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Division of Science and Mathematics, University of Minnesota-Morris, Morris, Minnesota 56267 USA
Received for publication June 10, 1999. Accepted for publication April 11, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Aconitum • genetic diversity • isozyme • monkshood • Ranunculaceae • RAPD • rare plant
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Its recognized range is restricted to areas in Iowa, Wisconsin, Ohio, and New York that remained unglaciated during the late Pleistocene. However, the taxonomic status of North American Aconitum populations has historically been problematic, as is the case among other members of this genus worldwide (Brink, 1982
; Kadota, 1987
). Aconitum reclinatum Gray (Sect. Lycoctonum), occurring in the mountains of the eastern United States, can be easily separated as a distinct species because of its white rather than blue flowers, distinctive flower shape, unique pubescence characteristics, and rhizomatous or fasicled rather than tuberous roots. However, defining relationships among the blue-flowered species (Sect. Napellus), including the species in this study, has been more difficult. Aconitum noveboracense was named as a distinct species by Asa Gray based upon type specimens collected in Chenango County, New York. However, it was later determined by Hardin (1964)
to be a subspecies of another species of the eastern mountains, A. uncinatum L. In more recent analyses, Brink (1982)
and Brink and Woods (1997)
found A. noveboracense populations to contain a subset of the total morphological variation present in A. columbianum Nutt., a much more common species that occurs widely in mountainous areas of the western United States. After measuring such characters as nectary depth, flower height, hood height, width and breadth, pendent sepal length, leaf dissection, taste of tubers, and tuber morphology, they concluded that Midwestern ("northern") Aconitum populations are merely disjunct populations of A. columbianum and are undeserving of distinction as a separate species. Iltis (1965
; and unpublished data) had earlier proposed this interpretation, noting that during or at the end of the Pleistocene, the progenitor of A. noveboracense was probably common along the glacial margin. As the glaciers retreated, it was left in the only suitable habitat remaining: unique areas with cool, moist microenvironments; places that are ecologically equivalent to the montane and periglacial environments occupied by other Aconitum species (Epling and Lewis, 1952
; Kadota, 1987
). This distribution pattern is shared by numerous species in the northeastern United States (Iltis, 1965
). Brink (1980, 1982)
thoroughly reviews the literature on the morphology of the above three species, noting that leaf and flower characteristics are highly variable within a species. The taxonomic affinity of Aconitum populations in the Black Hills of South Dakota has been questioned relative to management decisions in the Black Hills National Forest. Because they are geographically located between the range of the common Rocky Mountain species and the rare midwestern species, these populations could plausibly be members of either of the recognized taxa. Because morphological characters cannot reliably differentiate A. noveboracense and A. columbianum, we undertook this study to test the utility of molecular markers for distinguishing the taxa and providing information about their levels of genetic variation.
Some information regarding isozyme variation in Aconitum was available from the work of Dixon and May (1990)
. They examined the genetic relationship of 38 populations of A. noveboracense from Iowa, Wisconsin, and New York and found differences among river drainages within a state and between New York and midwestern populations. Also, in their preliminary analysis (unpublished data) they included data on six individuals from a single Aconitum population in the Black Hills of Wyoming. These limited data showed a fair degree of genetic affinity between the Wyoming population and an A. noveboracense population from Wisconsin as well as one from New York. However, because no Rocky Mountain populations of Aconitum columbianum were used in the analysis and because only two scorable polymorphic loci were found, it is impossible to interpret the implications of their findings for the question at hand. We extended the isozyme analysis of Aconitum and also used randomly amplified polymorphic DNA (RAPD) markers (Welsh and McClelland, 1990
; Williams et al., 1990
) to characterize levels of genetic variation among and within these taxa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and from A. noveboracense populations in New York (two populations). We also included plants from a population of A. uncinatum in Ohio. Specific locality information is included in Table 1. In large populations, plants were selected arbitrarily along one or more transects through the population. One or two leaves were collected from each of 20–30 individuals per population and stored on ice in individually labeled plastic bags until they could be transferred to a -85°C freezer, where they were kept until processed. If available, seeds were collected from mature capsules on these same individuals and stored in individually labeled paper envelopes, at room temperature and humidity, until they were homogenized.
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), a buffer modified from Yamada and Guries (1990)
by C. F. Williams (personal communication, Idaho State University) for use with the closely related genus Delphinium and a buffer described by Morden, Doebley, and Schertz (1987)
, which we ultimately used for population screening. Five stains gave clear band resolution of seven loci. These loci are considered putative, because the difficulty of growing A. noveboracense in captivity precludes formal genetic testing; however, the isozyme banding patterns observed were consistent with patterns observed in other species (e.g., Weeden and Wendel, 1989
). For the large-scale population screening, a single seed, weighing 40–80 mg, was taken from each of 20 plants per population (with the exception of A. uncinatum, for which only five plants were available). Seed tissue had been shown to work well for isozyme analysis of the closely related genus, Delphinium (Williams and Waser, 1999
) and was used here in an attempt to prevent interference with isozyme resolution by alkaloids and other compounds present in extracts of leaf material. Each seed was ground in 100 mL of extraction buffer (Morden, Doebley, and Schertz, 1987
). The extracts were frozen in microfuge tubes at -85°C until use. Stains revealing scorable loci included phosphoglucomutase (PGM, E.C. [Enzyme Commission] 5.4.2.2) and phosphoglucoisomerase (PGI, E.C. 5.3.1.9) on H gels, alcohol dehydrogenase (ADH, E.C. 1.1.1.1), malate dehydrogenase (MDH, E.C. 1.1.1.37), and 6-phosphoglucose dehydrogenase (6-PGD, E.C. 1.1.1.44) on T gels, triose phosphate isomerase (TPI, E.C. 5.3.1.1) on L gels, and malic enzyme (ME, E.C. 1.1.1.40) on N gels. On the initial screening, TPI and ME were monomorphic, so they were not used in further testing. Enzymes that showed activity but were not scorable included aspartate amino transferase, esterase, isocitrate dehydrogenase, mannose phosphate isomerase, and shikimate dehydrogenase, which appeared to be polymorphic but were not consistently scorable. Stains showing little or no activity included catalase, cytochrome oxidase, diaphorase, glucose-6-phosphate dehydrogenase, peroxidase, and xanthine dehydrogenase. Gel formulas follow Weeden and Wendel (1989)
for the H gels and buffer (Morden, Doebley, and Schertz, 1987
) for the T gels. Stain formulas follow Cardy et al. (1983)
for ADH, Soltis et al. (1983)
for PGI and PGM, and Vallejos (1983)
for MDH and 6-PGD.
RAPDs
Because of the greater effort and expense of assaying genetic information for each individual using RAPDs than using isozymes, we conducted RAPD assays on leaf tissue from a subset of the same individuals used for the isozyme assays. From each of the three major taxa (A. noveboracense, A. columbianum, and the unknowns from the Black Hills) we chose three populations, distributed to represent the geographic range of each taxon. The A. columbianum populations were from Colorado, Idaho, and Wyoming; the A. noveboracense populations were from New York, Wisconsin, and Iowa; and the Black Hills populations were taken from the south, middle, and north portions of their range. Amplification of samples from the Iowa population (St. Olaf) of A. noveboracense was poor, so these samples were not included further in the analysis presented here. We also included four samples of A. uncinatum from Ohio. In each population the 20 individuals used for the isozyme analysis were divided into two groups of ten; from each of these groups, three individuals were chosen at random for RAPD analysis. This stratified, random subsampling was done to reduce the chance of the subset chosen for RAPD analysis representing a statistically different portion of any population. While the seven primers chosen produced an average of 13.8 reliable bands for each individual, the DNA from a few individuals did not successfully amplify, so we have excluded those samples from the analysis. Since leaf tissue was used for the RAPD analysis and seed tissue was used for the isozyme analysis, this represents maternal and offspring genomes, respectively. Thus, the two methods do not represent exactly identical samples of the genetic constitution of the populations.
DNA extraction used a modification of the procedure described by Stewart and Via (1993)
. We ground 0.03–0.05 g of green leaf tissue in a microfuge tube using a polypropylene homogenizer (Kontes, Vineland, New Jersey, USA) mounted on an electric motor. This procedure recovers 2–4 µg of DNA from a 0.04-g sample. Comparison of these DNA isolates with samples further purified through resin extraction (Gene Clean II, Bio 101, Vista, California, USA) showed no difference in RAPD banding profiles (results not shown), so we did not use that extra step in our population comparisons. DNA concentration for each sample was measured using a Dynaquant 200 fluorometer (Hoefer, San Francisco, California, USA), and TE (Tris-EDTA) was added to bring each sample to a concentration of 10 ng/µL. Polymerase chain reactions were assembled using aerosol barrier pipet tips while wearing latex gloves and working in a laminar-flow hood. Water used for the reactions was purified through a Milli-Q filter system (Millipore, Bedford, Massachusetts, USA) to eliminate any water-borne contaminating organisms. These precautions enabled us to eliminate contamination so that negative control reactions produced no bands. Optimized reactions consisted of 1 µg/µL BSA, 2mmol/L MgCl2, 0.1 mmol/L of each dNTP, 10 ng target DNA, 5–6 pmol primer, 0.625 units Taq polymerase, in a buffer of 10 mmol/L Tris-HCl, pH 9.0, 50 mmol/L KCl, 0.1% Triton X-100, making a total volume of 25.2 µL. The reaction mixture was heated to 95°C for 3 min to denature the target DNA, after which 0.625 units of Taq polymerase (Promega, Madison, Wisconsin, USA; in storage buffer "B") and an overlay of 40 µL of mineral oil were added, followed by 35 cycles in a thermocycler (Integrated Separation Systems, Natick, Massachusetts, USA) modified to ensure equal heating and cooling of all samples. The temperature profile for the reactions had 1 min of denaturation at 92°C, 1 min annealing at 36°C, and 1 min extension at 72°C. To minimize errors arising from any variation in reaction conditions, we conducted all reactions for a given primer simultaneously on a 96-well PCR plate (Polyfiltronics, Rockland, Massachusetts, USA). For one individual of A. columbianum and one individual of A. noveboracense we ran multiple RAPD reactions on each plate using the same primer. While these provided a check on inter-reaction variation, their main function was to provide reference standards for band scoring (discussed in more detail below).
Testing 40 decamers (Genosys Biosystems, The Woodland, Texas, USA) with GC contents of 60, 70, or 80% showed that only primers with a GC content of 70% produced amplification products. Of these, we selected seven primers that produced the most useful banding profiles, based on the number and clarity of bands produced. These primers and their sequences are listed in Table 2. Optimum electrophoretic resolution was obtained using 2% Metaphor agarose in 1X TAE run at 3.4 V/cm for 6.5 h; this allows resolution of bands differing by 1% or less (
0.5–1 mm in migration). Gels were stained with 0.5 mg/L ethidium bromide for 40 min, illuminated with UV light, and photographed on Polaroid 667 film using an orange filter. Band migration was measured to the nearest 0.01 mm using digital calipers, and sizes were estimated using a linear regression method adapted from Schaffer and Sederoff (1981)
. Molecular size standards used were a combination of PCR markers (Promega) and a Hind III digest of lambda DNA, providing useful markers from 150 to 2332 bp in size. For each primer, we also ran reaction products from a single A. noveboracense and a single A. columbianum on each gel to ensure gel-to-gel scoring consistency.
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). We obtained estimates of the percentage polymorphic loci (P), the number of alleles per locus (A), and per polymorphic locus (Ap), and the observed and expected heterozygosity (Ho and He, respectively). For each polymorphic locus in each population we calculated Wright's (1978)
F statistics; gene flow among the populations was estimated as the number of migrants per generation (Nm) from the FST statistic as Nm = (1/4A)[(1/FST) - 1], where A = [n/(n - 1)]2 and n is the number of populations (Crow and Aoki, 1984
). We also conducted a similar analysis exclusively on the Black Hills populations. The measure of similarity among populations we report here is Nei's (1978)
unbiased estimator of genetic similarity, because of its suitability for small population sizes, though other measures produced essentially the same topology of relationships (results not shown). Cluster analysis of these similarity values using the unweighted pair group method (UPGMA) (described by Nei [1987
], as well as other authors) visually summarizes the degree of similarity among the populations analyzed.
For RAPD data, the presence/absence scores for each band in each individual were transformed into a matrix of pairwise distance measures using the RAPDPLOT program by Black and Antolin (1995)
. This matrix uses the similarity measure S developed by Dice (1945)
and applied to molecular data by Nei and Li (1985)
: S = 2NAB/(NA + NB), where NA is the number of bands in individual A, NB is the number of bands in individual B, and NAB is the number of bands they have in common. This measure varies from 0 when the samples compared have no bands in common, to 1 when they are identical. The measure is appropriate for traits that segregate independently, as is common for RAPD loci (Williams et al., 1990
). Since RAPDs are commonly dominant markers (Williams et al., 1990
; Heun and Helentjaris, 1993
), it would be possible to also score the absence of any particular band as a trait (Sokal and Michener, 1958
; Apostol et al., 1993
), but this might lead to an overestimate of the degree of relatedness between two distantly related organisms, since different mutations could have led to the loss of that RAPD band. Hilu (1994)
presents an empirical example of how the differences in these scores affect the resulting cluster analysis; there are only trivial differences in the resulting topology of the dendrogram, though the branch lengths indicate slightly higher levels of similarity when the matching coefficient is used. We used Felsenstein's PHYLIP package to conduct a UPGMA analysis of the resulting similarity values, and to construct the resulting phenogram tree illustrating the levels of similarity of all 47 individuals included in the analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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200 km to the west of the Black Hills, form a distinct cluster grouped within the Black Hills populations. The next most similar group includes the A. noveboracense plants from Hay Valley, Wisconsin. These plants, together with the Black Hills and Bighorn plants, form a cluster whose sister group is composed of the Rocky Mountain populations of A. columbianum and the New York population (Peekamoose) of A. noveboracense. Finally, the Ohio samples of A. uncinatum form a group that, based on the RAPD data, are distinctly different than all of the A. columbianum, A. noveboracense, and Black Hills populations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Most probably, this arises from the limited embryonic development characteristic of Aconitum and many related Ranunculaceae (Baskin and Baskin, 1998
). While the dendrogram of genetic identity values based on the isozyme data (Fig. 2) shows several results that might be expected, based on biogeography or taxonomy, other results are more surprising. For instance, the New York A. noveboracense populations, which are from a small area of the Catskill Mountains, show a very high degree of similarity. However, clustering with them are plants previously classified as three different species; this assemblage then connects to the Black Hills populations, which do not form a single, distinct cluster. The Black Hills populations have a higher level of genetic diversity than the other populations. For instance, the weighted average of the percentage of polymorphic loci for the Black Hills populations is 73.3%, compared to only 33.7% for the remaining populations. Similarly, the mean heterozygosity of the Black Hills populations is substantially higher (weighted average = 0.344) than for the other populations (0.133). The population from the Bighorn Mountains clusters tightly with two of the Black Hills populations. This population also shows levels of polymorphism (42.9%) and heterozygosity (0.279) more characteristic of the Black Hills populations than of the other populations of A. columbianum. Taken as a whole, the isozyme data do not resolve taxa corresponding to A. noveboracense, A. columbianum, and A. uncinatum; nor do they identify the Black Hills populations as being most closely allied with any of these three taxa. Instead, the isozyme data indicate a very high degree of similarity among all of the populations studied. All three taxa have populations that are similar at the level of 0.98 or higher. Conversely, the highest level of genetic divergence and diversity is among populations found within the Black Hills.
The high levels of heterozygosity observed in the Black Hills populations are also reflected in Wright's FIS statistic. Analyzed separately from the other populations (Table 6), these populations show an excess of heterozygotes at four of the six polymorphic loci and a mean value of FIS = -0.251. From these data alone, there is no way to determine whether the excess of heterozygotes arises from selection against homozygote survival, barriers to inbreeding, chance effects of sampling, or other causes. Prior research on the breeding system of Aconitum (Pyke, 1978
; Whitson, Stanislav, and Watson, 1986
) demonstrated that these plants are characteristically outcrossers, which would at least partially account for this heterozygosity.
Despite the impression from the cluster diagram based on isozymes (Fig. 3) that the Black Hills populations are quite distinct from one another, and despite the unsuitable habitat isolating the ravines in which these plants are found, there is evidently a substantial level of gene flow among the populations. This is evident in Wright's FST statistic for the Black Hills populations (0.129); the corresponding level of gene flow is Nm = 3.8. This level of genetic differentiation is lower than is characteristic of other species with similar breeding system, dispersal, and life form (long-lived perennial) (Hamrick and Godt, 1990
), but the level of gene flow is in the range found for other species with similar characteristics (Hamrick, 1987
). The effects of this exchange of genetic material are also seen in the results of the RAPD analysis, discussed below.
Other studies using isozymes find mean genetic identities of conspecific populations are usually above 0.90, whereas the genetic identities of congeneric taxa are usually much lower (mean = 0.67) (Gottlieb, 1977
; Crawford, 1983, 1990
; Giannasi and Crawford, 1986). One exception to this rule is for recently diverged progenitor-derivative pairs of species. Gottlieb (1973)
predicted that in progenitor-derivative taxa (1) the pair would exhibit a high degree of genetic similarity (2) the progenitor would contain more genetic variation than the derivative, (3) the progenitor would possess a number of alleles not present in the derivative, and (4) the derivative would possess few or no unique alleles. These patterns have been found in other studies of progenitor/derivative plant taxa found along the border of the Wisconsin-era glaciers (e.g., Soltis, 1982
; Loveless and Hamrick, 1988; Pleasants and Wendel, 1989
), and this explanation fits the favored scenario for the origin of A. noveboracense populations. If populations are glacial relicts derived from the widespread A. columbianum, they probably originated <15,000 yr BP, during or after the height of the Wisconsin glacial maximum (Frest, 1986
). This would not allow much time for genetic differentiation of isozyme alleles to have occurred, especially because these are slow-growing, long-lived perennial plants (Kuchenreuther, 1996
). However, A. noveboracense and Aconitum populations from the Black Hills do not have merely a subsample of alleles present in A. columbianum, which would make it clear that they are derivatives of that progenitor species, but instead possess unique alleles not present in our sample populations of A. columbianum. Because our sample of A. columbianum does not cover the entire range of the species, it is probable that we missed some of the genetic variation present within it. Alleles unique to Black Hills and Midwestern populations could either have evolved since isolation or be the result of founder effect from a progenitor population that had a different set of alleles than those now present in the A. columbianum populations we sampled. Alternatively, they could be alleles that occur in A. columbianum and were missed in our sample of individuals.
RAPDs
We were able to resolve many more loci (97) using RAPDs than isozymes (7); of these, 89.7% are polymorphic. Still, these genetic markers have some drawbacks. For instance, unlike isozymes, RAPD loci that have been examined act as dominant markers (Williams et al., 1990
; Carlson et al., 1991
; Bucci and Menozzi, 1993
; Lu, Szmidt, and Wang, 1995
). Since the presence of an amplifiable piece of template DNA, whether as one or two copies per cell, produces a PCR product that shows up as a band on a gel, heterozygotes are usually indistinguishable from homozygotes. This reduces the accuracy of estimates of frequencies of genotypes or alleles. Other drawbacks include the time required for optimizing the reaction conditions, the potential for comigration of nonhomologous bands (Ellsworth, Rittenhouse, and Honeycutt, 1993
; Xu, Wilson, and Bakalinsky, 1995
), the presence of bands that are variable in their intensity (perhaps reflecting different copy numbers of certain sections of the genome), and the large number of bands produced by some primers. However, with a large number of loci among which to choose, those bands that are not reliably amplified or separated can be excluded from the analysis, retaining a large amount of information useful for addressing our genetic questions.
The UPGMA phenogram arising from RAPD analysis (Fig. 3) indicates the similarity among all of the individuals analyzed. While the individuals might have been scattered widely at the tips of the diagram's branches, instead they usually cluster tightly as discrete populations. We also see greater diversity among the Black Hills populations than among the other populations, and only here do some individuals from different locations cluster together. Also notable is that the plants from the Bighorn Mountains form a tight cluster included within the cluster of Black Hills populations. Aconitum columbianum populations from Idaho and Colorado both formed tight clusters, closely related to each other, and the A. uncinatum population from Ohio also formed a cluster, most distinct from the rest of the populations included in this analysis. This distinction had even been noticeable while scoring the gels: banding patterns from the A. uncinatum samples were readily distinguishable from the other samples. More broadly, the phenogram distinguishes a "midwestern" group that includes the Wisconsin, Black Hills, and Wyoming populations, which is distinct from populations farther east and west, as well as from A. uncinatum. However, the level of similarity among all of these samples is high, especially among the A. noveboracense, A. columbianum, and Black Hills populations.
RAPDs have provided information useful for conservation of rare (Gustafsson and Gustafsson, 1994
; Rosetto et al., 1995
; Smith and Pham, 1996
; Palacios and Gonzalez-Candelas, 1997
; Maki and Horie, 1999
) and commercial (Gillies et al., 1997
) species. Yet in such studies, intraspecific similarity values are often lower than the lowest values seen in Fig. 3, with the possible exception of the level at which the Ohio A. uncinatum population connects to the other populations. For example, intrapopulation similarities in a cluster analysis of Gliricidia sepium, a leguminous tree from Central America, range from 0.55 to 0.75 (Chalmers et al., 1992
), while analysis of a single population of G. sepium in Guatemala (Dawson et al., 1995
) found that subpopulations were similar at the 0.7–0.8 level. Stylosanthes guianensis, a pasture legume from northern South America and southern Central America, forms a species complex interpreted as different varieties by some authors and as different species by others. Kazan, Manners, and Cameron (1993)
conducted a UPGMA analysis of pairwise RAPD similarity data that showed two main groups, similar to each other at the 0.6 level, which they interpret as corresponding to distinct species while other accessions clustering at the 0.75 level have been interpreted previously as distinct species. Accessions that were more similar, clustering at the 0.8 level and higher, have been interpreted as different varieties within a subspecies. Asphodelus, a Mediterranean herb, has within-species similarities that cluster in the range of 0.7–0.9, while similarities between closely related species cluster at the 0.55–0.65 level (Diaz Infante and Aguinagalde, 1996
). Paradoxically, an analysis of several millets (Echinochloa; Hilu, 1994
), including both tetraploid and hexaploid species, found that different accessions of a species would be similar at the 0.6–0.7 level, but that accessions representing different species, even of different ploidy levels, were similar at the 0.85 level. Thus, while there is no automatic correspondence between taxonomic similarity and RAPD similarity, we see that the Black Hills populations, A. noveboracense, and A. columbianum all cluster together at levels (0.80 and higher) characteristic of within-species variation in these other taxa examined.
Comparison of results from isozymes and RAPDs
Both the isozyme and RAPD data sets indicate a high level of similarity among all populations, despite their origins from locations as far apart as Idaho, Colorado, and New York. They also indicate that plants of the Bighorn Mountains are as similar to those of the Black Hills as the Black Hills populations are to each other, and that the Black Hills populations harbor high levels of genetic diversity, compared to other populations included in this analysis.
Comparison of the isozyme and RAPD dendrograms (Figs. 2 and 3) also reveals several differences between them. Particularly striking is the position of A. uncinatum in each. While this taxon is essentially indistinguishable from populations of A. columbianum and A. noveboracense based upon isozyme data, the RAPD data identify it as the most distinctive of the populations studied. Alhough only a small number of individuals represent this species in either analysis, the much larger number of loci resolved by RAPDs should result in a more accurate picture of its relationship to the other populations. Another interpretation, not conflicting with the first, is that there is a stronger conservation of alleles at the isozyme loci, preventing differentiation of A. uncinatum from other taxa.
A second difference between isozyme and RAPD results is that the Black Hills populations generally share a high level of similarity when compared using RAPD data, whereas they do not cluster tightly in the isozyme analysis. Again, the much larger number of RAPD loci probably present a more accurate picture of the relationships among these populations. This interpretation is actually confirmed by the isozyme data themselves: although the isozyme dendrogram separates the Black Hills populations, the statistical analysis indicates relatively low levels of population differentiation (FST = 0.129) and correspondingly high levels of gene flow among the populations (Nm = 3.8), underscoring the high levels of genetic identity among the populations.
While the A. columbianum populations from Idaho and Colorado cluster together on the RAPD phenogram, they separate more broadly in the isozyme analysis. However, the more significant message, since it is supported by both isozyme and RAPD loci, is that the populations in this study do not neatly fall into eastern ("noveboracense") and western ("columbianum") groups; instead, these populations have a high degree of similarity, clustering together at the 0.80–0.85 level with RAPDS and at the 0.85 level or higher with isozymes.
Several recent studies compare isozyme and RAPD variation in plants (Crawford, Wiens, and Haines, 1991
; Brauner, Crawford, and Stuessy, 1992
; Liu and Furnier, 1993
; Isabel, Beaulieu, and Bousquet, 1995
; Peakall, Smouse, and Huff, 1995
; Szmidt, Wang, and Lu, 1996
; Ayres and Ryan, 1997, 1999
; Buso, Rangel, and Ferreira, 1998
; Cole, 1998
; Sydes and Peakall, 1998
) and identify several general patterns. RAPDs produce many more loci than do isozymes, and a higher proportion are polymorphic. In some cases, even individual genets can be identified with RAPDs, or by combining the two classes of markers, while this is not possible with isozymes alone (Ayres and Ryan, 1997, 1999
; Sydes and Peakall, 1998
). RAPDs can provide useful measures of genetic variation even in populations that are monomorphic for isozymes (Buso, Rangel, and Ferreira, 1998
; Cole, 1998
). Our study finds little genetic differentiation among any of the populations sampled, either for isozyme or RAPD loci. By itself, the isozyme data set would be too small to be able to draw conclusions confidently. However, our RAPD analysis (based upon a much larger data set) is generally concordant with our findings from isozyme analysis, especially in terms of the degree of genetic similarity of populations, adding credence to this interpretation.
Thus, while our initial goal was to assign the Black Hills Aconitum populations to either A. columbianum or A. noveboracense, by using a variety of molecular markers to provide more information than is available based on morphology and biogeography, we found instead that the genetic information complicates the problem. The additional information provided by a large number of RAPD loci and a small number of isozyme loci mirrors the lack of morphological differentiation found between these two named taxa. We find a high degree of similarity between the Black Hills Aconitum populations and those of the Bighorns, as well as a high level of similarity between those populations and the A. noveboracense of the Midwest and A. columbianum of the Rocky Mountains. Most fundamentally, we find a high level of similarity among all of these populations tested, with the possible exception of the plants classified as A. uncinatum. These findings corroborate recent taxonomic treatments that, based on morphological grounds, treat these Aconitum populations as a single species (e.g., Brink and Woods, 1997
).
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FOOTNOTES |
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2 Author for correspondence (colect{at}mrs.umn.edu)
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LITERATURE CITED |
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———. 1982 Tuberous Aconitum (Ranunculaceae) of the continental United States: morphological variation, taxonomy and disjunction. Bulletin of the Torrey Botanical Club 109: 13–23[CrossRef][ISI]
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