636 AMERICAN JOURNAL OF BOTANY [Vol. 84 Figs. 1–9. Gametophytes illustrating promotion of antheridium formation, and dark spore germination by Polypodiaceae species. Note presence of antheridia and released sperm. 1–6. Antheridium promotion. 1. A 30-d-old gametophyte of Phymatosorus scolopendria on an inverted block of P. scolopendria. 2. A 45-d-old gametophyte of Campyloneurum phyllitidis on a cleared block of C. phyllitidis. 3. A 60-d-old gametophyte of Phymatosorus scolopendria on a cleared block of P. scolopendria. 4. A 75-d-old gametophyte of Polypodium pellucidum on an inverted block of Pteridium aquilinum. 5. A 15-d-old Onoclea sensibilis gametophyte on an inverted block of Polypodium pellucidum. 6. A 15-d-old O. sensibilis gametophyte on a cleared block of C. phyllitidis. 7–9. Spore germination promotion and antheridium formation in darkness. 7. A dark-grown
May 1997] CHIOU AND FARRAR—ANTHERIDIOGEN IN POLYPODIACEAE 637 TABLE 3. Average antheridium formation (%) by 45-d-old gametophytes of Onoclea sensibilis inresponse to secretions from mature gametophytes of Polypodiaceae species. Species tested Inverted Treatments* Cleared 10% extract Control Campyloneurum angustifolium 94 a 96 a 79 b 17 c Campyloneurum phyllitidis 100 a 98 a 77 b 17 c Lepisorus thunbergianus 100 a 100 a 92 b 17 c Microgramma heterophylla 100 a 100 a 92 b 17 c Phlebodium aureum 100 a 92 a 13 b 17 b Phymatosorus scolopendria 100 a 98 a 69 b 17 c Polypodium pellucidum 100 a 96 a 90 a 17 b * The same superscript letter in the same row indicates no significant difference in Duncan’s multiple test (95% CL). and respond to the antheridiogen of P. aquilinum. However, antheridiogenproductionand the antheridiogen concentration required to promote antheridium formation vary among species. Some of these species did not respond to same-species culture extracts or to culture extracts of P. aquilinum gametophytes, whereas they did respond to secretion by nearby mature, growing gametophytes. This suggests that the antheridiogen concentration required to promote antheridium formation is relatively high, which is one of the possible reasons why some previous workers failed to demonstrate the presence of an antheridiogen system in Polypodiaceae species or response of these species to antheridiogen A Pt (Voeller, 1971; Raghavan, 1989). Other possible explanations of nonpromotion of antheridium formation by extracts are that the extraction method might destroy antheridiogens or that the antheridiogen may retain activity for only a short time (Emigh and Farrar, 1977). However, the fact that antheridium formation by O. sensibilis was significantly promoted by culture extracts of all species tested, except P. aureum, indicates that these antheridiogens are stable. That GA3 did not promote antheridium formation in any species tested indicates that these antheridiogens may not be as closely related to GA3 as are the antheridiogens of the Schizaeaceae and Vittariaceae. Other indirect evidence of an antheridiogen system in these species comes from the pattern of male expression in multispore cultures. The male gametophytes in multispore cultures of every species were smaller, and male plants were produced later than female plants. This fits the model of antheridiogen function proposed by Naf (1963) who suggested that the fast-growing females secrete antheridiogen, which induces slow-growing gametophytes to produce antheridia. Promotion of dark germination by antheridiogen (or germinin)—All species tested, except P. aureum, germinated in darkness under one or more treatments. Apparently, a substance secreted from mature gametophytes of the Polypodiaceae species tested, and from gametophytes of P. aquilinum, substitutes for the light requirement for spore germination. Traditionally, this substance has been considered to be antheridiogen, although Voeller (1971) proposed a chemical different from antheridiogen, which he called germinin. In general, functions of the substance promoting antheridium formation and the substance inducing spore germination in darkness are parallel (Voeller, 1971), but Welling and Haufler (1993) hypothesized that these two substances might function independently. In this study, secretions of gametophytes promoted both spore germination in darkness and antheridium formation in the light in the same species, except for P. aureum, whereas culture extracts of gametophytes promoted both responses (P. pellucidum), or only antheridium formation in the light (C. phyllitidis, M. heterophylla, and P. scolopendria), or neither response (C. angustifolium and L. thunbergianus). These results suggest two possibilities. First, antheridiogen may be different from germinin, and the latter may be short lived or lose activity during the extraction process. Second, antheridiogenand germinin may be the same, but the threshold concentration for promoting antheridium formation may be lower than that for substituting for the light requirement for spore germination. On the other hand, secretions of P. aquilinum gametophytes promoted both antheridium formation in light and spore germination in darkness in Polypodiaceae (except in P. aureum in which only antheridium formation in light was promoted), whereas culture extracts of P. aquilinum either promoted both responses in Polypodiaceae (C. phyllitidis, M. heterophylla, P. pellucidum, and P. scolopendria), or induced spore germination in darkness but not antheridium productionin light (C. angustifolium and L. thunbergianus). These results suggest that antheridiogenand germinin are both stable in extraction and are possibly the same chemical, at least in P. aquilinum, but the threshold concentration for antheridium formation may be higher or lower than that for inducing spore germination in darkness depending on the species. Antheridiogen vs. inhibitor—In our antheridiogen experiments, gametophytes on treated media were considerably smaller than those on control media. Usually only spathulate or small cordate gametophytes developed on media containing extract of gametophyte cultures and only few-celled filaments to spathulate stages of prothalli developed on media already supporting other mature gametophytes. Only small sizes were attained regardless of whether gametophytes bore antheridia or remained asexual. Naf (1956) suggested that in gametophytes subjected to antheridiogen, potential vegetative growth is diverted to antheridium production. However, because this cannot explain the small asexual gametophytes, we suspected the activity of another growth-inhibiting substance. To test this hypothesis, we sowed spores of the Polypodiaceae on inverted and cleared agar blocks on which mature gametophytes of Anemia phyllitidis were growing. Anemia ← gametophyte of Phymatosorus scolopendria on a medium containing culture extract of P. aquilinum gametophytes. 8. A dark-grown gametophyte of Campyloneurum phyllitidis on an inverted block of C. phyllitidis. 9. A dark-grown gametophyte of Polypodium pellucidum on a cleared block of P. pellucidum. Bar 0.1 mm for Figs. 1–3, 5–6, 7, 9; 0.05 mm for Figs. 4, 8.