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Departamento de Biología Vegetal I, Facultad de Biología, Universidad Complutense, E-28040 Madrid, Spain
Received for publication January 17, 1998. Accepted for publication November 23, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: breeding system • Cryptogramma • ferns • gametophyte • genetic diversity • isozymes • Pteridaceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Alverson, 1993
). Cryptogramma acrostichoides R.Br. grows in the United States, Canada, and northeast Asia. It was divided by Alverson (1993)
into C. sitchensis (Ruprecht) Moore and C. cascadensis Alverson, a new species he described from northwestern United States and southwestern Canada (Alverson, 1989
). Cryptogramma brunoniana Hook. & Grev. grows in the mountains of southeast Asia, C. stelleri (Gmel.) Prantl is from eastern United States, Canada, and Asia, and C. crispa (L.) R. Br. has an euroasiatic distribution. Tryon and Tryon (1982)
and Tryon, Tryon, and Kramer (1990)
recognized only two species worldwide, C. stelleri and C. crispa, including all other described species as varieties of C. crispa. The only European species, C. crispa, usually grows in boulder fields or in wide stone crevices in mountains over 1800–2000 m, very often forming patches. The plants are apparently quite hardy and are covered by snow during the winter. They receive high solar radiation during the summer.
The breeding system of ferns is determined by the independent gametophyte generation. Homosporous fern gametophytes have the possibility to become bisexual and self-fertilize. This form of inbreeding (intragametophytic selfing) produces a completely homozygous sporophyte and allows a single dispersed spore to be an effective colonizer. This theoretical ability led Klekowski and Baker (1966)
to state that self-fertilization was the most frequent mode of reproduction of homosporous ferns in nature.
However, analyses of gametophyte development have shown that various mechanisms promote the formation of functionally unisexual gametophytes in many species. These mechanisms are ontogenetic sequences that produce asynchronous maturation of male and female gametes, actual unisexuality of prothalli, and control of antheridia initiation by the pheromone antheridiogen produced by maturing female gametophytes (Döpp, 1959
; Näf, 1979
; Schneller, Haufler, and Ranker, 1990
; Haufler and Welling, 1994
). Therefore, the above-mentioned mechanisms are means to promote outcrossing.
On the other hand, electrophoretic analyses of isozymes in natural populations allow exploration of levels and patterns of genetic variability and the breeding systems operating in those populations (Soltis and Soltis, 1989
). In fern populations most of these isoenzymatic studies about breeding systems have been carried out in forest species (D. Soltis and P. Soltis, 1987
; P. Soltis and D. Soltis, 1987
; Soltis and Soltis, 1988
), epiphytic species Ranker, 1992a
; Hooper and Haufler, 1997
), and colonizing species (Wolf, Haufler, and Sheffield, 1988
; Ranker, 1992b
; Ranker et al., 1996
). Isozyme analyses of natural populations of ferns have demonstrated that most diploid species investigated so far are outbreeders (Soltis and Soltis, 1992
).
Nothing is known about the reproductive biology of C. crispa, except for the developmental morphology of the prothallus (Momose, 1964
). Peck, Peck, and Farrar (1990)
studied C. stelleri and noted that although this species produced a large number of spores, no gametophytes were observed in the wild. We have also observed very high spore production in the Iberian populations and have noted that the spores are dispersed at the end of the summer. Because of this late spore release, we reasoned that germination would not occur until the next spring. However, neither gametophytes nor sporelings have been observed in the field, and only a few young sporophytes were found at the beginning of the summer and in the fall season.
Our goal was to study in the laboratory some aspects of the reproductive biology of this species that might explain its low frequency of reproduction in the wild. We studied spore germination (Pangua, García-Álvarez, and Pajarón, in press), gametophyte development, gamentangial ontogeny, and the breeding system. As an additional source of evidence concerning the breeding system and the genetic structure of sporophyte populations, we carried out an electrophoretic study of isozymes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and a 3:1 mixture of compost and sand soil, placed in 6-cm diameter petri dishes. The former was used to study initial development of gametophytes, and the latter was used to study morphology of gametophytes and sexual expression. Sowing of each sample was replicated twice on both agar and soil. Sterilization of both kinds of media was carried in an autoclave at 125°C for 20 min. Cultures were kept in a growth cabinet at 21°C and 30 µmol photons·m-2·s-2, and at 12 h of light and 12 h of dark. Fifty prothalli were randomly sampled weekly, fixed in a mixture of aceto-carmine and chloral-hydrate (Edwards and Miller, 1972
), and heated in a water bath at 50°C for 2 h. Gametophytes were rinsed with distilled water, mounted on a slide, and examined under a light microscope for the presence of antheridia and archegonia. Gametophytes were recorded as being "presexual," "male," "female," or "bisexual."
Isolate potential
Multispore cultures where all the spores came from the same sporophyte were established in mineral agar (Dyer, 1979
) to provide gametophytes for this experiment. To assay the isolate potential (see Peck, Peck, and Farrar, 1990
; Ranker et al., 1996
), presexual gametophytes, 
5 wk old, were isolated and grown on soil (same mixture as above), in small plastic boxes (3 x 3 cm). For each population there were 50 boxes: 25 containing only one prothallus and 25 containing paired prothalli (all 75 prothalli originated from the same plant). The prothalli were watered with several drops of tapwater weekly to facilitate fertilization and keep substrate humidity. Six months after the gametophytes were transplanted sexual expression and the appearance of sporophytes were quantified.
Antheridiogen
To assess whether C. crispa produces an antheridiogen and is sensitive to it, we carried out two experiments. A female prothallus from population LNE was placed in the center of a petri dish with agar, and spores from the same population were sown around it. A female prothallus from population MON was placed in the center of another plate, and spores from STI were sown around it. Two replicates of each were made. After 11 wk, a concentric sampling was made adjacent to (0 cm), at 0.5 cm, and at 1.5 cm distant from the central prothallus; 20, 50 and 50 prothalli were sampled, respectively. The sexual expression of each gametophyte was assessed.
Ploidy level
Cryptogramma crispa is a tetraploid, C. acrostichoides, treated as a variety of the former by several authors, is morphologically very similar but diploid. Up to now no diploid plants have been found in the Iberian Peninsula or in Europe. To check whether the populations studied by us fit this analysis at least one plant from each population was cytologically studied. Root tips were pretreated by placing them in a vial containing a saturated solution of paradichlorobenzene for 3 h and then fixed in a mixture of absolute ethanol and glacial acetic acid (3:1). Before staining with Wittman's hematoxylin, root tips were hydrolized in 1 mol/L HCl for 4 min, and a drop of Hoyer's medium was added before the cover slip was set in place and squashed. Spore size of these and ten plants of each population, randomly chosen, was measured. Spores were mounted in DePex, and the equatorial diameter, excluding the perispore, of 30 spores of each plant was measured.
Isozymes
Fresh sporophyte leaves from all populations except SCO and HU provided material for electrophoretic analysis. No living sporophyte material was available from populations SCO and HU. Starch gel electrophoresis was conducted following Soltis et al. (1983)
and Haufler (1985)
. The following ten enzymes were analyzed: phosphoglucoisomerase (PGI), triosephosphate isomerase (TPI), shikimate dehydrogenase (SKD), leucine aminopeptidase (LAP), aspartate aminotransferase (AAT), 6-phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), hexokinase (HEX), and malate dehydrogenase (MDH). When more than one activity zone was observed for an enzyme, these were identified as different loci. The loci were numbered sequentially with the most anodally migrating locus designated 1.
Following Schneller and Holderegger (1996)
, we performed an analysis of the isozyme-phenotype level. The fraction of polymorphic loci (P) has been used as a measure of genetic variability within populations. Frequencies of isozyme phenotypes were calculated for each locus at each population.
To evaluate the variability within and among populations, the data in the form of presence/absence of isozyme bands were analyzed. This allowed us to estimate the variability of each population and of all populations together in spite of lacking inheritance data. The method used was as follows. A matrix of similarity was generated using the Jaccard coefficient (see Rohlf, 1994
). The computation formula was J = a/ (n–d), where a is the number of bands common to both individuals compared, d is the number of bands not present in neither of both individuals, and n is the total number of bands. Following the UPGMA (unweighted pair-group method, arithmetic average) method, a dendrogram was constructed for each population and also for a combined analysis of all the studied populations. This method has been used with Random Amplified Polymorphic DNA (RAPD) data to compare population variability (Martín, González-Benito, and Iriondo, 1997
) and with isozyme data to examine phenetic relationships among species (Testolin and Ferguson, 1998
). Computations were carried out using the NTSYS-pc v.1.80 package (Rohlf, 1994
).
To evaluate the role of vegetative reproduction in determining the patchy structure of some populations, we undertook a detailed electrophoretic analysis of two small patches in GRE. One patch (GRE1) contained ten clumps of C. crispa; the other patch (GRE2) contained nine clumps. The clumps within each patch were growing sufficiently close to one another to suppose that they could have originated from the same creeping rhizome. This was tested by isozyme electrophoresis of frond tissue. We chose GRE because it was the best established population, with the largest number of plants and a clear patchy structure.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Ultimately a typical homosporous fern cordate prothallus is formed. The prothalli of Cryptogramma crispa are unisexual. Female gametophytes are larger than males and have well-developed wings on either side of a prominent notch. The male prothalli may also have wings (although these are not as well defined as in female prothalli) or may be ameristic with irregular shapes. The gametangia are typical of those in leptosporangiate ferns.
Sexual expression
Male and female prothalli appeared in all cultures, both multispore and single and paired gametophyte cultures. In LNE, PEÑ, and STI, antheridia appeared first (Fig. 1A–C), but in the other cultures male and female prothalli were simultaneous (Fig. 1D–F).
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The percentage of presexual prothalli decreased during the 5 mo of culture, while the percentage of males and females increased. Only occasionally were bisexual prothalli observed in some of the cultures (Fig. 1D,E).
After 10 mo, in multispore cultures young sporophytes were observed only in SCO and MON. The first sporophyte was observed in SCO after 6 mo of culture. After 10 mo there were 20 sporophytes in SCO and two in MON. No evidence of fertilization was detected in any other cultures.
Isolate potential
In the isolated gametophyte cultures, no sporophytes were observed over the entire 6-mo period of that experiment. Nevertheless, it is clear from the sexual expressions summarized in Table 2, that self-fertilization could only be possible in the bisexual prothalli of SCO.
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Isozymes electrophoresis
It is often difficult to interpret electrophoretic data from polyploid species (Tomiuk and Loeschcke, 1991
; Schneller and Holderegger, 1996
; Strefeler et al., 1996
). Due to the polyploid nature of C. crispa it was not possible to assign bands to specific alleles or even loci. However, in those enzymes clearly showing different activity zones, these have been considered to be different loci.
The frequencies of enzyme phenotypes are summarized in Table 4. From the ten enzymes assayed, only eight, apparently encoded by 11 gene loci, were interpretable (not IDH or PGM), although only two of the three activity zones detected in MDH could be clearly resolved. Three loci were monomorphic: HEX, MDH-3, and PGI-1. Seven loci were polymorphic: PGI-2, TPI, SDH, PGD, LAP, AAT, and MDH-2. With the exception of LAP (in which several enzyme phenotypes appeared with similar frequencies scattered among the different populations), the most frequent enzyme phenotype for each polymorphic locus was the same in all populations. In PGI-2 phenotype "e" was the most frequent; in TPI, SKD, PGD, AAT, and MDH-2 the "a" phenotype was the most frequent (Table 4). There were also some unique enzyme phenotypes, although with very low frequencies. This occurred especially in PGI-2 where each population, except MON, had at least one unique phenotype.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 1971
). Our results from the morphological development and observation of unisexuality are similar to those Momose (1964)
found studying Japanese plants of the same species. All spore collections were viable and within each population spore germination and gametophyte development were normal. Thus, spore viability and developmental problems do not appear to be limiting the reproductive potential of C. crispa.
All multispore cultures, whether initiated from spores from one or several plants, developed into a bigametophytic system (Klekowski, 1969
), with only male and female prothalli, except for the occasional presence of bisexual gametophytes in HU and SCO with very low percentages. Gametangial formation was slower than in other genera, e.g., Asplenium (Pangua, Lindsay, and Dyer, 1994
; Prada et al., 1995
), Cystopteris (Pajarón et al., 1996
), Athyrium (Schneller, 1979
), Pteridium (Lindsay, 1992
), and Blechnum (Cousens, 1979
).
The presence of male and female prothalli simultaneously implies an intergametophytic breeding system. The presence of a large number of male gametophytes surrounding the female ones suggests that an antheridiogen system influences gametangial production. Moreover, this conclusion was supported by the results of sowing spores around a mature female prothallus. The number of male gametophytes decreased as the distance from the female prothalli increased. Döpp (1959)
showed that this species was sensitive to the antheridiogen type "A" of Pteridium aquilinum, as were other homosporous fern species (Döpp, 1959
; Haufler and Gastony, 1978
; Chiou and Farrar, 1997
).
From the results of the isolated and paired gametophyte cultures, the following general conclusions can be made. Most of the prothalli remain unisexual, and most of these are female, but in all the cultures a significant proportion remained presexual. Gametangia formation is slower in isolated and paired gametophytes than in multispore cultures. Although unknown factors could be operating, the fact that gametangia development was too slow in the isolated and paired cultures might be the reason why so few sporophytes were produced. Thus, gametophytes of Cryptogramma crispa need a long time to produce gametangia, at least when more or less isolated, which is the way they probably grow in nature. If spores germinate in autumn, just after release, or in the next spring (see Pangua, García-Álvarez, and Pajarón, in press
), the development of antheridia and archegonia will be under the worst climatic conditions, freezing temperatures in winter or water lacking in summer, which could affect fertilization. On the other hand, in multispore cultures, as can be seen in Fig. 1D–F, even when a large number of antheridia producing gametophytes are present (and have been present continuously for at least 2 mo, alongside archegonia producing gametophytes), the first evidence of fertilization is not detected until after 6 mo of culture. Anyway, whether gametangia ontogeny, unknown factors, or both are responsible, there is a fertilization delay, and then the growing season plays an important role in the establishment of sporophytes. The growing season at the localities where C. crispa grows in Spain is reduced to 4 mo, usually June to September, or even less at the highest altitudes (Rivas-Martínez, 1987
). Thus, although other factors or genetic load may be affecting too, the long time needed by C. crispa to produce gametangia and to fertilize, together with the short growing season at the localities where it grows must be related with the unsuccessful sporeling establishment in the wild. Moreover, the scarcity in these rock boulder fields of safe sites for spore germination and gametophyte establishment adds difficulties to the reproduction process.
The mode of sexual expression provides evidence for outcrossing as the main breeding system in this species. This hypothesis has been tested by starch gel electrophoresis of isozymes. Although we have not been able to calculate the average individual heterozygosity, or the average number of alleles per locus, a measure of genetic variability has been obtained from the percentage polymorphic loci (P). All five populations showed high levels of polymorphic loci, ranging from 40% in MON to 70% in LNE. The MON population, which shows the lowest value and the highest levels of intrapopulational similarity (Fig. 3), is a small population growing at the lower altitudinal limit of this species in the Iberian Peninsula. This population is established in a siliceous protruding mountain surrounded by a great calcareous range, therefore it is more or less isolated. This isolation and the small size of the population may have led to the loss of genetic variation through genetic drift (Soltis and Soltis, 1990a
).
The high levels of similarity found in patches GRE1 and GRE2 (Figs. 4 and 5) can be explained by the small number of individuals, 10 and 9, respectively. Although they were similar, they were not identical as would be expected in vegetatively produced individuals. From the analysis of the whole GRE population shown in Fig. 9, in which the similarity matrix was obtained with plants from GRE and from both patches GRE1 and GRE2 excluding the MDH results, can be deduced the fact that genotypes seem to be randomly distributed through the population. Thus, it seems that no pattern in the distribution of genotypes exists. Although only two patches were tested, we think that the patchy structure of this population is not related to the existence of a mainly vegetative reproduction system, and outcrossing should be the main reproduction system in it (Soltis and Soltis, 1990a
). Habitats of rock-inhabiting species are characterized by the occurrence of very restricted safe sites (Holderegger and Schneller, 1994
). Thus, ecological factors, such as limited suitable habitats for sporophytes and safe sites for spore germination and gametopyte establishment, due to the rock boulder fields where it usually grows, determine the patchy structure in this species. However, the spatial structure of plant distribution is not accompanied by a genetic patchiness in the population.
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found population-level values of P between 54.5 and 69.1% (average 62.1%) for outcrossing species in the genus Pleopeltis. These values are similar to the highest values reported by Soltis and Soltis (1990a)
for two other outcrossing fern species, Pellaea andromedifolia (Kaulf.). Fée and Bommeria hispida (Kuhn) Underwood. Moreover, as can be seen in the dendrogram of all populations, the clusters observed consist of plants from all or nearly all populations. Thus, each individual population contains most of the variability detected in the Iberian range of this species; this again suggests that this species has an outcrossing breeding system (Hamrick, Linhart, and Mitton, 1979
; Haufler and Soltis, 1984
; Watano, 1988
). The fact that the most common isozyme phenotypes are the same in all populations indicates that there is probably a high level of gene flow among populations. Nevertheless, each population, again except MON, which is the less variable, shows a unique combination of bands that can be related to the presence of unique alleles. These peculiar band patterns, and therefore these rare alleles, are present in low frequencies, which is compatible with the existence of an intergametophytic breeding system.
To date there have been few comparative studies of the breeding systems in diploid and polyploid fern species. However, the general conclusion from these studies (e.g., Masuyama, 1979
; Masuyama and Watano, 1990
; Soltis and Soltis, 1990b
) is that diploid fern species tend to be outcrossers and that polyploid fern species tend to be inbreeders. The results obtained in this study have clearly demostrated that C. crispa, a tetraploid species, is an exception to this general trend. Moreover, C. crispa shows a "type A" ontogenetic sequence of gametangia formation (Masuyama and Watano, 1990
), which until now has been a character more frequently associated with diploid species. Moreover, there is good evidence that the species has retained an antheridiogen system to promote outcrossing and the gametophytes are primarily unisexual, which have been also more frequently related with diploid than with polyploid species.
The only data available for other Cryptogramma species relate to C. stelleri, the diploid from eastern United States, Canada, and Asia. Peck, Peck, and Farrar (1990)
, discovered in a study of this species in the United States that although 50% of gametophytes were bisexual, they were carrying a high genetic load (98%) and had a correspondingly low selfing potential (2%). Thus, C. stelleri appears to follow the general trend of being a diploid outcrosser. Compared with C. stelleri, the proportion of bisexual prothalli produced by C. crispa in this study was notably lower, and no sporophytes were observed in isolated bisexual gametophytes.
The diploid breeding behavior of C. crispa points toward the possibility that this could be another example of a polyploid fern species that has undergone, or is undergoing, genetic diploidization (Werth, Guttman, and Eshbaugh, 1985
; Werth, 1989
; Werth and Windham, 1991
). This fact, perhaps not surprising given the apparent antiquity of this species, must be studied in more detail.
Analysis of the origin of tetraploid Cryptogramma crispa and, as a consequence, the knowledge of its diploid parents, or parent, would help to clarify the interpretation of the isozyme results. This interpretation will give more accurate information about its genetic variability, reproductive behavior, and the ocurrence of genetic diploidization.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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———. 1993 Cryptogramma. In Flora of North America editorial committee [eds.], Flora of North America, vol. 2. Oxford University Press, New York, NY.
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