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Initial investigation into the reproductive biology of the antelope-horn milkweed, asclepias viridis walter (asclepiadaceae).

ABSTRACT--A large population of antelope-horn milkweed, Asclepias viridis Walter, (ASCLEPIADACEAE) was studied during the growing seasons of 1997 and 1998. Hand-pollination experiments suggested that this species exhibits at least some level of self compatibility. Mature fruit set was approximately 1.5% (95% confidence interval 1.4-1.7%). The average number of seeds per follicle was estimated at 72.2 (95% confidence interval 66.4-78.0). Both values are comparable to those determined by other milkweed studies. Examination of excavated plants and detection of genetic markers via Random Amplified Polymorphic DNA (RAPD) technology suggested that this species is not clonal.

Key words: Asclepiadaceae, Asclepias, plant reproductive biology, pollination biology.

INTRODUCTION

Members of the genus Asclepias L. (ASCLEPIADACEAE) package pollen into hardened aggregates termed "pollinia." Individual flowers bear five pairs of pollinia; each pair is connected by translator arms to a corpusculum. Pollination is effected when a pollinium is inserted into one of five stigmatic chambers by an insect vector (Queller 1983, Morse 1987, Wyatt and Broyles 1994). Three adjacent stigmatic chambers provide growing pollen tubes access to one ovary; the remaining two stigmatic chambers provide pollen tube access to the second ovary (Sage et al. 1990).

Considered a derived pollination mechanism, fruit set in the genus is low and typically averages less than 5% (Moore 1947; Woodson 1954; Willson and Rathcke 1974; Wyatt 1976, 1980, 1981; Lynch 1977; Willson and Price 1980; Chaplin and Walker 1982; Stephenson 1981; Queller 1985; Shannon and Wyatt 1986; Wyatt and Shannon 1986; Wyatt and Broyles 1990, 1994; Wyatt et al. 1996). Asclepias pollinators are typically generalists and include bees, wasps, moths and butterflies (Willson and Rathke 1974, Lynch 1977, Willson and Bertin 1979, Willson et al. 1979, Bertin and Willson 1980, Chaplin and Walker 1982, Morse 1982, Kephart 1983, Morse and Fritz 1983, Morse 1986, Southwick 1983, Shannon and Wyatt 1986, Wyatt and Shannon 1986, Broyles and Wyatt 1991, Jennersten and Morse 1991, Wyatt and Broyles 1994, Fishbein and Venable 1996). Gene flow within and between populations may be extensive due to generalist pollinators, long-distance pollen dispersal and wind-dispersed comose seeds (Broyles and Wyatt 1991, Broyles and Wyatt 1993, Broyles et al. 1994, Wyatt and Broyles 1994).

Over the past half century, numerous studies have addressed various aspects of milkweed reproductive biology. Unique among dicots, the milkweed's pollination system is an important reproductive characteristic that has facilitated these studies. Specifically, results of pollinator activity can be directly quantified, pollen units carried by pollinators can be counted, seed numbers are consistently high, and all seeds in a particular fruit share the same father (Wyatt and Broyles 1994).

Low fruit set is another prominent characteristic of milkweed reproduction. Although numerous follicles may be set initially, very few develop and produce mature seed. This high level of follicle abortion appears to be due to an unusual form of late-acting self-incompatibility (Kahn and Morse 1991, Wyatt and Broyles 1994, Wyatt 1996). Investigators have focused on two hypotheses to explain this low level of fruit set: (1) effective levels of pollination are limiting and (2) that resources for fruit and seed are limiting (Wyatt and Broyles 1990).

Proponents of effective pollination limitation argue that early fruit abortion is due largely to insufficient numbers of compatible pollinia for species that are mostly self incompatible (Wyatt and Broyles 1994). However, this hypothesis may not be valid for those species of milkweed that are mostly or wholly self compatible. Proponents of resource limitation argue that because pollination levels are generally high in milkweed populations, early abortion is largely due to resource limitation (Willson and Rathke 1974; Willson and Price 1977; Willson and Price 1980; Queller 1983, 1985).

The ability to reproduce asexually is another important reproductive characteristic of some milkweed species. Although asexual reproduction is common in milkweeds, it is not universal. Whether and to what extent individual species form asexual clones has important consequences on milkweed reproductive biology studies since self pollinations among wholly or partially self-incompatible clones may affect levels of fruit set.

Asclepias viridis typically inhabits open locations in fields, roadsides and wood margins. Its range extends from Florida to Texas north into Nebraska, Illinois, Ohio and West Virginia (Woodson 1954, Duncan and Foote 1975). Individuals typically occur in colonies. Plants often produce a single stem (Woodson 1954, Ajilvsgi 1979) but they can branch and form several stems from the caudex (Woodson 1954).

The purpose of this study was to perform an initial investigation into certain fundamental aspects of the reproductive biology of A. viridis. Specifically, this study sought to determine if A. viridis is self compatible, to measure fruit set and seed production and to investigate if A. viridis is clonal. This was the first such study of this species.

MATERIALS AND METHODS

An urban lot was chosen as the field site for this study. The site is located in southeastern Lake Charles, Louisiana. (93 [degrees] 12'16" W X 30 [degrees] 10'58" N) and measures 373 m by 73 m.

On 11-12 April 1997, during the period of maximum anthesis, all stems with flowers present were tagged. The total number of flower-bearing stems in the population was 720. Seventy-three stems were sampled to estimate the total number of flowers in the population. Observed insect visitors included an unidentified species of bumblebee (Bombus sp.) and monarch butterflies (Danaus plexippus L.).

Each follicle from tagged plants was subsequently harvested during June at the point each began dehiscence. At this stage, seeds were sufficiently developed so that they could be counted. Because milkweed follicles are subject to predation (Willson and Rathcke 1974, Wilbur 1976, Willson and Price 1977, Price and Willson 1979, Queller 1985), follicles were collected as quickly as possible to avoid loss and to make accurate seed counts. Two such herbivores, milkweed bug (Oncopeltus fasciatus Dallas) and monarch butterfly larvae (D. plexippus), were observed in the study site. Of 139 follicles collected, 25 were randomly selected and their seeds were counted.

Hand-pollinations were made on individuals in the study site during the period from 18-26 April 1998. Because A. viridis produces large gynostegia that typically measure 4 mm in diameter, hand pollinating in the field was feasible. The umbels of sampled plants were protected from natural pollinators prior to anthesis by covering them with mesh bags. A single flower from the lowest umbel was chosen for hand pollination. Because smaller plants may be unable to set fruit (Chaplin and Walker 1982), we chose for pollination only plants that exhibited at least two umbels. Under the magnification of a dissecting microscope, pollinia were removed from a donor flower by grasping the corpusculum with small forceps. Anther flaps from the recipient flower were gently splayed so that a pollinium could be inserted into the stigmatic chamber with its convex surface innermost. Following insertion, the anther flaps were pushed back together. A single pollinium was inserted into each recipient flower and only one flower per plant was pollinated. The umbel was again covered with a mesh bag to prevent subsequent natural pollination. After perianth senescence, the bags were removed so follicles could develop normally. All sample plants were continuously monitored from the time of pollination until follicles matured. Additionally, the umbels of ten control individuals were covered with mesh bags prior to anthesis. These control individuals were observed from anthesis through floral senescence to ensure that the bags prevented natural pollination.

Test samples consisted of 30 self pollinations and 30 cross pollinations. All cross pollinations were made from a single individual that had been determined in the previous year to produce fertile pollinia. Specifically, trial hand pollinations using pollinia from this individual donor resulted in a single mature pod on two other individuals in 1997.

Experiments were conducted to determine whether or not this species is clonal. Because the donor plant was a member of the population under study, results from the clonal tests were necessary to ensure that donor pollinia represented outcrossing gametes. To that end, 20 sample sites were selected for examination. Specifically, all plants within a 30 cm radius of 20 target plants were exhumed and examined for asexual structures. Additionally, individuals from four of the twenty samples were selected for molecular marker analysis using Random Amplified Polymorphic DNA (RAPD) fingerprinting (Williams et al. 1990). Specifically, two individuals each from one of the four sample sites (a total of eight individuals) were considered a "clone-candidate pair" and their fingerprint patterns were compared.

Total DNAs were extracted from fresh leaf tissue using the CTAB method of Doyle and Doyle (1987). DNA template was amplified via polymerase chain reaction (PCR) (Mullis and Faloona 1987, Saiki et al. 1988) with Tfl enzyme (Epicentre Technologies, Madison, WI), using the following thermocycling protocol: (94 [degrees] C, 1 min, 35 [degrees] C, 1 min, 72 [degrees] C, 1 min) X 45 cycles; (72 [degrees] C, 7 min) X 1 cycle: (4 [degrees] C soak). Each of the four clone-candidate pairs were fingerprinted according to standard RAPD protocol. Twenty replicates, each amplified by a different ten-base-pair random primer (Set OPA, Operon Technologies, Inc., Alameda, California) were used. Amplified DNA markers were separated and resolved in a 1% agarose gel containing ethidium bromide. Fingerprint patterns were visualized under UV light and recorded using Polaroid film.

RESULTS

The rate of successful mature follicle fruit set was estimated at 1.5% (95% confidence interval 1.4 and 1.7%) (Table 1). Of the 139 stems that set mature fruit, the average number of follicles per stem was 1.4 (Table 1).

From the seed count of randomly selected follicles, a mean of 72.2 [+ or -] 5.76 seeds per follicle was determined at a confidence interval of 95%. Based on the 195 follicles collected, we estimated that the total number of seeds produced by the entire population was 14,079 [+ or -] 1,123.

No individual extracted from the soil had any vegetative connection to any other individual. The fusiform caudexes were found to support one to several stems but no stolons or other asexual vegetative structures were observed.

Tests using RAPD fingerprints detected polymorphisms in all four of the clone-candidate pairs. Specifically, at least three polymorphic bands were detected for each clone-candidate pair (Table 2). These results suggest that A. viridis is not clonal and, therefore, hand pollinations between plants within this population represent outcrosses.

Of the 30 flowers that were cross pollinated by hand, three (10%) produced mature fruit with developed seeds. Of the 30 flowers that were self pollinated, four (13.3%) produced mature fruit with developed seeds. These data suggest that this species exhibits at least some level of postzygotic self compatibility and, therefore, is not an obligate outcrosser. Of the ten control individuals, none produced mature follicles. Therefore, we conclude that the mature follicles produced in this experiment were the result of the test pollinations only.

DISCUSSION

Morphological examination and molecular techniques performed in this study suggest that A. viridis is not clonal. Establishing this was necessary to ensure that donor pollinia used in the hand-pollination experiment were composed of outcrossing gametes. There are conflicting data in the literature concerning whether particular milkweeds are clonal. Specifically, Wilbur (1976) reported that there was no evidence of vegetative propagation in A. verticillata; however, Willson and Price (1977) reported that slender rhizomes often connect several stems. Additionally, Shannon and Wyatt (1977) cite Wilbur (1976) and state that A. exaltata does not reproduce vegetatively. However, Broyles and Wyatt (1993) and Queller (1985) reported limited vegetative reproduction in A. exaltata. These two critical conflicts demonstrate the necessity of establishing whether a species is clonal by both molecular techniques and morphological examination. In this case, RAPD technology provided a fast and convenient method to conclude that A. viridis is not clonal.

As previously stated, 10% of cross pollinations produced mature fruits with developed seeds. This value is similar to those of some previous studies that also found low fruit set rates among compatible hand-crossed pollinations: 15.2% in A. subulata (Wyatt 1976); 14.8% in A. tuberosa (Wyatt 1976, 1981); 19.7% in A. exaltata (Queller 1985).

The success rate of 13.3% in the hand pollination selfing experiment suggests that this species exhibits at least some level of self compatibility. Of the milkweed species that have been studied previously, about half are reported to exhibit some level of self compatibility. Only A. incarnata (Kephart 1981), A. curassavica (Wyatt and Broyles 1997) and A. fruticosa (Wyatt and Broyles 1997) appear to be completely self compatible.

Successful fruit set of approximately 1.5% in A. viridis falls within the typical range of <5% for other milkweeds that have been studied (Moore 1947; Woodson 1954; Willson and Rathcke 1974; Wyatt 1976, 1980, 1981; Lynch 1977; Willson and Price 1977, 1980; Stephenson 1981; Chaplin and Walker 1982; Queller 1985; Shannon and Wyatt 1986; Wyatt and Shannon 1986; Wyatt and Broyles 1990, 1994; Wyatt et al. 1996). Investigators have focused primarily on two hypotheses to explain this low level of fruit set: (1) effective levels of pollination are limiting and (2) resources for fruit and seed are limiting (Wyatt and Broyles 1990).

Proponents of effective pollination limitation argue that early fruit abortion in self-incompatible species is due largely to insufficient numbers of compatible pollinia reaching stigmatic chambers. For example, Morse and Fritz (1983) and Morse (1994) suggested that the scarcity of pollinia limited the production of mature pods in the largely self incompatible A. syriaca. A similar argument was made by Wyatt (1980) in the case of A. tuberosa.

In the self-compatible Asdepias speciosa, Bookman (1984) contended that insufficient pollination apparently did not explain low fruit set. Bookman's 1984 study indicated that pods fathered by some donors were more often produced than pods of other donors. Bookman (1984) was unclear whether this difference between donors in pollen vigor was primarily due to genetic or to environmental factors. In the self-compatible A. exaltata, results from the study by Queller (1985) led him to state that low fruit set was not due to insufficient pollination, nor to genetic-developmental incompatibilities nor to herbivory. Queller (1985) concluded that fruit production was constrained by the availability of nutrient resources.

Another relevant factor in the reproductive biology of milkweeds is seed production. The estimated total of 14,079 seeds in this population of A. viridis exceeds the estimated total of 12,902 flowers. These data demonstrate that low fruit set is not synonymous with low seed production. A. viridis allocates its reproductive resources to generate high seed content in rare mature follicles rather than on generating high numbers of mature follicles.

There is no universal reproductive strategy among milkweeds with respect to clonality nor compatibility. For example, A. syriaca is mostly self incompatible (Sparrow and Pearson 1948, Kephart 1981) and clonal (Sparrow and Pearson 1948, Wilbur 1976, Kephart 1981), but A. viridis is self compatible and not clonal. However, evidence to date suggests that all milkweeds set fruit at low levels and produce follicles with large number of seeds.
TABLE 1. Summary of fruit set data.

 Category Value

A. Flowers:
 Total number of stems tagged 720
 Number of tagged stems sampled for
 flower counts 73
 Total number of flowers counted on
 sampled stems 1,308
 Mean number of flowers per stem @ 95%
 confidence 17.92 (16.07-19.77)
 Estimated number of flowers in the
 population 12,902 (11,568-14,236)

B. Rate of fruit set:
 Total number of mature follicles
 harvested 195
 Mature follicles to estimated flowers @
 95% confidence 0.015 (0.014-0.017)

C. Average number of follicles per stem
 that set fruit:
 Total number of mature follicles
 harvested 195
 Total number of stems that set fruit 139
 Average number of follicles per stem
 that set fruit 1.4
TABLE 2. Polymorphisms detected from RAPD analysis on eight individuals
representing four clone-candidate pairs. The detection of polymorphic
bans are indicated by an "X".

 Clone candidates

OPA primer # Pair 1 Pair 2 Pair 3 Pair 4

 1 -- X -- --
 2 -- -- -- --
 3 -- -- -- --
 4 -- -- -- --
 5 -- -- -- --
 6 -- X -- X
 7 X X X X
 8 -- -- -- X
 9 -- -- -- --
 10 -- -- -- --
 11 -- -- -- --
 12 X -- X --
 13 -- -- -- --
 14 -- -- -- --
 15 -- -- -- --
 16 X X -- --
 17 -- -- X X
 18 -- -- -- --
 19 -- -- -- --
 20 -- -- -- --


ACKNOWLEDGMENT

We thank M. Paulissen for his previous review of this paper.

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Ray Neyland, Billie J. Hoffman, and Harry A. Meyer

Department of Biological and Environmental Sciences
McNeese State University
Lake Charles, LA 70609-2000

Robin Lowenfeld

Louisiana Environmental Research Center
Lake Charles, LA 70609
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Author:Neyland, Ray; Hoffman, Billie J.; Meyer, Harry A.; Lowenfeld, Robin
Publication:The Proceedings of the Louisiana Academy of Sciences
Article Type:Statistical Data Included
Geographic Code:1U7LA
Date:Jan 1, 1999
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