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(American Journal of Botany. 1998;85:1316-1323.)
© 1998 Botanical Society of America, Inc.

A mating system conundrum: hybridization in Apocynum (Apocynaceae)1

Samuel A. Johnson2,a, Leo P. Bruederle3,a and Diana F. Tombacka

a Department of Biology, Campus Box 171, University of Colorado at Denver, P.O. Box 173364, Denver, Colorado 80217-3364


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Based upon an intermediate morphology, Apocynum x-floribundum Greene has long been considered a hybrid involving A. androsaemifolium and A. cannabinum. The floral morphology in this genus, however, appears to prohibit both import and export of pollen, and observations of numerous insect visitors reveal that pollen is not routinely carried from flower to flower. Furthermore, reproductive success as measured by fruit set is very low in most populations. Hybridization was thus called into question, with allozyme evidence used to test the hypothesis of a hybrid origin for A. x-floribundum. Six diagnostic loci, as well as two loci exhibiting highly disparate allele frequencies, were resolved for each parent. All examined populations of A. x-floribundum were heterozygous at these loci, thus supporting the hypothesis of hybridization. Evidence from additional loci indicated that all populations tend to be strongly clonal. Observed heterozygosity was very low in the parental species, suggesting a history of inbreeding or a severe bottleneck. There was no support for earlier assumptions that some intermediates are derived from backcrosses or "secondary hybrids." Statistical analyses of plant height, leaf shape, petal length, sepal length, follicle length, seed length, and seed number per follicle supported these conclusions. The persistence and vegetative spread of hybrid clones may contribute to the illusion that hybridization is common.

Key Words: allozymes • Apocynaceae • Apocynum • hybridization • pollination • population genetics


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The taxonomy, phylogeny, pollination system, and ecological role of the strictly North American genus Apocynum (Apocynaceae) have long been in debate. In 1930 Woodson revised the genus, reducing over 80 described species to seven: A. pumilum (A. Gray) Greene, A. androsaemifolium L., A. medium Greene, A. jonesii Woodson, A. suksdorfii Greene, A. cannabinum L., and A. hypericifolium Ait. The remaining taxa were relegated to subspecific or hybrid rank. He assumed that most of his subspecies had arisen by hybridization, noting numerous intermediate morphological characters, a high degree of pollen sterility, and low to no fruit set. Furthermore, he concluded that all species were self-incompatible in view of the fact that "otherwise they would yield theoretically 100% fruit, since the anthers press so closely about the stigma that self-pollination is unavoidable." He failed to explain how cross-pollination, let alone hybridization, could arise in view of the curious floral structure in this genus (discussed later). Anderson (1936) showed that the offspring of putative parental species (A. cannabinum L. and A. androsaemifolium L.) were very similar to the parents, while some of the intermediates produced variable offspring. He regarded this as evidence of hybridization, but at that time neither Anderson nor Woodson had the genetic tools to test the hybridization hypothesis.

While Colorado's Front Range hosts many forms of Apocynum, contemporary field guides (e.g., Weber, 1990) distinguish only three "species:" A. cannabinum, A. androsaemifolium, and A. x-medium Greene, the last named being an intermediate, highly variable, putative hybrid taxon. Greene also described A. lividum (1901) and A. floribundum (1893) from among the same general group of intermediates. However, the characters that have been used to separate A. x-medium from A. floribundum and A. lividum (mainly extent and placement of pubescence) are considered inconsistent. Woodson (1930) considered both A. floribundum and A. lividum as varieties of A. x-medium. McGregor et al. (1986) also considered these names synonymous, but noted that the specific epithet A. floribundum has priority. Characters that separate these and other taxa were found to be unreliable; furthermore, forms that seem to be distinct are not consistently assignable outside of A. floribundum. Therefore, we follow the nomenclature of McGregor et al. (1986) and adopt the conventional "x-floribundum" to denote the assumption of hybrid origin. Apocynum sibiricum Jacq. is recorded from the Front Range also. It is supposedly distinguished from A. cannabinum by having sessile cordate-clasping leaves, smaller follicles, and subfoliaceous inflorescence bracts (Harrington, 1954), but we were unable to distinguish it. This taxon is regarded by McGregor et al. (1986) and Weber (1990) as synonymous with A. cannabinum.

Apocynum flowers appear to be ideally suited to selfing. A full, detailed description of floral morphology in the family can be found in Woodson (1930) and Cronquist (1981). The most striking feature of the flower is its morphology, which appears to prevent both the import and export of pollen. The androecium is connivent about the gynoecium, shielding it from contact with potential pollinators. The anthers dehisce introrsely and are connate by the edges, so that neither the receptive stigmatic surfaces of the stigmatic head nor the pollen is readily accessible to most flower visitors. In fact, pollen has been reported to shed onto the surface of its stigmatal head (Woodson, 1930; Rosatti, 1989), below which there is a mucus ring that adheres the anthers onto the style thereby catching any pollen that may fall. Anthers never splay upon maturation of the flower, but remain adherent to the pistil until the corolla falls from the fertilized ovary. This presents a troubling conundrum: how can such a floral structure produce hybrids? And the obvious question arises: are the intermediates really hybrids?

Apocynum is apparently a diploid genus with n = 11 (Darlington and Wylie, 1956). It is known to reproduce asexually by rhizomes (Woodson, 1930). Dates of flowering and fruit set vary widely, probably as a function of temperature and rainfall (S. A. Johnson, personal observation). In the Front Range of Colorado A. x-floribundum is normally the first to bloom, with flowers first opening during the middle of June (but as early as 18 May) and all species in full flower by the end of June. Flowering may continue into mid-September, but usually finishes by the first week of August. In dry years, populations of this genus appear to be the leading producers of nectar in some areas during late June and July. Some extraordinarily dense populations of A. x-floribundum seem to act as ecological keystones, attracting huge numbers of insect visitors when flowers of other species are few and scattered.

Although the putative parental species are broadly sympatric along the Front Range, populations are separated by habitat preference. Apocynum cannabinum occurs on prairie river floodplains, terraces, and roadside ditches, usually in fine sandy or loamy soils. Apocynum androsaemifolium inhabits valleys and slopes in the transition zone, usually on gravel soils in the partial shade of oaks or pines. Apocynum x-floribundum inhabits roadsides and streambeds, usually on sandy soils. Although A. x-floribundum can occasionally be found in proximity to the other species, the two putative parental species were never found within a half kilometre of each other, and are usually much more distant.

For the above reasons we questioned the hybrid hypothesis. A polytypic genus of highly inbred species might respond to a selection gradient (from the prairie to the higher foothills) in the same way. The fact that intermediate morphs are found geographically between the extreme morphs could be explained in either way—as hybrids thriving in marginal habitats or as several species adapted specifically to a selection gradient. We addressed the hybrid hypothesis by analyzing morphological and allozyme data from parental and putative hybrid populations. The objectives of this study were to determine whether A. x-floribundum is a hybrid of A. cannabinum and A. androsaemifolium, or whether it is a related species of more distant origin. If it is a hybrid, is there evidence for introgression? Population dynamics of these taxa are also described.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study populations
We located over 60 populations of this genus between the towns of Fountain, Colorado, to the south and Boulder, Colorado, to the north. Of these, 11 from the southern portion of this range were selected for study (Table 1). Three stands (c1, c2, and c3) were clearly assignable to A. cannabinum [according to Woodson (1930) and subsequent keys (Rydberg, 1932; McGregor et al., 1986; Weber, 1990)]; three stands (a1, a2, and a3) were clearly assignable to A. androsaemifolium according to the same authorities; and five stands were selected to illustrate a wide range of A. x-floribundum variants (x1, x2, x3, x4, and x5).


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  		Table 1. Size, location, elevation, and associated species for each of 11 Colorado populations of Apocynum.

 
Reproductive biology
Exclusionary cages were made to enclose 0.44-m plots of plants or ramets to study the efficiency of pollinators. Three replicates each of diurnally available, nocturnally available, never available, and always available plots were caged or, in the cases of always available controls, marked with string. Each morning and evening the cages enclosing the diurnally and nocturnally available plots were switched to exclude the unwanted pollinator sets. This experiment was only done in the largest and densest patch of flowering Apocynum found, population x1. Reproductive success was measured in numbers of fruit set in each plot. In order to quantify reproductive success, records were made of fruit set as a percentage of the estimated total numbers of flowers in a given population. Numbers of flowers were estimated as: total flowers = flowers per ramet x ramets per square metre x population area in square metre. The actual (or estimated) total number of developed or developing follicles found was divided by this number to derive percentage fruit set. Seed viability was verified by germination of seeds in petri dishes with damp filter papers. Seeds were taken from representative populations of each taxon. In each population tested, follicles were collected from numerous ramets, and seed samples were amassed by taking three or four seeds from each follicle.

Field studies also included observation, collection, and identification of flower visitors; examination of visitors for pollen load assessment; and observation of insect behavior on inflorescences. Insects were collected between 0800 and 2300 in June and July from 1993 through 1996. All specimens were taken by aerial net or killing jar directly from flowers. All flower-visiting insects were examined using a stereoscope, and pollen that was suggestive of Apocynum was examined using a compound light microscope or scanning electron microscope. A few ramets of A. androsaemifolium and A. x-floribundum were dug up to determine the depth of the connecting rhizomes; clonal growth was verified in this way.

Morphology
In order to compare morphological evidence with allozyme evidence (see below), both reproductive and vegetative characters were measured. Characters chosen were petal length, ratio of petal length to sepal length, follicle length, number of seeds per follicle, seed length, plant height, and ratio of leaf length to leaf width. The first three of these and plant height have been used in taxonomic keys as diagnostic features of the taxa. The remainder are measures that appeared to vary greatly among populations, but which have apparently not been reported. Data were collected from each population along haphazardly placed transects that criss-crossed stands, so that all extremes of each population were represented. This was done to increase the probability of obtaining different individuals rather than clonal ramets. For vegetative characters, sample sizes were >=20. Several reproductive characters were represented by smaller samples due to low fruit set in some populations.

Morphological data were analyzed by one-way ANOVA whenever the data were normally distributed, followed by Tukey's tests. Ratios (petal/sepal length and leaf length/width) were arcsine transformed. In the case of numbers of seeds per follicle, data were not normally distributed and could not be improved by transformations. These data were analyzed with the nonparametric Kruskal-Wallis test. All statistical analyses were performed using StatMost (version 2.50, DataMost, Salt Lake City, Utah).

Population genetics
Allozyme data were obtained for the 11 populations described above using horizontal starch gel electrophoresis coupled with substrate-specific staining. Twenty-four samples of mature leaf tissue were collected from each of the populations of A. cannabinum and A. androsaemifolium (except c3, for which N = 20), with ten samples from each population of A. x-floribundum. All samples were again collected along haphazard transects that criss-crossed the populations. This increased the probability of obtaining different individuals rather than clonal ramets. Soluble enzymatic proteins were extracted by grinding mature leaves in a 0.1 mol/L Tris-HCl buffer at pH 7.5, modified from Werth (1985) to 10% polyvinylpyrrolidone (PVP-40) and 1% 2-mercaptoethanol. Extracts were adsorbed onto Whatman Number 17 chromatography paper wicks (3 x 13 mm) and stored at -70°C until electrophoresis.

The gel (10.5% starch) and electrode buffer systems used in this electrophoretic study were morpholine citrate, pH 6.1 (Clayton and Tretiak, 1972) and lithium borate, gel buffer pH 8.5/electrode buffer pH 8.1 (Cheliak and Pitel, 1984). Following electrophoresis, morpholine citrate gels were stained for PGI-1, PGI-2 (phosphoglucoisomerase); PGM-1, PGM-2 (phosphoglucomutase); MNR-1, MNR-2 (menadione reductase); MDH-3 (malate dehydrogenase); PGD-1, PGD-2 (phosphogluconate dehydrogenase); and SDH (shikimate dehydrogenase). Lithium borate gels were stained for PGI-1, PGI-2; TPI-1, TPI-2 (triose-phosphate isomerase); SOD (superoxide dismutase); AAT (aspartate aminotransferase); and ADH (alcohol dehydrogenase). Staining involved minor modifications of protocols summarized in Vallejos (1983) and Soltis et al. (1983). Data were collected as individual genotypes based on relative mobility of enzymes. Genotypic deviations from Hardy-Weinberg expectations were calculated using a chi-square test to compare expected and observed frequencies of all classes of homozygotes and heterozygotes. Descriptive statistics were calculated for the number of alleles per locus (A), number of alleles per polymorphic locus (Ap), percentage loci polymorphic (P), observed (Ho) and expected heterozygosity (He), and clonality (G). The latter was derived by preparing a ratio of unique genotypes for each species to the total number of individuals sampled (Pleasants and Wendel, 1989). Values for clonality range to a maximum of one for those populations in which each individual comprises a unique genotype.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reproductive biology
Caged treatments in population x1 all failed to set fruit, as did the population as a whole. In fact, most populations of the putative hybrid A. x-floribundum failed to set fruit from 1993 through 1996. Follicles were found in small numbers in populations of the putative parental taxa. Percentage fruit set in all populations studied ranged between zero and 0.017% (Table 2). The highest success that we observed (18%) was in an A. androsaemifolium population in lower Bear Creek. Germination experiments revealed that seeds from all taxa are viable (Table 2), the rates ranging from 20 to 94%, with the two A. x-floribundum populations exhibiting the poorest germination.


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  		Table 2. Estimated percentage fruit set and germination for Apocynum populations (1995). N = number of seeds tested for each population.

 
Extensive collections of Apocynum-visiting insects by Robertson (1928), Wilson, Moffett, and Harrington (1958), and the present study indicate that little or no pollen is carried by most floral visitors. Robertson noted pollen on various appendages of visitors, but apparently did not microscopically analyze it to prove that it was from Apocynum. The persistent tetrads of spheroidal Apocynum pollen grains are distinctive. No such pollen was found in our examination of 391 insect visitors representing ~176 species. Many visitors, especially bees and some beetles, carried abundant pollen from other plants. Apocynum x-floribundum was by far the most heavily visited of the three taxa, in terms of both numbers and diversity. We recorded 163 species from this taxon alone, mostly from a single population. Apocynum androsaemifolium also attracted a diverse community, ~71 recorded species, but in smaller numbers, while A. cannabinum attracted a very large number of insects representing only ~25 species.

Close observation of bees representing many families of Hymenoptera and flies representing several families of Diptera indicated that bees and flies were nectar feeders on Apocynum flowers and seemed unconcerned with access to pollen. Butterflies perched on the cymes and directed their proboscises along the inner walls of the petals. They appeared to be nectar thieves. Several predaceous insect species visited flowering Apocynum patches in search of prey, but their incidental contact with anthers did not appear to bring them into contact with pollen.

Woodson (1930) discussed an hypothesis that flies and bees that become trapped by the sagittate bases of the anthers might contact pollen as they struggle to escape. If they did escape, this pollen might be transferred to other flowers. Woodson offered no evidence in support of this hypothesis. Several examples were found of insects that had died after being trapped thus in flowers (S. A. Johnson, personal observation). However, there is no pollen available at the bases of anthers, and the trapped insects that were found (all bees and flies) did not appear to carry pollen. The scarcity of Apocynum pollen on the insects examined indicates that these plants are not mutualists, but "keystone prey."

Morphology
Five of the seven morphological characters studied provide support for a hybrid origin of A. x-floribundum (Tables 3–5GoGo, Fig. 1). Only leaf length:width ratio was not useful in separating the taxa, or was not suggestive of hybridization. One vegetative character studied, plant height, was significantly different among taxa, with A. x-floribundum intermediate (Fig. 1a). All of the reproductive characters differed significantly among taxa. The petal length:sepal length ratio, long used to identify Apocynum taxa in the field, is a demonstrably good diagnostic character. Tukey's test indicated that all three taxa are separable in this way, A. x-floribundum being intermediate. Petal length taken alone was an even better diagnostic character, the differences between taxa being highly significant, with A. x-floribundum intermediate to the putative parental species (Fig. 1b).


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  		Table 3. Mean, standard deviation, and range values for morphological measures for three Apocynum species.

 

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  		Table 4. ANOVA and Tukey's test results from analysis of morphological characters among Apocynum cannabinum, A. x-floribundum, and A. androsaemifolium.

 

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  		Table 5. (A) Kruskal-Wallis test results for seed number per follicle in Apocynum species, where H (test value) = 65.9, df = 2, and P = 0.000. (B) Box-and-whisker plots follow.

 


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  		Fig. 1. Variation for three morphological characters in 11 Apocynum populations. Bars represent the mean (white line), the mean ±1SD (black bar), and the range (gray bar). ( a.) Follicle length. (b.) Petal length. (c.) Plant height.

 
The putative parent species have large follicles compared to the fruits of A. x-floribundum (Fig. 1c). Seed numbers and size are also distinctive, with A. androsaemifolium producing huge numbers of small seeds compared with A. cannabinum's smaller number of large seeds. Apocynum x-floribundum produces a still smaller number of small seeds. The Kruskal-Wallis test revealed significant differences in seed number (P = 0.000, Table 5); ANOVA's and Tukey's tests indicated that seed length is also statistically different except between A. androsaemifolium and A. x-floribundum (Table 4).

Population genetics
Three loci (Pgi-1, Mnr-2, and 6Pgd-1) were monomorphic among the parental and hybrid populations and therefore uninformative. Whereas six loci (Pgm-2, Mdh-3, Sdh, 6Pgd-2, Tpi-1, and Aat) were fixed for different alleles in the parent species, two loci (Pgi-2 and Sod-1) exhibited highly disparate allele frequencies (Tables 6, 7).


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  		Table 6. Allozyme data for 11 Apocynum populations, where A = number of alleles per locus, A p = number of alleles per polymorphic locus, P = polymorphism, H o = observed heterozygosity, H e = expected heterozygosity, and G = clonality.

 

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  		Table 7. Allele frequencies for diagnostic and supportive loci for Apocynum populations. Deviations from Hardy-Weinberg with significant {chi} scores are footnoted.

 
The A. cannabinum populations all had very low levels of heterozygosity (Hobs = 0.043) and polymorphism (P = 20%). The species mean number of alleles per locus (A) was 1.19, with no more than two alleles found at any polymorphic locus. The largest stand, Monument Creek (c2), exhibited no variation at 11 loci, with one locus (Pgm-2) having five out of 24 sampled ramets heterozygous; the same five ramets were the only homozygotes at another locus (Sod). These loci are in Hardy-Weinberg equilibrium. However, because only two genotypes were represented and because the five distinctive ramets were adjacent to one another along the transect, it is probable that the sample represented only two genets. The second largest stand, Fountain Creek Regional Park (c1), was monomorphic for all loci resolved. The Fountain Creek at Nevada Avenue population (c3) had an Hobs = 0.08 and P = 17%. Of the two polymorphic loci, only Pgm-2 was out of Hardy-Weinberg equilibrium ({chi} = 16.3, P < 0.001), with an overabundance of heterozygotes and the complete absence of one homozygote class (aa).

Apocynum androsaemifolium showed similar trends, with species mean Hobs = 0.065, P = 31%, and A = 1.44. The Larkspur population (a1) had three polymorphic loci (Pgi-1, Sod-1, and Tpi-2). In the South Cheyenne Canyon population (a3), the only polymorphic locus (Sdh) was heterozygous for all 24 individuals, and therefore grossly out of Hardy-Weinberg equilibrium ({chi} = 24.0, P < 0.001), again suggesting that the population is a single genet. The Bear Creek population (a2) had heterozygotes at two loci (Pgi-2 and Sdh). These loci were in Hardy-Weinberg equilibrium, but as in all previous cases the heterozygotes were adjacent ramets on the transect.

The A. x-floribundum populations, in spite of their great morphological diversity, were very similar genetically. Relative to A. androsaemifolium and A. cannabinum, heterozygosity was elevated in all populations (Hobs = 0.657) and polymorphic loci accounted for 63% of the loci studied. Number of alleles per locus averaged 1.69, considerably higher than the other populations. In A. x-floribundum, four of the loci were entirely heterozygous for alleles for which A. cannabinum and A. androsaemifolium were diagnostically fixed (i.e., Mdh-3, Pgd-2, Tpi-1, and Aat); the only exception was the appearance of a "c" allele at Mdh-3 in population x5. Additionally, four supportive loci (Pgi-2, Pgm-2, Sdh, and Sod-1), at which allele frequencies were highly disparate between the parental taxa, were also 100% heterozygous for the alleles most common in A. cannabinum and A. androsaemifolium. At every locus at which A. x-floribundum populations were heterozygous, there were significant deviations from Hardy-Weinberg equilibrium ({chi} = 10.0, P < 0.01).

As suggested by the aforementioned data, populations of all three species exhibited high levels of clonality (G), that is, few unique genotypes. Species means ranged from 0.060 for A. x-floribundum to 0.097 for A. androsaemifolium with most population samples comprising only one or two genotypes (Table 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Speciation by hybridization is well documented in plants (Abbot, 1992; Arnold, 1992; Rieseberg, 1995), although there have been many misconceptions about the hybrids that arise from these events. Rieseberg (1995) noted that hybridization does not always result in intermediate morphological characters. In a study of hybridization in Asclepias, Wyatt and Broyles (1992) showed that morphological analysis of reproductive characters alone did not detect hybrids, but that vegetative characters and isozyme evidence could easily identify hybrids. Molecular evidence was far more reliable in detection of hybrids. The present study indicates that both morphological and molecular evidence are strongly indicative of hybridization in Apocynum.

Apocynum cannabinum and A. androsaemifolium are fixed for alternate alleles at six loci for which A. x-floribundum is heterozygous. Allozyme data leave no doubt that the A. x-floribundum populations are hybrids of A. androsaemifolium and A. cannabinum. Even if the six parental populations are interpreted as comprising ten clonal individuals, which is entirely possible given these data, it is highly unlikely that all of the intermediate "individuals" would be almost exclusively heterozygous, while the other ten "individuals" would be homozygous. Furthermore, all of the intermediates are probably F1 hybrids. Homozygotes at Pgi-2 ("dd") and Sod-1 ("bb") in some A. x-floribundum populations could result from backcrossing with parent species. However, these genotypes could just as easily result from F1 hybridization—Pgi-2d and Sod-1b are present in both of the parent species, albeit at different frequencies. Thus, there is no compelling evidence for Woodson's (1930) hypothesis of "secondary hybridization." The remaining four of his seven specific taxa should be similarly analyzed.

Analyses of morphological data also strongly support these conclusions. Measures of follicle length, seed number, and seed length are consistent with partial hybrid sterility in A. x-floribundum. Petal length, petal:sepal length ratios, and plant height all place A. x-floribundum as an intermediate taxon. In general, reproductive characters provide more definitive evidence than do vegetative characters, although plant height also clearly places A. x-floribundum as an intermediate taxon. Characters used in field guides and floras (petal:sepal ratios and plant height) appear to be reliable characters for field determinations. Petal length taken alone, however, may be an even better indicator.

Allozyme evidence suggests that clonal growth is common. As Cook (1983) pointed out, cloning should be regarded as growth rather than reproduction. None of the populations examined had more than four genotypes (G = 0.17) among the samples, and seven of the 11 populations revealed only one (0.04 < G < 0.09). This is consistent with sampling ramets of large genets. The fact that all variants found in these populations lay adjacent to one another along their respective transects is further evidence for clonality. That vegetative reproduction is operative here is most strongly indicated by the fact that heterozygosity, when encountered [for example, Sdh in South Cheyenne Canyon (a3) A. androsaemifolium, Pgm-2 in Upper Fountain Creek (c3) A. cannabinum, and several loci in all of the A. x-floribundum populations], occurred at or near 100%, and sometimes at more than one locus. The extremely high {chi} values associated with long series of heterozygotes verify beyond a doubt that all of these populations consist of one or a very few large genets.

That Apocynum spreads by cloning is well known. Excavating two or more adjacent shoots from populations of all three species revealed that they were connected underground. The connecting rhizomes lay 15–32 cm beneath the surface, although on steep, unstable gravel scree they may be as shallow as 10 cm. Even in populations such as a3, in which sexual reproduction was demonstrably successful and the resultant seeds were viable (Table 2), the population was composed of genetically identical individuals for the loci studied.

Clonality alone, however, cannot account for the extremely low heterozygosity found in the parental species. The fact that so many loci are fixed for the same alleles in these populations suggests a history of inbreeding. Selfing, however, appears unlikely. In fact, support for Woodson's (1930) assertion that this genus is self-incompatible has been provided by S. Lipow (personal communication) at the University of Georgia. In a sample of 123 hand-pollinated selfed A. cannabinum flowers, not a single fruit was set. In contrast, cross-pollinated flowers set fruit, albeit at a low rate, i.e., 10.6%. A severe bottleneck somewhere in the history of these species could also explain the low levels of genetic variation observed in these populations. Because populations separated by many kilometres are fixed for the same alleles, random genetic drift is an unlikely cause of this effect. Overdominance selection could account for the occasional cases of excess heterozygotes, but the total eradication of both homozygotes is a less parsimonious interpretation than the cloning of a highly inbred species. The fact that populations are composed of one or a very few clones, and that populations are scattered, indicates that effective population size may be very small in any given region. Inbreeding would be an inevitable consequence.

Wright's F values are not presented because they are misleading in samples taken from a single genet. When a locus is 100% heterozygous (i.e., where p = q = 0.5), F equals -1, indicating obligate outcrossing. But a single genet with a heterozygous locus will also produce an F value of -1 at that locus. Due to the clonal nature of these species and the fact that the inflorescences of ramets tend to flower simultaneously, each clone might be interpreted as a single enormous inflorescence. Nakamura, Stanton, and Mazer (1989) hypothesized that very large inflorescences tend to reduce male reproductive success due to a high likelihood of self-pollination and that selection might therefore be expected to favor a reduction in the size of the inflorescence to enhance outcrossing. If this is so, clonal growth in Apocynum may be overpowering selection forces that would reduce inflorescence size. It is also possible that the floral structure, which severely limits pollen accessibility, is a response to specialized pollinators that fly long distances to disseminate pollen. This, too, would enhance male reproductive success.

It is apparent that the Apocynum species studied herein represent two easily characterized parent species and a series of F1 hybrid populations that are genetically very similar. The extremely low fruit set in these hybrids, lower even than in the parent species, may be attributed to partial hybrid sterility. The rarity of backcrossed individuals is probably an artifact of this sterility, allotopy (occupation of different macrohabitats) among species, and the apparent rarity of hybridization events. It is interesting that seeds that are occasionally produced by some hybrids show some viability (Table 2).

The problem of pollen vectors remains an inscrutable mystery, wherein must lie the answer to the mating conundrum in Apocynum. Vectors of pollen between A. androsaemifolium and A. cannabinum would have to be highly motile and move great distances. Collections and observations of floral visitors (S. A. Johnson, unpublished data), while not exhaustive, have thus far only identified seven species (out of the 176 recognized) on both parent Apocynum species. These seven were honey bees (Apis melifera), an halictid bee (Lasioglossum sp.), four species of skipper butterflies (Epargyreus sp., Polites sp., Oarisma sp., and Piruna sp.), and the common European cabbage white butterfly (Pieris rapae). Of these, only honey bees and cabbage whites are ubiquitous and wide-ranging. Most halictid bees are social to some degree, and are known to feed on nectar and pollen (Wilson, 1971). But their tiny size suggests that they do not travel as far as honey bees in foraging. According to Scott (1986), Epargyreus and Polites are quasiterritorial, the males adopting perches from which to spot and pursue females. These would tend to be localized species. By contrast, Oarisma and Piruna males are patrollers, possibly prone to moving greater distances along paths or in swales. Apocynum pollen was not observed on any of the seven insects. Nevertheless, to offset inbreeding among the few clones, pollinators would have to be able to access individual plants across areas spanning many kilometres at least. While it is possible that the highly apparent white flowers are pollinated by moths, the moth community is poorly represented in collections due to collector bias in favor of diurnal species. However, nocturnal observations of three of the 33 species of moths found visiting hybrid Apocynum flowers (Autographa californica, Neleucania sp., and Crambus lecheallus) revealed nothing differing from the habits of diurnal visitors (S. A. Johnson, personal observation). Furthermore, no moth species were found with Apocynum pollen.

There is a much greater overlap in the floral visitor communities between hybrids and parental species than between the two parental species (24 insect species were common to A. x-floribundum and A. androsaemifolium, and 18 were common to A. x-floribundum and A. cannabinum). However, if cross-pollination was occurring, backcrossed populations would be expected.

Anderson's (1936) assertion that hybridization is common was apparently based on the abundance of hybrid plants in collections. This assumption must be questioned. The present research indicates that hybridization might be rare, but that the viability of hybrids is very good. There is evidence that once a hybrid becomes established, it may persist for many decades and grow into large, discontinuous stands, creating the illusion of multiple hybridization events. The A. x-floribundum population in Palmer Park (x3) spans a large, improved gravel road that was built several decades ago, as well as a heavily eroded streambed that is periodically scoured by flash flooding. This population, which appears to be genetically homogeneous, must predate both the road and the deep streambed. The illusion of frequency of hybrid events would be further enhanced if hybrid progeny from a single outcross produced phenotypic variants as they crept across environmental gradients. Broyles, Vail, and Sherman-Broyles (1996) demonstrated that F1 hybridization in milkweeds may be much rarer than it appears, even in taxa in which hybrids are readily diagnosed. They found that in one site where Asclepias exaltata L. and A. syriaca L. were sympatric, only ~0.4% of the stems represented hybrids. They also found that fruits that contained hybrid seeds had significantly depressed seed set, a result similar to our findings in Apocynum (Table 2).

It is not surprising that over 80 species were originally described in this genus. Each hybridization event from the seven currently recognized species may produce a unique genotype that grows clonally and presents itself as a distinct taxonomic entity. Despite the curious floral morphology and the scarcity of pollen on potential pollinators, morphology and allozymes together show that A. x-floribundum is a result of hybridization between A. cannabinum and A. androsaemifolium.


   FOOTNOTES
 
1 The authors thank Sara E. Hill for assistance in the laboratory and Dr. James Koehler for advice regarding statistical analyses. This paper represents a portion of a thesis for the Master of Arts degree submitted to the Department of Biology at the University of Colorado at Denver. Funding for portions of this research was provided from The Colorado Springs School Parents' Association (SAJ) and the Buechner-Herbst Opportunity Fund (LPB). Back

2 Present address: The Colorado Springs School, 21 Broadmoor Ave., Colorado Springs, CO 80904. Back

3 Author for correspondence. Back


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