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. 2006 Sep;98(3):609–618. doi: 10.1093/aob/mcl137

Spore Fitness Components Do Not Differ Between Diploid and Allotetraploid Species of Dryopteris (Dryopteridaceae)

LUIS G QUINTANILLA 1,*, ADRIÁN ESCUDERO 1
PMCID: PMC2803563  PMID: 16845140

Abstract

• Background and Aims Although allopolyploidy is a prevalent speciation mechanism in plants, its adaptive consequences are poorly understood. In addition, the effects of allopolyploidy per se (i.e. hybridization and chromosome doubling) can be confounded with those of subsequent evolutionary divergence between allopolyploids and related diploids. This report assesses whether fern species with the same ploidy level or the same altitudinal distribution have similar germination responses to temperature. The effects of polyploidy on spore abortion and spore size are also investigated, since both traits may have adaptive consequences.

• Methods Three allotetraploid (Dryopteris corleyi, D. filix-mas and D. guanchica) and three related diploid taxa (D. aemula, D. affinis ssp. affinis and D. oreades) were studied. Spores were collected from 24 populations in northern Spain. Four spore traits were determined: abortion percentage, size, germination time and germination percentage. Six incubation temperatures were tested: 8, 15, 20, 25 and 32 °C, and alternating 8/15 °C.

• Key Results Allotetraploids had bigger spores than diploid progenitors, whereas spore abortion percentages were generally similar. Germination times decreased with increasing temperatures in a wide range of temperatures (8–25 °C), although final germination percentages were similar among species irrespective of their ploidy level. Only at low temperature (8 °C) did two allotetraploid species reach higher germination percentages than diploid parents. Allotetraploids showed faster germination rates, which would probably give them a competitive advantage over diploid parents. Germination behaviour was not correlated with altitudinal distribution of species.

• Conclusions 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.

Keywords: Allopolyploidy, Dryopteris, fitness component, northern Spain, spore abortion, spore germination, temperature

INTRODUCTION

Polyploidy is an important mode of speciation in vascular plants. A recent estimate in angiosperms indicates that approx. 70 % of all species are polyploids (Masterson, 1994). In pteridophytes, polyploidy has been suggested to occur in 95 % of species (Grant, 1981; but see also Soltis and Soltis, 1989). While the understanding of the genetic and genomic consequences of polyploidy has increased dramatically in the last two decades, the adaptive consequences of polyploidy remain poorly known (for reviews, see Ramsey and Schemske, 2002; Soltis et al., 2003). The establishment of rare polyploids within diploid populations may be constrained by a frequency-dependent mating disadvantage, a principle referred to by Levin (1975) as minority cytotype exclusion. On the other hand, theoretical studies have identified some of the factors that may enhance the establishment and persistence of polyploids among their diploid progenitors. Most authors consider that hypothetical higher fitness of polyploids, together with intercytotype reproductive barriers, are the main determinants of polyploid success (see Rodríguez, 1996, and references therein).

Fitness comparison of related cytotypes should consider the consecutive different life stages of individuals (Campbell, 1991). The independent gametophytic generation of pteridophytes comprises several sequential stages connected by transitional processes, i.e. sporogenesis, spore dispersal and germination, gametophyte survival and fecundity, each process constituting a fitness component (van Tienderen, 2000). Polyploids, at least those of recent origin, must overcome some initial drawbacks, such as the incidence of meiotic aberrations (e.g. irregular disjunction resulting from univalent and multivalent formation) and other processes (reviewed in Ramsey and Schemske, 2002) which lead to the production of non-viable spores. Consequently, neopolyploid pteridophytes might have a fitness disadvantage at sporogenesis. Unfortunately, the available information on this possibility is scarce compared with the abundant data on pollen viability and seed set in angiosperms (Ramsey and Schemske, 2002). For example, Wagner and Chen (1965) noted that Dryopteris species have some abortive spores and suggested that polyploids show ‘more irregular’ spores than diploids. In contrast, Whittier and Braggins (1994) found similar abortion percentages in spores from diploid and tetraploid Psilotum nudum populations. Sheffield et al. (1993) obtained high germination percentages for apparently aneuploid spores of triploid Pteridium aquilinum.

In the context of plant polyploidy, one of the most thoroughly investigated fitness components is diaspore (seed or spore) germination (e.g. Whittier and Braggins, 1994; Burton and Husband, 2000). Phenotypic differences between polyploids and related diploids at any life stage may be the consequence of three mechanisms. First, chromosome doubling leads to an increase in nucleus and cell sizes, which in turn may affect whole-plant morphology (Stebbins, 1971). Pteridophyte spores (e.g. Moran, 1982) and angiosperm seeds (e.g. Bretagnolle et al., 1995) are frequently larger in polyploids than in diploid parents. The relatively slower germination rate of some polyploids may, in part, result from lower metabolic rates and slower mitotic division rates of larger cells with more chromosomes (Gottschalk, 1976; Cavalier-Smith, 1978). Secondly, in addition to these biophysical effects of increased DNA content, polyploidy usually involves important genetic and epigenetic modifications (Wendel, 2000; Liu and Wendel, 2003). Among them, increased heterozygosity has been correlated with increased vigour of polyploids in diaspore germination, and other fitness components (e.g. Soltis and Rieseberg, 1986; Tomekpe and Lumaret, 1991). Thirdly, phenotypic differences between related cytotypes may reflect evolution since the time of polyploid formation (Bretagnolle and Lumaret, 1995), i.e. genetic differentiation via natural selection, genetic drift, etc.

Comparative investigations on spore germination of allopolyploid ferns and their progenitors show taxa-specific results. For example, spore germination of some allotetraploid Polypodium (Kott and Peterson, 1974), Polystichum (Pangua et al., 2003) and Dryopteris (Whittier, 1970) was faster and reached higher percentages than that of their diploid ancestors. In contrast, two allotetraploid Dryopteris, D. corleyi and D. guanchica, and their common diploid parent, D. aemula, showed similar germination percentages (Quintanilla et al., 2002). Several allotetraploid Asplenium also had germination traits similar to those of their diploid parents (Prada et al., 1995). The main goal of the present study was to determine the consequences of allopolyploidy on some spore fitness components and to identify any adaptive implications in a model group of Dryopteris species.

Dryopteris in northern Spain constitutes a suitable experimental set to analyse the effects of genetic and environmental factors on spore germination. This reticulate complex includes diploids, triploids and tetraploids with well established genomic relationships (e.g. Gibby et al., 1977; Fraser-Jenkins, 1982; Viane, 1986; Widén et al., 1996; Geiger and Ranker, 2005), growing in a variety of habitats. In addition, the sporophytes of most species lack extensive clonal growth and thus depend on the establishment and mating of gametophytes. In this report, two fitness components are studied, i.e. spore abortion percentage and germination percentage; and two spore traits with potentially adaptive consequences, i.e. spore size and germination rate. Four hierarchical factors are considered: ploidy level, altitudinal distribution, species and population. Differences both among species and among populations have been reported in fern spore germination (e.g. Cousens, 1981; Pangua et al., 1994; Quintanilla et al., 2000). The following questions are specifically addressed. (a) Do diploids and allotetraploids differ in sporogenesis success or spore size? (b) What are the effects of temperature on germination percentage and germination rate of the species, considering their interpopulation variation? (c) Do species with the same ploidy level have similar germination behaviour? (d) Are there common germination responses to temperature among species with similar altitudinal distribution?

MATERIALS AND METHODS

Studied taxa

Six Dryopteris taxa (buckler-ferns) naturally growing in Spain were included: D. aemula, D. affinis ssp. affinis, D. corleyi, D. filix-mas, D. guanchica and D. oreades. Taxa were identified on the basis of macro- and micromorphological characters (see Salvo and Arrabal, 1986; Viane, 1986). These ferns are diploids (2n=2×=82) or allotetraploids (2n=4×=164) in the sporophyte generation so their spores contain a haploid or diploid complement of chromosomes, respectively (Table 1). The only exception is D. affinis ssp. affinis (hereafter referred to as D. affinis), in which both sporophyte and spores are diploid with two distinct genomes (oreades and an unknown ‘pure’ affinis ancestor; Fraser-Jenkins, 1980). This fertile hybrid is obligately apogamous: sporophytes arise from somatic cells of the gametophyte (Sheffield et al., 1983). The sporophytes of studied taxa lack extensive clonal growth, the only exception being D. oreades which has spreading, freely branched rhizomes. The studied taxa were classified into three groups according to their altitudinal distribution in Spain (Table 1): ‘thermophiles’ live at altitudes below 1000 m, preferably near the coast; ‘cryophiles’ grow at high altitudes in the mountains, usually above 1500 m; and ‘mesophiles’ have wide altitudinal ranges. Thermophiles and mesophiles live in a variety of forest and scrub habitats, whereas cryophiles prefer steep screes and rocky ledges (Fraser-Jenkins, 1982; Page, 1997). In most sites, several species co-exist. Most potential hybrid combinations have been described (e.g. D. × asturiensis = D. affinis × corleyi), including triploid backcrosses (e.g. D. ×arecesiae=D. aemula × corleyi).

Table 1.

Putative genome constitution and altitudinal distribution of the Dryopteris taxa studied

Taxon Sporophyte genome constitution* Spore genome constitution (spore ploidy) Altitude range in m (habitat)†
D. aemula AA A (1×) 0–900 (thermophile)
D. oreades OO O (1×) 600–2400 (cryophile)
D. affinis ssp. affinis OH OH (2×) 0–2000 (mesophile)
D. corleyi AAOO AO (2×) 50–650 (thermophile)
D. filix-mas CCOO CO (2×) 100–3100 (mesophile)
D. guanchica AAII AI (2×) 0–1000 (thermophile)
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*A, aemula genome; O, oreades genome; H, hypothetical ‘pure’ affinis genome; C, caucasica genome; I, intermedia genome.

†Altitudes taken from Flora iberica (Salvo and Arrabal, 1986).

Plant material

Four populations per species were sampled in northern Spain (Table 2). Spores were obtained from ten individuals randomly selected per population. Fragments of lamina were collected with mature but closed sporangia (the only exception being D. oreades, which had started spore release at the collection date). Spore release was promoted by drying the fragments on sheets of smooth paper for 2 weeks in the laboratory. Spores from the ten individuals of each population were then pooled prior to beginning germination tests. Samples of these pools were mounted in DePeX (BDH Chemicals, Poole, UK) to determine spore abortion and spore size. Spore abortion percentage was estimated by counting the number of aborted spores in four random samples of 100 spores per population. Spores were considered aborted when they lacked a protoplast or were collapsed. Spore size was based on a measurement of the longest axis excluding the perispore, i.e. exospore length. Thirty non-aborted spores were measured from every population. Measurements were made using a light microscope (600×) equipped with a drawing tube, connected to a digitizing tablet (Intuos A3, Wacom, Saitama, Japan) (see Viane, 1990), and the program ImageTool (version 3·0, UTHSCSA, http://ddsdx.uthscsa.edu/dig/itdesc.html). With this method, the accuracy reached was 0·6 µm.

Table 2.

Populations from which spores were collected

Species, with province*, location and altitude Collection date, and voucher†
D. aemula
    C. Eume river near to Caaveiro, 50 m 2 August 2003, PQ 706
    C. Mera river near to Soutochao, 250 m 7 August 2003, PQ 711
    C. Castro river near to Igrexafeita, 250 m 7 August 2003, PQ 713
    C. San Xusto river near to Toxosoutos, 160 m 11 August 2003, PQ 715
    D. oreades
    Le. Ancares range, Laguna Ferreira, 1790 m 13 August 2003, PQ 662
    Le. Ancares range, Ancares pass, 1710 m 13 August 2003, PQ 663
    Le. Ancares range, between Ancares pass and Miravalles peak, 1700 m 14 August 2003, PQ 664
    Lu. Caurel range, Faro peak, 1440 m 15 August 2003, PQ 674
    D. affinis
    C. Mandeo river near to Chelo, 40 m 2 August 2003, PQ 701
    C. Eume river near to Caaveiro, 50 m 2 August 2003, PQ 705
    C. Belelle river near to Fervenza power station, 100 m 7 August 2003, PQ 708
    C. Castro river near to Igrexafeita, 250 m 7 August 2003, PQ 712
D. corleyi
    O. N-634 road between Buelna and Santiuste, 80 m 20 August 2003, PQ 678
    O. N-634 road near to Pendueles, 70 m 21 August 2003, PQ 679
    O. Purón river near to El Peñatu, 40 m 21 August 2003, PQ 680
    O. Purón river near to Candal, 40 m 21 August 2003, PQ 681
D. filix-mas
    Lu. Caurel range, Devesa da Rogueira forest, 1250 m 15 August 2003, PQ 671
    Lu. Caurel range, Devesa de Fonteformosa forest, 1360 m 15 August 2003, PQ 673
    Lu. Caurel range, Carbedo, 820 m 15 August 2003, PQ 675
    Lu. Ancares range, Pedrafita pass, 1100 m 17 August 2003, PQ 676
D. guanchica
    C. Mandeo river near to Chelo, 40 m 2 August 2003, PQ 700
    C. Eume river near to Caaveiro, 100 m 2 August 2003, PQ 704
    C. Belelle river near to Fervenza power station, 100 m 7 August 2003, PQ 707
    C. Castro river near to Igrexafeita, 250 m 7 August 2003, PQ 714
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*Abbreviations of Spanish provinces according to Castroviejo et al. (1986).

†Voucher specimens are deposited at the herbarium of the Real Jardín Botánico de Madrid. PQ, Pías & Quintanilla.

Germination tests

Spores were sown on mineral agar (see Dyer, 1979, p. 282) containing the fungicide Nystatin (100 U mL−1) in 5·5 cm diameter plastic Petri dishes subsequently sealed with Parafilm. Spores were incubated with a 12 h light/12 h dark photoperiod (daylight fluorescent tubes, photon irradiance 30–45 μmol m−2 s−1 in the 400–700 nm region). Germination trials were conducted at five constant temperatures: 8, 15, 20, 25 and 32 °C, and an 8/15 °C alternating temperature. In this latter treatment, the lower temperature corresponded to the 12 h period of darkness. This set of temperatures was selected according to field conditions (Fig. 1). For each treatment, four Petri dishes (replicates) of each population were incubated. Every 2 d, 100 non-aborted spores were selected at random on each dish to determine the germination percentage. The criterion of germination was the protrusion of the rhizoid initial out of the spore coat (Turnwald et al., 1999). The germination percentages became stable—no change in final germination—before 1 month at 20, 25 and 32 °C; in the rest of the treatments, a second month of monitoring was necessary. The variables gathered were the final germination percentage and the number of days needed to reach 50 % of final germination (‘germination time’ hereafter).

Fig. 1.

Fig. 1.

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Monthly temperatures at two localities representative of the warmest (A) and the coldest (B) conditions among the studied populations. Shade temperatures were recorded from 1·5 m above the ground with an HOBO Pro logger (Onset Computer Corporation, Pocasset, MA, USA). Mean and absolute values are based on temperatures recorded every hour for each month. The studied species present in these localities are Dryopteris aemula (A), D. affinis (A, B), D. filix-mas (B), D. guanchica (A) and D. oreades (B). The horizontal lines mark the temperatures used in the germination tests.

Statistical analyses

Generalized linear models (GLMs; McCullagh and Nelder, 1989) were built for the following spore traits: abortion percentage, size, germination time and germination percentage using S-PLUS (version 7, Insightful Corporation, http://www.insightful.com/products/splus/). GLMs were used because response variables clearly departed from standard normality assumptions, and because of the hierarchical nature of the statistical models used (see below). It is known that hierarchical data structure implies correlation between data points at different scales, inflating the error degrees of freedom and increasing the chance of making a Type I error. In order to overcome these problems, the data were analysed using a multilevel GLM approach. Two types of hierarchical GLMs (Escudero et al., 2002) were prepared. First the so-called genomic model, in which the variability accounted for by populations was nested within species and these within the corresponding spore ploidy (1× or 2×). Specifically, an analysis was carried out of whether or not the level of ploidy exerts a control on the modelled variables once the interpopulation and interspecies variability are partialled out. The second model, applied only to germination time and germination percentage, is the so-called ecological model in which species appear nested within the type of habitat range (thermophilous, cryophilous or mesophilous). Another term related to the germination temperature was added to this model. Two of the tested temperatures were not included: 32 °C (because germination was nil) and 8 °C (because most species germinated at extremely slow rates reaching very low percentages). Since it was expected that the effect of temperature would be species dependent, the corresponding term was nested within species.

A binomial response function was set for the two percentage variables (aborts and total germination) and a logit link function. A gamma error with an inverse link was used for the germination time because under these conditions the explained variation was maximal (Guisan et al., 2002). Finally the exospore length was modelled with a Gaussian response and an identity link. χ2 tests were conducted to evaluate whether or not selected predictors explained a significant fraction of the total deviance (Guisan et al., 2002).

RESULTS

The spore abortion percentages and the exospore lengths of each population are shown in Fig. 2. The models used to test the effects of ploidy, species and population on these variables are presented in Table 3. Ploidy and species had no significant effect on spore abortion percentage. Almost all populations had low abortion percentages (means <10 %), with the exception of two D. corleyi populations (16 and 20 %). Exospore lengths showed significant differences on all analysed levels: ploidy, species andpopulation. Diploid spores (corresponding to allotetraploids and apogamous diploid D. affinis) were bigger than haploid spores, with wider variation in the former. Exospores of the allotetraploids D. guanchica (species means ± 1 s.e., n=120: 40·3 ± 0·3 µm) and D. filix-mas (40·4 ± 0·3 µm) were slightly longer than those of diploid parents tested, D. aemula (36·4 ± 0·3 µm) and D. oreades (39·0 ± 0·2 µm), respectively. However, the allotetraploid D. corleyi (49·1 ± 0·5 µm) had significantly larger spores than diploid parents, D. aemula and D. oreades. Dryopteris affinis spores (44·6 ± 0·4 µm) were larger than those of the related species tested, D. oreades and D. filix-mas.

Fig. 2.

Fig. 2.

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Mean values (± 1 s.e.) of the spore abortion percentage and of the exospore length for the six Dryopteris species. Each dot corresponds to one population. Open and filled symbols are for species with haploid and diploid spores, respectively. Each mean abortion percentage is for four samples (n = 4) of 100 spores and each mean exospore length is for 30 non-aborted spores (n = 30).

Table 3.

Results of hierarchical GLMs to explore the effects of spore ploidy (1× or 2×), species and population on spore abortion percentage and exospore length of the six Dryopteris species

Spore abortion percentage
 Exospore length
Effect in model d.f. Change in deviance P for χ2 test on deviance d.f. Change in deviance P for χ2 test on deviance
Ploidy 1 4·10 0·6774 1 17 095·15 0·0000
Species (ploidy) 4 1·81 0·6829 4 10 428·50 0·0000
Population [species (ploidy)] Not tested 18 9965·10 0·0220
Null 95 4·27 719 22 637·68
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Abortion percentage was modelled assuming binomial distribution, with logit link function. Exospore length was modelled using a Gaussian distribution, with identity link. Significant effects (P < 0·05) are indicated in bold. The population level could not be tested for abortion percentage due to convergence problems related to the binomial nature of this variable and the relatively small sample size.

d.f., degrees of freedom.

The germination percentages per population under different temperature conditions are presented in Fig. 3, and the germination times are given in Fig. 4. One of the four D. oreades populations was not included in germination tests due to fungal contamination of the culture media. The models used to analyse the effects of ploidy or habitat preference, species, population and temperature on these variables are summarized in Table 4. Overall, except for the most extreme temperatures 8 and 32 °C, the final germination percentages were high, with populations of all species exceeding 85 % at any of the temperature treatments. None of the species germinated at 32 °C. At 8 °C there was a marked decline in germination, but the differences among species were great. Most species germinated slowly and reached low percentages, with population means generally <10 %, and D. aemula showed zero germination. In contrast, D. corleyi and D. guanchica germinated successfully, with most population means >70 %. The effects of 8 and 32 °C were not lethal but inhibitory, as the same spores transferred to adequate conditions (20 °C) did germinate (results not shown). Given the marked inhibitory effects of 8 and 32 °C, both temperatures were excluded from the models to improve statistical power. Habitat, species and population had no significant effects on germination traits (Table 4). Ploidy and incubation temperature significantly affected germination time but not germination percentage (Table 4). Specifically, diploid spores germinated faster than haploid spores and germination times increased with decreasing temperatures (Fig. 4). The times of 8/15 °C treatment were only slightly longer than those at constant 15 °C.

Fig. 3.

Fig. 3.

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Mean values of the final germination percentage for each population of the six Dryopteris species cultured at different temperatures. The same symbol and line style in Figs 3 and 4 correspond to the same population. For clarity, standard errors are not shown. Each mean is for four Petri dishes (n = 4), on each of which 100 non-aborted spores were evaluated.

Fig. 4.

Fig. 4.

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Mean values of the germination time for each population of the six Dryopteris species cultured at different temperatures. The same symbol and line style in Figs 4 and 3 correspond to same population. For clarity, standard errors are not shown. Germination time is the number of days needed to reach 50 % of final germination (n = 4 Petri dishes per population).

Table 4.

Results of hierarchical GLMs to explore the effects of spore ploidy (1× or 2×), habitat (thermo-, meso- or cryophilous), species and population on germination percentage and germination time of the six Dryopteris species

Germination percentage
 Germination time
Model, and effect in model d.f. Change in deviance P for χ2 test on deviance d.f. Change in deviance P for χ2 test on deviance
Genomic
    Ploidy 1 46·39 0·0517 1 81·26 0·0186
    Species (ploidy) 4 43·20 0·5277 4 76·93 0·3633
    Population [species (ploidy)] 17 27·33 0·5329 17 74·33 1·0000
    Temperature [species (ploidy)] 6 23·91 0·7539 6 14·38 0·0000
    Null 367 50·17 367 86·80
Ecological
    Habitat 1 50·17 0·9833 1 84·91 0·1698
    Species (habitat) 4 43·20 0·1376 4 76·93 0·0922
    Population [species (habitat)] 17 27·33 0·5329 17 74·33 1·0000
    Temperature [species (habitat)] 6 23·91 0·7539 6 14·38 0·0000
    Null 367 50·17 367 86·80
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Germination percentage was modelled assuming binomial distribution, with logit link function. Germination time was modelled using a gamma distribution, with inverse link. Significant effects (P < 0·05) are indicated in bold.

d.f., degrees of freedom.

DISCUSSION

Spore abortion

The present results indicate that success of sporogenesis in Dryopteris is not constrained by allopolyploidy. Spore abortion percentages were low in most populations, and differences owing to ploidy level and species were not significant. This is consistent with the regular bivalent formation observed in the two allotetraploids D. filix-mas (Manton, 1950) and D. guanchica (Gibby et al., 1977). Two populations of the other allotetraploid species, D. corleyi, had the highest abortion percentages (Fig. 2). These may be due to meiotic abnormalities and other genotypic and phenotypic effects that reduce the fertility of polyploids, especially recently formed polyploids (reviewed by Ramsey and Schemske, 2002). There is low chloroplastic molecular divergence between D. corleyi and its parent D. aemula (Geiger and Ranker, 2005), suggesting that the origin of D. corleyi is a relatively recent event. A cytogenetic survey of this species found only bivalents in sporogenesis (Fraser-Jenkins and Gibby, 1986). However, chromosome pairing behaviour in tetraploids can vary at the inter- and intrapopulation level (e.g. Cubas and Sleep, 1994; Stuessy et al., 2004) and the study populations may have meiotic abnormalities. Studies on pollen viability and seed set show that fertility of neopolyploids is increased rapidly by natural selection (Ramsey and Schemske, 2002). Relatively lower fertility in some D. corleyi populations could be due to lack of time for selection against causes of spore abortion.

Spore abortion percentages were similar in the apogamous D. affinis and the related sexual species D. oreades and D. filix-mas (the three taxa share the D. oreades genome; Table 1). In the 16-meiocyte sporangia of D. affinis, there is virtually no bivalent formation probably due to the presence of two different genomes, one from D. oreades and one from a hypothetical ‘pure’ D. affinis ancestor (Manton, 1950; Corley, 1967). Consequently, all the spores from such sporangia are abortive. However, the majority of sporangia in this hybrid taxon have eight tetraploid meiocytes, giving rise to 32 viable diplospores (Manton, 1950). Sheffield et al. (1983) did not find evidence that sporangial development of D. affinis was in any way imperfect, in agreement with the low abortion percentages obtained here.

Spore size

Diploid spores are longer than haploid spores in the studied buckler-ferns. There are also significant differences at the species and population levels. Assuming that the shape of spores approximates a prolate spheroid (see Beyer, 1987), and that the length : width ratio is a constant, the relative increase in volume with polyploidy is equal to the cube of the relative increase in length. Similarly, the relative increase in surface area is equal to the square of the relative increase in length. For example, the volume of the longest spores (Fig. 2; mean = 50·4 µm in a D. corleyi population) is 2·8 times greater and the surface area is twice that of the shortest spores (mean = 35·8 µm in a D. aemula population).

The increase in volume and reduction in surface-to-volume ratio associated with polyploidy has been described in various cell types and diverse plant groups (Stebbins, 1971). In ferns, the abundant information on polyploid cell sizes, especially spores and guard cells, confirms these findings (reviewed by Barrington et al., 1986). However, a similar spore size between polyploids and their diploid progenitors has been reported in several species assemblages, including some Dryopteris (e.g. Britton, 1968; Wagner, 1971; Viane, 1985). It was found that D. guanchica and D. filix-mas have spores slightly bigger than their diploid parents included here, D. aemula and D. oreades, respectively. In contrast, D. corleyi, the allotetraploid derived from these diploids, has exceptionally big spores not only in the context of the results presented here but in the whole European tetraploid Dryopteris (see, for example, lengths in Salvo and Arrabal, 1986). There is evidence that the cell volume of some polyploid plants tends to decrease over time, towards the levels of related diploids (Butterfass, 1987). If this tendency was followed by the spore size of allotetraploid Dryopteris, it would support the above-proposed recent origin of D. corleyi.

Spore size also differed among populations within the studied species. The evolutionary significance of intra- and interspecific variation in spore size is not well known, and selective pressures on this character may be complex and even conflicting. For example, the effect of small size may be beneficial to dispersal far from the parent sporophyte but may have adverse effects on gametophyte establishment. Cox and Hickey (1984) concluded that different climatic conditions among populations explain megaspore size variation in Isoetes. However, since spores of each species were collected within a narrow area with small local variation in climate, differences among populations are probably not a consequence of different ecotypes, but are the outcome of the parent sporophyte environment (see Andersson and Milberg, 1998, and references therein).

Germination time

Incubation temperature had a marked effect on germination time of all populations from all species. This effect consisted of an increase in time taken for germination to occur with decreasing temperature, especially at 8 °C. The treatment alternating between 8 °C (darkness) and 15 °C (light) yielded germination rates only slightly slower than those at constant 15 °C, indicating that germination rate is mainly constrained by day temperatures.

It was found that diploid spores germinate faster than haploid spores, in agreement with previous studies (e.g. Kott and Peterson, 1974). Timing of diaspore germination plays a critical role in plant fitness, with earlier germinators usually showing a competitive advantage (reviewed by Verdú and Traveset, 2005). The leaves of the studied buckler-ferns have a similar phenological pattern, emerging in spring and completing spore maturation during the summer (Willmot, 1989; Page, 1997). Spore dispersal also occurs synchronically among these species in late summer (the only exception being D. guanchica, which produces leaves and disperses spores throughout the year; personal observation). Cousens et al. (1985) suggested that microsites suitable for the growth of gametophytes are rare and may lead to the clustering of many different species in a single small space. In this scenario, differences in rate of germination might increase competitive ability of polyploids vs. diploid parents. Cavalier-Smith (1978) proposed that chromosome doubling alone (i.e. autopolyploidy) slows down metabolism and growth, probably due to the change in cell geometry. The studied allotetraploid Dryopteris have larger spores than their diploid ancestors, as seen above, and faster germination rates. Thus, hypothetical detrimental effects of increased nucleus and cell sizes may have been counteracted by genetic modifications induced by hybridization, such as increased heterozygosity (Wendel, 2000; Liu and Wendel, 2003), or by conventional evolution since the time of allopolyploid origin (Bretagnolle and Lumaret, 1995).

Germination percentage

Owing to the virtual absence of asexual reproduction in the studied species, spore germination is a prerequisite for sporophyte recruitment. Any competitive advantage of polyploids at germination would favour their establishment and persistence among diploid parents (Rodríguez, 1996). Nevertheless, allotetraploid and diploid Dryopteris showed similar high germination percentages at incubation temperatures of 8/15, 15, 20 and 25 °C. In contrast, none of the species germinated at 32 °C. Such a type of inhibition has been found previously in the genus (see Haupt, 1992, and references therein). Extreme temperatures in the spore collection sites are below this value, even on the hottest days of the summer (Fig. 1). However, germination is probably also prevented by lower temperatures between 32 and 25 °C. This could be an adaptation to avoid gametophyte exposure to harmful conditions such as dehydration.

At the other extreme, germination percentages at a constant 8 °C varied considerably among species. Most species germinated slowly and reached very low percentages at this temperature. In the mountains of northern Spain, temperatures drop abruptly after the summer (Fig. 1B), the season of spore dispersal. Thus, many spores will become dormant and will be incorporated into the soil spore bank. Viable spores of Dryopteris spp. have been found in soil samples (Schneller, 1988; Dyer and Lindsay, 1992). Spores in the soil bank could avoid freezing temperatures and germinate in the following spring. Dryopteris corleyi and, above all, D. guanchica are the only species that germinated successfully at 8 °C, in contrast to their diploid parents. For example, D. aemula, their common ancestor and, like them, thermophilous (Table 1), was the only species with zero germination at this temperature. The ability of both allotetraploids to germinate at low temperatures may imply a fitness advantage over sympatric congeners. Dryopteris corleyi and D. guanchica occur at low altitudes, where temperatures are mild throughout the year, and winters are almost frost free (Fig. 1A). Earlier gametophyte development of D. corleyi and D. guanchica during the winter would increase competitive success over diploids and reduce hybridization risk. Both allotetraploids form contact zones with their parent D. aemula. Given that the corresponding triploid backcrosses are sterile (Gibby and Widén, 1983; Pérez and Díaz, 1990), asynchrony in gametophyte development would prevent ineffectual matings. Consequently, minority cytotype disadvantage (Levin, 1975) could be reduced or, in other words, the co-existence of diploids and allotetraploids is favoured.

The present study considered only data on the relative fitness components spore set and spore germination percentages. If relative measures of reproductive success are poorly correlated with absolute figures expressed on a per individual basis, then evolutionary inferences drawn from the former may be misleading (see Herrera, 1991, and references therein). A similar relative success was found among cytotypes in sporogenesis and spore germination. However, allotetraploid Dryopteris have larger leaves than diploid parents (Salvo and Arrabal, 1986; Rünk et al., 2004), and, apparently, produce more spores per sporophyte (personal observation). This absolute reproductive advantage may favour polyploid performance.

Conclusions

Of the species studied, allotetraploids have slightly bigger spores, whereas spore abortion percentages are not significantly different from those of diploid ancestors. The only exception is the narrow endemic D. corleyi, with high abortion percentages and giant spores that may indicate recent allopolyploidization. Germination times decreased as temperatures increased over the range 8–25 °C, although final germination percentages were generally similar among species irrespective of their habitat preference or ploidy level. At 8 °C, however, only the allotetraploids D. corleyi and D. guanchica reached high germination percentages. It can thus be concluded that any realistic assessment of intercytotype differences at spore germination must consider a broad range of conditions, as has been suggested for a variety of plant fitness components (e.g. Heywood and Levin, 1984; Stratton and Bennington, 1998; Herrera, 2000). Further culture experiments are needed to assess the effects of other environmental factors, such as light and nutrient availability, on the germination of related cytotypes. In addition, although allotetraploids show faster germination rates, which would probably give them a competitive advantage, allopolyploidy lacks a general and constant effect on percentage of germination. Recent studies found many genic and genomic changes within the early generations after polyploidy formation (e.g. Song et al., 1995; Shaked et al., 2001; Adams et al., 2003). Consequently, hypothetical fitness constraints of allopolyploidization on sporogenesis or spore germination may have been counteracted by natural selection. Gametophyte populations of several fern species exist beyond the distributional range of the sporophyte generation (see Rumsey and Sheffield, 1996, and references therein). This observation and the present results indicate that other gametophytic or sporophytic fitness components following germination are more important in determining the competitive balance between polyploids and related diploids.

Acknowledgments

We thank Dr A. Dyer for insightful comments on the manuscript and linguistic advice, B. Pías for help with spore collection, and Dr B. Dove for useful information on his program ImageTool. We are also grateful to Dr E. Sheffield and an anonymous referee for suggestions which greatly improved the manuscript. This research was supported by University Rey Juan Carlos Project PPR-2004-53.

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