Emilia Pangua

Spore Viability Under Different Storage Conditions in Four Rupicolous Asplenium L. Taxa

Abstract Spore germination of four rupicolous taxa of Asplenium (A. adiantum-nigrum. var. adiantum-nigrum, A. adiantum.nigrum. var. silesiacum, A. septentrionale subsp. septentrionale and A. ruta-muraria. subsp. ruta-muraria) was determined after 1, 6, and 12 months of storage in Eppendorf tubes (dry storage) or on agar plates (wet storage) at −20, 5 and 20°C. In general, technique and temperature factors and the moisture-temperature interaction, had a significant effect on germination percentage. In all cases, except for A. ruta-muraria, germination percentage was maintained in wet and dry storage, but in the dry storage method percent germination was higher. These results indicate some capacity of Asplenium spores to withstand desiccation, and that ecological requirements of species may influence spore viability and should be taken into account when designing spore conservation programs. Spores of A. ruta-muraria yielded better results in wet storage. In dry storage its response was different from that of the other three taxa. Wet storage at −20°C killed all or most spores of all taxa.

A Comparative Study of the Gametophytic Generation in the Polystichum aculeatum Group (Pteridophyta)

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.

Allozyme variation in the sympatric ferns Culcita macrocarpa and Woodwardia radicans at the northern extreme of their ranges

Isozymes were used to study genetic variation in the clonal ferns Culcita macrocarpa and Woodwardia radicans in the northwestern Iberian Peninsula, their northern distributional limit. Despite their high chromosome numbers, both species were isozymic diploids. In C. macrocarpa all 18 resolved loci were monomorphic, with the same allele in all ramets from all populations. In W. radicans only two of the 16 interpreted loci were polymorphic, with two alleles per locus; ramet-level genotypes showed Hardy-Weinberg equilibrium, indicating an intergametophytic mating system; the number of genets distinguished was 1–3 per population; and among-population variation was low (FST = 0.231), suggesting effective gene flow (i.e. spore exchange). More generally, the very low (W. radicans, HT = 0.012) or zero (C. macrocarpa) genetic diversity detected in the present study may be due to genetic drift associated with the reduction of populations in the last glaciation, and to founder effects in the subsequent Holocene expansion.

Genetic variation in the allotetraploid Dryopteris corleyi (Dryopteridaceae) and its diploid parental species in the Iberian Peninsula

Studies on genetic diversity help us to unveil the evolutionary processes of species and populations and can explain several traits of diploid-polyploid complexes such as their distributions, their breeding systems, and the origin of polyploids. We examined the allozyme variation of Dryopteris aemula and D. oreades, diploid ferns with highly fragmented habitats, and the allotetraploid D. corleyi to (1) analyze the putative relationship between both diploids and the tetraploid, (2) compare the levels of genetic variation among species and determine their causes, and (3) assess the breeding system of these taxa. The allozymic pattern of D. corleyi confirms that it derived from D. aemula and D. oreades. The lack of genetic diversity in D. aemula, a species of lowland habitats, may be due to genetic drift associated with the contraction of populations in the last glaciation. By contrast, the alpine D. oreades had moderate intrapopulation genetic variation, which may derive from the expansion of populations during the last glaciation. In the latter species, low interpopulational variation suggested effective gene flow (spore exchange), and genotype frequencies in Hardy-Weinberg equilibrium indicated cross-fertilization of gametophytes. Evolutionary history appears to be an essential element in the interpretation of genetic variation of highly fragmented populations.

Studies on Cryptogramma crispa Spore Germination

In order to study the germination capacity of Cryptogramma crispa, spores were cul- tured on sterilized Petri dishes with nutritive medium solidified with agar. Germination was checked at 10, 15, 20 and 25?C, and, as in most of homosporous ferns, the germination optimum was at temperatures above 20?C. Two light intensities were used, 10 and 40 iLEm-2s-, to reproduce the possibilities of the spores falling on open sites or in rock cracks or hollows. A lower light intensity accelerates germination. After sowing, some plates were kept at chilling and other at freezing temperatures to check the effect of low storage temperatures on the germination capacity of the spores. After these processes, the spores are able to germinate and reach similar germination rates, although the frozen spores delay the beginning of germination and show a decreased ger- mination rate. The results of these experiments point toward the possibility that the spores of C. crispa are dispersed at the end of the growing season and go through a dormancy until next spring. In the Iberian Peninsula (Spain and Portugal), Cryptogramma crispa R. Br. grows on ecologically very particular and well defined habitats. It occupies especially siliceous stone fields, such as granite, gneiss, sandstones, quartzites, or slates in high mountain zones, usually above the timber-line. In the Iberian Peninsula, its optimum is about 2000 m. The plants grow preferentially in rock fissures or cracks and in hollows between rock blocks. In these habitats, which are mostly over 2000 m elevation, the growing season may be very short, scarcely two months in extreme conditions, and usually no more than four months (Rivas-Martinez, 1987). The considerations of the distribution of pteridophytes suggest the need for more detailed investigations on the life-cycles of species to determine the im- portance of specific variations in the life-cycle in limiting the distributions of plants. Variations in the distributions of species might be accounted for by random processes, such as dispersal, or in a more deterministic manner by subtle and specific variations in life cycle characteristics (Woodward, 1987). In ferns, it is important to study the factors that can affect the development of the gametophyte that would lead to the establishment of the sporophytic gen- eration.

Reproductive and competitive interactions among gametophytes of the allotetraploid fern Dryopteris corleyi and its two diploid parents.

BACKGROUND AND AIMS Several models predict that the establishment of polyploids within diploid populations is enhanced by non-random mating (i.e. selfing and assortative mating) of cytotypes and by a higher relative fitness of polyploids. This report assesses the role that antheridiogens (i.e. maleness-inducing pheromones) and intercytotype differences in growth rate have on polyploid performance. METHODS Three buckler-fern species were studied: the allotetraploid Dryopteris corleyi and its diploid parents, D. aemula and D. oreades. In one experiment, gametophytes of these species were cultured under rich growth conditions to compare the timing of gametangia production. The substrata on which these gametophytes had grown were used as antheridiogen sources in a second experiment. The three species were combined as source and target of antheridiogen (i.e. nine species pairs). Timing of antheridia production and gametophyte size were determined after those antheridiogen treatments. KEY RESULTS Under rich growth conditions the allotetraploid produced archegonia earlier than those of diploid parents. Female gametophytes of the three species produced antheridiogens that inhibited growth and favoured maleness both within and among species. Gametophyte size was similar in the three species but antheridia formed earlier in the allotetraploid. CONCLUSIONS Unisexuality, promoted by non-specific antheridiogens, enhances random mating both within and among species. The resulting hybridization can favour the reproductive exclusion of the allopolyploid in sites where it is outnumbered by diploids. However, the earlier production of gametangia in the allotetraploid favours assortative mating and may thus counterbalance reproductive exclusion.

SEXUAL EXPRESSION AND GENETIC DIVERSITY IN POPULATIONS OF CRYPTOGRAMMA CRISPA (PTERIDACEAE)

The reproductive biology of Cryptogramma crispa, a tetraploid species with a broad circumboreal and alpine distribution, growing mainly in siliceous boulder fields and crevices, was studied in the laboratory by growing gametophytes in plates with both solidified agar media and sterilized soil. In addition, an electrophoretic study of isozymes was carried out on frond samples from five natural populations, as an additional source of evidence concerning the breeding system and the genetic structure of sporophyte populations. Populations throughout the Iberian range of the species were selected for this study, and a Scottish population was included to represent plants from outside our local area and ecology. The morphological development of gametophytes is of the Adiantum type. All multispore cultures developed into a bigametophytic system, consisting in most cases of male and female prothalli. This pattern of sexual expression provides evidence for outcrossing as the main breeding system in this species. Moreover, there is good evidence that the species possesses an antheridiogen system to promote outcrossing. The long time needed by gametophytes to produce gametangia, and afterwards to fertilize and produce sporophytes, might be the primary reason why so few young sporophytes are found in the wild. The values of the percentage of polymorphic loci and the similarity levels obtained from the isozyme analyses indicate a level of genetic variability that would be expected in an outcrossing species. All these characteristics are usually associated with diploid fern species rather than polyploid species.

Asplenium ceterach and A-octoploideum on the Canary Islands (Aspleniaceae, Pteridophyta)

Abstract Isozyme and plastid DNA analysis prove that true A. ceterach occurs on the Canary Islands, in addition to A. aureum and an octoploid taxon. Combining morphological and cytological observations leads to correct determination, but the exospore length alone also allows reliable identification of these Canarian species. Our allozyme data suggest that the Canarian A. ceterach population is not completely genetically isolated from the European ones. The holotype of Ceterach aureum var. parvifolium, formerly regarded as an octoploid taxon, proved to be A. ceterach, leaving the octoploid without a correct name. The recently described A. octoploideum shows monomorphic, presumably fixed heterozygosity for a combination of the patterns seen in A. ceterach and A. aureum at four loci (Aat, Skdh, Me, and Pgi-2) confirming its allo-octoploid nature. It most probably originated by chromosome doubling in a tetraploid hybrid between A. aureum and A. ceterach or via the union of their unreduced gametes. Pgi-2 indicates multiple origins of the allo-octoploid, implicating recurrent gene flow from tetraploids to octoploids.

Isozymic Contribution to the Systematics of the Asplenium seelosii Group

Abstract The systematics of the Asplenium seelosii complex have been debated for a long time. This complex includes strictly rupicolous plants that live on limestone cliffs mainly in mountains of southwest Europe: the Alps, the Pyrenees, and several mountain ranges of the Eastern Iberian Peninsula. The disjunct distribution of the populations and several morphological characters, i.e., leaf indumentum and the structure of the perispore, have been used to distinguish species and subspecies. The goal of this study was to evaluate the different systematic treatments of this complex by means of isozyme electrophoresis. Seventeen populations throughout the range of the complex were studied, and 15 enzymatic systems were assayed. There was no within population genetic variation and genetic identity between populations varied widely. Analysis of isozymic data clearly differentiated two groups corresponding to the species proposed, Asplenium seelosii and A. celtibericum, but these data do not support the recognition of subspecies in this complex.

Gametophyte Features in a Peculiar Annual Fern, Anogramma leptophylla

Anogramma leptophylla is one of few homosporous ferns with ephemeral sporophytes and perennial gametophytic tubercules. This rare aspect of the life cycle has considerable bearing on the reproductive features of the gametophyte. Four populations in the Iberian Peninsula were selected to provide spores for laboratory cultures and to sample natural populations of gametophytes for comparative studies. Sexual expression and the role of the tubercule in the reproductive biology of this fern were studied. No significant differences were found between percentages of sexual versus asexual prothalli in natural populations or laboratory cultures, or between percentages of unisexual and bisexual prothalli. These results may indicate that sex in A. leptophylla is genetically determined instead of environmentally controlled as in most ferns. The presence of a gametophytic tubercule with nutrient reserves seems to be most favourable for the development of archegonia, and for the establishment of young sporophytes. This tubercule, which can resist stressful environmental conditions, allows for the development of new gametophytic outgrowths and of dormant embryos as soon as environmental conditions improve. This bryophyte-like behaviour gives this fern some advantages in colonisation and has probably contributed to its wide geographic range.