Spore germination of five globally threatened fern species [Culcita macrocarpa C. Presl, Dryopteris aemula (Aiton) O. Kuntze, D. corleyi Fraser-Jenkins, D. guanchica Gibby and Jermy and Woodwardia radicans (L.) Sm.] was determined after 1, 6 or 12 months of storage in glass vials (dry storage) or on agar (wet storage) at -20, 5 or 20 degrees C. In all species, storage technique, storage temperature and the technique-temperature interaction all had a significant effect on germination percentage. In most cases, the germination percentage was best maintained by wet storage at 5 or 20 degrees C. In the case of the hygrophilous species C. macrocarpa and W. radicans, 6 or 12 months' dry storage killed most spores. Only Woodwardia radicans germinated in the dark during wet storage at 20 degrees C. Wet storage at 5 degrees C prevented dark germination, and reduced bacterial and fungal contamination. Wet storage at -20 degrees C killed all or most spores in all species. In the three Dryopteris species, the differences among the storage conditions tested were smaller than in C. macrocarpa and W. radicans, and the decline in spore viability during storage was less marked, with high germination percentages being observed after 12 months of dry storage at all three temperatures. Dry storage, which has lower preparation time and space requirements than wet storage, was generally more effective at the lower temperatures (-20 or 5 degrees C).

Aim:To disentangle the influence of environmental factors at different spatial grains (regional and local) on fern and lycophyte species richness and to ask how regional and plot-level richness are related to each other.Location: Global.

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The results of this study suggest that (i) relative fitness of allopolyploids at sporogenesis does not differ from that of diploid parents and (ii) neither does allopolyploidization involve a change in the success of spore germination.

Summary 1.In environmental sex determination (ESD) gender is decided after conception, depending on the environment, rather than being genetically fixed. ESD in plants has been mainly studied in angiosperms, where the dominant form of ESD is sexual lability. Surprisingly, ESD has rarely been studied in homosporous ferns, the only plants in which ESD is the rule, rather than the exception. 2. Here, we address the mechanism underlying ESD for the fern Woodwardia radicans by experimentally manipulating nutrient availability and gametophyte density. 3. Stress (limited nutrient supply, crowding) affected sexual expression in W. radicans gametophytes in a way compatible with ESD. Under good growth conditions (low density or high nutrient), gametophytes matured sexually at a relatively large size and turned into females and subsequently into bisexuals. Under harsh growth conditions, gametophytes matured sexually at a smaller size and turned into males. 4. Interestingly, gametophyte sexual expression was consistent with the size-advantage model, because the number of archegonia increased with gametophyte size, but not the number of antheridia. 5. The sex switch threshold size was variable and decreased with increasing stress, as predicted by age and size to maturity models. 6. Synthesis. Sexual expression in fern gametophytes can be fruitfully studied within the ESD theoretical framework. Stress induced male expression in gametophytes in a way compatible with ESD. In addition, size-related patterns of sexual expression were consistent with the size-advantage model, because female function benefited more from a larger size than did male function. Finally, the sex switch threshold size was variable and decreased with increasing stress, a result predicted by age and size to maturity models but seldom empirically tested before.

The gametophytic generation of the allotetraploid Polystichum aculeatum and its diploid parents, Polystichum setiferum and Polystichum lonchitis, was studied in order to compare their morphology, gametangial ontogeny, and breeding system. Six populations, two of each species, were selected for spore collection. Germination, gender expression, and antheridiogen experiments were established on agar and soil culture media. Germination percentage in the tetraploid was higher, and the only morphological difference was found in the length of marginal hairs that were also longer in P. aculeatum. Gender expression in the allotetraploid was a mixture of the diploids. Differences in gender expression of both diploids, with many male prothalli in P. lonchitis and many female ones in P. setiferum, may favor the formation of the hybrid that originated the allotetraploid. An antheridiogen system was observed in both P. aculeatum and P. setiferum, and each species responded to one another's antheridiogen. In contrast, exudates from P. lonchitis failed to induce precocious maleness within the species but did induce an antheridiogen response in gametophytes of P. setiferum.

The higher genetic diversity observed in the Azorean population studied suggested a possible refugium in this region from which mainland Europe has been recolonized after the Pleistocene glaciations. High among-population genetic differentiation indicated restricted gene flow (i.e. lack of spore exchange) across the highly fragmented area occupied by D. aemula. The deviations from Hardy-Weinberg equilibrium reflected strong inbreeding in D. aemula, a trait rarely observed in homosporous ferns. The absence of spatial genetic structure indicated effective spore dispersal over short distances. Additionally, the cross-amplification of some D. aemula microsatellites makes them suitable for use in other Dryopteris taxa.

For many plants, sex is not fixed by genotype but determined by environmental conditions during development. In homosporous pteridophytes, sex is environmentally determined by the presence or absence of antheridiogens, maleness-inducing pheromones. It has been proposed that antheridiogens primarily reduce growth rate, with small gametophyte size responsible for maleness. To test this hypothesis, the effects of antheridiogen and intergametophytic competition on gender expression and gametophyte size were studied in a culture experiment with Woodwardia radicans. We found that (1) antheridiogen inhibited growth of gametophytes; and (2) slow growth favored maleness, whereas fast growth favored femaleness, irrespective of the presence or absence of antheridiogen. Both conclusions are consistent with the hypothesis that, in W. radicans, antheridiogen effect is mediated by size. They also agree with the "size-advantage" hypothesis in which energetic limitations associated with relatively small individual size impose a less severe limitation for male reproductive success than for female reproductive success. The results are also discussed with regard to a genetic sex-determining pathway that has recently been identified.