Interpopulational Comparison of Dose-Mediated Antheridiogen Response in Onoclea sensibilis

-Intraspecific variation in antheridiogen response has been reported in several fern species. Here, we characterized and compared dose-mediated antheridiogen response among six populations spanning the range of Onoclea sensibilis, a species widely used for antheridiogen assays. A crude aqueous filtrate (CAF) of antheridiogen APT was obtained from cultures of Pteridium aquilinum and introduced into mineral agar at six concentrations ranging from 0 (control) to 10-1. Response was initially characterized for one population using three criteria: percentage of male gametophytes, number of antheridia per gametophyte, and mean surface area of gametophytes. Percent male was consistently the best descriptor of the response profile and was used to compare responses among populations. In all populations, gametophytes exhibited little or no response at CAF concentrations 10-5 and lower; a saturated response, almost all gametophytes being male at 10-3 and higher; and an intermediate response, usually between 50% and 80% males, at 10-4. With few exceptions, response levels and profiles were very consistent among populations. Data and methodology provided herein indicate that use of the percent male criterion can provide a reliable quantitative assay using Onoclea that can facilitate future comparative research in antheridiogens. Gametophytes of numerous and diverse taxa within the Polypodiaceae sensu lato share response to antheridiogen A (sensu Voeller 1964; in this paper antheridiogen A refers to the general class of antheridiogens produced by and biologically active in taxa belonging to Polypodiaceae sensu lato, whereas antheridogen ApT, sensu Nif [1979], refers specifically to antheridiogen A produced by Pteridium aquilinum (L.) Kuhn). However, antheridiogen response is neither pervasive nor equivalent among polypodiaceous ferns. Species representing different genera may differ by orders of magnitude for the minimum APT dose required for response (Niif et al., 1975; Nif, 1979), and congeneric species may also differ in antheridiogen response, as shown in Bommeria (Haufler and Gastony, 1978), Cystopteris (Haufler and Ranker, 1985), and polystichoid ferns (Yatskievych, 1993). Variation in response may also occur within species, either among populations or even among individuals within populations. In Hemionitis palmata L. , lower response was detected in populations with lower genetic load (Ranker, 1987), suggesting that reduced response to antheridiogen may facilitate a higher selfing rate, thereby eliminating recessive lethals within populations (Schneller et al., 1990). A genetic basis for variation in antheridiogen sensitivity was determined in Ceratopteris. Genotypes with lowered antheridiogen response were obtained experimentally in C. richardii Brongn. through application of mutagens, and a simple twogene basis of inheritance for antheridiogen sensitivity was determined (Scott and Hickock, 1987; Warne et al., 1988). These observations combine to indicate that antheridiogen response is subThis content downloaded from 157.55. 39.35 on Wed, 31 Aug 2016 04:08:53 UTC All use subject to http://about.jstor. org/terms AMERICAN FERN JOURNAL: VOLUME 89 NUMBER 4 (1999) ject to evolutionary change. Because antheridiogen may be of importance in determining or facilitating the mode of sexual reproduction by gametophytes, evolutionary changes in antheridiogen response may represent variation in an important life history attribute. Of the species compared by Naf (1979), the greatest sensitivity to antheridiogen ApT was exhibited by Onoclea sensibilis L. , which was therefore used as a reference for interspecific comparisons and has become the standard fern species for assaying antheridiogen A. This species is broadly distributed, occurring over most of eastern North America and disjunct in eastern Asia (Gastony and Ungerer, 1997). Although geographic sources of 0. sensibilis have varied among studies, the possibility of geographically based variation in antheridiogen response has not specifically been addressed. Most workers utilizing 0. sensibilis (e.g., Haufler and Ranker, 1985; Miller, 1968) have reported highly sensitive antheridiogen response and lack of spontaneous (i.e., without exposure to antheridiogen) antheridium formation, comparable to that indicated by Naf (1979). However, unusual responses have been reported from occasional frond collections (Nif, 1979), for example spontaneous antheridium formation (Klekowski and Lloyd, 1968; Miller and Miller, 1970; Rubin and Paolillo, 1983). Furthermore, the way in which response levels in relationship to antheridiogen doses have been quantified has varied among studies and sometimes has not been fully explicit. The objectives of this study were to determine consistent criteria for quantifying antheridiogen response of 0. sensibilis and to determine whether there are differences in response among populations from different regions. MATERIALS AND METHODS The antheridiogen exudate used in this study was obtained from gametophytes of Pteridium aquilinum, a standard source of antheridiogen A due to the high activity of its exudate. Spores from P aquilinum accession Sheffield 56 from England were sown under axenic conditions as follow. Spores were separated from sporangia by rinsing through a Nitex screen with 100 pm openings, using a wetting solution (2 drops of Tween-80 per 100 ml distilled water). Spores were collected in a cylindrical "basket" made from an inverted plastic 30 mm centrifuge tube. The conical bottom of the tube was cut off, and the cap was perforated with numerous holes using a hot dissecting needle. A finemeshed Nitex screen (5 ,um openings) was placed between the cap and the centrifuge tube opening to form a "basket" bottom that would retain spores but allow passage of liquids. Spore samples, wet with the Tween solution, were rinsed three times with distilled water and set in the dark overnight to allow germination of fungal spores. The following day, the baskets were placed in a sterilizing solution of 5% commercial hypochlorite for 90 sec, then placed in three rinses of sterile distilled water. Spores were then sown by pipetting onto mineral agar in densities ranging from 5-30 spores per cm2 in plastic petri dishes. Mineral agar contained Parker's macronutrients and Thompson's micronu222 This content downloaded from 157.55. 39.35 on Wed, 31 Aug 2016 04:08:53 UTC All use subject to http://about.jstor. org/terms STEVENS & WERTH: ANTHERIDIOGEN RESPONSE IN ONOCLEA TABLE 1. Collection localities for spores of Onoclea sensibilis used in this study. Collectors are indicated in parentheses.