ARTÍCULOS ORIGINALES
Light and temperature effects on seed germination of four native species of Mediterranean high mountains (Spain) (With 1 Table & 1 Figure)
Efectos de la luz y de la temperatura sobre la germinación de semillas de cuatro especies autóctonas de alta montaña Mediterránea (España) (Con 1 Tabla y 1 Figura)
Serrano-Bernardo F1, JL Rosúa1, M Díaz-Miguel2
1 Departamento de Botánica. Edificio Politécnico. Universidad de Granada. Campus
Fuentenueva, s/n. 18071-Granada, España. e-mail: fserber@ugr.es
2Departamento de Fisiología Vegetal. Facultad de Ciencias. Universidad de Granada.
Campus Fuentenueva, s/n. 18071-Granada, España.
Recibido/Received: 02.11.2006. Aceptado/Accepted 30.03.2007.
Abstract. The Mediterranean region is considered one of the most important zones worldwide in terms of plant diversity. Sierra Nevada (S Spain) is a unique mountain massif within the high-mountain systems of the Mediterranean. It is of great floristic importance and very rich in endemic species. However, it requires numerous tasks for restoring plant cover due to the constant anthropic degradation, especially because of the ski station located near the second-highest peak of the mountain. One of the restoration efforts consists of trying to reproduce, in the laboratory, the ideal conditions to pre-germinate seeds of some of the species most threatened by these perturbations. This is to transplant the seedlings afterwards in the mountain, thereby contributing to the recovery of these species. With this purpose, a germination experiment was performed, under controlled conditions, with seeds of Genista versicolor Boiss. Hormathophylla spinosa (L.) P. Küpfer, Reseda complicata Bory, and Thymus serpylloides Bory. These four species, native to the Mediterranean high mountains, were germinated under constant darkness, 12 or 24 h light followed by constant darkness, and constant light, at temperatures of 15 ºC or 25 ºC. The germination process was monitored during 15 days. Results were species-dependent: some species germinated easier under darkness at low temperatures, such as Genista versicolor, whereas Thymus serpylloides presented maximum germination under constant light to a higher temperature.
Key words: Germination; Plant-cover restoration; Mediterranean high mountains; Sierra Nevada; Light; Temperature.
Resumen. La región mediterránea es considerada como una de las zonas más importantes a nivel mundial en cuanto a diversidad vegetal. Sierra Nevada (Sur de España) constituye un área importante dentro de esta región. Es un macizo montañoso único dentro de los sistemas de alta montaña mediterránea, de gran importancia florística y muy rico en endemismos. Esto hace necesarias numerosas tareas de restauración de la cubierta vegetal, debido a las constantes alteraciones antrópicas que sufre, especialmente como resultado de la estación de esquí que hay en él. Una de estas tareas de restauración consiste en intentar reproducir, en laboratorio, las condiciones ideales para pregerminar semillas de algunas de las especies más amenazadas por estas alteraciones. Esto permitiría posteriormente transplantar dichas especies al campo, contribuyendo a su recuperación. En el presente trabajo, se llevó a cabo un experimento de germinación, en condiciones controladas, con semillas de Genista versicolor Boiss. Hormathophylla spinosa (L.) P. Küpfer, Reseda complicata Bory, and Thymus serpylloides Bory. Estas especies, autóctonas de alta montaña mediterránea, fueron puestas a germinar en condiciones de Oscuridad Constante, 12 ó 24 horas de luz y posterior traslado a Oscuridad Constante, y Luz Constante, a temperaturas de 15 ºC o 25 ºC. Durante 15 días se siguió la evolución de la germinación. Los resultados obtenidos fueron heterogéneos. Hubo especies con mayor facilidad para germinar en oscuridad a bajas temperaturas, como Genista versicolor, en oposición a Thymus serpylloides, que presentó un máximo porcentaje germinativo con luz constante a la temperatura más elevada.
Palabras clave: Germinación; Restauración de la cubierta vegetal; Alta montaña Mediterránea; Sierra Nevada; Luz; Temperatura.
INTRODUCTION
The Mediterranean region is considered one of the most important zones
worldwide regarding plant diversity (Médail & Quézel, 1997; Myers et al., 2000;
Malcolm et al., 2006; Médail & Diadema, 2006; Vogiatzakis et al., 2006), including
Mediterranean high-mountain areas (Väre et al., 2003; Giménez-Benavides
et al., 2005). One of these mountain systems is Sierra Nevada (S Spain), with the
only high-mountain national park in Europe, covering 86,208 ha. Also, this is the most southern European massif, and its proximity to Africa gives it particular
climatic characteristics. These characteristics, in combination with its altitude
(among other factors) make it the only Mediterranean mountain of Spain that
has five bioclimatic levels (Rivas-Martínez, 1990). This contributes to explain
the high biodiversity of this massif. Thus, Sierra Nevada has more than 2000
vascular plant species and subspecies, constituting more than 30% of the flora
of the Spanish peninsula. Of this high number of vascular plant species, more
than 80 are endemic, especially in the peak zones, where the percentage of endemic
species exceeds 30%. In exclusive ecological niches such as the rocky or
gravely areas, over 80% of the species are endemic (Blanca, 2002). In these fragile
areas, stands the Sierra Nevada ski station, the most southern ski resort of
Europe. It receives a great number of skiers and visitors year round: roughly a
million people per year for a surface area of around 350 ha. Works preparing the
runs have seriously perturbed the ecosystems of these slopes, resulting in a
major loss of biodiversity and increased soil erosion. It is essential to restore the
plant cover for preventing further ecosystem degradation and loss of biodiversity.
Chambers (1997) and Macyk (2000) have described the alpine ecosystem
as one of the most difficult for restoring disturbances, because of the
exclusive physical determinants of the environment, such as low temperatures,
high levels of solar radiation, persistent winds, irregular topography
and steep slopes. Among these determinants, temperature and light play a
major role. Temperature regulates germination in three ways (Bewley &
Black, 1994): 1) by determining the capacity and percentage of germination;
2) by eliminating primary and/or secondary dormancy; and 3) by inducing
secondary dormancy. The optimal germination temperature for most
seeds which are not in dormancy is 25 to 30 ºC. However, temperature ranges
for germination depend largely on the (1) period of the year in which
plants complete their life cycle and (2) geographic origin of the species
(Besnier, 1989). Thus, seeds usually require a thermal periodicity to germinate:
a cold season (of activation), between 0 and 10 ºC (stratification),
alternating with another one where the embryonic axis grows at intermediate
temperatures (20-30 ºC). In fact, low temperatures can break dormancy
of some seeds (e.g. woody plants, cereals), and are necessary for germination
at higher temperatures (Bewley & Black, 1982). High temperatures
sometimes reduce dormancy of some seeds but induce secondary dormancy
in others. Therefore, the response of seeds to these conditions can be essential
for the regeneration of communities after a perturbation.
In addition, light is also important for seed germination. Many species
respond to the environment with optimal growth and development according to
the light they receive (Maloof et al., 2000). Some seeds germinate similarly in
light and darkness (Baskin & Baskin, 1988), while others do it more readily
either under light (Colbach et al., 2002) or darkness conditions (Thanos et al.,
1989). Also, light requirements for germination can vary with temperature. It
has been demonstrated that some species need a constant temperature and light
to germinate and others can germinate either under light or darkness conditions
but need temperature fluctuations (Felippe, 1978). In other species, stratification
(Farmer et al., 1984) or high temperatures (Amritphale et al., 1989) replace
light requirements for germination.
Our objective was to determine some of the light and temperature requirements
for germination of the study species under laboratory conditions. This
would contribute to know the required light and temperature conditions necessary
for optimum germination of these species in soils of Sierra Nevada. After seed
germination in the laboratory, seedlings were transplanted to the natural environment
in an effort to improve methods of restoring plant cover in degraded areas.
MATERIALS AND METHODS
Seeds from four endemic species of the Betic Sierras and Sierra Nevada
were used in this study: Genista versicolor Boiss., Hormathophylla spinosa (L.) P.
Küpfer), Reseda complicata Bory, and Thymus serpylloides Bory. They were
collected from different areas near the ski station, between 2000 and 3000 m in
altitude. Before the study initiation, similar-size seeds from each species were
kept in darkness at 4 ºC for at least three months. Thereafter, seeds were sorted
out for uniform size and similar external characteristics, discarding those with
obvious alterations or malformations.
Previous to the germination test, a sample of seeds was tested for viability,
applying the tetrazolium test (AOSA, 2000). It showed a positive staining reaction in 80-90% of the seeds. All seeds were surface sterilized by immersion in
1% sodium hypochlorite for 5 min, and washed afterwards with distilled water.
Thereafter, petri dishes 10 cm in diameter were prepared with filter paper (both
previously sterilized). Sixty seeds were placed on each of 6 petri dishes (replicates;
10 seeds x dish), 10 ml of distilled water was added, and the following four
treatments were applied: Constant Darkness; Light 12 h followed by constant
darkness; Light 24 h followed by constant darkness; and Constant Light.
Germination tests were conducted in a cultivation chamber having fluorescent
tubes of white light and a light/darkness timer, at 15 ºC or 25 ºC. Dishes were
sampled daily during 15 days. A seed was considered germinated when the radicle
emerged. Days to reach half the maximum germination percentage (T50 : estimate
of germination rate) was determined in each case. Distilled water was
added whenever moisture loss was detected.
Data were analyzed using one-way ANOVA. When F tests were significant,
means were compared using the Duncan's multiple-range test (Duncan, 1955).
RESULTS
1) Temperature: 15 ºC
In G. versicolor, germination percentages under Darkness or with a pre-treatment
of 12 h Light were more than 18% greater than those under a pre-treatment
of 24 h Light (Fig. 1A). Germination rate of seeds germinated in Darkness
was greater than that in the other treatments; these seeds presented a T50 value
about half of that on seeds germinated with Constant Light (Table 1).
Hormathophylla spinosa seeds, in all cases where light was involved, registered
germination values greater than those of seeds germinated under Constant
Darkness. Values increased by 10% in the case of 12 h Light, 16% under the
pre-treatment with 24 h Light, and 28% under Constant Light, in relation to
seeds kept in Darkness (Fig. 1B; Table 1). Seed germination was significantly
greater (p<0.05) under Constant Light than under Constant Darkness (Table 1).
Similarly, results for R. complicata showed that seeds in the light treatments reached
higher germination percentages than under Constant Darkness, although
differences were not significant in any case (p>0.05). Treatments with 12 h and
24 h Light reached the higher germination rates (48% and 52% germination) than under Constant Light and Constant Darkness, respectively (Fig. 1C). Treatments with 12 and 24 h Light also showed faster germination (T50= 3.8 days)
than those under Constant Light and Constant Darkness (T50=8.1 and 3.9 days,
respectively; Table 1). Finally, T. serpylloides reached 82% germination under
any light pre-treatment. This value was 24% higher than that for seeds germinated
in Constant Darkness. However, differences were non-significant (p>0.05)
in any case. When the evolution of germination was studied, no significant differences
(p>0.05) were found among Constant Light, Constant Darkness and
Light pre-treatments of 12 and 24 h (Figure 1D).
2) Temperature: 25 ºC
No differences (p>0.05) in germination percentages were found among
treatments in any species at this temperature (Table 1). When G. versicolor seeds
were germinated at 25 ºC, or 15 ºC, there were no significant differences (p>0.05)
in final germination percentage in the Constant Light and 24 h pre-treatment
(Table 1). Seeds in the 12 h pre-treatment showed greater (p<0.05) germination
percentages at 25 than 15 ºC (Table 1). The reverse was true (p<0.05) under
Constant Darkness (Table 1). All treatments with light presented curves situated
above those corresponding to seeds germinated in Constant Darkness (Fig. 1E).
Seeds of H. spinosa had greater (p<0.05) germination percentages at 15 than 25 ºC
under all study treatments (Table 1, Fig. 1F). Germination percentages for R.
complicata were greater (p<0.05) at 25 than 15 ºC under the Constant Light or
Constant Darkness treatment (Table 1; Fig. 1G). In addition, at 25 ºC, all treatments
had lower T50 values than at 15 ºC (Table 1). Seeds of T. serpylloides under
Constant Light and Constant Darkness showed greater (p<0.05) germination percentages
at 25 than 15 ºC (Table 1; Fig. 1H). However, seeds of T. serpylloides
pre-teated with 12 h Light showed greater (p<0.05) germination percentages at
15 than 25 ºC (Table 1).
DISCUSSION
Mediterranean high-mountain ecosystems are especially sensitive to disturbances,
such as overgrazing and agriculture, increase in tourist pressure or
factors which act synergycally (Lorite et al., 2007). They are also vulnerable to
natural climatic variations (Hódar & Zamora, 2004). In this sense, interactions
between light and temperature can determine the germination time and
seed capacity to germinate of many species. Thus, some seeds require light at a
certain temperature but not at others (Pons, 1992). Light characteristics that
affect germination include photon length, quality, and irradiance reaching the
seed (Casal et al., 1998). For many species in different habitats, light responses
are measured by perception via the phytochrome (Vázquez-Yanes et al., 1996;
Casal & Sánchez, 1998).
In addition, it is generally recognized that high-mountain species do not
need a specific temperature to promote germination (Körner, 2003), although
most of them usually prefer high temperatures (Billings & Mooney, 1968). This
also seems to occur in the case of Mediterranean high-mountain species in the
oromediterranean level (Giménez-Benavides et al., 2005). However, this does
not apply to the four species of the present study, which is situated also at this
same bioclimatic level. This is very likely because of the extraordinary combination
of natural factors that mix and interact in the ecosystems of this type of
Mediterranean mountain. In this sense, Sierra Nevada is a zone where the solar
radiation is especially intense during the dry months. This produces great thermal
gradients during the day between the atmosphere, which is always cold, and
an overheated soil. This is especially true because rocky soils present dark
colours, which reflect little the incident radiation (Ortega, 1989). The fruiting
period of the study species spans from the end of July (H. spinosa) to the end of
September (R. complicata). This implies, as occurs in other Mediterranean highmountain
species (Angosto & Matilla, 1994), that light sensitivity of each species
can be a major factor inducing possible secondary dormancy (Baskin &
Baskin, 1998). This is because of the few weeks that the seeds have to germinate
after snowmelt, and the harsh climatic conditions of the Mediterranean high
mountains (Giménez-Benavides, 2005). Except in specific cases, light can also
slow down the germination process. The fact that seeds of G. versicolor need light
at 25 ºC but not at 15 ºC to activate germination may be related to an increase of
the ABA levels at this latter temperature, which can inhibit growth that precedes
germination (Karssen, 1976).
The study species are chamephytes (the replacement buds are near the
ground surface during the unfavourable season). This, and factors such as seed
weight and size, can cause any amount of light to be sufficient for promoting germination
in a very short response-time period (Casal et al., 1997). This happened
with R. complicata and T. serpylloides seeds at 15 ºC, and can make differences
hardly appreciable between the exposure to light for 12 or 24 h. The exception
was G. versicolor at 15 ºC, where the germination percentages reached after the
seeds were exposed to light for 24 h (identical to the results under Constant
Darkness) did not appear to follow the general pattern of the other species.
The cold stratification in darkness, used to increase germination percentages
in many high-mountain species (Bewley & Black, 1982; Baskin & Baskin,
1998) helps to increase light sensitivity, especially in seeds with permeable and
semi-permeable coats (Hartmann et al., 1997). This can be explained by the greater
easiness of the seeds of H. spinosa, R. complicata, and T. serpylloides to germinate in the presence of light. It could be that G. versicolor seeds do not respond
efficiently to darkness stratification because either the lack or excess of
time, and this could have determined secondary dormancy (skotodormancy).
This would involve less light sensitivity (Steadman, 2004), giving rise to the germination
percentages found when seeds were exposed to light. Nevertheless, it
would be necessary to increase knowledge of these four species, specially in
those that require light to germinate, and in particular, in the study of the
phytochrome response (specially Pfr). This, when present in sufficient quantities,
can stimulate germination (Hillhorst, 1998; Vleeshouwers & Bouwmeester,
2001). Phytochrome may also be one of the factors responsible for the relationship
between temperature and light-exposure time in the germination process
of these Mediterranean high-mountain plants.
AKNOWLEDGEMENTS
Our appreciation to Oficina del Parque Nacional-Natural de Sierra Nevada for donating the seeds of the different study species, and to Mr. David Nesbitt for translating the original manuscript into English. This work has been granted by the Plan Propio de Investigación de la Universidad de Granada to Dr. Francisco Serrano-Bernardo.
REFERENCES
1. Amritphale, D., S. Iyengar & R.K. Sharma (1989). Effect of light and storage temperature on seed germination in Hygrophila auriculata (Schumach.) Haines. Journal of Seed Technology 13: 39-43.
2. Angosto, T. & A.J. Matilla (1994). Modifications in seeds of Festuca indigesta from two different altitudinal habitats. Seed Science Technology 22: 319-328.
3. AOSA, Association of Official Seed Analysts (2000). Tetrazolium Testing Handbook, no. 29, Las Cruces, New Mexico.
4. Baskin, C.C. & J.M. Baskin (1988). Germination ecophysiology of herbaceous plant species in a temperature region. American Journal of Botany 75: 286-305.
5. Baskin, C.C. & J.M. Baskin (1998). Seeds: Ecology, biogeography, and evolution of dormancy and germination. Acad. Press, San Diego, California. 666 p.
6. Besnier, F. (1989). Semillas: Biología y Tecnología. Ed. Mundiprensa, Madrid. 524 p.
7. Bewley, J.D. & M. Black (1982). Physiology and biochemistry of seeds in relation to germination. Vol. 2. Viability dormancy and environmental control. Springer, Berlín.
8. Bewley, J.D. & M. Black (1994). Seeds: Physiology of development and germination. 2nd ed. Plenum Press, NY. 445 p.
9. Billings, W.D. & H.A. Money (1968). The ecology of artic and alpine plants. Biological Reviews 43: 481-529.
10. Blanca, G. (2002). Flora amenazada y endémica de Sierra Nevada. Junta de Andalucía. Ed. Univ. Granada, Granada. 410 p.
11. Casal, J.J. & R.A. Sánchez (1998). Phytochromes and seed germination. Seed Science and Research 8: 317-319.
12. Casal, J.J., R.A. Sánchez & J.F. Botto (1998). Modes of action of phytochromes. Journal of Experimental Botany 49: 127-138.
13. Casal, J.J., R.A. Sánchez & M.J. Yanovsky (1997). The function of phytochrome A. Plant, Cell and Environment 20: 813-819.
14. Chambers, J.C. (1997). Restoring alpine ecosystems in the western United States: environmental constraints, disturbance characteristics, and restoration success. In: Urbanska, K.M., Webb, N.R. and Edwards, P.J. (eds), pp. 161-187. Restoration Ecology and Sustainable Development, Cambridge Univ. Press, UK. 397 p.
15. Colbach, N., B. Chauvel, C. Dürr & G. Richard (2002). Effect of environmental conditions on Alopecurus myosuroides germination. I. Effect of temperature and light. Weed Research 42: 210-221.
16. Duncan, D.B. (1955). Multiple range and multiple F tests. Biometrics 11: 1-42.
17. Farmer, R.E., P. Charrette, I.E. Searle & D.P. Trajan (1984). Interaction of light, temperature and chilling in the germination of black spruce. Canadian Journal of Forest Research 14: 131-133.
18. Felippe, G.M. (1978). Estudos de germinacao, crescimento e floracao de Bidens pilosa L. Revista do Museo Paulista 25: 183-217.
19. Giménez-Benavides, L., A. Escudero & F. Pérez-García (2005). Seed germination of high mountain Mediterranean species: altitudinal, interpopulation and interannual variability. Ecological Research 20: 433-444.
20. Hartmann, K, C. Kroo â & A. Mollwo (1997). Phytochrome-mediated photocontrol of the germination of the Scentless Mayweed, Matricaria inodora L. and its sensitization by nitrate and temperature. Journal of Photochemistry and Photobiology B-Biology 40: 240-252.
21. Hillhorst, H.W.M. (1998). The regulation of secondary dormancy. The membrane hypothesis revisited. Seed Science and Research 8: 77-90.
22. Hódar, J.A. & R. Zamora (2004). Herbivory and climatic warning: a Mediterranean outbreaking caterpillar attacks a relict, boreal pine species. Biodiversity and Conservation 13: 493-500.
23. Karssen, C.M. (1976). Two sites of hormonal action during germination of Chenopodium album seeds. Physiologia Plantarum 36: 264-270.
24. Körner, C. (2003). Alpine plant life - Functional Plant Ecology of High Mountain Ecosystems. Springer, Heidelberg. 344 p.
25. Lorite, J., F.B. Navarro & F. Valle (2007). Estimation of threatened orophytic flora and priority of its conservation in the Baetic range (S. Spain). Plant Biosystems 141: 1-14.
26. Macyk, T.M. (2000). Reclamation of alpine and subalpine lands. In: Barnhisel, R.L., Darmody, R.G. and Lee Daniels, W.L. (eds), pp. 537-566. Reclamation of drastically disturbed lands. Am. Soc. Agr. Publ., Madison, WI.
27. Malcolm, J.R., C. Liu, R.P. Nelson, L. Hansen & L. Hannah (2006). Global warming and extinctions of endemic species from biodiversity hotspots. Conservation Biology 20: 538-548.
28. Maloof, J.N., J.O. Borevitz, D. Weigel & J. Chory (2000). Natural variation in phytochrome signaling. Seminars in Cell and Developmental Biology 11: 523-530.
29. Médail, F. & K. Diadema (2006). Mediterranean plant biodiversity and human impact: Macro and micro-regional approaches. Annales de Geographie 115: 618-640.
30. Médail, F. & P. Quézel (1999). Biodiversity hotspots in the Mediterranean Basin: setting global conservation priorities. Conservation Biology 13: 1510-1513.
31. Myers, N., R.A. Mittermeier, C.G. Mittermeier, G.A. Fonseca & J. Kent (2000). Biodiversity hotspots for conservation priorities. Nature 403: 853-858.
32. Ortega, F. (1989). Principales aspectos climáticos de Sierra Nevada. In: Chacón, J., Ortega, F., Pulido, A. & Rosúa, J.L. (eds), pp. 25-31. Avance del estudio de impacto ambiental del anteproyecto de reparación y acondicionamiento de la pista del río para el Campeonato del Mundo de esquí alpino con instalación de nieve artificial y nuevos remontes, Univ. Granada.
33. Pons, T.L. (1992). Seed responses to light. In: Fenner, M. (ed), pp. 259-284. Seeds: the ecology of regeneration in plant communities. CAB Int., UK.
34. Rivas-Martinez, S (1990). Bioclimatology and biogeography of West Europe. In: Duplessey, V.C., Poms, A. and Fantecli, R. (eds), pp. 225-246. Climate and global change. Proceedings of European School of Climatology and Natural Hazards. European Commission, Brussels.
35. Steadman, K.J. (2004). Dormancy release during hydrated storage in Lolium rigidum seeds is dependent on temperature, light quality, and hydration status. Journal of Experimental Botany 55: 929-937.
36. Thanos, C.A., K. Georghious & F. Skarou (1989). Glaucium flavum seed germination: An ecophysiological approach. Annals of Botany 63: 121-130.
37. Väre, H., C. Lampinen, C. Humphries, P. Williams (2003). Taxonomic diversity of vascular plants in the European alpines areas. In: Nagy, L. Grabherr, G., Körner, C., and Thompson, D.B.A. (eds), pp. 133-148. Alpone biodiversity in Europe. Ecological studies, vol. 167. Springer, Berlín.
38. Vázquez-Yanes, C., M. Rojas-Arechiga, M.E. Sánchez-Coronado & A. Orozco-Segovia (1996). Comparison of light-regulated seed germination in Ficus spp. and Cecropia obtusifolia: ecological implications. Tree Physiology 16: 871-875.
39. Vleeshouwers, L.M. & H.J. Bouwmeester (2001). A simulation model for seasonal changes in dormancy and germination of weed seeds. Seed Science and Research 11: 77-92.
40. Vogiatzakis, I.N., A.M. Mannion & G.H. Griffiths (2006). Mediterranean ecosystems: problems and tools for conservation. Progress in Physical Geography 30: 175-200.