American Fern Journal 94(l):28-38 (2004J Under Spore Storage Conditions Viability Different Four Asplenium Taxa Rupicolous in L. Aragon^ and Pangua^ Cristina Emilia F. Departamento de Biologia Vegetal Facultad de Biologia, I, Universidad Complutense, Ciudad 28040 Madrid Universitaria, — Abstract. Spore germination of four rupicolous taxa of AspJenium [A. adiantum-nigrum. var. adiantum-nigrum, A, adiontum. nigrum, var. silesiacum, A. septentrionale subsp. septentrionale and A. ruta-muraria, subsp. ruta-muraria) was determined after and 12 months of storage in 1, 6, Eppendorf tubes (dry storage] or on agar plates (wet storage) at -20, 5 and 20°C. In general, technique and temperature factors and the moisture-temperature interaction, had a significant on effect germination percentage. In all cases, except for A. ruta-muraria, germination percentage was maintained in wet and dry storage, but in the dry storage method percent germination was higher. These results indicate some capacity of Asplenium spores to withstand desiccation, and may that ecological requirements of species influence spore viability and should be taken into when account designing spore conservation programs. Spores of A, ruta-muraria yielded better results in wet storage. In dry storage response was from Wet its different that of the other three taxa. storage at -20°C killed all or most spores of all taxa. Interest in the conservation of pteridophyte spores has become evident in recent decades, because they are easy to obtain, can be stored in large quantities, and can germinate rapidly in simple media (Dyer, 1979). Spores are of interest not only in ex situ conservation programs, but also, as Page et ah show, taxonomic (1992) in studies in the broadest sense, and as a commercial source However, in horticulture. in contrast to seed conservation (Baskin and known spore during affect viability storage. Lloyd & Klekowski (1970) calculated the variation in viability of chloro- phyllous and (green) non-chlorphyllous spores over storage periods of 2 months marVpH to 3 VearS. notinp tTlP rnntract VioHAroon Frrjj.'co/iim 9 tn 94 f 1 ears The conditions under which spores are stored have a notable impact on their viability. Generally, to avoid deterioration, they are stored in dry, ambient or low temperatures, although some in cases has resuUed this in loss of viability and (Beri Bir, 1993; Camloh, Another 1999). option has been that tried is storage of spores in a hydrated (Lindsay state et al, 1992), analogous to conditions prevailing in natural spore banks which some in spores of species can remain viable for long periods (Lindsay and Dyer, has been 1990). It Current address: Area de ^ Biodiversidad y Conservacion. Escuela Superior de Ciencias Experimentales Tecnologia. Universidad Rey y Juan Carlos. E-28933 Mostoles, Madrid. ^ Corresponding Author. ARAGON & PANGUA: SPORE VIABILITY ASPLENIUM IN 29 may more observed that this type of storage be effective than dry storage for certain species. may Pteridophyte spores remain viable, in a metabolically inactive state, when The conditions are not adequate for germination [Page, 1979]. length of which time over spores can maintain enormously from viability varies species to species [Miller, 1968} and has been shown that other characters, such as it spore age (Raghavan, 1989 and references therein], ploidy level (Kott & & and Peterson, 1974; Kott Britton, 1982], the presence of chlorophyll are Even though they can survive desiccation [Lloyd and Klekowsky, influential. 1970; Lebkuecher, 1997], chlorophyllous spores have generally limited much compared with pteridophytes with longer-lived non-chloro- viability, phyllous spores. Page et aL [1992] point out the need to investigate storage conditions that guarantee the maintenance of spore viability for the longest possible time, their and developmental Having genetic integrity, their capacity. available collec- tions of adequately stored spores of interest in order to avoid the loss of is same species in nature, in the case of threatened species, while the time at having subsequent developmental phases with offering the possibility of which investigate other aspects of the biology of the species. to were on In the present study, various storage conditions tested the spores of four Asplenium A, septentrionole Hoffm. subsp. septentrionale, A. taxa: [L.] adiantum-nigrum adiantum- ruta-muraria L. subsp. ruta-muraria. A, L. var. & nigrum, and A, adiantum-nigrum silesiacum [Milde] Viane Riechstein. var. homogeneous and These taxa constitute a biogeographical ecological group. They are circumboreal species [Pichi SermoUi et aL, 1998] orophyllous, Asplenium rupicolous, and are tetraploids. septentrionale subsp. septen- all trionale an autotetraploid derived from subspecies caucasicum Fraser- is & and widely throughout Eurasia Jenkins Lovis [Lovis, 1964] distributed as is well as disjunctly in North Africa and on the Pacific coast of North America, Asplenium where preferentially inhabits acid substrates. ruta-muraria it an but derived from subspecies subsp. ruta-muraria also autotetraploid is & and broad dolomiticum Lovis Reichst. [Lovis, 1964] distributed across a is and northern hemisphere, in Europe, Asia America, having belt in the its southern limit in North Africa. prefers basic substrates. Finally, A. It adiantum-nigrum an allotetraploid arising from crossing and subsequent is and chromosomal duplication of cuneifolium Viv. A. onopteris L. [Shivas, /I. has wide range throughout Europe, Macaronesia, Asia, Africa. North 1969). a It and America and where colonizes cracks fissures preferentially in Australia, it Two recognized: the typical variety described siliceous rocks. varieties are above, and the variety silesiacum [serpentinicolous ecotype), of ultrabasic northern and western Europe [Salvo, 1990). substrates in and have been combined hydration (wet dry) with Different degrees of temperature regimes in order to analyse the percentage germination different This same methodology has been used varying periods of storage. in after group a previous study (Quintanilla et aL, 2002) of a of relict Macaronesian These authors aimed optimise method to the species that inhabit forest floors. AMERICAN FERN VOLUME NUMBER 30 JOURNAL: 94 1 (2004) Table 1. Location of populations studied, collector and date of collection. Coordinates Collection UTM Taxa Location Altitude data Collector(s) A. septentnonale Madrid. Manzanares 1300 30TVL2310 Aragon & July C. F. El Real. La Pedriza 2000 Camacho R. G. A A A, adiantum- Coruna. Bana, 225 29TNH2153 October G. Quintanilla L. nigrum var. 2000 adiantum- nigrum A A. adlanfum- Coruna. Sierra 280 29TNJ8139 October Amigo & J. nigrum var. de la Capelada. 2000 L. G. Quintanilla silesiocum Chao do Monte ' A. ruta-muroria Guadalajara. 1000 30TVL9263 Pangua & July E, Somolinos. 2000 Pajaron S. of storage of viable spores as part of a conservation Although strategy. the taxa included in this study present no problem from a conservation point of view, was our objective to establish whether the optimal spore method storage varies amone the taxa. Material and Methods Spores were obtained from populations of each taxon (Table Fertile 1). fronds of 15 sporophytes were collected per population and transported the to where laboratory they were washed under water and two weeks tap pressed for until the spores were Each sample was released. obtained from mixture a of from spores sporophytes all gathered in each population. The spores of these four taxa were subjected to different storage conditions which in different degrees of hydration (wet and dry techniques] and temperatures were combined. With wet sown the technique, spores were on medium directly a mineral agar (Dyer, 1979) that had been sterilized in an autoclave 20 atm and at 125^C 20 minutes, on for sterile plastic Petri dishes cm which diameter), were (5.5 sealed with Parafilm (American National Can, Chicago) avoid To to desiccation. prevent contamination, the antifungal agent U Nystatin mV^] was (100 added medium to the culture autoclaving and after sowings also, in all the spore samples were passed through two layers of lens cleaning (Whatman tissue International Maidstone, Ltd. 2105841) n" to eliminate impurities, remains of sporangial With walls, etc. the dry technique, spores were kept Eppendorf in tubes germination were until tests carried out at which time they were plated out as above. The dishes and tubes were stored at temperatures and of 20, 5 -20°C. They were kept in the dark by wrapping them aluminium in avoid foil to germination during the storage period. Germination were tests carried out 6 and 12 months after 1, of storage. All 21^ m -2 _-l s intensity of photonic flow, with a 16 h 8 h dark photoperiod. Four light: were replicates incubated for each combination of technique and temperature ARAGON & PANGUA: SPORE VIABILITY IN ASPLENIUM 31 and the percentage germination was assessed after 30 days. This same was germination test carried out before storage to estabhsh a control group. Germination was considered to have occurred the spore wall was broken and if the rhizoid had emerged. Germination rate [%] was calculated on the basis first of a count of 100 randomly chosen spores from each dish. To determine the effects of technique (wet and dry) and temperature 5 [20, and -20°C) on germination the percentages were arcsine-transformed and rate, means compared by two-way The their a analysis of variance [Zar, 1999). analyses were repeated for 6 and 12 months of storage. The multiple com- 1, among means homogeneous parisons for the identification of groups, wherein < was made the effect of a factor significant were using the Tukey [p 0.05), test < All analyses were done with the SPSS program [p 0.05). statistical (1999). Results As in the majority of pteridophyte species, spores of the taxa studied The required the presence of light to germinate. effect of technique and temperature and upon was factors, their interaction, spore germination statistically significant in cases, except for the hydration factor one all after = month of storage in the case of A, ruta-muraria (F 0.059; Table The 2), between existence of a significant interaction factors implies that the effect of each each Thus, multiple comparisons between media is different for level. made were for each possible combination of hydration and temperature. The response of A. adiantum- nigrum var. adiantum-nigrum, A, adiantum- nigrum and var. silesiacum A, septentrionaJe to the different storage was B and whereas conditions similar lA, those of ruta-muraria (Fig. C), ^4. were more variable ID), In general, high percentages of germination were (Fig, found in the three taxa, irrespective of the storage conditions, except first for which wet storage -20°C, in case only a small percentage of spores of these at three taxa germinated after the month. In these three cases, dry storage was first was fairly effective, although there a slight decrease in the percentage of viable when — months B and Dry spores after 12 of storage kept at 20''C (Fig. lA, C). < was storage at 5°C (Table 3) significantly higher [p 0.05) than for dry-storage — at 20 and 20''C, in the case of A. adiantum-nigrum var. silesiacum^ and at — months 20°C for A. septentrionaJe, after 12 storage (see Table 3), With respect A. ruta-muraria, the best results were obtained with wet to storage at 20 and no spores germinated at -20^C. Percentages achieved S^'C; with dry technique were generally lower than under humid conditions and the months' lower than in the other taxa, except for the results after 6 storage at 5 — and 20°G ID), where similar percentages were obtained to those with wet (Fig. storage (Table 3). Discussion Our results indicate that for the taxa studied, except A, ruta-muraria, storage under any of the tested conditions allowed relatively high percentages of Under viable spores, except with wet storage at -20°C. those conditions there CO N3 and months Table Levels of significance of the effects of technique and temperature factors on percentage spore germination after 6 12 of storage. 2. 1, < < < MS, mean squared; df, degrees of freedom; *, 0.05; **, p 0.001; * * * p 0.001. /> ri Storage time month months n 6 12 lonths 1 MS F MS F MS F Tax Source a of variation d.f. 91,188*** 77.284*** 23,600*** A. septentrionale Technique 2,303,118 1,583,659 849,024 1 > 85,803*** 154,609*** 115,463*** Temperature 2 2,167,125 3,168,146 3,727.413 X 77,013*** 89,262*** 46,565*** Technique Temperature 2 1,945,107 1.829,107 1,503,217 Error 18 25,257 20.491 32,282 f * t » fe * [CAN 88.865*** 214.497*** 207,121*** A. adiantum-nigrum Technique 1,983,658 2,361,546 1.960,820 1 112.603*** 345,977*** 350,271*** var. adiantum nigrum Temperature 2 2.513.572 3,809,133 3,316.035 tr X 81,737*** 259,725*** 274,695*** Technique Temperature 1,824,572 2,859.518 2,600,584 2 § J Error 18 22,322 11,010 9,467 n P > • * V + 4 t c 75,106*** 126.386*** 207,865*** A. adiantum-nigrum Technique 1 2,963.416 3,358,753 2,335,769 64,104*** 109.782*** 237,922*** silesiacum Temperature 2 2,506,259 2.917,486 2.674.307 var. L^ Technique X temperature 1,605,239 41.058*** 1,899,082 71,460*** 1,775,800 158,032*** ^ 2 # • Error 18 39,097 26,575 11.237 < 14 • B 4 ft o 22,904*** 8,684** A. ruta-niuraria Technique 15,918 0.059 ns 455,188 2,263,204 1 7.371** 37.032*** 32.557*** Temperature 2 1,977,344 1,941.044 3,208,584 UME X 42,274*** 15,139*** Technique 'temperature 1,305,054 4,805* 2,477,879 1,491,976 2 18 268,264 52,415 98,553 CO Error * « * i 4 * t 1 NUMBER 1 (2004 ARAGON & PANGUA: SPORE VIABILITY ASPLENIUM IN 33 B 100 100 80 80 60 60 40 40 - 20 20 W20 W5 W20 W5 control W-20 D20 D5 D-20 Oontrol W-20 D20 D5 D-20 c D 100 100 80 80 - 60 60 I 6 40 40 D12 20 20 - T T control W20 W5 W-20 D20 D5 D-20 control W20 W5 W-20 D20 D5 D-20 Fig. 1. Germination percentage after 1, 6, and 12 months' storage with different techniques (VV. wet; D, dry) at temperatures of 20°, 5°, and -20°C. (A) Asplenium adiantum- nigrum var. adiantum- nigrum; [B] A. adiantum-nigrum var. slleslacum; [C] A, septentrionale; [D] A. ruta-muraria. was month practically no germination after one of storage. This combination of hydration and temperature was generally inefficient at maintaining spore viability, confirming observations under identical conditions by Quintanilla et on This implies combination al. (2002) relict forest species. that this is not Pangua efficient irrespective of species ecology. et (1999) also noted al. Cryptogramma a decrease in germination for spores of crispa (L.) R. Br. kept in — the wet at 18°C. Germination percentage in that species varied among populations. In this species wet spores subjected to a temperature of 70*^0 when yielded higher germination percentages than did dry spores subjected to the same temperatures after 24 h of treatment (Simpson and Dyer, 1999). Given that a dry -20''C treatment did not result in such drastic reduction in germination, obvious that previous hydration renders the spores more it is sensitive to freezing. showed Adiantum pedatum and Hill (1971) that spores of L. Thelypteris month medium, had palustris Schott, after a of freezing in liquid higher when percentages of germination the characteristic periods of time than for shown spores were kept ambient temperature. Although has not been that at it same longer preservation times yielded the results, nevertheless appears that it may these spores require chilling in order to germinate. Hill (1971) did not specify whether the spores were frozen immediately after their inclusion in the medium was or there a time of imbibition. if Wet and maintained number storage 20 the viability of a large of at S^'C and hence an method spores of taxa studied represents effective of storage. all CO 4^ Tabu: 3. Percentage germination [mean ± standard error) of spores without previous storage (control) and after 1, 6, and 12 months storage, with wet (W] and dry (D) techniques at temperatures of 20°, 5° and -20°C. The vertical linos indicate those groups with homogenous means, between which no there are significant difft^rences. Storage time month months months 6 12 1 Tax Control Germination Treatment Cormination Treatment Germination Treatment a W W W ± ± -20 ± -20 ± -20 A. septantrionale 77.8 3.1 0.5 0.5 0.0 0.0 0.0 0.0 W ± ± D -20 D -20 70.2 1.5 20 60.5 6.8 47.0 ±8.7 ± D -20 ± D ± D 70.7 5.8 71.5 3.6 20 66.7 5.7 20 W W W5 ± ± ± > 71.0 5.5 5 74.0 1.4 20 73.0 1.6 D5 ± D ± ± D 72.5 4.0 20 76.0 3.4 73.5 4.4 5 W W5 ± D ± ± 77.0 3.1 5 77.5 2.6 79.2 1.7 20 n W W W-20 ± ± -20 ± ± -20 > A. nHinntiim-nigrum 92.0 1.1 4.5 1.9 0.0 0.0 0.0 0.0 Z adiantum ± D -20 ± D -20 ± D -20 var. 79.5 4.1 79.7 3.1 74.5 1.6 nigrum 84.0 ± 0.7 \V 20 84.7 ± 1.8 D 20 80.0 ± 2.0 D 20 D5 D5 ± D ± ± 84.7 2.4 20 87.7 2.9 80.5 3.0 W5 W5 W5 ± ± ± o 85.7 2.6 88.2 1.0 83.2 2.3 c D5 W20 W20 ± ± ± 86.7 1.1 89.2 1.5 84.5 2.1 W W-20 W-20 ± ± ± -20 ± A. adiantuni-nigrum 79.0 0.6 2.0 1.7 0.0 0.0 0.0 0.0 > W W ± ± ± D -20 var. silesiacum 74.2 3.5 20 69.7 6.4 5 64.2 1.2 m > ± D -20 ± D -20 ± D < 73.5 7.2 72.5 3.7 66.7 2.1 20 O W W ± ± D ± 76.2 2.8 5 76.7 3.9 20 67.0 2.8 5 W W20 ± D ± ± 80.2 2.3 20 76.7 3.6 20 72.5 2.6 D5 D5 ± ± ± D 86.2 2.5 87.2 2.7 81.0 4.1 5 W W W-20 CD ± ± ± -20 ± -20 A. ruta-iniiraria 73.0 2.6 0.0 0.0 0.0 0.0 0.0 0.0 ± D -20 ± D ± D -20 25.5 17.5 31.7 11.5 20 7.2 4.7 a ± D ± D -20 ± D 20 28.7 6.1 54.7 6.8 10.5 7.4 5 ± D ± D ± D 34.5 17.1 5 65.2 3.3 5 26.0 10.7 20 tX3 W5 W W ± ± ± 55.0 10.4 65.5 1.0 20 69.5 3.2 5 W5 W W20 ± ± ± 56.5 13.2 67.2 5.1 70.5 1.8 20 O o 4^ AR.\GON & PANGUA: SPORE VIABILITY IN ASPLENIUM 35 Lindsay et aL (1992] studied the response to spore hydration of four species with non-chlorophyllous spores and one with chlorophyllous spores, of all which were hygrophilous. Fully hydrated spores were capable of germinating much at ambient temperature after two years of storage at 20''C at higher percentages than those preserved dry but under otherwise identical con- ditions. Other hygrophilous species, such as Woodwardia radicans Smith [LJ and macrocarpa show marked Culcita C. Presl., a sensitivity to desiccation, such only those spores had been maintained wet medium that that in a 60% 84% germinated months' and compared 12 after storage, respectively, 1% 0% with those kept in the dry, and respectively, the same temperature at These (Quintanilla et aL, 2002). results are interesting because spores of would natural spore banks be in a wet state (Page et aL, 1992], especially those from species that inhabit places where the soil very wet throughout the is entire year. Dyer and Lindsay have shown (1992) the persistent presence of ^4. adiantum-nigrum, A. ruta-muraria and A. septentrionale Hoffm. in British (L.) spore banks. However, in the latter two strictly rupicolous species, the presence of spores in these banks of low importance, because, although the is new gametophyte already established, sporophytes cannot be established, is possibly due to a problem of competition with other species. Furthermore, minimum two these species require a temperature for germination (Young, may Pangua Dyer and which 1985; et aL, 1994; Lindsay, 1996], they encounter within cracks or other protected places. Nevertheless, they can tolerate temperatures of up to 70''C for at least 24 h (Simpson and Dyer, 1999). These may results represent an adaptive efficiency in these species the media they for — inhabit exposed rocks with large temperature differences throughout the day and the year. In our study, for A, septentrionale, A. adiantum-nigrum var. adiantum- nigrum and A. adiantum-nigrum var, silesiacum dry storage gave results similar to those with wet storage, although perhaps longer-term storage would have revealed greater differences. Nevertheless, in light of the results, it appears that these rupicolous taxa, have a relatively high capacity to withstand desiccation. Therefore appears that ecological requirements of species can it indeed result in taxa specific adaptations in terms of spore viability, although mind must be born few species have been studied and considerable in that it may variability exist in this respect. In A. ruta-muraria wet storage, with the general exception of-20°C for all the taxa studied, was significantly more effective than dry in maintaining spore viability. Results obtained at 5 and 20°C are essentially the same, showing a germination capacity that increased slightly with time of storage. However, the response obtained with dry storage at different sampling times is difficult was months to explain. Only after 6 of storage at 5°C the germination comparable wet These might be explainable by need to that of storage. results a go through cold period before germination. For A. ruta-muraria, despite to a method most wet not being a hygrophilic species, the suitable preservation is at 5 or 20°C. AMERICAN FERN VOLUME NUMBER 36 JOURNAL: 94 (2004) 1 Woynar setiferum (Forsskal) and Athyrium filix-ft typical results in increased spore viability 12 and 24 months, after respectively, whereas at 20°C, spore viability is practically lost (Lindsay and Dyer, in Simpson and Dyer, Spores Cyathea 1999]. of delgadii Sternb. (Simabukuro et and Kuhn ah, 1998) Pteridium aquilinum remain viable years dry [L.) for after and storage 4°C [Ashcroft which at Sheffield, 2000), for reason the authors have proposed the routine use of this storage technique and temperature. Some with species chlorophyllous such Osmunda, spores, as those of also retain their viability after years of dry storage temperatures of 2° and 6°C at (Stokey, our 1951). In case, therefore, appears that the dry technique would be good it a option for the conservation of the spores although two since, the techniques have yielded favourable results, the dry technique has some advantages, such as saving space, time and The materials. ideal storage temperature, in this case, would be mind 5°C, bearing in the results of our experiments, where the spores kept dry temperature had somewhat at this higher germination percentages in A. adiantum-nigrum var. adiantum- nigrum, and significantly higher percent- ages in A. adiantum-nigrum and silesiacum var. A. septentrionale. Recent studies (Agrawall ef 1993; Pence, 2000] have demonstrated al., the effectiveness of preservation of dried chlorophyllous and non-chlorophyllous spores at-196°C in liquid nitrogen. Pence observed (2000) germination rates of spores of A. ruta-muraria under stored these conditions were that similar to may that of the control population. These imply results imbibed that spores are by low affected very temperatures, but keeping them that dry good a is conservation technique. The time and of storage the processes of sterilization bring about alterations in germination and subsequent development of the gametophytes. Smith and Robinson (1975) studied germination Polypodium of vulgare using spores L. dry-stored 4°C They at for 7 years. observed a decrease in germination and an increase in the proportion of abnormal gametophytes. Similar were results obtained by and Beri Bir (1993) for Pteris vittata stored room temperature L., at for 100 days; spores germination lost capacity in association with total loss of amino sugars, acids and Camloh proteins. (1999) observed in Platycerium bifurcatum (Cav.) C. Chr. that sterilized spores and lost viability that with age there were fewer and would shorter rhizoids. be interesting, addition It in to germination studies for conservation, to study the impact on development the of the gametophyte. In our work, although we have not carried out thorough a post-germination study, the plates used in the various experiments remained chambers in culture 20°C months and at for 6 the gametophytes appeared to develop normally. This may suggests that storage time not affect the subsequent development of the prothalli. ACKNOWISDGMENTS Stuart We early draft of this paper. also thank two anonymous reviewers and Dr. R. James Hickey for ARAGON PANGUA: SPORE ASPLENIUM & VIABILITY IN 37 and PB97 the Universidad Complutense de Madrid Project PR78/02-11020. Literature Cited Agrawal, D. C., S. S. Pawar and A. F. MascarenHAS. 1993. Cryopreservation of spores of Cyathea An spinulosa Wall. Ex. Hook. F. endangered tree fern. Plant Physiol. 142:124-126. J. 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