SEED DORMANCY OF MICROCEPHALA

Mattana, E., Daws, M.I. and Bacchetta, G. (2009), Seed Sci. & Technol., 37, 491-497 Research Note

Seed dormancy and germination ecology of : a mountain endemic species of Sardinia (Italy)

E. MATTANA1, M.I. DAWS2 AND G. BACCHETTA1

1 Centre for the Conservation of Biodiversity (CCB), Department of Botanic Sciences, University of Cagliari, 09123, V.le S. Ignazio da Laconi 13, Cagliari, Italy (E-mail: [email protected]) 2 Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK

(Accepted November 2008)

Summary

Lamyropsis microcephala only occurs in a very restricted area in the Gennargentu massif (CE-Sardinia, Italy) at 1450-1700 m a.s.l. This rare species has been inserted in the International Union for Conservation of Nature (IUCN) Red Lists under Critically Endangered. In this study, the effect of a pre-chilling period (90 days at 5°C) on germination was investigated as well as the ability of this species to form a soil seed bank. L. microcephala seeds exhibited type 2 non-deep physiological dormancy. Pre-chilling increased the final germination percentages, the germination speed and the temperature range at which high levels of germination were observed. The high optimal germination temperatures (ca. 70% germination at 30°C) demonstrate that this species is well adapted to a temperate climate and the lack of a persistent soil seed bank represents a rarity trait that likely exacerbates the threats to the survival of this species.

Experimental and discussion

Lamyropsis (Charadze) Dittrich is a genus of the and a member of the tribe Cardueae Cass. subtribe Carduinae Dumort, that comprises 8 perennial species distributed from S Europe eastwards to SW Asia (Mabberley, 2002) and in particular the Caucasus region, where the temperate continental bioclimate prevails (Rivas-Martínez et al., 2004). Lamyropsis microcephala (Moris) Dittrich & Greuter only occurs in a very restricted area in the Gennargentu massif, or more precisely on the south-western and north-eastern slopes of M.te Bruncu Spina at 1450-1700 m a.s.l. (NU, CE-Sardinia, Italy). Available climatic data (Fonni, NU, 992 m. a.s.l.) show a typical Mediterranean annual trend for temperature and precipitation with a hot, dry summer and cool, wet winter. This rare species has been inserted in the International Union for Conservation of Nature (IUCN) Red Lists under Critically Endangered CR B1ab(iii)+2ab(iii) (IUCN, 2008). L. microcephala

491 E. MATTANA, M.I. DAWS AND G. BACCHETTA has been reported to have only a limited sexual reproductive capacity by virtue of both a low seed set and low germination percentages, largely relying instead on vegetative propagation (Diana Corrias, 1977; IUCN, 2008). However, in a recent study, although Bacchetta et al. (2007) confirmed a low production of fertile seeds, they obtained higher percentages of seed viability (76% by X-ray analysis) and germination (50% at 20°C and 8/16 h light and dark) than reported in previous studies. Finch-Savage and Leubner-Metzger (2006) classified seeds of the Asteraceae as either non-dormant (ND) or physiologically dormant (PD). PD can be divided into three levels; the majority of seeds with PD have a non-deep PD level. Non-deep PD can be broken by scarification, after-ripening in dry storage, hormone (GA) application and cold or warm stratification (Baskin and Baskin, 2004). Seeds of species that require pre-chilling for breaking dormancy become non-dormant during winter and germinate in spring (Baskin and Baskin, 2003). Seeds of many Asteraceae, require pre-chilling to break their physiological dormancy (e.g. Buchele et al., 1991; Baskin et al., 1998; Brändel, 2004) and light to germinate (Merritt et al., 2006). The effect of light on germination on Asteraceae can also be correlated with their seed mass: germination becomes less dependent on light with increasing seed mass (Milberg et al., 2000). Soil seed banks can be classified as transient (TSB) or persistent (PSB) and to distinguish between TSB or PSB, knowledge of the presence of ungerminated viable seeds after 1 year of burial is required (Thompson and Grime, 1979). The ability to form a PSB is crucial to the survival of many rare or declining species (Keddy and Reznicek, 1982; Rowell et al., 1982; Thompson et al., 1993; Lutz Eckstein et al., 2006). Baskin and Baskin (1998) report several studies where persistent soil seed banks have been identified for species in the Asteraceae. Knowledge of germination requirements and seed persistence in the natural environment will help facilitate effective ex situ and in situ conservation of this species. Consequently, we investigated the germination ecology of L. microcephala in particular focusing on (1) the effects of a pre-chilling period on germination to test if seeds of this species show a type (sensu Baskin and Baskin, 2004) of non-deep PD and (2) the ability of L. microcephala seeds to form either a TSB or PSB. Cypselas (hereafter seeds) were collected after obtaining permits as required by European and national laws for the species listed in the appendices of the Habitat Directive (DIR.92/43/CEE). Harvesting was conducted in September 2007 at the time of natural ripening / dispersal from the main population of L. microcephala in the Bruncu Spina Mountain (N 40° 01’ E 09° 17’), along the Rio Aratu river (Desulo, NU). Seeds were stored at the Sardinian Germplasm Bank (BG-SAR) where, once cleaned by removing the papus and separated from the empty seeds, they were placed in a dry room at 15% R.H. and 15°C until germination experiments started in November 2007. Seed dry mass was 1.8 ± 0.5 mg, calculated by drying three replicates of five seeds each in an oven at 103°C for 17 h, followed by weighing to seven decimal places. Three replicates of 25 seeds per treatment were sown on the surface of 1% water agar in Petri dishes and incubated at both constant (5, 10, 15, 20, 25 and 30°C) and alternating temperatures (20/05, 25/10 and 30/15°C). In all treatments, seeds were exposed to irradiance for 8 h per day. In the alternating temperature regimes the 8 h light

492 SEED DORMANCY OF LAMYROPSIS MICROCEPHALA period coincided with the elevated temperature period. To verify the effect of light on germination, a preliminary test was carried out by sowing three extra replicates at 25°C in the dark, achieved by wrapping dishes in aluminium foil. Seeds in this experiment were only scored once, at the end of the test, to avoid any exposure to irradiance. To investigate the effect of pre-chilling, the same number of Petri dishes of seeds on agar was put at 5°C, in the light, for 3 months before transfer to the above-mentioned conditions (except for 5°C). Germination was considered to be visible radicle or cotyledon emergence. After 75 days at the end of the germination tests the viability of any remaining seeds was checked by a cut test (ISTA, 2006) and the final germination percentage calculated on the basis of the total number of filled seeds. Experimental seed burial was carried out following a modification of the protocol in Arroyo et al. (2004). Sets of 5 replicates containing 10 seeds each were introduced into fine grain nylon mesh envelopes which were placed in plastic nets. The envelopes were filled with sieved local soil and buried so that the seed envelopes were at a depth of 5 cm at the original population location. The burial sites were the four vertices and the centre of a square (1 m sides). Seed burial was performed at the time of natural seed dispersal. After 1 year, the replicates were exhumed and analyzed. L. microcephala seeds did not require light for germination (preliminary germination test at 25°C both in the light and dark; One-way ANOVA, p > 0.05) and, without any pre- treatments they germinated best at the highest temperature regimes tested, both at constant and alternating temperatures (figure 1A). The cut test carried out on non-germinated seeds showed a total viability (germinated plus viable seeds) of 69.2 ± 16.7% (± 1 SD). The germination tests carried out after pre-chilling resulted in significantly higher levels of germination (One-way ANOVA, p < 0.001) than without any pre-treatment (figure 1B). In particular, not only the maximum germination percentages increased compared to the tests without pre-chilling, but seeds also germinated at high percentages (> 40 %) at a wider temperature range, including temperatures lower than 25°C (at constant temperature regimes) and with a minimum temperature lower than 15°C (at alternating temperature regimes). There were significant differences in percentage germination (One-way ANOVA, p < 0.05) at each germination condition with or without pre-chilling, except for 25, 30 and 20/05°C (figure 1). Pre-chilling also had a positive effect on germination rate: the final germination percentage was reached more quickly after pre-chilling than without pre-chilling (figure 2). For example, at 25°C time to maximum germination was reduced from 49.0 to 6.0 days for control and pre-chilled seeds respectively. After one year of burial 26.0 ± 20.7% (mean ± SD) of the seeds were not retrieved having decayed completely. In all five envelopes only split empty seed coats were retrieved, suggesting that the seeds had germinated in situ in the soil and that this species is unable to form a persistent soil seed bank (PSB). No viable seedlings were retrieved. L. microcephala seeds, following the dormancy classification system (Baskin and Baskin, 2004), showed a type 2 of non-deep physiological dormancy (PD): after pre- chilling the temperature range at which seeds could germinate to high percentages increased from high to low temperatures (Baskin and Baskin, 2004). Pre-chilling increased the final germination percentages, the temperature range at which seeds can germinate (figure 1) and the germination speed (figure 2) confirming the results found in previous

493 E. MATTANA, M.I. DAWS AND G. BACCHETTA

100 A B b c b 80 b c ab c ab 60 a a 40 b germination (%) b a 20 b b a a 0

5°C 10°C 15°C 20°C 25°C 30°C 20/05°C 25/10°C 30/15°C 10°C 15°C 20°C 25°C 30°C 20/05°C 25/10°C 30/15°C Figure 1. Final germination percentage after 75 days at each temperature regime in the light (A) without any pre-treatments and (B) after pre-chilling at 5°C for 3 months; in each graph values with the same letters are not significantly different at p > 0.05 (One-way ANOVA followed by post hoc Fisher’s LSD test). Data are the means of 3 replicates (± 1 standard deviation). studies for several Asteraceae species (Baskin and Baskin, 1988; Buchele et al., 1991; Baskin et al., 1998; Brändel, 2004). Alternating temperature regimes and light did not improve final germination percentages, which is consistent with a seed dry mass of 1.8 ± 0.5 mg. Probert (1992) suggested a response to alternating temperatures represents an adaptation of small- seeded species which ensure that germination occurs only close to the soil surface. In addition, as found in several studies, both in tropical and temperate climates (Milberg et al., 2000; Jankowska-Blaszczuk and Daws, 2007), small seeds are more likely to require light to germinate. Thus, seedlings of this light insensitive (for germination) species are presumably able to emerge from greater soil depths than light can penetrate to, which is likely given a seed mass of 1.8 mg (Bond et al., 1999). The ability of the seeds to germinate in darkness has been confirmed by the results of the experimental seed burial, which showed that this species is unlikely to form a PSB. Persistent seeds tend to be small and compact, while short-lived seeds are normally larger and either flattened or elongated (Thompson et al., 1993). However the lack of a PSB may increase the vulnerability of this species to further habitat disturbance / loss. Indeed an ability to form a PSB is a trait that has been associated with widespread / common species in comparisons of common and rare species (e.g.: Burne et al., 2003; Williams et al, 2005). The high optimal germination temperatures found for L. microcephala seeds, as well as its pre-chilling requirement, demonstrate that this species is well adapted to a temperate climate which is consistent with the centre of origin for this genus, being the temperate continental regions of SW Asia (Mabberley, 2002). This suggests that L. microcephala may not be well adapted to the current Mediterranean climate in Sardinia. The optimal germination temperature for Mediterranean species is typically within the range 5-15°C and these species are also characterized by having a low germination rate and being negatively affected by prolonged chilling (Thanos et al., 1989; Skordilis and Thanos, 1995; Doussi and Thanos, 2002).

494 SEED DORMANCY OF LAMYROPSIS MICROCEPHALA

80 100

25°C 30°C p < 0.01 p < 0.05 80 60 germination (%)

60 40

40 time (days)

20 20

0 0 no pre-chilling after pre-chilling no pre-chilling after pre-chilling Figure 2. Time to achieve the final germination percentages at 25 and 30°C, with and without pre-chilling at 5°C for 3 months (squares). Data are the mean of three replicates ± 1 standard deviation (p < 0.01 at 25°C and p < 0.05 at 30°C by One-way ANOVA followed by post hoc Fisher’s LSD test). The final germination percentages for each condition are shown by the bars (+1 standard deviation).

Seed dispersal take place in late summer (Diana Corrias, 1977) and L. microcephala seeds, having a transient soil seed bank (TSB), presumably germinate in the following late spring, after experiencing the low winter temperatures that accelerate germination. In contrast, ‘typical’ Mediterranean species germinate in early winter thereby maximising the length of the growing season during the rainy period, before the onset of summer drought (Doussi and Thanos, 2002). Consequently, newly established seedlings of L. microcephala will face the summer drought that is typical of the Mediterranean climate which may explain why this species only occurs in comparatively rare moist micro-sites such as catchment areas, where water is still available during the main part of the summer (Bacchetta et al., 2007). In fact, seed dispersal by wind might result in high seedling mortality after seeds being dispersed to a range of unsuitable drier micro-sites. This loss of seed may explain why vegetative reproduction is mainly responsible for population persistence/growth in these rare moist micro-sites, as reported by Diana Corrias (1977), Snogerup (1985) and IUCN (2008).

Acknowledgements

The Centre for the Conservation of Biodiversity (CCB) is supported by the “Provincia di Cagliari - Assessorato Tutela Ambiente”. This research was also supported by the “RAS - Assessorato Difesa dell’Ambiente – Servizio tutela della natura”. The support of the Seed Conservation Department, Royal Botanic Gardens, Kew is gratefully acknowledged. G. Fenu (CCB) helped with fieldwork.

495 E. MATTANA, M.I. DAWS AND G. BACCHETTA

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