Protoplasma

, Volume 243, Issue 1, pp 117–128

Ranunculus glacialis L.: successful reproduction at the altitudinal limits of higher plant life

Authors

    • Institute of BotanyUniversity of Innsbruck
  • Gerlinde Steinacher
    • Institute of BotanyUniversity of Innsbruck
  • Ursula Ladinig
    • Institute of BotanyUniversity of Innsbruck
Original Article

DOI: 10.1007/s00709-009-0104-1

Cite this article as:
Wagner, J., Steinacher, G. & Ladinig, U. Protoplasma (2010) 243: 117. doi:10.1007/s00709-009-0104-1

Abstract

Biodiversity decreases with increasing altitude, mainly because of the increasingly adverse climate. In the European Alps, only a few plant species occur above 4,000 m a.s.l., among these is Ranunculus glacialis L. Current studies have shown that R. glacialis has a highly conservative growth strategy and low developmental plasticity in response to different dates of snowmelt. Therefore, it was of particular interest to observe whether this strategy is maintained at higher altitudes and to reveal the reproductive limits. We examined the effect of the date of snowmelt on reproductive development and reproductive success in R. glacialis over several years at two subnival sites (2,650 and 2,880 m a.s.l.) and at a nival site (3,440 m a.s.l.) in the Austrian Alps. At the subnival sites, reproductive performance was relatively stable (prefloration period, i.e. snowmelt to onset of anthesis, 2–3 weeks; postfloration period, i.e. onset of anthesis until fruit maturity, 4–5 weeks). Depending on the date of flowering, the mean seed/ovule (S/O) ratio was 0.5–0.8. The temporal safety margin between seed maturation and the onset of winter conditions was at least 1 month. The situation was quite different in the nival zone: the prefloration period usually lasted 1 month, anthesis up to 2 weeks, and seed development 6–7 weeks; when seeds matured in time, the S/O ratio was 0.4–0.6. Overall, R. glacialis shows a high developmental plasticity. At higher altitudes, R. glacialis can double the time taken for seed development but runs a high risk of seeds not maturing in time.

Keywords

Ranunculus glacialisDevelopmental plasticityEmbryologyMountain plantPrefloration periodReproductive successSeed development

Abbreviations

S/O ratio

Seed/ovule ratio

DAA

Days after onset of anthesis

SN-L

Subnival low

SN-H

Subnival high

doy

Day of year

Introduction

In mountain systems, species richness diminishes with altitude. In the European Alps, most alpine plant species occur between 2,000 and 3,000 m a.s.l. (Grabherr et al. 1995). As altitude increases, fewer species are adapted to the prevailing environmental extremes. Along the alpine–nival ecotone (Pauli et al. 1999), species richness decreases from more than 200 species in the alpine zone to about 30–40 species in the nival zone (Grabherr et al. 1995). Only about a dozen species still occur above 4,000 m. There, isolated individuals inhabit microhabitats where the thermal regime is probably not very different to that some hundred metres lower (Körner 2003). Among the highest ascending vascular plants in the Alps is Ranunculus glacialis L. along with some saxifrages such as Saxifraga biflora, Saxifraga bryoides, Saxifraga moschata, and species of the genus Androsace, Gentiana, Achillea, and Draba (Heer 1885; Anchisi 1985; Ozenda 1988; Grabherr et al. 1995; Körner 2003).

The main constraints at higher altitudes are large temperature fluctuations, an increased number of days with snow and regular night frosts during the growing season, which lasts about 10 weeks (Körner 2003; Larcher and Wagner 2009). Therefore, plants adapted to such environments must be snow resistant and to a certain extent frost resistant in their active state (Larcher and Wagner 1976; Taschler and Neuner 2004; Larcher et al. 2010). Additionally, they must exhibit a high developmental flexibility in this climatically stochastic environment. In spite of a certain degree of adaptation, it is a life at the margin (Crawford 2008), permanently at risk of suffering damage and loss of the seed crop in climatically adverse periods (Kudo 1991; Ladinig and Wagner 2007).The same situation holds true for plants growing in arctic environments (Molau 1993, 1997). Unfavourable weather conditions additionally reduce the abundance of pollinators and restrict their activity to short favourable periods (Arroyo et al. 1985; McCall and Primack 1992). This can lead to pollen limitation and a reduced seed set in predominant outcrossing species (Muñoz and Arroyo 2006; García-Camacho and Totland 2009).

Short vegetation periods are not only restricted to higher altitudes but are also characteristic of snow-beds in lower mountain regions. In hollows, huge snow packs can accumulate during winter, which melt 1–2 months later than in the surrounding terrain. These late-melting microhabitats are colonised by snow-bed specialists, which tolerate the constraints of late flowering: growth and reproduction take place at a decreasing day length and at decreasing temperatures in a mostly wet and therefore already cool environment. Snow-bed species, as a rule, show a high degree of phenological plasticity (e.g. Kudo 1991; Galen and Stanton 1991, 1995; Kudo and Suzuki 1999; Inouye et al. 2002; Totland and Alatalo 2002; Huelber et al. 2006) and may condense phenophases when emerging later (Billings and Bliss 1959; Inouye and Wielgolaski 2003, and citations therein). However, late-emerging individuals usually set fewer seeds or may even fail to mature fruits when winter conditions set in too early (Kudo 1991; Wagner and Reichegger 1997). Galen and Stanton (1991) demonstrated fitness differences in Ranunculus adoneus individuals flowering at different times along a snowmelt gradient: early-flowering individuals set larger seeds than late-flowering individuals, and seed size positively affected the survival rate of seedlings.

The arctic–alpine pioneer species R. glacialis L. grows in snow-beds and sparsely vegetated alpine fellfields at higher altitudes in the European mountains and in the Arctic. In the European Alps, R. glacialis occurs in genetically different groups (Schönswetter et al. 2004). The main distribution range is between 2,400 and 3,200 m a.s.l. (Landolt 1992). In climatically favourable microhabitats, the species ascends over 4,000 m a.s.l.; the highest locality in the Swiss Alps is documented at 4,275 m a.s.l. (Zimmermann 1975). In the High Arctic, the species is still found in the far north of Greenland up to 80°N (Meusel et al. 1965) and in Spitsbergen, where it grows at sea level (Rønning 1996). Like many alpine Ranunculaceae, this species prefers moist sites; sprouting and flowering occur when the meltwater runoff at the growing sites is highest. Vegetative and reproductive development takes only a few weeks (Moser et al. 1977). These traits are most advantageous for colonising cool and wet sites where the growing season is short.

Previous studies have shown that R. glacialis has a highly conservative growth strategy in that the number of phytomers in a shoot sympodium remains unaffected by changing environmental conditions (Järvinen 1989; Prock and Körner 1996; Totland and Alatalo 2002). Moreover, Totland and Alatalo (2002) observed, in an open top experiment in the alpine zone of south Norway, that neither an increase in temperature nor a year-to-year variation in melting dates significantly affected the speed of phenological development or the reproductive output. These findings are consistent with our observations in the main distribution range in the Alps. Therefore, we were particularly interested in finding out whether the seemingly low developmental plasticity is maintained under suboptimal climatic conditions at higher altitudes. We further aimed to reveal where the limits of reproductive performance of this species are.

Over several years, we examined the effect of the date of snowmelt on reproductive development and reproductive success in R. glacialis at two subnival sites and at a nival site in the Austrian Alps. The specific objectives were to explore at the different altitudes: (1) the variation in the length of the prefloration period with respect to the date of snowmelt, (2) the species-specific pattern of seed development and the plasticity of this developmental phase in response to the date of flowering, (3) the relationship between the date of flowering and reproductive success, and (4) the safety margin between seed maturity and the end of the growing season. In addition, we assessed the seed mass for subnival and nival individuals and speculated about consequences for offspring fitness.

Materials and methods

Morphology of R. glacialis

R. glacialis forms a rhizome system, consisting of individually rooted, loosely connected ramets. During the short active period of a few weeks, new ramets form belowground, developing four to six leaves, and depending on the ramet size, a uni- to multiflorous inflorescence (Totland and Alatalo 2002; J. Wagner, personal observations). These ramets become functional in the following growing season. The bowl-shaped flowers contain up to 180 extrorsely opening anthers and 108 ± 47 standard deviation (SD) carpels (Steinacher and Wagner 2010). Carpels of a flower mature into an aggregate fruit consisting of single-seeded achenes, which disperse independently. Soon after fruit dispersal, aboveground tissues (leaves, reproductive shoots) senesce.

Study areas and sites

The investigations were carried out in the Tyrolean Central Alps at two subnival sites and at one nival site. The subnival sites were on the northeast-facing forelands of the Hintertux Glacier (designated as subnival low, SN-L, 2,650 m a.s.l., 47°04′N, 11°39′E, Zillertal Alps) and of the Stubai Glacier (designated as subnival high, SN-H, 2,880 m a.s.l., 46°59′N, 11°07′E, Stubai Alps). The coverage of vascular plants is medium to low and shows typical elements of a subnival association (Androsace alpina, Cerastium uniflorum, R. glacialis, and Saxifraga bryoides, and in the Hintertux Glacier foreland, also Geum reptans and Oxyria digyna). The study areas were about 100–200 m below the rims of the glaciers in disturbed scree habitats interspersed with larger pieces of rock. Most investigations were performed on individuals growing naturally in this area. At the Stubai Glacier, in addition, 15 randomly selected individuals were excavated with root balls at the end of the 2003 growing season and immediately replanted in a plain area of about 1.5 x 1.0 m (referred to as subnival Ranunculus plot, SN-H plot). This was to facilitate the determination of the prefloration period (see below). Investigations in the nival zone were carried out on Mt. Brunnenkogel in the Ötztal Alps (3,440 m a.s.l., 46°55′N, 10°52′E). This mountain rises from the Pitztal Glacier area and is fully glaciated on its northern side. A small ridge on the summit and the steep southern flank are ice free. R. glacialis individuals were distributed along the crest, with an additional 20 individuals found growing in a flat area of about 2 x 1.5 m (referred to as nival plot).

Climate measurements

Plant temperatures were recorded at all sites at hourly intervals throughout the investigation period, using small data loggers (Tidbit, Onset, Bourne, MA, USA). Loggers were installed on the soil surface and shaded by leaves or white plastic grids to avoid overheating due to high radiation. Periods with snow cover and the exact melting dates could be derived from the logger data. To reveal the impact of temperature on the speed of development, the length of the respective developmental phase was correlated with the frequency of days with mean temperatures at ≤2°C (which is, according to Körner (2003), the lower temperature limit for effective growth in mountain plants).

Prefloration period

The prefloration period (time span from snowmelt until first flowering; Cleve 1901) was determined in plants for which the date of snowmelt was known. At the SN-L site, 10 individuals were marked with colour-coded skewers as they emerged from the winter snow along snow fronts. At the SN-H site and at the nival site, prefloration periods were studied in the individuals of the Ranunculus plots; temperature loggers in the plots indicated the exact melting dates. Plants were regularly monitored, and for each individual, the day of first flowering was assessed.

Seed development

To investigate the duration of seed development, about 30 flowers from different, randomly chosen individuals at the subnival sites, and about 15 flowers in the nival Ranunculus plot were labelled with coloured plastic rings at the beginning of anthesis in each study year. For those flowers that attained fruit maturation, the postfloration period, i.e. the period between onset of anthesis and onset of seed dispersal (Molau 1993; in the present study: achenes start to fall out) was recorded.

Additionally, the dynamics of seed development was investigated at the SN-H site during the 2003 growing season. From the date of labelling (n = about 200 randomly chosen flowers in at least 120 individuals) until fruit maturity, 10 labelled flowers or fruits from different individuals were sampled at 2–4 day intervals. Samples were fixed in Carnoy solution (96% ethanol, acetic acid, 3:1) and stored in the fixative until further processing. Ovules and developing seeds were dissected from the ovaries, mounted in the clearing solution according to Herr (1971), and microscopically examined using differential interference contrast optics. The length (longest axis between chalaza and micropyle) of the seed, endosperm, and embryo were measured with image analysing software (Optimas 6.5, Optimas Corp., Seattle, WA, USA). On average, 60 seeds from 10 flowers were investigated per sampling date. From the embryological state on each date, the duration of the following developmental phases was determined: onset of anthesis until fertilisation, histogenesis (fertilisation until formation of cellular endosperm), and maturation (end of histogenesis until release of achenes).

Fruit mass was determined at the SN-H site and at the nival site during four study years. For each site, mature achenes from at least 10 randomly collected flowers from different individuals were pooled and stored in paper bags at 5°C. The hundred grain weight was assessed with an analytical balance.

Reproductive success

To determine the relationship between reproductive success and flowering time, flowers from 20 to 30 randomly chosen individuals per subnival site (SN-L, SN-H) and year were labelled at onset of anthesis, harvested shortly before fruit maturity, and fixed in Carnoy solution. Under a dissecting microscope, the number of intact and undeveloped (unfertilized or aborted) seeds per flower was counted, and the seed/ovule ratio (S/O ratio, i.e. the proportion of ovules that successfully developed into seeds; Wiens 1984) was calculated. At the nival site, it was not possible to label a sufficient number of flowers on a certain day. Therefore, 44–50 maturing fruits of 24–25 individuals were harvested along the whole summit ridge and fixed. The S/O ratio and additionally the seed size, the seed consistency, and the embryological state were determined. From these data, the approximate onset of flowering for each aggregate fruit could be estimated.

The investigations were carried out in the years 2001–2008. Data could not be collected each year at each site (see Table 1). In particular, the nival site was not always accessible, and experiments were repeatedly hampered or destroyed through feeding damage (snow voles, mountain hares) at all sites.
Table 1

Overview of investigations at the different sites in the different study years

 

2001

2002

2003

2004

2005

2006

2007

2008

Prefloration period

SN-L

  

SN-H, Nival

SN-H, Nival

SN-L, SN-H

SN-L

SN-H, Nival

Seed development

SN-L

 

SN-H

SN-H

Nival

SN-H

SN-L

Nival

100-grain weight

    

SN-H, Nival

SN-H, Nival

SN-H, Nival

SN-H, Nival

Reproductive success

SN-L

SN-L, Nival

SN-H, Nival

SN-L, SN-H

Nival

 

SN-L

SN-H

SN-L lower subnival site, SN-H higher subnival site, Nival nival site

Statistics

Differences in the length of the prefloration period and in the duration of seed development among sites were analysed with one-way analysis of variance (ANOVA) followed by Bonferroni post hoc comparison. As the sample size varied greatly and to avoid pseudoreplication, we used sample means of each variable. The t test was applied to detect site differences (subnival versus nival) in the seed mass. To study relationships between the duration of different developmental phases and the frequency of days with snow and frost, a correlation analysis (Pearson) was carried out. All analyses were made with the statistical package SPSS (SPSS Inc., Chicago, IL, USA). The critical level of significance was α = 0.05.

Results

Climate

Depending on the year and the snow accumulation in winter and spring, the study sites became snow free between the end of May and early July (Table 2). The mean melting day during the investigation period was 10 June (day of year (doy) 161) at the SN-L site, the 18 June (doy 169) at the SN-H site, and the 14 June (doy 165) at the nival site. The vegetation period mostly ended in early (nival site) to mid-September (subnival sites). The length of the vegetation period decreased with altitude from in average 101 days at the lowest and 85 days at the highest site. Furthermore, plants in the nival zone were more frequently covered by snow and experienced more days with night frost than plants in the subnival zone. Mean temperatures during the growing season were around 8°C at the subnival sites and around 4°C at the nival site.
Table 2

Climatic parameters for the different study sites during the investigation period 2001–2008

Locality

Onset of the growing season (doy)

End of the growing season (doy)

Length of the growing season (days)

% days with snow cover

% days with minimum temperature <0°C

Mean temperature during the growing season (°C)

Hintertux Glacier (SN-L) 2,650 m a.s.l. mean (min–max)

161 (144–185)

262 (246–276)

101 (80–120)

8 (0–13)

12 (3–26)

8.1 (6.6–9.6)

Stubai Glacier (SN-H) 2,880 m a.s.l. mean (min–max)

169 (136–190)

260 (246–276)

91 (69–116)

8 (3–16)

19 (10–33)

7.9 (6.5–10.2)

Mt. Brunnnenkogel (nival) 3,440 m a.s.l. mean (min–max)

165 (152–179)

250 (227–275)

85 (56–107)

23 (3–37)

49 (35–58)

4.5 (3.5–6.6)

Data are based on boundary layer temperatures recorded by temperature loggers in a melting front at the Hintertux Glacier, and in the Ranunculus plots at the Stubai Glacier and on Mt. Brunnenkogel. Growing season is the period between melting of the winter snow cover and daily mean temperatures below freezing or the formation of a continuous snow cover in autumn.

doy day of year

Prefloration period

At the subnival sites, the prefloration period lasted on average 15–20 days (Fig. 1A). The total mean over the years was 15.6 ± 1 days standard error (SE) for the SN-L and 16 ± 2.8 days SE for the SN-H site; the differences were not statistically significant. The shortest individual period was 13 days, and the longest was 23 days. Individuals at the nival site had a prefloration period more than twice as long (total mean 35 ± 1.4 days SE) as subnival individuals with similar thawing dates. From bud burst (corolla visible) to onset of anthesis took up to 2 weeks at the nival site but only a few days at the subnival sites. Differences in the prefloration period found within a site for different thawing dates were not significant while differences between the subnival sites and nival site were (p < 0.001, one-way ANOVA). This can mainly be explained by differences in temperatures during the prefloration period (Fig. 1B): there was a clear correlation (Pearson r = 0.984, p < 0.001) between the period length and the frequency of days with a mean temperature of 2°C and lower.
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Fig. 1

Length of the prefloration period of R. glacialis at the two subnival sites (SN-L, SN-H) and at the nival site, A depending on the date of snowmelt, and B when correlated with the frequency of days during the prefloration period with daily temperature means ≤2°C. Values are means ± SD of n = 10 (SN-L), 11–16 (SN-H), and 6–7 (nival) individuals for which the exact thawing date was known

Anthesis and seed development

Flowers of R. glacialis show weak protandry. Anthers open extrorsely, with the outermost whorl dehiscing on the first day of anthesis. At that time, stigma lobes are still short (Fig. 2A), but their tips are already receptive (Steinacher and Wagner 2010). At the subnival sites, under favourable weather conditions, stigmas were fully unfolded and papillate 3–4 days after onset of anthesis (DAA). By day 6, most pollen was dispersed, and stigmas were pollinated (Fig. 2B). During seed development, the corolla turns from white to pink (Fig. 2C) but remains fresh for up to 2 weeks and longer. Later, the corolla becomes brown and dry, and forms an envelope for the maturing achenes (Fig. 2D).
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Fig. 2

State of embryonic development in relation to phenology in R. glacialis. A–D Phenological phases, A onset of anthesis, B advanced anthesis; about 2/3 of the anthers dehisced, stigmas pollinated. C Postanthesis; pollen dispersed from all anthers; stigmas are fully expanded and pollinated, achenes develop; the corolla has turned pink. D Fruit maturity, dispersal of achenes. a–d Embryonic phases. a Seven-celled embryo sac (esa) with polar nuclei fusing, b early embryo development: proembryo (pem) and nuclear endosperm (nes), c end of histogenesis: globular embryo (gem) embedded in cellular endosperm (ces), d seed maturity: poorly differentiated embryo (em) with a single cotyledon. Scale: 100 µm

At the onset of anthesis, about 60% of ovules contained a seven-celled embryo sac (Fig. 2a) of the Polygonum type (Yakovlev 1981; Johri et al. 1992). In the remainder of ovules, gametogenesis was not yet complete (four- to eight-nucleate embryo sac, pole nuclei not fused). In R. glacialis, the 2n central cell nucleus approaches the egg cell before fertilisation, and moves towards the antipodes after fertilisation. Fertilised ovules could be observed from 2 DAA on. Histogenesis lasted about 20 days (Fig. 3). During this phase, nuclear endosperm (Fig. 2b), later cellular endosperm, and the seed coat formed, and the seeds increased greatly in size. The zygote divided soon after fertilisation. Already 4 DAA, four-celled proembryos had developed. In spite of the early onset of embryogenesis, 70% of embryos were in an early globular state at the end of histogenesis; only 30% of embryos had attained a late globular state (Fig. 2c). Seed maturation (reserve deposition and maturation drying) took about 10 days (Fig. 3). Mature seeds contained late-globular or asymmetric heart-shaped embryos with a single cotyledon (Fig. 2d) embedded in albuminous endosperm. During the final maturation drying the testa becomes firm and the pericarp scarious. Achenes already start to release from the receptacle in a premature state (pericarp still green), but at first, they remain enclosed in the bowl-shaped dry corolla (Fig. 2D). The dry and brown achenes are dispersed during the following weeks.
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Fig. 3

Dynamics of seed development in R. glacialis at the higher subnival site SN-H in the year 2003 expressed as the increase in the length of the entire seed, the endosperm, and the embryo. Values are means ± SD of 60 seeds on average from 10 flowers per sampling date. Arrow Achenes started to release from the receptacle

The period between onset of anthesis and seed maturity (release of achenes) lasted between 30 and 35 days at the subnival sites irrespective of the date of anthesis (Fig. 4A). In the nival zone, seeds needed around 50 days to become mature. As for the prefloration period, the time required for seed development strongly correlated with the frequency of days with a mean temperature of 2°C and lower (Fig. 4B, Pearson r = 0.953, p < 0.001).
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Fig. 4

Length of the period (days) between onset of anthesis and seed maturity in R. glacialis at the subnival sites (SN-L, SN-H) and at the nival site, A depending on the date of flowering, and B correlated with the frequency of days during seed development with daily temperature means ≤2°C. Values are means ± SD of n = 5–10 (SN-L), 6–15 (SN-H), and 7–8 (nival) flowers for which the date of anthesis and maturation was known

Subnival individuals (SN-H site) produced heavier achenes than nival individuals in most years. Only in 2008 were achene weights the same (Fig. 5). Because of the pooled fruit harvest, it was not possible to statistically analyse the results for individual years. Calculated over the years, the difference in the 100-grain weights between the subnival and nival site were statistically significant (p = 0.018, t test).
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Fig. 5

Hundred grain weight (achenes) in different study years at the subnival higher site (SN-H) and at the nival site. Values based on pooled samples from 10 to 20 randomly collected aggregate fruits per site and year

Reproductive success

Reproductive success varied greatly among sites and different dates of flowering but also among flowers within a sample (Fig. 6). At the subnival sites, earlier flowering led to a higher seed set than later flowering (Pearson correlation between date of flowering and S/O ratio r = 0.934, p = 0.006). The highest reproductive success (mean S/O = 0.87) was attained at the subnival SN-H site in the exceptionally warm growing season of 2003 when anthesis had already started in early June. In the remaining years, the mean S/O ratio was between 0.7 and 0.8 when flowering took place in June and early July but dropped to about 0.5 when anthesis started in late July. At the nival site, the mean S/O ratio was between 0.42 and 0.63 and did not correlate with the flowering date in June and July. When flowering occurred later than mid-July, the risk of losing the seed crop was high. As a rule, nival individuals that flowered in August matured no seeds. August-flowering subnival individuals attained seed maturity when the growing season was long enough but failed when autumn snow falls set in too early as was the case in 2001 at the SN-L site (Fig. 6 double symbol).
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Fig. 6

Reproductive success of R. glacialis expressed as S/O ratio depending on the date of flowering at the subnival sites (SN-L, SN-H), and at the nival site. Values are means ± SD of n = 16–32 (SN-L), 30 (SN-H), and 11–33 (nival) flowers for which the date of anthesis was known. For June and July, the S/O ratio strongly correlates with the date of flowering at the subnival sites (r = 0.934, p = 0.006, Pearson; full line), but not at the nival site (dotted line). Flowers opening in August matured no seeds. Double symbol Late-flowering cohort at the subnival SN-L site in the atypical short growing season 2001

There was a strong correlation between the date of anthesis and the safety margin between the date of seed maturity and end of the growing season (Fig. 7; Pearson r = 0.992, p = 0.001 for the subnival sites excluding data from 2001, and r = 0.982, p = 0.003 for the nival site). In a climatically normal year, the safety margin in the nival zone approached zero when flowering started later than mid-July. At this time, the leeway was still 1 month at the subnival sites. Only in the particularly short growing season of 2001 were the situations in the subnival zone similar to those in the nival zone (see Fig. 7, double symbols).
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Fig. 7

Safety margin in days between the date of seed maturity and the end of the growing season depending on the date of flowering at the subnival sites (SN-L, SN-H), and at the nival site. Values are the difference in days between the date of seed maturity in flowers labelled at onset of anthesis and the date of onset of autumn snow falls in the respective year. Double symbol Safety margin for an early- and late-flowering cohort at the SN-L site in the unusual short growing season 2001

Discussion

Our investigations over several years have shown that sexual reproduction of R. glacialis is at its optimum in the subnival zone. At the subnival study sites, the snow-free period (period between snowmelt and the onset of winter snow cover) lasted about 3 months. During this period, mean plant temperature was about 8°C, temperatures fell below zero on only an average of 16% of days, and plants were temporarily covered by snow on an average of 8% of days (see Table 2). Under these conditions, the duration of phenological stages of R. glacialis was rather stable: first individual flowering started 2–3 weeks after snowmelt, anthesis lasted about 1 week, and seed development took 4 to 5 weeks. In total, propagation and vegetative growth proceeded over 7–8 weeks, usually leaving a safety margin between seed maturation and the onset of winter conditions of at least 1 month. The high developmental stability coincided with a high phenological flexibility in that the date of emergence in June and July had hardly any influence on the temporal sequence of reproductive phenophases, but it has to be added that vegetative and reproductive growth in R. glacialis is under the control of photoperiod, as Prock and Körner (1996) could show in a cross-continental comparison study. Ecotypes in the Alps are adjusted to the photoperiodic regime in Central Europe and undergo only one development cycle per growing season. When transplanted to the Arctic, however, plants show a considerable phenorhythmic disorder and may flower several times per growing season. Obviously, extended long-day conditions provoke a cascade of sympodial shoots on which flowers open proleptically. This would mean that an earlier start to the growing season in a warmer climate would not necessarily be beneficial for R. glacialis, but on the contrary, could cause developmental disorder. Evidence for such disturbances comes from our observations in the particularly long, warm growing season in 2003: R. glacialis in the subnival zone had already flowered by the end of May, and we found several Ranunculus individuals flowering a second time in August.

R. glacialis finishes its yearly reproductive cycle faster than other nival plant species studied to date. The remainder of species mostly show both a longer prefloration and postfloration period. For example, Saxifraga bryoides needs about 6 weeks for prefloration, about 10 days for anthesis, and another month for seed development, giving a total of around 12 weeks (Ladinig and Wagner 2007). A similar reproductive period has been observed for S. moschata (Ladinig and Wagner 2005). Androsace alpina requires 3–4 weeks for prefloration and 7–8 weeks for seed formation; and Cerastium uniflorum needs 6 weeks to form new shoots with terminal flowers and another 5 weeks for anthesis and seed formation (J. Wagner, G. Steinacher, S. Erler, and S. Widmann, unpublished results). The comparatively short reproductive period in R. glacialis became particularly evident at sites where individuals of the different species grew side-by-side and thus had the same melting day. We observed that R. glacialis, as a rule, had already dispersed its seeds by the time S. bryoides and C. uniflorum started to flower and A. alpina was in late anthesis.

Two weeks of prefloration and 4 weeks postfloration seems to be the shortest period for reproductive development of R. glacialis with no ability to further accelerate the development. This also became apparent in the open top experiment at an alpine site in Southern Norway (Totland and Alatalo 2002): warming had no significant effect on vegetative growth and reproductive success. Despite large differences in the dates of snowmelt among years, average reproductive output and ramet size differed little among years. Obviously, the given ambient temperature conditions did not constrain growth (Totland and Alatalo 2002), which is consistent with our findings in the subnival zone. The only possible constraint is a lack of time in late melting sites when a permanent snow cover forms too early in autumn. But this situation is not the rule and arose only once within the study years.

The situation was quite different in the nival zone. Here, the snow-free period lasted only about 85 days with mean plant temperatures of around 4°C and about 50% of days with snow and frost. Days with snow cover were more frequent, and summer snow cover lasted longer than in the subnival zone. Particularly, the longer periods with snow produced a stop–start situation, markedly retarding reproductive growth in R. glacialis. The prefloration period usually dragged on for more than 1 month, anthesis lasted up to 2 weeks, and seed development 6–7 weeks—giving a total of at least 3 months. The particularly long anthesis period at lower temperatures can be attributed not only to a slower progamic flower development (anther dehiscence, stigma expansion) but might also be the effect of low pollinator visitation rates. Low pollinator activity retards pollen removal from stamens and pollen deposition on the stigma, and thus prolongs flowering (Arroyo et al. 1985; Robertson and Lloyd 1993; Itagaki and Sakai 2006).

Thus, with increasing elevation, not only the shorter growing season becomes limiting but also the delay in reproductive development. This further signifies that, at higher altitudes, seed formation in R. glacialis is only safe at early melting sites. For melting dates later than mid-June, there is a high risk of the plants not attaining seed maturity. Nevertheless, the reproductive development of R. glacialis appears more efficient than in other nival species which take as long to mature seeds in the subnival zone (see above) as R. glacialis in the nival zone.

In the subnival zone, seeds matured each year. Early- and midflowering individuals attained mean S/O ratios of around 0.8. In late-flowering individuals, the S/O ratio decreased to about 0.5, which was in the range usually found at the nival site (S/O ratio 0.4–0.6) and reported by Totland and Alatalo (2002) for the alpine population in Southern Norway. Remarkably, Järvinen (1989) found S/O ratios of between 0.6 and 0.8 in Finnish Lapland (69°N) where the growing season is even shorter than at the nival site in our study. One reason could be that under extended long-day conditions, pollinator visits are more frequent. Most arctic insects are active whenever it is warm enough, independent of the time of day (Danks 2004). One might further speculate that, at higher latitudes, ecotypes of R. glacialis have evolved that under continuous daylight grow and reproduce especially quickly and thus compensate for the short growing season. Ecotypic variation in response to different lengths of the growing season has been reported for a number of species growing in the Arctic (McGraw and Antonovics 1983; Crawford et al. 1993; Crawford and Smith 1997; Crawford 2008). Ecotypes adapted to short growing seasons in a low temperature regime, inter alia, show higher rates of photosynthesis and metabolism than ecotypes that grow at warmer and earlier thawing sites (Crawford and Smith 1997).

A high seed set is typical for inbreeding plant species and those with a mixed mating system (Wiens 1984; Molau 1993; Richards 1997; Totland and Schulte-Herbrüggen 2003). According to this, the high S/O ratios of R. glacialis would suggest a high selfing capacity. However, pollinator exclusion experiments at the subnival site yielded only a low selfing rate (S/O ratio 0.08 for spontaneous selfing, and 0.12 for selfing by hand; G. Steinacher, unpublished). Emasculated and bagged flowers showed an S/O ratio of 0.05 ± 7.4 SD, which suggests a small contribution from apomixis, but this still has to be investigated in more detail. These data thus indicate predominant outcrossing as was reported for the R. glacialis population in Scandinavia (Totland and Alatalo 2002). There are only a few studies of the breeding systems of Ranunculus species growing in cold climates. Species investigated so far propagate sexually (Philipp et al. 1990, Totland 1994, Pickering 1997a, b) and/or asexually (Hörandl et al. 2005; Paun et al. 2005; Hörandl and Paun 2007, Cosendai and Hörandl, 2010). Most sexual species are facultatively xenogamous but they are self-compatible to varying degrees; e.g. five Ranunculus species cooccurring in the Australian alpine region promote outcrossing by incomplete protogyny, though most of them are fully self-compatible (Pickering 1997a,b). Self-compatibility can provide reproductive assurance when due to a harsh and stochastic climate, pollinators are sparse or uncertain. Similarly, those species which have evolved pseudogamous apomicts are self-compatible and thus independent of pollinators. By contrast, most sexual ancestors are largely self-incompatible (Hörandl 2008). It is remarkable that R. glacialis in spite of its extreme habitat has only a low potential for uniparental reproduction. Obviously, xenogamy is favoured to maintain genetic diversity, particularly since cross-pollination seems not to be a limiting factor in this species.

In our study, the high reproductive success in early- to midflowering subnival individuals points to sufficient pollinator activity. The main pollinators of R. glacialis are Muscid and Syrphid flies whose pollen carry over is not very effective (Escaravage and Wagner 2004). However, a high frequency of visiting insects, which can be regularly observed on bright days (G. Steinacher, J. Wagner, personal observations) and an extended flower longevity (Steinacher and Wagner 2010) may compensate for the low pollination effectiveness of a single insect. At the nival site, pollinator activity seems to become more limiting as shown by generally lower S/O ratios. Similarly, the lower reproductive success of late-flowering individuals at the subnival sites might be due to a decrease in flight activity of insect pollinators later in the season as has been reported for Scandinavian mountain habitats (Totland 1993, 2001). Even if the seed output of R. glacialis is reduced later in the season or at higher elevations, an average of between 40 and 70 seeds per flower mature when the growing season is long enough.

Achenes of R. glacialis—as in many Ranunculus species—are released from the receptacle in a premature state when the pericarp and seed coat are still green. The photosynthetically active tissues enable seed maturation to be completed independently of the mother plant (Boesewinkel and Bouman 1995). Maturing achenes are protected by the bowl-shaped perianth, which can be seen as an adaptation to the harsh conditions in cold climates (Hörandl et al. 2005).

An early independence of the offspring may be advantageous in short growing seasons; however, in the case of R. glacialis, it is combined with a low degree of embryo differentiation. Mature seeds contain a firm endosperm and a morphologically undifferentiated embryo with only one poorly developed cotyledon which is common in basal eudicots (Förster 1997; Engell 1995). Thorough investigation of the germination physiology of R. glacialis is lacking, but seeds may be dormant and need afterripening to become germinable, as is the case in several Ranunculus species (Stanton and Galen 1997; Baskin and Baskin 1998). Preliminary investigations (J. Wagner, unpublished results) suggest a morphophysiological dormancy similar to that of Caltha leptosepala (Forbis and Diggle 2001). It is possible that the rudimentary embryo is dormant during winter, and then undergoes further development during the following growing season, becoming ready to germinate in the second year after dispersal.

The germination capacity of seeds seems to be high in R. glacialis—at least in the main distribution zone. In a study on the population dynamics in an upper alpine habitat, Diemer (1992) found a high seedling density particularly on south-facing slopes (more than 200 seedlings per square metre on average). However, the high seedling density did not lead to a corresponding increase in the abundance of juvenile stages, suggesting that seedling mortality is substantial. There are no investigations of the seedling dynamics of R. glacialis in the nival zone. Given the lower reproductive success, a lower seedling density would be expected. Additionally, nival individuals produce significantly lighter seeds with less storage reserve than subnival individuals in most years. A decrease in seed weight with altitude has been reported for different high mountain species and points to limiting conditions for producing and filling seeds (Pluess et al. 2005; Zoller et al. 2005). Smaller seeds might negatively affect fitness of the offspring as demonstrated by Galen and Stanton (1991) for Ranunculus adoneus. However, we could also show that nival individuals of R. glacialis are capable of producing normally sized seeds in climatically favourable periods. Possibly, these seeds play a crucial role in population turnover at higher altitudes; in that, offspring is mainly recruited from this pool of more vigorous seeds.

Concluding remarks

The reproductive performance of R. glacialis is quite stable and largely independent of the emergence date within the main habitat in the subnival zone. Because of the short reproductive period, there is usually no risk of losing the seed crop. Though flowers are mainly pollinated by small flies which are weak pollinators, a long flower lifetime increases the frequency of pollinator visits and thus assures reproductive success. The stable performance found in earlier studies suggested a lack of developmental plasticity. Our investigations have shown that the response of R. glacialis is plastic only at higher altitudes, where climatic conditions become increasingly limiting. Under the far lower temperature regime in the nival zone, Ranunculus adoneus is capable of markedly prolonging all phases of reproductive development. The total reproductive period can be twice as long as in the subnival habitat. This also illustrates that growth in R. glacialis is rather insensitive to permanently low temperatures as long as tissues do not suffer frost damage (first damage between −6°C and −8°C; Larcher and Wagner 1976; Taschler and Neuner 2004). The adaption to cooler growth temperatures is also apparent in the pronounced heat sensitivity of R. glacialis (Larcher et al. 1997) and its poor ability to adjust dark respiration when temperatures increase (Cooper 2004). Thus, high respiration rates and the inability to thermally acclimate seem to be a fundamental reason why R. glacialis is restricted to cold environments. At higher elevations, however, time for growth and reproduction may become limiting as a longer development time coincides with a markedly shorter growing season. This signifies that, in the nival zone, only early-flowering cohorts have the chance to mature seeds. For late-flowering individuals, this becomes increasingly unlikely.

Acknowledgements

This study was funded by the Austrian Science Foundation (FWF) as part of the project “Diversity of sexual reproduction in high mountain plants” (P15595-B3). We thank Stefanie Erler, Daniela Hosp, and Stephanie Widmann for their help with fieldwork and for providing data. We thank E. Hörandl and M. Akhalkatsi for valuable suggestions on the manuscript. Thanks also to the Stubaier Gletscherbahn and the Zillertaler Gletscherbahn for free transportation by cable car.

Conflict of interest

The authors declare that they have no conflict of interest.

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© Springer-Verlag 2010