AOBPreview originally published online on September 4, 2002
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Annals of Botany 90: 461-467, 2002
© 2002 Annals of Botany Company
Effect of Storage Method on Spore Viability in Five Globally Threatened Fern Species
1 Departamento de Biología Vegetal I, Facultad de Biología, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain and 2 Departamento de Botánica, Facultade de Farmacia, Universidade de Santiago, Campus Sur, 15782 Santiago de Compostela, Spain
* For correspondence. Fax +34 91 394 5034, e-mail lugarqui{at}universia.es
Received: 13 March 2002; Returned for revision: 23 May 2002; Accepted: 28 June 2002 Published electronically: 4 September 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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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 °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 °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 °C. Wet storage at 5 °C prevented dark germination, and reduced bacterial and fungal contamination. Wet storage at –20 °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 °C).
Key words: Culcita macrocarpa, dark germination, Dryopteris aemula, Dryopteris corleyi, Dryopteris guanchica, ex situ conservation, spore germination, Woodwardia radicans.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Spores of many pteridophyte species have characteristics that make them ideal for ex situ conservation. Notably, they are easy to obtain in large quantities, require little storage space, and germinate rapidly without stringent culture requirements (Dyer, 1979). Conservation plans for a number of threatened fern species include propagation from spores (e.g. Estrelles et al., 2001; Lusby et al., 2002). In addition, pteridophyte spores typically maintain viability (i.e. ability to germinate) for a long time, although this characteristic varies considerably among species. The feature that has the greatest impact on spore viability is the presence of chlorophyll. A minority of pteridophytes produce chlorophyllous spores, which germinate faster but also decline in viability faster than non-chlorophyllous spores (Lloyd and Klekowski, 1970). Various hypotheses have been proposed to explain the reduced viability of chlorophyllous spores, including a higher respiratory rate (Lloyd and Klekowski, 1970) or inability to recover photosynthetic competence after desiccation (Lebkuecher, 1997).
Spore viability is clearly of key relevance for spore-based ex situ conservation efforts. Nevertheless, and in contrast to the extensive information available on seed conservation techniques, relatively little is known about the factors that affect spore viability. Traditionally, pteridophyte spores have been stored under dry conditions, either at ambient or low temperature (Dyer, 1979). Viable hydrated spores have, however, been found in soils many months after dispersion (for reviews, see Lindsay and Dyer, 1990; Dyer and Lindsay, 1992). Lindsay et al. (1992) found that spores of several fern species maintained under wet conditions showed a greater ability to germinate than spores maintained under dry conditions.
The aim of the present study was to identify suitable storage conditions for spores of five globally threatened fern species, all of which produce non-chlorophyllous spores: Culcita macrocarpa C. Presl (Dicksoniaceae), Dryopteris aemula (Aiton) O. Kuntze, D. corleyi Fraser-Jenkins, D. guanchica Gibby and Jermy (Dryopteridaceae) and Woodwardia radicans (L.) Sm. (Blechnaceae). These species constitute an ecologically and biogeographically homogeneous group. With the exception of D. corleyi, which is endemic to the coast of northern Spain, all are Macaronesian relicts (Pichi Sermolli, 1979; Pichi Sermolli et al., 1988) with a highly fragmented distribution in the Azores, Madeira and Canary Islands, and on the southern European coast. These species are considered rare not only because of their small geographic range, but also because of their narrow habitat specificities (see Rabinowitz, 1981). All five species require high relative humidity and mild temperatures throughout the year, conditions that typically occur in riverine woodland in steep-sided and frequently north-oriented valleys (Amigo and Norman, 1995). This is likewise an ideal habitat for certain species required for forestry, notably Eucalyptus globulus Labill.; as a result, the hazel- and alder-dominated woodlands of the northern Iberian Peninsula in which these fern species live are increasingly being felled for planting of eucalypts. All five species are included in Annex II of the Habitats Directive (Anon., 1992) and/or the Spanish Vascular Flora Red Data List (Aizpuru et al., 2000).
For identification of suitable storage conditions, spores were wet- or dry-stored at –20, 5 or 20 °C for 1, 6 or 12 months, with subsequent determination of the germination percentage over 30 d.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plant material
Five fern species were studied: Culcita macrocarpa, Dryopteris aemula, D. corleyi, D. guanchica and Woodwardia radicans. For each species, spores were obtained from ten individuals at one site in the north-west Iberian Peninsula (Table 1). In each case, fragments of lamina were collected with mature but closed sporangia. To prevent premature spore release, the fragments were transported to the laboratory in moist paper. In the laboratory, they were washed with abundant running water, and dried on sheets of smooth paper for 2 weeks. Spores of the different individuals of each species were then pooled prior to beginning experimental storage.
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Spore storage conditions
Spores were stored under wet or dry conditions. For wet storage, spores were sown directly on to 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 (American National Can, Chicago, IL, USA). For dry storage, spores were placed in hermetic glass vials. The Petri dishes and glass vials were wrapped in aluminium foil, and stored for 1, 6 or 12 months at –20, 5 or 20 °C. Dry-stored spores were sown onto mineral agar in Petri dishes immediately before the spore germination tests, as for wet storage.
Spore germination tests
After storage for 1, 6 or 12 months, Petri dishes sown with wet- or dry-stored spores were transferred to a room with a temperature of 20 ± 2 °C and a 16 h light photoperiod (daylight fluorescent tubes, photon irradiance 30–45 µmol m–2 s–1 in the 400–700 nm region). These germination conditions have previously been shown to be suitable for C. macrocarpa and W. radicans (Quintanilla et al., 2000); no data were available on conditions suitable for the other species. Germination tests were performed with four Petri dishes (replicates) for each of the 18 treatments. After 30 d, we selected 100 spores at random on each dish and determined how many had germinated, providing an estimate of germination percentage. A spore was considered to have germinated if its wall had ruptured and the first cell had started to emerge. As a pre-storage control, we performed identical germination tests with four Petri dishes sown with spores obtained before storage.
In wet storage at 5 or 20 °C, germination may occur during storage, despite the absence of light. To account for this possibility, in all six such treatments the germination percentage (100 randomly selected spores) was also determined at the start of the 30-d germination period, immediately after removal of the foil wrapping.
Statistical analyses
The results obtained for each species and each storage period (1, 6 or 12 months) were analysed by fixed-factor analysis of variance with two factors, storage technique (wet or dry) and storage temperature (–20, 5 or 20 °C), and germination percentage (after arcsine transformation) as the dependent variable. Subsequent pairwise comparisons were made using Tukey tests (P < 0·05). All statistical analyses were performed using SPSS (1999).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For all species and all storage periods, the effects of storage technique, storage temperature and the technique x temperature interaction were all statistically significant (in almost all cases with P < 0·001; Table 2). The only exception was W. radicans after 1 month’s storage, for which storage technique had no significant effect. The significant interaction between technique and temperature indicated that the effect of temperature differed between the two storage methods. In view of these results, pairwise comparisons were performed considering all combinations of technique and temperature, rather than considering each factor separately (see Zar, 1999).
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In general, wet storage at 5 or 20 °C was the procedure that best preserved viability (Table 3, Fig. 1). Indeed, for spores of W. radicans and C. macrocarpa, these were the only storage procedures that avoided a decline in germination percentage after 12 months’ storage. Except for a small proportion of D. guanchica spores, wet storage at –20 °C was lethal for all spores. In the three Dryopteris species, differences between the storage methods were minor, and the decline in germination percentage over time less pronounced; good results were likewise achieved with dry storage at all three temperatures. In general, dry storage at –20 or 5 °C led to higher germination percentages than dry storage at 20 °C.
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The only species that germinated in the dark during storage was W. radicans, which germinated only at 20 °C (means ± s.e.m., n = 4: 47 ± 1 %, 57 ± 3 %, 60 ± 4 %, after 1, 6 and 12 months, respectively). Dark-germinated spores showed filaments of one–three elongated colourless cells, as well as a rhizoid. When the same Petri dishes were maintained for another 30 d in the light (see Materials and Methods), the overall germination percentage scarcely increased (68 ± 1 %, 65 ± 2 % and 61 ± 1 %, respectively; Table 3), but some gametophytes acquired the typical cordate shape and produced archegonia. Other filaments that formed in the dark died, especially after 12 months’ storage.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Correct evaluation of the results of these experiments requires recognition of the design limitations common to studies of this type. First, at the start of our germination trials, the water content of the wet-stored spores was probably higher than that of the control spores, which in turn was probably higher than that of dry-stored spores. Spores of Pteris vittata stored dry at about 20 °C lose volume and imbibition capacity (Beri and Bir, 1993). In addition, it is possible that wet storage of spores initiates germination processes besides imbibition. This would at least partially explain the higher germination percentages obtained for wet-stored spores than for dry-stored spores, and in some cases even for control spores (Fig. 1), since in all cases the percentages were obtained after a relatively short period (30 d germination trial). It is possible that the observed differences might be reduced if a longer germination period were allowed. Furthermore, the rate of change in spore moisture content and temperature was not controlled either at the start or the end of storage. This variable has been shown to be important for seed germination (e.g. González-Benito et al., 1998), and is worth considering in future studies of fern spore storage.
Secondly, in many cases the mean germination percentages obtained for the different storage methods did not differ significantly at the 5 % level (Table 3): greater discrimination among methods could no doubt be achieved by using larger sample sizes (though note that in the context of ex situ conservation a statistically significant but small difference is of limited practical relevance).
Thirdly, greater discrimination among methods might also be achieved by investigating longer storage periods (the maximum in the present study was 12 months). Smith and Robinson (1975) monitored germination of Polypodium vulgare spores stored for several years, and found that the death rate was lower in the earlier years of storage than in the later years. Furthermore, in this and other studies (e.g. Towill and Ikuma, 1975; Beri and Bir, 1993; Camloh, 1999), it has been found that older spores often generate gametophytes with developmental abnormalities. This constitutes a fourth limitation of the present study, in that the only criterion of viability evaluated was germination percentage.
The present results do support the view that fern spores stored wet deteriorate more slowly than spores stored dry. This conclusion was reached by Lindsay et al. (1992), who suggested that this is attributable to turnover mechanisms that counteract the deteriorative process of ageing. Page et al. (1992) suggested that dry-stored spores may suffer from chromosome mutations. Beri and Bir (1993) reported that levels of reserve substances declined with storage under dry conditions, though to date no comparable information is available for storage under wet conditions. Sheffield et al. (2001) found that germination of spores of four fern species stored dry at 4 °C was enhanced by the addition of sucrose to the germination medium. The magnitude of the positive effect of sucrose declined with storage time.
Wet storage at 5 or 20 °C was the only method that maintained the viability of C. macrocarpa and W. radicans spores after 1 year (Table 3 and Fig. 1). This may be related to the autecology of these two species, both of which are hygrophilous, i.e. require very high levels of soil moisture and relative humidity. Dry storage is probably inappropriate for many hygrophilous species. Likewise, Lindsay et al. (1992) concluded that for ferns with chlorophyllous spores, many of which occur in wet-mesic habitats (Parihar, 1965; Lebkuecher, 1997), wet storage may be a more effective method. Recently, both chlorophyllous and non-chlorophyll ous spores have been successfully stored at –196 °C in liquid nitrogen (Agrawal et al., 1993; Pence, 2000). The present and previous findings also suggest that for species with spores sensitive to desiccation, such as C. macrocarpa and W. radicans, herbarium sheets are likely to be an inadequate source of spores; by contrast, for many other ferns, particularly species of xeric habitats, herbarium sheets are a useful source of spores (see Windham and Haufler, 1986; Windham et al., 1986).
Wet storage at –20 °C killed the spores of the species studied within only 1 month of storage. Pangua et al. (1999) stored spores of Cryptogramma crispa (a species that can grow at very high elevations) under similar conditions and found that the decline in germination percentage at freezing temperatures varied among the populations from which the spores had been obtained. Simpson and Dyer (1999) noted that imbibed spores are more sensitive to freezing than non-imbibed spores. These findings suggest that frost may have a lethal effect on imbibed spores in natural spore banks near the surface. Air temperatures below 0 °C are exceptional at weather stations close to our spore-collection sites (Carballeira et al., 1983; Anon., 1995), but are more common in neighbouring areas, and may thus play an important role in restricting distribution. By contrast, some fern species appear to be highly tolerant of freezing: Hill (1971) found that Adiantum pedatum, Thelypteris palustris and Woodwardia virginica showed high germination percentages after freezing in liquid medium for 1 month.
Ashcroft and Sheffield (2000) have proposed routine use of dry storage, in view of marked savings of time and space. These advantages are important in the context of ex situ conservation programmes. As in programmes based on seed storage (e.g. Brown and Marshall, 1995), spores should ideally be obtained from several populations, in each case from several individuals together representative of that population’s genetic variability. As regards temperature for dry storage, our results indicate that spores stored at –20 or 5 °C maintain their viability better than spores stored at 20 °C (Table 3 and Fig. 1). Similar results have been reported in numerous studies (see Simpson and Dyer, 1999, and references therein). At lower storage temperatures, dehydration is reduced (Raghavan, 1989), as is metabolic rate, so that cellular deterioration rates are slowed. After 1 month of storage at –20 °C, germination percentages remained relatively high (Table 3), which suggests that putting herbarium sheets in a freezer for a few days to kill insects is acceptable with regard to spore viability, as pointed out by Windham et al. (1986).
The three species of Dryopteris studied are closely related: D. guanchica and D. corleyi are allotetraploids sharing the D. aemula genome (Gibby et al., 1978; Fraser-Jenkins, 1982). Therefore, it is not surprising that these species showed marked similarities as regards spore storage: after 1 year the spores retained high viability, with less marked among-method differences than for C. macrocarpa and W. radicans. Unlike in the genus Polypodium (see Kott and Peterson, 1974), the initial viability of spores of the diploid taxon (D. aemula) was no lower than that of spores of the tetraploid taxa (D. guanchica and D. corleyi) (Table 1, around 80 % for all three taxa). Windham et al. (1986) suggested that spores of polyploids should be longer-lived in view of their greater size (implying a lower relative exposure of the cytoplasm to unfavourable environmental conditions) and their lower respiratory rate. However, results obtained by these authors for the genus Pellaea, and the present results for Dryopteris, do not support this relationship between ploidy and spore viability. For example, D. aemula spores dry-stored at 20 °C (the least effective method for preserving viability, together with wet storage at –20 °C) maintained a constant germination percentage of about 67 % over the 1-year storage period, while D. guanchica spores stored in this way showed a marked decline over the same period, from 76 % to only 48 % (Table 3).
Finally, two potential disadvantages of wet storage need to be borne in mind: loss of spores in species capable of germinating in the dark (Ashcroft and Sheffield, 2000), and the higher risk of bacterial or fungal contamination. Simabukuro et al. (1998) proposed certain techniques for avoiding contamination of this type, including the addition of Nystatin to the medium. This fungicide showed marked efficacy in our experiments, since scant development of bacteria and fungi was detected even after 1 year of wet storage at 20 °C. In addition, and as reported in previous studies (Dyer, 1979, 1983), Nystatin did not appear to affect either germination or gametophyte development, though note that we did not compare germination without fungicide. The second potential disadvantage of wet storage is germination in the dark during storage. In the present study, this was only detected in W. radicans spores wet-stored at 20 °C. These spores did not show any additional germination after 30 d of light treatment, indicating that all viable spores germinated in the dark. Some filaments died during storage, but others showed normal development (starting with an archegoniate phase; see Klekowski, 1969) on exposure to light. Given the high germination percentages obtained at 20 °C, it seems likely that germination without light would likewise occur at lower temperatures. Furthermore, the available climatic data (Carballeira et al., 1983; Anon., 1995) indicate that air temperature (and possibly soil temperature near the surface) frequently exceeds 20 °C in the north-west Iberian Peninsula (where the W. radicans population was collected). All this suggests that germination in the dark is biologically important for W. radicans. Lindsay et al. (1994) hypothesized that the capacity to germinate in the dark shortly after dispersal constitutes a key competitive advantage with respect to species that are ‘trapped’ in the spore bank. In some ferns with photosynthetic gametophytes, germination without light can be induced by antheridiogens or gibberellins (see references in Schneller et al., 1990; Dyer and Lindsay, 1992). Spore germination in the dark without other stimuli has previously been reported in various species, but germination percentages similar to those obtained under light have only been reported for Pteridium aquilinum (Lindsay et al., 1994).
Thus, to conclude, wet storage at 5 °C is best for storage of spores of the species considered here since it maintains high viability, minimizes bacterial and fungal contamination, and prevents germination in the dark.
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ACKNOWLEDGeMENTS |
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We thank Dr A. Escudero for help with experimental design, G. Norman for English translation and useful comments on the manuscript, Dr A. F. Dyer for suggestions which greatly improved the manuscript, and Dr I. Iglesias and Dr J. Izco for permission to use the controlled temperature room and seed bank of the Botany Department of the University of Santiago. This research was supported by Xunta de Galicia Project PGIDT99PXI20301A and Spanish Ministry of Science and Technology Project PB97-0307. During this study, L.G.Q. was in receipt of an FPI grant from the Spanish Ministry of Science and Technology.
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