Annals of Botany 90: 461±467, 2002
doi:10.1093/aob/mcf224, available online at www.aob.oupjournals.org
Effect of Storage Method on Spore Viability in Five Globally Threatened Fern
Species
 N1
L U I S G . Q U I N T A N I L L A 1 , * , J A V I ER A M I G O 2 , E M I L I A P A N G U A 1 and S A N T I A G O P A J A R O
de BiologõÂa Vegetal I, Facultad de BiologõÂa, Universidad Complutense, Ciudad Universitaria, 28040
Madrid, Spain and 2Departamento de BotaÂnica, Facultade de Farmacia, Universidade de Santiago, Campus Sur,
15782 Santiago de Compostela, Spain
1Departamento
Received: 13 March 2002 Returned for revision: 23 May 2002 Accepted: 28 June 2002 Published electronically: 4 September 2002
Spore germination of ®ve 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 signi®cant 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).
ã 2002 Annals of Botany Company
Key words: Culcita macrocarpa, dark germination, Dryopteris aemula, Dryopteris corleyi, Dryopteris guanchica,
ex situ conservation, spore germination, Woodwardia radicans.
INTRODUCTION
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
* For correspondence. Fax +34 91 394 5034, e-mail lugarqui@
universia.es
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 ®ve 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 speci®cities (see Rabinowitz, 1981). All
®ve 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
ã 2002 Annals of Botany Company
462
Quintanilla et al. Ð Spore Storage for Five Fern Species
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 ®ve
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 identi®cation 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.
MATERIALS AND METHODS
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.
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 Para®lm (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 6 2 °C and a 16 h light photoperiod
(daylight ¯uorescent tubes, photon irradiance 30±45 mmol
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 ®rst 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 ®xed-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).
RESULTS
For all species and all storage periods, the effects of storage
technique, storage temperature and the technique 3
temperature interaction were all statistically signi®cant (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 signi®cant effect. The
signi®cant 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).
TA B L E 1. Sites from which spores were collected, and pre-storage germination percentages
Species
Location
C. macrocarpa
D. aemula
D. corleyi
D. guanchica
W. radicans
Spain:
Spain:
Spain:
Spain:
Spain:
A CorunÄa Province, Fragas do Eume.
A CorunÄa Province, Fragas do Eume.
Asturias Province, N-634 road near to Pendueles.
A CorunÄa Province, Mariaqueira stream valley.
A CorunÄa Province, Fragas do Eume.
Collection date
Mar. 2000
Jul. 2000
Aug. 2000
Jul. 2000
Mar. 2000
Pre-storage
germination %
(mean 6 s.e.m.)
69
79
79
81
46
6
6
6
6
6
3
3
2
1
5
Quintanilla et al. Ð Spore Storage for Five Fern Species
In general, wet storage at 5 or 20 °C was the procedure
that best preserved viability (Table 3, Fig. 1). Indeed, for
463
spores of W. radicans and C. macrocarpa, these were
the only storage procedures that avoided a decline in
TA B L E 2. Results of analyses of variance for spores of the ®ve fern species, with dependent variable germination
percentage (arcsine transformed) and two factors, storage technique (wet or dry) and storage temperature (±20, 5 or 20 °C)
Storage time
1 month
Species
Source
C. macrocarpa
Technique
Temperature
Technique 3
temperature
Error
Technique
Temperature
Technique 3
temperature
Error
Technique
Temperature
Technique 3
temperature
Error
Technique
Temperature
Technique 3
temperature
Error
Technique
Temperature
Technique 3
temperature
Error
D. aemula
D. corleyi
D. guanchica
W. radicans
d.f.
MS
F
1
2
2
404´383
3006´932
3039´579
9´638
71´663
72´441
18
1
2
2
41´959
6402´074
1910´629
3355´587
18
1
2
2
6 months
12 months
MS
F
MS
F
**
***
***
1857´955
662´964
8745´382
22´465
8´016
105´743
***
**
***
8899´989
3585´502
2591´607
451´134
181´747
131´367
***
***
***
450´112
134´331
235´922
***
***
***
82´704
3394´540
2167´088
3589´821
20´937
13´367
22´142
***
***
***
19´728
2659´992
3111´730
2622´802
14´489
16´950
14´287
**
***
***
14´223
1391´085
2202´427
2492´928
113´524
179´737
203´444
***
***
***
162´127
1069´160
2170´888
2980´129
128´932
261´790
359´378
***
***
***
183´585
1009´212
2367´428
3158´473
253´728
595´200
794´078
***
***
***
18
1
2
2
12´254
1456´469
358´714
538´328
43´837
10´797
16´203
***
***
***
8´292
948´841
1642´193
2344´091
30´068
52´040
74´283
***
***
***
3´978
453´006
2085´401
3181´091
20´572
94´703
144´460
***
***
***
18
1
2
2
33´225
3´618
1839´226
1783´730
0´024
12´308
11´936
NS
***
***
31´556
432´408
2199´530
1660´728
76´764
390´477
294´824
***
***
***
22´021
1546´747
1547´771
2415´483
273´197
273´378
426´639
***
***
***
18
149´436
5´633
5´662
d.f., Degrees of freedom; MS, mean square; NS, not signi®cant (P > 0´05); *, P < 0´05; **, P < 0´01; ***, P < 0´001.
TA B L E 3. Germination percentages (mean 6 s.e.m.) of spores of the ®ve fern species stored wet (W) or dry (D) for 1, 6 or
12 months at different temperatures (±20, 5 or 20 °C)
C. macrocarpa
1 month
6 months
12 months
94 6 1 (W 20)a
64 6 5 (D 20)b
62 6 3 (D ±20)b
49 6 3 (W 5)b
43 6 11 (D 5)b
0 6 0 (W ±20)c
87 6 1 (W 20)a
84 6 2 (W 5)a
70 6 2 (D ±20)a
26 6 14 (D 5)b
0 6 0 (D 20)c
0 6 0 (W ±20)c
84 6 2 (W 20)a
84 6 1 (W 5)a
9 6 5 (D 5)b
1 6 1 (D ±20)c
0 6 0 (D 20)c
0 6 0 (W ±20)c
D. aemula
87 6 2 (W 20)a
83 6 1 (D 5)a
80 6 2 (D ±20)ab
67 6 5 (D 20)b
10 6 3 (W 5)c
0 6 0 (W ±20)d
93 6 1 (W 20)a
84 6 1 (D 5)a
80 6 4 (D ±20)ab
65 6 4 (D 20)ab
42 6 22 (W 5)b
0 6 0 (W ±20)c
93 6 1 (W 20)a
83 6 2 (D 5)ab
69 6 3 (D 20)ab
63 6 6 (D ±20)ab
42 6 23 (W 5)b
0 6 0 (W ±20)c
D. corleyi
78 6 4 (W 20)a
71 6 2 (D 5)ab
67 6 1 (D ±20)ab
67 6 2 (W 5)ab
59 6 4 (D 20)b
0 6 0 (W ±20)c
78 6 1 (W 20)a
78 6 3 (W 5)a
72 6 2 (D ±20)ab
64 6 2 (D 20)b
63 6 3 (D 5)b
0 6 0 (W ±20)c
85 6 2 (W 5)a
77 6 2 (W 20)b
73 6 1 (D ±20)bc
68 6 2 (D 20)cd
63 6 1 (D 5)d
0 6 0 (W ±20)e
D. guanchica
81 6 1 (D ± 20)a
78 6 3 (D 5)a
76 6 3 (D 20)a
73 6 3 (W 20)a
61 6 7 (W 5)a
26 6 6 (W ±20)b
80 6 3 (W 5)a
80 6 4 (D 5)a
79 6 3 (W 20)a
77 6 6 (D ±20)ab
58 6 1 (D 20)b
4 6 2 (W ±20)c
84 6 3 (W 5)a
79 6 5 (W 20)a
72 6 2 (D ±20)a
72 6 2 (D 5)a
48 6 4 (D 20)b
1 6 1 (W ±20)c
W. radicans
68 6 1 (W 20)a
54 6 4 (W 5)a
42 6 13 (D 20)a
34 6 4 (D ±20)a
33 6 15 (D 5)a
0 6 0 (W ±20)b
65 6 2 (W 20)a
58 6 2 (W 5)a
34 6 1 (D 5)b
15 6 3 (D ±20)c
11 6 0 (D 20)c
0 6 0 (W ±20)d
61 6 1 (W 20)a
59 6 2 (W 5)a
20 6 1 (D 5)b
14 6 2 (D ±20)b
1 6 0 (D 20)c
0 6 0 (W ±20)c
Each mean is for four Petri dishes (n = 4), on each of which 100 randomly selected spores were evaluated. Within each species and storage period,
values with the same superscript indicate that means that do not differ signi®cantly at the 5 % level (Tukey tests).
464
Quintanilla et al. Ð Spore Storage for Five Fern Species
F I G . 1. Time course of germination percentage for spores of Culcita macrocarpa (A), Dryopteris aemula (B), D. corleyi (C), D. guanchica (D) and
Woodwardia radicans (E) stored under different conditions.
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.
The only species that germinated in the dark during
storage was W. radicans, which germinated only at 20 °C
(means 6 s.e.m., n = 4: 47 6 1 %, 57 6 3 %, 60 6 4 %, after
1, 6 and 12 months, respectively). Dark-germinated spores
showed ®laments 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 6 1 %, 65 6 2 % and 61 6 1 %, respectively;
Quintanilla et al. Ð Spore Storage for Five Fern Species
Table 3), but some gametophytes acquired the typical
cordate shape and produced archegonia. Other ®laments that
formed in the dark died, especially after 12 months' storage.
DIS CUS S ION
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. GonzaÂlezBenito 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 signi®cantly 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 signi®cant 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. Shef®eld 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
465
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-chlorophyllous spores have been successfully stored at ±196 °C in
liquid nitrogen (Agrawal et al., 1993; Pence, 2000). The
present and previous ®ndings 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
Hau¯er, 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 nonimbibed spores. These ®ndings 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 Shef®eld (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
466
Quintanilla et al. Ð Spore Storage for Five Fern Species
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; FraserJenkins, 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 Shef®eld, 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
ef®cacy 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 ®laments 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.
ACKNOWLEDGEMENTS
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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