with the random-trees option in the test version 4.0d64 of PAUP provided by D. L. Swofford.
Parsimony analyses were performed using the branch-and-bound search strategy. Bootstrap analyses (Felsenstein, 1985
) were run, using 1000 replications to obtain estimates of reliability for nodes.
Cytology
Two living plants of D. ladina from Fuorcla Val dal Botsch (population code VDB, Table 1) were transferred to the greenhouse for cytological investigation, which was done using root tips. These were pretreated for 0.5 h with colchicine (0.05%), then fixed in ethanol/acetic acid (3:1), and stained and squashed in lacto-propionic orcein. For the determination of chromosome numbers, 5–10 metaphases were counted for both individuals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T, position 524; C A, position 541). Whether this group is sister to D. dubia (DD2) or D. tomentosa (DT1) remains unresolved in the parsimony analysis (Fig. 3). ITS and cpDNA sequences have been deposited in GenBank (accession numbers AF120721–120726, AF120738–120744).
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), the two plants are therefore tetraploid, which corresponds with the indication in Merxmüller and Buttler (1965)
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and Buttler (1969)
, because the presence of two distinct ITS sequences in the tetraploid D. ladina is not compatible with an autopolyploid origin. The rDNA sequences found in D. ladina are most closely related to those of the presumed (Buttler, 1969
) parental species, D. aizoides and D. tomentosa. Additivity of rDNA sequences in the allopolyploid is intuitively expected and has often been found (for a review see Rieseberg and Brunsfeld, 1992
). However, as shown by Wendel, Schnabel, and Seelanan (1995)
, bidirectional interlocus concerted evolution may effectively remove one of the parental rDNA repeats from the hybrid genome. Although the exact nature of this process is not well understood, it appears that homogenizing of ITS sequences via bidirectional interlocus concerted evolution requires many sexual generations for completion (Wendel, Schnabel, and Seelanan, 1995
; Brochmann, Nilsson, and Gabrielsen, 1996
). The presence of two parental ITS sequences in D. ladina may indicate a relatively recent origin of the species. The absence of one parental rDNA repeat in one of the studied individuals of D. ladina is most likely a consequence of the small number of clones sequenced and is thus no evidence for the loss of one parental repeat due to bidirectional interlocus concerted evolution. The cpDNA data per se do not provide conclusive evidence about the maternal progenitor of D. ladina. Intraspecific cpDNA sequence divergence in D. aizoides was found to be extensive, with three genetically well-differentiated groups of cpDNA haplotypes occurring in Switzerland (unpublished data). Intraspecific cpDNA sequence divergence among different D. aizoides haplotypes is similar or larger than interspecific divergence among D. aizoides, D. dubia, and D. tomentosa. Draba aizoides can be excluded as the maternal progenitor of D. ladina because all known haplotpyes are more distant from D. ladina than are either D. tomentosa or D. dubia. The topology of the cpDNA tree (Fig. 3) suggests that the chloroplast genome found in D. ladina shared a common ancestor with that found in both D. dubia and D. tomentosa. Given the small genetic differences between cpDNA haplotypes this could implicate that D. dubia instead of D. tomentosa was involved in the formation of D. ladina. However, strong evidence for the hypothesis that D. tomentosa is in fact the maternal species of D. ladina comes from indel I1 (Table 3), which is present in both D. dubia haplotypes, although in different length variants, but is absent from the chloroplast genomes of D. tomentosa and D. ladina. Further evidence for the identification of D. tomentosa as the maternal species comes from the analysis of ITS sequences, which excludes D. dubia from the pool of potential progenitors of D. ladina, since the two ITS types found in the hybrid clearly belong to D. aizoides and D. tomentosa. We therefore conclude that D. ladina is of allopolyploid origin with D. aizoides as the paternal parent and D. tomentosa the maternal parent.
Single vs. multiple hybridization events and age of D. ladina
Allopolyploidy results from chromosome doubling following hybridization between two genetically distinct diploid species. This important mechanism of speciation in flowering plants (Grant, 1981
) was considered to produce genetically depauperate species because instant reproductive isolation precludes influx of genetic variation from the parental diploid taxa.
Recently, molecular studies on polyploid evolution have found extensive genetic diversity indicative of multiple origins of many polyploid taxa (for a review see Soltis and Soltis, 1993
). Recurrent hybridization events have been documented occurring over relatively short time spans and geographic distances (Ashton and Abbott, 1992
; Arft and Ranker, 1998
). If polyploidization is less rare than has been assumed (Soltis, Doyle, and Soltis, 1992
; Soltis and Soltis, 1993
), then significant genetic variation may be incorporated into allopolyploid species from genetically distinct parental individuals or populations. In arctic Draba, multiple origins of polyploids appear to be the rule rather than the exception (Brochmann, Nilsson, and Gabrielsen, 1996
; Brochmann, Soltis, and Soltis, 1992b,c
), making polyploid Draba a textbook example for recurrent speciation through hybridization (Briggs and Walters, 1997
).
In D. ladina, however, no intraspecific ITS or chloroplast variation was detected despite extensive intraspecific cpDNA variation in D. aizoides and moderate intraspecific variation in D. dubia and D. tomentosa, the presumed progenitor group. Lack of variation in D. ladina therefore does not result from choice of an inappropriate cpDNA fragment or from low variation in the progenitor species. Also, the two populations examined represent two geographically distinct locations of this narrow endemic, for which less than 12 populations are known. We therefore propose a single allopolyploid origin for this narrow-endemic species. Otherwise, we would expect to find variation at least among populations, such as in the autopolyploid Saxifraga osloensis (Brochmann, Nilsson, and Gabrielsen, 1996
).
Sequence divergence between ITS and cpDNA sequences found in D. ladina and its progenitor species may be due to sampling error (i.e., insufficient sampling of progenitor sequences), or because the genome of D. ladina has diverged from the progenitor's genomes. The observation that both parental rDNA repeat types are still present in the allopolyploid and that none has yet been eliminated from the genome through bidirectional interlocus concerted evolution should allow us to estimate the age of D. ladina. Unfortunately, however, estimates of mutation rates for noncoding cpDNA sequences and for ITS sequences (Baldwin, 1992
; Suh et al., 1993
) are either unknown or differ widely among lineages. Thus one cannot reliably estimate the age of D. ladina based on the sequence divergence observed. Similarly, the time scale necessary for bidirectional interlocus concerted evolution to remove one parental rDNA repeat from the hybrid genome is unknown. There seems to be general agreement, however, that many sexual generations are necessary for homogenization to be complete (Wendel, Schnabel, and Seelanan, 1995
; Brochmann, Nilsson, and Gabrielsen, 1996
). The generation time of D. ladina is unknown. Our personal observations, however, suggest that plants can grow to a very old age, probably >20 yr. Taking all this circumstantial evidence into account, we propose a relatively recent, probably postglacial origin for D. ladina.
Geographic range
D. ladina is a narrow endemic. This pattern is in marked contrast to many other polyploids, for which a wider geographic range relative to their diploid progenitors has been reported (Soltis and Soltis, 1991
). Three factors may account for the restricted range of D. ladina: the presumed single origin, low dispersal capacity of its seeds, and its restriction to dolomite crevices and scree on limestone at elevations between 2600 and 3000 m. A similar conclusion was reached in the allopolyploid Cardamine schulzii, which evolved within the last century and failed to expand its geographic range because of specific habitat requirements (Urbanska et al., 1997
).
Repeated evolution, which has often been reported in the arctic-alpine genus Draba (Brochmann, Soltis, and Soltis, 1992a, b, c
) and has been suggested for the alpine D. dolomitica (Buttler, 1969
), could also lead to a wide geographic distribution. Indeed, the parental species of D. ladina, D. aizoides, and D. tomentosa, are widely distributed in the Alps and co-occur frequently (Welten and Sutter, 1982
, and personal observations). Hybrids between the two species have been reported from various regions in the Alps (Markgraf, 1958
). However, our molecular results provide no evidence that the polyploidization necessary for the repeated evolution of D. ladina has occurred more than once.
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FOOTNOTES |
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2
Author for correspondence (e-mail: alex.widmer{at}geobot.umnw.ethz.ch
).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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