The Journal of Heredity 2001:92(5)
© 2001 The American Genetic Association 92:404-408
Morphological, Cytogenetic, and Molecular Evidence for Introgressive Hybridization in Birch
From the Department of Biology, University of Iceland, Grensásvegi 12, Reykjavík 108, Iceland (Thórsson and Anamthawat-Jónsson), and Department of Biology, Ecology Section, University of Turku, Turku, 20014 Finland (Salmela).
Address correspondence to K. Anamthawat-Jónsson at the address above or e-mail kesara{at}hi.is.
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Abstract |
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Extensive morphological variation of tetraploid birch (Betula pubescens) in Iceland is believed to be due to gene flow from diploid dwarf birch (B. nana) by means of introgressive hybridization. A combined morphological and cytogenetic approach was used to investigate this phenomenon in two geographically separated populations of natural birch woodland in Iceland. The results not only confirmed introgressive hybridization in birch, but also revealed bidirectional gene flow between the two species via triploid interspecific hybrids. The populations showed continuous morphological variation connecting the species, but karyotypically they consisted of only three types of plants: diploids, triploids, and tetraploids. No aneuploids were found. Some of the tetraploid plants had B. pubescens morphology as expected, but most of them had intermediate characters. Most of the diploid plants were B. nana, but some were intermediates and a few had B. pubescens morphology. The triploid plants were either intermediates or they resembled one of the two species. Similar introgressive variation was observed among the diploid and triploid progeny of open-pollinated B. nana in a garden. Birch samples including field plants and artificial hybrids were further examined using a molecular method based on genomic Southern hybridization. The experiments verified introgression at the DNA level.
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Introduction |
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Betula L. (birch) is a genus of some 50 species distributed throughout northern temperate regions. In Europe, three species are generally recognized (Atkinson 1992; de Groot et al. 1997): silver birch (B. pendula Roth., diploid 2n = 28, Eurasian), downy birch (B. pubescens Ehrh., tetraploid 2n = 56, Eurasian), and dwarf birch (B. nana L., diploid 2n = 28, circumpolar). Although there is a considerable overlap between the geographical and altitudinal distributions of these species, B. pendula extends further south into the Balkans than B. pubescens, while B. pubescens is more prevalent in the north and west of Europe including Fennoscandia and Iceland. B. nana, an arctic-alpine species, is more northerly and is found at higher altitudes than the two tree birch species. In the areas where the distributions of birch species overlap, plants with intermediate morphology (presumed hybrids) have been frequently recorded (reviewed in Atkinson 1992; de Groot et al. 1997; Walters 1964). As these natural putative hybrids are either not convincingly confirmed by cytotaxonomic investigation or they have turned out to be a part of the large range of variability within species, the occurrence of introgressive hybridization in birch evolution is speculative. We have therefore conducted a systematic investigation using molecular and cytogenetic methods to determine whether introgression occurs in nature.
In Iceland, B. pubescens is the only tree species forming natural woodlands and forests in different geographic regions and habitats, from coastal to inland areas up to 600 m above sea level. In these woodlands, B. pubescens and B. nana occur sympatrically, but in the central highland, B. nana is the predominant birch species (Thórhallsdóttir 1998). Birch woodland/forest in Iceland began about 9000 years ago (Hallsdóttir 1995), and until the first settlement about 1000 years ago, was estimated to cover some 30% of the land area. Due to human habitation this has diminished and now only 1% of the land area is covered with birch forest. It has therefore become urgent to protect and regenerate this woodland in order to increase its extent and diversity. This requires a good understanding of the population dynamics of birch, its genetic structure and evolution, and the factors and processes involved in making the woodlands and forests of today.
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Materials and Methods |
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Plant Materials
Two birch woodland populations were chosen for this investigation: "Bifröst" in western Iceland at 64.45°N/21.30°W and "Brekkuskógur" in southwestern Iceland at 64.15°N/20.30°W, both being about 200 m above sea level. At Bifröst, 42 plants were selected, regardless of species identity (i.e., from both B. pubescens and B. nana) at a distance of about 50 m from one plant to the next. At Brekkuskógur, 30 plants were chosen in the same way. Plants were marked in early spring (May/June), their growth form and habit was recorded, and newly formed leaves were taken for DNA and chromosome analyses. The plants were visited regularly during the growing season: in midsummer to collect leaf and branch samples for morphological analysis, and in the late season to examine catkins and to collect seeds. In addition to the woodland materials, 24 plants germinated from seeds of two open-pollinated B. nana plants were also examined. The mother plants were grown in a garden in Reykjavík but originated from Úthlid, the woodland connected to Brekkuskógur. The plant materials used in the Southern hybridization experiments included samples from Iceland and Finland, as well as artificial hybrids (see legend of Figure 1).
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Morphology Index
Discrete species-specific morphological characters including growth form, growth habit, leaf shape, leaf tip, leaf base, and leaf margin (Clapham et al. 1962; Elkington 1968; Kenworthy et al. 1972; Pelham et al. 1988) were examined. Walters (1964) described leaves of B. nana as orbicular with crenate margins and those of B. pubescens as cordate with dentate margins. The growth form and habit was evaluated in the field, but the leaf characters were scored from 30 randomly collected leaves per plant, except from the crossing progeny, where only a few leaves from each plant were analyzed. The leaf shape characters were highly uniform within plants. Scores were assigned to place B. nana at the lowest ranks and B. pubescens the highest (Table 1). For each plant, the scores of all characters were combined into a single value called "morphology index."
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Chromosome Analysis
Mitotic chromosomes were isolated from leaf tissues using a modification of the root-tip method by Busch et al. (1996). Chromosomes were stained with fluorescent dye DAPI (4,6-diaminophenylindole) and visualized in an epifluorescence microscope using 1000x magnification. Chromosome counts were made from individual plants, 10 to 50 metaphases per plant. Successful chromosome counts were obtained from 33 plants from Bifröst, 28 plants from Brekkuskógur, and 21 plants from the garden.
Genomic Southern Hybridization
Total genomic DNA was isolated from samples of the two birch species and their hybrids as previously described (Anamthawat-Jónsson and Heslop-Harrison 1995). Genomic Southern hybridization experiments were conducted according to Anamthawat-Jónsson et al. (1990) and Anamthawat-Jónsson and Heslop-Harrison (1996). The total genomic DNAs were digested with restriction endonucleases DraI, BamHI, and EcoRI, electrophoresed, transferred to nylon membrane, hybridized with labeled total genomic DNA from one of the birch species, and simultaneously blocked with sonicated, unlabeled total genomic DNA from the other birch species. ECL (chemiluminescence method; Amersham International, Denmark) was used for labeling and hybridization. The hybridization stringency was 90%, based on 0.1 M NaCl in the hybridization buffer. The blocking with B. pubescens DNA was 50x, and with B. nana was 10x the probe amount. Only the DraI blot is shown in this article (Figure 1).
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Results |
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Morphological and Cytogenetic Studies
Total scores of species-specific characters (morphology index on a scale of 0–13, shown in Figure 2) were obtained from individual plants in the woodland populations and from crosses. The lowest value (0) represented characters taxonomically specific to B. nana and the highest value (13) represented characters specific to B. pubescens. Based on these morphological characters, the population at Bifröst (Figure 2A) consisted of two peaks, one having B. nana morphology (score 0–1) and the other having B. pubescens morphology (score 8–10), although none had all of its species-specific characters. Other plants were distributed between these two peaks, as they possessed intermediate characters or they had characters from both species. For example, the plant Bifröst-76 (total score 6) had the growth form and habit, leaf base, and leaf shape of B. nana, and the petiole and leaf margin of B. pubescens, whereas leaf tip was intermediate of both species. Chromosome numbers in 33 of 42 plants at Bifröst were determined, and as expected, the results (Figure 2B) showed that most diploid (2n = 28) plants had B. nana morphology and the majority of the tetraploid (2n = 56) plants had the morphology of B. pubescens. Plants having intermediate morphological indices (score 2–7) were diploid, triploid (2n = 42), and tetraploid. None of the plants examined here were aneuploid. The morphological variation at the woodland Bifröst and the cytogenetic results clearly suggest the occurrence of introgressive variation via the intermediacy of triploid hybrids.
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The population at Brekkuskógur (Figure 2C) appeared to have been through more intensive introgression than the Bifröst population. Within the same scale of morphology index, Brekkuskógur had no apparent peak typical of B. nana, and the B. pubescens peak shifted toward the hybrid range of morphology (score
9). None of the plants examined had all or most of the morphological characters specific to B. pubescens (score 10–13). Furthermore, all three genomic groups of plants (diploid, triploid, and tetraploid; Figure 3) were found to be extremely variable morphologically. While some of the diploid plants were B. nana (score 0–1), the others were B. pubescens-like (score 8–9) or had intermediate morphology. This in particular is strong evidence indicating bidirectional introgression between B. nana and B. pubescens, not only to the tetraploid species as previously believed, but also the opposite.
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Similar morphological variation was observed among the progeny of open-pollinated B. nana plants from Úthlid, in the same area as the woodland Brekkuskógur (Figure 2D). In this case, triploid progeny were more abundant than diploids due to the relative richness of B. pubescens pollen and the earliness of its anthesis in this area (Anamthawat-Jónsson and Tómasson 1999). But more importantly, the variation among the diploid and the triploid progeny was surprisingly high; the diploids had morphological scores of 1–7 and the triploids had scores of 2–9. None of the diploid plants had all the specific characteristics of B. nana. Most of the triploid plants had the morphology of B. pubescens, some were intermediates, but a few resembled B. nana. These results indicated that the mother plants were introgressed B. nana and the pollen pool most likely came from introgressed plants as well as fertile hybrids. The results supported bidirectional introgression in birch.
Molecular Genetic Experiments
Molecular evidence supporting introgressive hybridization in birch is also presented. The method of genomic hybridization (Anamthawat-Jónsson et al. 1990) was used to differentiate B. nana and B. pubescens from one another by means of detecting essentially species-specific DNA sequences. Total genomic DNA isolated from one of these two birch species was used as a labeled probe to hybridize to a Southern blot of restriction enzyme-digested genomic DNA of birch samples. Simultaneously, unlabeled total genomic DNA from the birch species not used as probe was applied to the blot to block common DNA sequences. Figure 1 shows the result of one such genomic hybridization experiment, whereby blot A received B. nana probe and B. pubescens block and blot B received the opposite. Three pairs of samples were analyzed: B. nana (lanes 3 and 4), B. pubescens (lanes 5 and 6), and artificial hybrids of these two species (lanes 1 and 2). All the DNA digests loaded in gel were of relatively equal amounts. In both blots, most hybridization signal came from B. pubescens and the hybrids, whereas B. nana showed positive signal on one lane (Icelandic sample, lane 4) but no signal on the other lane (southern Finnish sample, lane 3). The hybridization signal on these blots clearly was B. pubescens specific. The same results were obtained from many more Southern experiments conducted with a vast range of samples, and the amount of hybridization signal was found to be relatively quantitative to its proportion in the genome (unpublished results).
In blot A, where Icelandic B. nana DNA was used as probe, the B. pubescens blocking DNA competitively hybridized to common sequences in the probe and in the target lanes. The B. nana genome most likely had very little or no species-specific sequences, that is, the genome being highly conserved, all or most of its DNA was therefore blocked by sequences shared with B. pubescens. As a result, the probe had no labeled B. nana DNA to hybridize with the target lanes (due to the blocking having higher hybridization kinetics and hence being more effective). The "pure" B. nana target from southern Finland (lane 3) was therefore blank. On the contrary, the positive signal on the Icelandic B. nana target was apparently the result of hybridization of labeled B. pubescens-specific DNA that existed in the probe and in the target. This probe produced a strong hybridization signal on the B. pubescens and the hybrid lanes, which contained a significant amount of B. pubescens-specific sequences in their genomes. It can be concluded that (1) the probe, Icelandic B. nana from Brekkuskógur, contains B. pubescens DNA as a result of introgressive hybridization; (2) the Icelandic B. nana target (from the woodland Úthlid adjacent to Brekkuskógur) also has B. pubescens DNA in its genome; and (3) the southern Finnish B. nana seems to be relatively pure—at least this particular sample.
Blot B showed exactly the same result as blot A, although the probe and block combination was the opposite. The B. nana block competitively hybridized to its common sequences in both species and therefore all the hybridization signal detected was due mostly to B. pubescens-specific DNA. The distinctive ladder-like pattern characteristics of DraI restricted DNA (also seen in agarose gel; Anamthawat-Jónsson and Heslop-Harrison 1995) was part of the B. pubescens-specific DNA sequences. This highly repetitive 200 bp satellite DNA was observed in more than 100 B. pubescens samples collected so far, and thus could be useful in finding the origin of this birch.
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Discussion |
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It was proposed that extensive morphological variation of tree birch in Iceland, B. pubescens (tetraploid, 2n = 56) was the result of gene flow from dwarf birch (B. nana, diploid, 2n = 28) by introgressive hybridization between these two coexisting species (Gröntved 1942; Elkington 1968). It was later shown by our cytogenetic study (Anamthawat-Jónsson and Tómasson 1990) that such introgression could take place in nature the same way as with plants in controlled crosses, via backcrossing of the triploid interspecific hybrids. Although lacking chromosome data, introgressive hybridization between these two species has also been reported to have occurred in northern Scandinavia and Fennoscandia (Jetlund 1994; Kallio et al. 1983). But cytogenetic and molecular evidence for introgressive hybridization in birch species in nature is presented for the first time here. Furthermore, our results also reveal for the first time bidirectional gene flow between these two species, not only from the diploid to the tetraploid species but also the opposite.
We have identified triploid birch plants in woodland populations and these plants are morphologically similar to triploid hybrids obtained from crosses. Interspecific hybridization between B. nana and B. pubescens has undoubtedly occurred in nature. The proportion of triploid plants in the samples from these woodlands is 9% and 14% at Bifröst and Brekkuskógur, respectively. This is likely to consist of both the first generation (F1) interspecific hybrids and those derived from later generations of backcrossing. Based on our study of the material from crosses (Anamthawat-Jónsson and Tómasson 1990), triploid progeny could also originate from backcrossing of the F1 hybrids with either B. nana or B. pubescens parents. As the triploid birch does not always have intermediate morphology, especially at Brekkuskógur, this birch probably consists essentially of later-generation (introgressed) triploids. In addition, the F1 hybrids would not always have intermediate morphology simply because the parents are introgressed plants. The present study indeed finds no "pure" B. pubescens in these woodlands and very little "pure" B. nana at Brekkuskógur. Our molecular study confirms that B. nana plants at Brekkuskógur have B. pubescens DNA in their genome, evidently due to introgressive hybridization. In the northwestern part of Iceland, Elkington (1968) found that Icelandic birch plants were more hybrid-like compared to B. pubescens from England and B. nana from Scotland. A few plant samples from southern Finland have been used in a study related to the present investigation, and these samples, both B. nana and B. pubescens, are more molecularly and phenotypically pure compared to the Icelandic samples. In Iceland, however, nonintrogressed B. pubescens birch may be found in the eastern part of the country and pure B. nana is likely to exist in the central highland.
The cytogenetic data, when correlated with the morphology index, have established interspecific hybridization in nature and revealed introgression as the most likely consequence of hybridization and repeated backcrossing. According to Anderson (1949), introgressive hybridization (introgression) is genetic modification of one species by another through the intermediacy of hybrids. But to accept that introgressive hybridization has occurred and is the cause of genetic variation in natural populations, there must be evidence of the transfer of genetic material from one species into another (Heiser 1973). Out of 165 well-documented cases of introgression covering many plant species of all different growth form types, not more than 40% of these cases could be considered having strong evidence for introgression (Rieseberg and Wendel 1993). The difficulties have been in basing an analysis of introgression on morphological characters alone. Morphological characters typically have an unknown, but presumably complicated genetic basis, and a nonheritable component that is difficult to estimate. Introgression must therefore be studied using other tools as well, especially chemical, biochemical, genetic, and molecular methods. Molecular approaches, in particular, have been successfully applied (reviewed in Briggs and Walters 1997; Rieseberg et al. 2000), and the consensus is that species-specific markers are the most effective in probing introgression. Our molecular approach is indeed based on species-specific markers, because common sequences are blocked in a competitive hybridization condition. Using this method we are able to confirm the presence of introgression into the genome of birch species, at least from B. pubescens to B. nana. The introgression in this direction has not been reported before, although introgression of B. nana into B. pubescens has been investigated often (reviewed in Atkinson 1992). Together with the morphological and cytogenetic studies, it can therefore be concluded that introgression in Icelandic birch species is bidirectional and is the source of variation found in both birch species.
The method of genomic hybridization is simple, as no knowledge of DNA sequences is required and it is easy to use. It is also able to detect genomewide introgression, and therefore it should be possible to give an estimate of the extent of introgression and its localization in the genome by in situ hybridization. Examples of intergenomic or interspecific introgression in many plant species have been revealed by the genomic in situ hybridization (GISH) method, as well as by genomic Southern hybridization, as used in the present study (e.g., Heslop-Harrison et al. 1990; Pasakinskiene et al. 1998). As shown by the pattern and intensity of Southern hybridization signal in the present study, the overall amount of introgressed DNA is quite extensive and is likely to be composed of different classes of species-specific DNA sequences. Further work with birch is therefore needed to identify DNA sequences that are involved in the introgressive hybridization, its molecular mechanisms, and cytogenetic events that may or may not have influenced survival and adaptability of birch in Iceland.
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Acknowledgments |
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We are grateful for the financial support provided by the Icelandic Research Council, the University of Iceland and the University of Turku, Finland. We thank Thorsteinn Tómasson, Adalsteinn Sigurgeirsson, Vignir Sigurdsson, Irma Saloniemi, and Erkki Haukioja for valuable discussions on the subject of birch introgression.
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Footnotes |
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Corresponding Editor: James L. Hamrick
Received August 23, 2000
Accepted July 12, 2001
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References |
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