Germination requirements in a population of Typha latifolia

Aquatic botany ELSEVIER Aquatic Botany 56 (1997) 1- 10 Germination requirements in a population of Typha latifolia Tiziana Lombardi *, Tiziana Fochetti, Andrea Bertacchi, Antonino Onnis Department of Agronomia e Gestione dell'Agro-Ecosistema, Via S. Michele degli $calzi, 2, UniversiChdegli Studi, 56124 Pisa, Italy Accepted 22 October 1996 Abstract The physio-ecological requirements of germination and early achene growth of a population of Typha latifolia L. were studied. The effects of constant (10, 20 and 30°C) or alternating (10/20 °, 20/30 ° and 10/30°C) temperatures, photoperiod (12/12, 8/16, 6/18, 4/20, 2/22 and 0 / 2 4 h day/night), after-ripening time and culture medium salinity (NaCI) on germination were assessed. Results showed that germination required light and alternating temperatures (optimal results at 20/30°C with 12/12 h photo-thermoperiod). In these conditions the germination trend showed no appreciable variation during the first year of achene after-ripening. The presence of more than 0.1 M NaC1 in the culture medium caused a significant reduction in the percentage of seeds germinating, and inhibited further seedling growth. Typha latifolia can, therefore, be considered a helio-thermophilic and glycophilic species, at least during early growth stages. © 1997 Elsevier Science B.V. All rights reserved. Keywords: Typhaceae; Seeds; After-ripening; Salinity; Autecology 1. I n t r o d u c t i o n An important aspect o f macrophytes in wetland ecology is the function of these species as bioindicators o f the conditions of the water body in which they are at least partially submerged. Such species include Althenia filiformis (Onnis, 1981), Typha * Corresponding author. 0304-3770/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved. PII S0304-3770(96)01096-0 2 T. Lombardi et al./Aquatic Botany 56 (1997) 1-10 latifolia (Taylor and Crowder, 1983), Juncus acutus (Stefani et al., 1991), Eichornia crassipes (Ak~in et al., 1994, Lenzi et al., 1994). This function assumes particular significance since wetlands are increasingly subjected to direct a n d / o r indirect man-related pollution. In this context one of the more important problems is actually the increasing salinization of water bodies, a phenomenon that could be due both to the rise of saltwater from deeper levels of groundwater as a result of an excessive water extraction for drinking or agricultural purposes, and to badly planned hydraulic engineering works in coastal lagoon areas. We, therefore, undertook an in-depth study of the autecology of one of the most representative components of wetlands in the circumpolar boreal hemisphere, T. latifolia L. This species is characterized by the ability to form dense mono- or oligo-specific populations rapidly, thanks to its extensive rhizomatous root system. Each of its spikes is capable of producing thousands of seeds. In nature, however, it is rare to find young T. latifolia seedlings, and hardly ever in the vicinity of such populations (McNaughton, 1968; Bonnewell et ai., 1983). Sharma and Gopal (1978) and Grace (1983) hypothesized that the non-germinability of seeds in nature within and near the areas already occupied by T. latifolia is to be attributed to the absence of ecological conditions favourable to germination, rather than to allelopathic effects. Such conditions would above all include the absence of cover vegetation, in order to make easier the subsequent rapid expansion by vegetative propagation (Gopal and Goel, 1993). Previous studies on germination in this species showed seed photosensitivity and high temperature requirements. Thus Morinaga (1926), followed later by Sifton (1959) and Bonnewell et al. (1983), reported that optimal results are achieved with elevated alternating temperatures, but only if accompanied by light and very low pO 2. Factors external to normal T. latifolia ecology may affect germination and dispersal, leading to a decrease of this species in stands. We therefore addressed this issue by examining an Italian T. lattfolia population, in order to obtain experimental data on (1) ecological requirements for germination and early growth stages, (2) the natural physio-ecological conditions of the habitat in which plants producing the trial fruits had developed. 2. Materials and methods Achenes of T. latifolia were harvested at maturity (October) from plants forming a monospecific population along the drainage ditch 'della Bigattiera' near Tirrenia (Pisa, Italy). Achenes were stored in a dark room at 20 + 2°C until they were used in germination and growth tests. Fruits were placed in 9-cm diameter Petri dishes each containing 30 ml culture medium. One hundred fruits were used for each trial, subdivided into four replications each consisting of 25 fruits. The batches were then placed in growth chambers set to the required temperatures and regulated to the required photoperiod (70 p,m o l m -2 s- ~, Philips T2 40 W / 3 3 lamp). Each experimental test lasted for 10 days. Seeds were considered germinated upon rupture of the pericarp by the coleoptile. All data were analyzed using one- or two-way ANOVA followed by Duncan's multiple range comparison test. T. Lombardi et aL / Aquatic Botany 56 (1997) 1-10 3 2.1. Germination and after-ripening time In the months of November, January, May and October subsequent to date of harvesting (30, 100, 205 and 360 days of after-ripening), Typha achenes stored at 20 + 2°C were placed in Petri dishes with deionized water in controlled temperature chambers at constant (10°C, 20°C and 30°C) and alternating (10/20°C, 10/30°C, 20/30°C, with a 12/12 h thennoperiod) temperatures, in darkness or light with a 12/12 h photoperiod. During the alternating temperature trials, the light period was always accompanied by the highest temperatures. 2.2. Germination and photoperiod Six-month-old achenes stored at 20 + 2°C were also tested in deionized water as described above, at alternating temperatures of 20/30°C and with the following photoperiods (light/dark): 12/12 h, 8 / 1 6 h, 6 / 1 8 h, 4 / 2 0 h, 3/21 h, 2 / 2 2 h, 0 / 2 4 h. 2.3. Germination and salt stress The germination trend of 6-month-old seeds grown in NaC1 solutions with different concentrations (0.0 M, 0.1 M, 0.2 M, 0.3 M, corresponding to 0.0%o, 5.8%o, 11.6%o and 17.4%~, respectively) was studied. Trials were set up as described above, and were carried out both at constant (10°C, 20°C, 30°C, with 12/12 h photoperiod) and alternating (10/20°C, 20/30°C, 10/30°C, with 12 h thermo-photoperiod) temperatures. At the end of each trial, on the tenth day of culture, young seedlings were evaluated for determination of shoot length and assessment of possible presence of chlorosis and necrosis. 3. Results 3.1. Germination and after-ripening time Dark germination was found to be virtually absent (consistently below 10%) at the end of the 10 day culture period, both for seeds subjected to constant (10°C, 20°C, 30°C) or alternating (10/20°C, 20/30°C, 10/30°C) temperatures. In the presence of a photoperiod, germination was strongly influenced by the different temperatures utilized. At constant temperatures, germination never reached 50%. Thus, values varied from a minimum of 0% at 10°C to a maximum of 34% at 30°C (Fig. 1). At alternating temperatures of 10/20°C, seeds usually began to germinate on Day 3. On Day 10 of culture, germination was close to 100% in all months investigated. At 10/30°C, germination was in excess of 50% as early as Day 3 of culture (in November 1993, a value of 72.5% was obtained). Germination capacity reached 100% on Day 4. At the alternating temperatures of 20/30°C, seeds began to germinate on Day 2, reaching a mean percentage of roughly 70% on Day 3, 100% germination was obtained as early as Day 4. To Lombardi et al./ Aquatic Botany 56 (1997) 1-10 []Day3 50 []Day10 a tO ! "~ ¢-. 25 a . . . 30 . c 100 205 I'~, ,d 360 d 30 , cHi b , 100 205 360 after-dpening time 20"C 30"C Fig. 1. Mean percent germination (%) of Typha latiJblia L. seeds at 20°C and 30°C after 30-360 days of after-ripening. No germination was observed at 10°C. Different letters within the same day of culture and temperature indicate significant differences at the 0.05 probability level among days of after-ripening. 3.2. Germination and photoperiod Six-month-old seeds subjected to a 12/12 h, 8/16 h or 6 / 1 8 h (light/dark) photoperiod showed a mean germination of roughly 77% on Day 3 (Fig. 2). In all cases, 100% germination was achieved by Day 4. With a decrease in photoperiod (4/20 h, []Day2 aaa []Day3 aaa •Day4 aaa •Day7 •Day10 a 100 75 o~ v E .9 c" 50 E 25 12/12 h 8/16 h 6/18 h 4•20 h 2/22 h 0/24 h photoperiod Fig. 2. Mean percent germination (%) of Typha latifolia L. seeds at different photoperiod and after 2, 3, 4, 7 and 10 days of culture. Different letters within the same day of culture indicate significant differences at the 0.05 probability level among different photoperiod. T. Lombardi et al./ Aquatic Botany 56 (1997) 1-10 5 3/21 h light/dark), a marked reduction in the germination energy was observed. Thus, with a 4 / 2 0 h photoperiod only 12.5% germination was obtained on Day 3, and 100% germination was not achieved until Day 10 of culture. With 3 / 2 1 h, germination decreased further, so that no more than 3.75% germination was obtained after 72 h, and 87% on Day 10. With only 2 h of light, even lower germination percentages were observed; thus germination was virtually absent after 72 h (2.5%), and rose to 70% by Day 10. In the dark conditions no germination occurred. --am--0.0 M -,,O--0.1 M --o--0.2 M -,-0--0.3 M 10/20"C a 100, m......lm--m~...~l a o~ v c 0 50 E 2 3 4 5 6 7 8 9 10 days 20/30"C 100 • / v c" ii~ln_i_n_ll_i_ n a .o_ r- 50 E 2 3 4 5 6 7 8 9 10 days 10/30°C 100 - o~ tO 50 ._= • C 01 2 3 4 5 6 7 8 9 10 days Fig. 3. Mean percent germination (%) of Typha latiJblia L. seeds at alternating temperatures during 10 days of culture in NaCI (M). Different letters at 10 days indicate significant differences at the 0.05 probability level among different salinity treatments. 6 T. Lombardi et al./Aquatic Botany 56 (1997) 1-10 Table 1 Mean length ( c m ) + S E of T. latifi~lia seedlings in different NaCI solutions and at various alternating temperatures (°C) Temperature (°C) 10/20°C 10/30°C 20/30°C Length of seedlings (cm) 0 NaCI 0.1 M NaCI 0.2 M NaCI 0.3 M NaCI 8.4+ 1.3 a 17.1 _+2.1 b 15.9 5:1.2 b 1.5+0.5 c 1.6-+0.1 c 2.0 -+ 0.3 d 1.1 +0.1 e 1.25:0.2 e 1.5 5:0.1 f 0.05:0.0 g 1.0+0.3 h 1.3 5:0.4 h Different letters within the columns and the lines indicate significant differences at the 0.05 probability level. 3.3. Germination and salt stress Germination trials conducted in the presence of NaCI showed that under constant temperatures germination was clearly compromised. At the highest salt concentrations (0.2 and 0.3 M) no germination was achieved in any trial. In 0.1 M NaC1, germination reached 6.7 ___2.7% at 20°C and 4.0 ___2.3% at 30°C by Day 10 compared, respectively, to 18.7 _ 2.1 and 34.0 ___4.4% in the control. At 10°C germination was totally absent even at the lowest NaCI concentrations. In the presence of altemating temperatures, on the other hand (Fig. 3), it was found that germination decreased proportionately to increasing NaCI concentration. Thus with a 10/20°C thermoperiod, germination of achenes subjected to 0.2 M NaC! did not rise above 20% on Day 10, and germination was absent in achenes cultured on 0.3 M NaC1. With a 10/30°C photoperiod, an appreciable reduced rate of germination was observed, but percent germination on Day 10 was consistently elevated for 0.1 and 0.2 M NaC1 (97.3% and 81%, respectively). In contrast, the 0.3 M concentration exerted a marked inhibitory effect on germination (14.7%). With a 20/30°C thermoperiod, germination on Day 10 of culture on 0.1 and 0.2 M NaCI was little above 60%, while only 33% germination was observed in the presence of 0.3 M. T. latifolia seedlings (shoot + root) grown on culture media containing NaC1 at various molarities were also examined on Day 10 of culture in order to assess growth. A marked decrease in growth as compared to controls cultured on deionized water was observed at each saline concentration assayed (Table 1). Analysis of variance showed that shoot length recorded at 10/20°C and 10/30°C differed significantly from lengths Table 2 Mean percent+ SE of vegetative tip necrosis of T. latifolia seedlings in different NaC1 solutions and at various alternating temperatures (°C) Temperature (°C) 10/20°C 10/30°C 20/30°C Length of seedlings (cm) 0 NaCI 0.1 M NaCI 0.2 M NaCI 0.3 M NaCI 0.0 _+0.0 a 0.0-+0.0 a 0.0 _+0.0 a 17.5 5:2.2 b 2.2_+ 1.0 c 12.1 _+3.0 d 38.9 -+ 0.4 e 5.7-t-0.3 f 14.3 _+3.2 d 0.0 ___0.0 h 5.6_+ 1.7 f 20.8 _ 1.3 g Different letters within the columns and the lines indicate significant differences at the 0.05 probability level. T. Lombardi et al./Aquatic Botany 56 (1997) 1-10 7 obtained at 20/30°C. The latter temperatures were found to give the greatest seedling lengths. In addition, a high percentage of stressed seedlings showed clear signs of vegetative tip necrosis, and this percentage increased with increasing saline concentration. Statistical analysis (two-way ANOVA) showed significant temperature-related differences in percent necrosis at each NaC1 concentration utilized (Table 2). Thus at 10/30°C no statistically significant percentage differences were found at 0.2 and 0.3 M, whereas at 20/30°C significant differences were observed at 0.1 and 0.2 M NaCI. 4. Discussion Observations carried out in the first year of research led to a fairly detailed picture of the climatic and environmental requirements of an Italian T. latifolia population. Attention focused in particular on analysis of seed germination trends and early growth of seedlings, which to date have not formed the subject of in-depth study by any author. Results obtained showed that alternating temperatures were necessary for germination. Under constant temperatures, germination consistently failed to reach 50% by the tenth day of culture. Furthermore, at 10°C germination was found to be close to zero. At all three alternating temperatures considered, on the other hand, maximum germination was observed (100% throughout the entire trial year). It was also noted that among the three thermo-periods considered, the two in which a 30°C phase was present (10/30°C and 20/30°C) were more favourable to germination. The favourable effect of alternating temperatures on seed germination has long been known, and has been reported for many species such as Cynodon dactylon (Morinaga, 1926), Bidens tripartitus (Rollin, 1956), Lycopus europaeus (Thompson, 1969), Chenopodium album and Panicum maximum (Murdoch et al., 1989). Sensitivity to periodic temperature fluctuation constitutes an important sensory system enabling plants to respond to daily variations in soil surface characteristics, ensuring, for instance, that germination of deeply buried seeds is prevented. In addition, since seeds are subject to fewer temperature fluctuations when submerged or floating, they can more successfully perceive the pronounced changes in thermoperiod once they reach land (Thompson, 1974). It can therefore be suggested that such a mechanism enables T. latifolia germination to occur on land rather than in water. Our results confirm previous findings concerning the importance of light for germination of Typha spp. achenes. Results obtained in our trials, showing a marked decrease in germination rate and capacity when the photoperiod fell below 6 h day -~, supported the above findings (Fig. 2). These results indicated that germination was dependent not only on the sum of light hours to which seeds were exposed, but also on the cumulative number of dark hours that neutralize the previous light-derived positive impulse. So it is not only light intensity but also the number of hours of light that regulate T. latifolia seed germination. It can, therefore, be hypothesized that the principal mechanism triggering germination of this species is the presence of phytochrome, as already demonstrated for other species with intermittent light germination trigger, such as some species of Pawlonia, Limonium, Epilobium, Hypericum, Ludwigia (Bonnewell et al., 1983). In this way seeds avoid the risk of germination if they are buried or shaded by other 8 T. Lombardi et al./Aquatic Botany 56 (1997) 1-10 vegetation, including cases when they fall in the vicinity of dense monophytic populations from which the seeds themselves are derived (Sharma and Gopal, 1978). Light requirement can, therefore, guarantee seedling viability because of insufficient levels of Typhaceae seeds reserves, above all oleic and linoleic acid (Meara, 1957); in this family germination indeed initiates with emergence of the photosynthesizing coleoptile (Lang, 1965). During the first year, no appreciable changes in germination rate and/or capacity were noted, with the exception of the 20°C and 30°C trials, in which a germination peak was observed on the tenth day of culture. This peak, which occurred in May (205 days of after-ripening), disappeared in the following trial (at 360 days of after-ripening) (Fig. 1). Such a result could be explained by the fact that in nature, T. latifolia seeds germinate roughly at this time of the year (in spring), when water bodies still have adequate water levels, external temperatures are very mild and hours of daylight are increasing. In such conditions, an endogenous biorhythm could favour seed germination even under non-optimal thermoperiod conditions (constant 20°C) inasmuch as the photoperiod is markedly favourable (12 h / 1 2 h). In India, on the other hand, it is reported in the literature that Typha angustata seeds germinate in January (Sharma and Gopal, 1978). Typha latifolia is considered a typical freshwater species (Fassett and Calhoun, 1952; McMillan, 1959; McNaughton, 1966; Grace and Wetzel, 1982). However, observations carried out in nature to investigate species distribution showed this species also to be present in wetlands or drainage channels that undergo frequent influx of brackish and/or salt water. NaC1 had a negative effect on germination rate and capacity, and seedling growth and development. This effect was proportionate to culture medium NaCi concentration, but it was detected under optimal photoperiodic conditions as well, so that even the lowest NaC1 concentration was already found to exert an adverse effect. Furthermore, seedlings were also adversely affected by changes in culture medium composition during the thermoperiod previously found to be the most favourable in the presence of deionized water (20/30°C). It would therefore appear that the 'heat'-'NaCl' synergy accentuated the salt-related effect of ionic and/or osmotic stress (Onnis and Pelosini, 1976; Cremonini et al., 1992). Additional effects recorded during the trial included shoot tip necrosis, exhibiting percent values that increased with increasing solution salinity. Here, too, it was the 20/30°C thermoperiod that induced the greatest percentage of necrotic tips out of the total number of germinated seeds. Therefore, T. latifolia should be considered a glycophilic species, at least during germination and early growth, and it is unable to colonize saltwater environments. This finding is in agreement with previous reports in the literature concerning other species of the same genus, such as Typha glauca (Galinato and Van der Valk, 1986). This research into the germination behaviour of T. latifolia allowed a number of conclusions on the ecology of this species to be drawn. Its seeds are easily transportable over long distances by anemochory, and thanks to endogenous control mechanisms, they succeed in germinating in areas that prove to be optimal for the formation of dense monospecific or oligospecific populations. This development takes place extremely rapidly: during our observations we found that plants were able to spread vegetatively T. Lombardi et a l . / Aquatic Botany 56 (1997) 1-10 9 through rhizomes as early as the third month after germination. In addition, Sobrero et al. (1993) noticed that in the Argentinian species Typha subulata, sexual reproduction became possible during the plants first year of life ( 7 - 8 months). Indeed colonization is so rapid that the species can easily become a weed infesting water bodies and irrigation channels, thereby inflicting considerable damage on agriculture (cf. T. angustata in India, in Gopal and Sharma, 1983; T. subulata in Argentina, in Sobrero et al., 1993). In conclusion, the decrease in 7". latifolia stands in coastal areas affected by the presence of saltwater of marine or mainland origin can thus be explained by the extreme sensitivity of this species to variations in water body salinity values that can range from 14.5%~ in winter to above 89%o in summer (Onnis and Pelosini, t976)--particularly during the germination stage and early seedling growth. As a result of this sensitivity, colonization by seed becomes unlikely, whereas survival of an existing population may be possible, albeit with difficulty, through vegetative propagation. On-going research on other species has shown that fertility is markedly depressed in plants grown on salt-rich media (Lombardi, 1991). Such a situation may also hold for Typha, so that preservation and dispersal of the species could become even more difficult if the trend towards salinization of water bodies continues. References Ak(jin, G., Saltabas, O. and Afsar, H., 1994. 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