Flora (2003) 198, 377–388 http://www.urbanfischer.de/journals/flora Evidence of the possibility of natural reciprocal crosses between Pinus sylvestris and P. uliginosa based on the phenology of reproductive organs Adam Boratyński1*, Krystyna Boratyńska1, Andrzej Lewandowski1, Zbigniew GoŁĄb2 & Piotr Kiciński3 1 2 3 Polish Academy of Sciences, Institute of Dendrology, Parkowa 5, 62-035 Kórnik, Poland Stołowe Mts National Park, Słoneczna 31, 51-350 Kudowa, Poland Forestry Faculty of Agricultural University, Department of Forest Botany, Wojska Polskiego 71d, 60-615 Poznań, Poland Submitted: Feb 13, 2003 · Accepted, in revised form: Apr 30, 2003 Summary Phenological observations of development of generative organs of Pinus sylvestris and P. uliginosa (= P. rotundata) were carried out in spring of 1999, 2000 and 2001. The aim of the research was to verify the hypothesis that both taxa have a possibility of reciprocal crossing. Two populations of P. uliginosa were tested, one on the Silesian Lowland (We ˛ gliniec) (altitude of 180m), the other in the Stołowe mountains (Batorów) (at altitude of 750 m). The lowland population of P. uliginosa is not numerous and surrounded by extensive P. sylvestris forests, the mountain one is 3 times more numerous, and the nearest population of P. sylvestris is at a distance of 2km. Observations in the field were carried out twice a week during the period of development of micro- and macro-strobili of both taxa. The results show that the P. sylvestris strobili developed every year earlier than those of P. uliginosa. Nevertheless, this did not eliminate the possibility of cross pollination and gene flow from P. sylvestris toward P. uliginosa on both localities of the last species. The possibility of pollination of P. sylvestris macrostrobili by P. uliginosa pollen was found also possible, but more restricted. Normally, on lowlands the same phenological phases take place about 10 days earlier than in the mountains. This is documented for two years of observations of more or less typical whether conditions. An earlier development of micro- and macrostrobili of both species in their mountain populations was observed in an observation year characterized by an extremely early and warm spring. Key words: “Pinus mugo complex”, microstrobili development, macrostrobili development Introduction Relationships among pine species of the subsection Sylvestres, including Pinus sylvestris L., P. mugo Turra, P. uncinata Ramond and P. uliginosa Neumann (= P. rotundata Link), have been the subjects of taxonomic studies for the last several decades. Biometric, chemotaxonomic, biochemical genetic investigations, and lately molecular methods have been used to determine the relationships among the four taxa (e.g., Staszkiewicz & Tyszkiewicz 1969, 1972; Szweykowski & Bobowicz 1983 ; Prus-GŁowacki & Szweykowski 1983 ; Bobowicz 1990 ; Neet-Sarqueda 1994 ; PrusGŁowacki & Stephan 1998 ; Prus-GŁowacki et al. 1998 ; Lewandowski et al. 2000 ; Wachowiak et al. 2000). The taxonomic position of P. uliginosa has been discussed in the studies cited above. This taxon, originally described from the peat-bogs of the Stołowe and Bystrzyckie Mountains in the Central Sudethians (Neumann 1837 ; Wimmer 1837), is morphologically intermediate between P. mugo Turra and P. sylvestris L. (Staszkiewicz 1985, 1993), but also between P. mugo * Corresponding author: Adam Boratyński, Polish Academy of Sciences, Institute of Dendrology, Parkowa 5, 62-035 Kórnik, Poland, e-mail : borata@man.poznan.pl 0367-2530/03/198/05-377 $ 15.00/0 FLORA (2003) 198 377 and P. uncinata (Christensen 1987). Consequently, P. uliginosa is considered to be of hybrid origin, developed as a result of hybridization between those pairs of species (Lewandowski et al. 2000). Participation of all three species in the hybridization process cannot be excluded (Lauranson-Broyer et al. 1997). Because of its intermediate features, P. uliginosa is sometimes identified as the hybrid between P. mugo and P. sylvestris, named P. ×rhaetica Brügger (Staszkiewicz 1985, 1993 ; Mirek et al. 1995 ; ZajĄc & ZajĄc 2001). Hybrids between P. mugo and P. sylvestris occur naturally in places where the species grow together (Staszkiewicz & Tyszkiewicz 1969, 1972; BoratyŃski 1978 ; Szweykowski & Bobowicz 1983 ; Staszkiewicz 1985, 1993; Bobowicz 1990 ; Siedlewska 1994 ; Christensen & Dar 1997). Evidence for gene flow between these species has been established on the basis of biometric and genetic studies. Investigations using isoenzymatic markers and DNA have shown that gene flow between P. sylvestris and P. mugo is possible, but restricted (Filppula et al. 1992; Lewandowski et al. 2002). The affinity of P. uliginosa to both P. sylvestris and P. mugo has been recently confirmed on the basis of isoenzyme systems (Lewandowski et al. 2000). This also suggests the possibility of gene flow between P. sylvestris and P. mugo and, consequently, between P. sylvestris and P. uliginosa. However, to date it remains unknown if the in situ phenology of strobili, pollen release, and pollen reception enable their reciprocal cross-pollination. The main goal of the present study was to evaluate the possibility of P. sylvestris and P. uliginosa cross-pollination on the basis of phenological observations of the development of reproductive organs in locations where they are growing together or close to each other. Another aim was to compare the phenology and development of reproductive organs of both taxa in spring both on a lowland and mountain sites. Material and methods Two populations of P. uliginosa were chosen for the study (Table 1). The first is located at We ˛ gliniec, in the western part of the Silesian Lowland, and consists of less than 100 individuals (Ambroży 1999), whereas the second is located at Batorów, in the Stołowe Mountains (central Sudethians), and includes about 300 individuals (GoŁĄb 1998). The population of P. uliginosa at We ˛ gliniec is its northernmost location, isolated from other populations of the taxon and surrounded by extensive forest stands of P. sylvestris. The population of P. uliginosa at Batorów is isolated from other populations of the taxon, and also from the nearest population of P. sylvestris. P. sylvestris does not form extensive forest stands there and the nearest population is about 2 km away from the P. uliginosa stand. Temperature and relative air humidity was measured on both stands of Pinus uliginosa and on Szczeliniec Mt in spring of the year 2000, using data loggers HOBO PRO RH/Temp. H08-03208 (MicroDAQ, New Hampshire). The phenological observations were carried out in 1999, 2000 and 2001, starting in early spring and ending with the end of pollen release and reception on the last tested specimen. Every year, 10 or more individuals of each of P. uliginosa and P. sylvestris were observed at both locations (Table 1). The trees ranged between 5–18m in height, but only the lowestgrowing micro- and megastrobili were sampled. About 10 conglomerations of microstrobili were observed to describe the phenology of every individual male plant. Not in all cases sufficient megastrobili were formed to observe ten on every individual in a particular year, especially on P. uliginosa. In those cases the smaller number of megastrobili was used to characterize the female phenology. Observations of the development of P. sylvestris megastrobili and pollen release carried out mostly in seed-orchards by many authors (Sarvas 1962, 1967, 1972; Jonsson et al. 1976; Boydak 1977; ChaŁupka & Fober 1977; WesoŁy 1982; Koski 1991; ChaŁupka 1993; Burczyk & ChaŁupka 1997; Krizo 2001) were very helpful to form the basis of our investigations. The dates of the first observations were adjusted to the development of P. sylvestris, which started earlier than P. uliginosa. The frequency of observations depended on weather conditions, chiefly temperature and precipitation Table 1. Sampled populations of Pinus sylvestris and P. uliginosa No. Taxon Location Longitude E Latitude N Altitude [m] Number of individuals analysed 1 Pinus sylvestris 15°14⬘ 51°17⬘ 180 12 2 Pinus uliginosa 15°14⬘ 51°17⬘ 180 14 3 Pinus sylvestris 16°19⬘ 50°29⬘ 900 10 4 Pinus uliginosa Silesian Lowland, area adjacent to nature reserve at We ˛ gliniec Silesian Lowland, nature reserve at We ˛ gliniec Stołowe Mts, Mt Szczeliniec Wielki, on sandstone rocks Stołowe Mts, Great Peat-bog at Batorów 16°19⬘ 50°29⬘ 750 10 378 FLORA (2003) 198 (ChaŁupka 1993), but observations were usually made twice a week with the use of binoculars. Four developmental stages of megastrobili and six of microstrobili were distinguished (Lännerholm 1976) during the field observations (Table 2). The field research was preceded by examination of the development of megastrobili on specimens of P. sylvestris, P. uliginosa and P. mugo in the collections of the Kórnik Arboretum. The duration of pollen release by microstrobili and pollen reception by megastrobili were calculated separately for P. sylvestris and P. uliginosa in all four observed populations. Results Characteristics of phenology of micro- and megastrobili of P. sylvestris and P. uliginosa Springtime climate of 2000 was different on both observed localities and characterized by a specific course of temperature and relative air humidity (Figs 1 and 2). Both measured climatic factors were much more stable in the mountains than in the lowland. Day and night temperatures and relative air humidity in the Stołowe Mts showed a much narrower range than on the lowland site. There were also more dry days in the mountains than in lowland (Fig. 2). Timing of successive phenophases of micro- and megastrobili of P. sylvestris and P. uliginosa at We˛gliniec and Batorów (Table 3) was different every year, depending on weather conditions. The differences in dates of phenophases between 1999 and 2001 (with a late and cold spring) and 2000 (with an extremely early, dry and warm spring) were as great as 30 days in the case of P. sylvestris and even 35 days in P. uliginosa in the Stołowe Mts. The differences between years, however, were reduced at We˛gliniec and were 15 –18 days for P. sylvestris and 10 days for P. uliginosa (Table 3). Fig. 1. Course of air temperature in April and first days of May of 2000 at the Silesian Lowland (We ˛ gliniec) and Stołowe Mts (Batorów) sites In both species the megastrobili developed earlier than the microstrobili (Figs 3 and 4). The first scales of megastrobili were clearly visible and receptive a few days before pollen release occured on the same individuals. The differences between starting dates of pollen release and reception were greater in the Stołowe Mts than on the Silesian Lowland (Tables 4 and 5). P. sylvestris and P. uliginosa exhibited similar differences between dates of development of micro- and megastrobili. The differences depended on weather conditions in particular years. The differences were the smallest in 2000 and on average amounted to only about 0.5 days for P. uliginosa and 1.4 days for P. sylvestris on the Silesian Lowland, and 10 and 7 days, respectively, in the Stołowe Mts. In the years 1999 and 2001, characterized by cooler and more humid springs, megastrobili developed and were receptive up to 2 weeks before pollen release occurred by the same individuals. In both localities pollen release by P. sylvestris and P. uliginosa started later than pollen reception and ended at the same time or a few days later or earlier, depending on year (Table 3). Table 2. Developmental stages of micro- and megastrobili of Pinus sylvestris and P. uliginosa (after Johnsson et al. 1976) Phase Microstrobili Megastrobili 0 1 Bud covered with scales, microstrobili invisible Visible microstrobili 2 Microstrobili developed, without pollen release 3 Start of pollen release – at least one microstrobilus dehiscent and releasing pollen grains when vibrating the twig Intensive pollen release; most microstrobili release pollen when vibrating the twig End of pollen release; most of microstrobili completely opened, without pollen, only a singular release pollen when vibrating the twig Microstrobili without pollen Bud covered with scales, megastrobilus invisible Start of pollen reception; upper scales of megastrobilus are visible as receptive Intensive pollen reception; complete megastrobilus is visible with most scales receptive End of pollen reception – scales thickened, closed access to the pollen chambers, but megastrobili not deflected 4 5 6 Megastrobili deflected FLORA (2003) 198 379 generally shorter and varied between 5 and 18 days (12.0 on average). Somewhat lower values were recorded at We˛gliniec (Table 6). The periods of pollen reception of particular individuals of P. uliginosa in the Stołowe Mts ranged from 5 to 28 days (17.4 on average), and pollen release from 6 to 18 (12.6 on average), whereas at We˛gliniec pollen reception lasted 7– 29 days (14.9 on average), and pollen release 5 –17 days (10.0 on average) (Table 6). Fig. 2. Course of relative air humidity in April and first days of May of 2000 at the Silesian Lowland (We ˛ gliniec) and Stołowe Mts (Batorów) sites The duration of pollen reception by particular individuals of P. sylvestris in the Stołowe Mts ranged from 7 to 28 days (15.0 on average) and was different in length in particular years. The duration of pollen release was Differences in development of strobili between P. sylvestris and P. uliginosa Pinus sylvestris released pollen and showed pollen reception before P. uliginosa in the Stołowe Mts and at We˛gliniec, but these differences show a considerable year-to-year variability. At We˛gliniec the dates of successive phenological phases of P. sylvestris were earlier than those of P. uliginosa (Table 7), but their duration was usually similar in both species (Table 6). The Table 3. Average dates of pheno-phases of development of micro- and macrostrobili of Pinus sylvestris and Pinus uliginosa in We ˛ gliniec and Batorów Location We ˛ gliniec Species Pinus sylvestris Year 1999 2000 2001 Pinus uliginosa 1999 2000 2001 Batorów Pinus sylestris 1999 2000 2001 Pinus uliginosa 1999 2000 2001 380 FLORA (2003) 198 Micro- and macrostrobili phases Date of phenophases [mm.dd] start intensive end Pollen release Pollen reception Pollen release Pollen reception Pollen release Pollen reception 05.14 05.02 05.02 05.01 05.14 05.04 05.21–05.30 05.14–05.28 05.06–05.24 05.05–05.16 05.18 –05.28 05.06–05.26 06.04 06.02 05.28 05.19 05.30 06.06 Pollen release Pollen reception Pollen release Pollen reception Pollen release Pollen reception 05.11 05.02 05.11 05.10 05.16 05.06 05.21–06.04 05.21–06.04 05.15–05.24 05.15–05.25 05.26–06.02 05.23–06.06 06.11 06.12 05.28 05.29 06.10 06.11 Pollen release Pollen reception Pollen release Pollen reception Pollen release Pollen reception 05.24 05.13 04.26 04.20 05.28 05.14 05.28–06.05 05.27–06.03 04.30–05.08 04.26–04.30 06.01–06.09 05.21–06.01 06.13 06.15 05.11 05.07 06.14 06.14 Pollen release Pollen reception Pollen release Pollen reception Pollen release Pollen reception 05.28 05.26 04.30 04.24 05.28 05.17 06.04–06.11 06.01–06.12 05.05–05.11 04.26–05.04 06.05–06.18 05.24–06.15 06.18 06.20 05.18 05.08 06.24 06.20 Fig. 3. Differences in development of reproductive organs of Pinus sylvestris at a Silesian Lowland (We ˛ gliniec) and Stołowe Mts (Szczeliniec) site release of pollen by P. sylvestris took place every year at the time of intensive reception of pollen by megastrobili of P. uliginosa (Fig. 5). Pollen release in P. uliginosa occurred at the end of pollen reception of megastrobili of P. sylvestris (Fig. 6). In the Stołowe Mts the first phases of the development of micro- and megastrobili of P. sylvestris took place on average 6 days earlier than those of P. uliginosa (Table 7). Intensive pollen release by P. sylvestris from Mt. Szczeliniec near Batorów lasted 9 days. The megastrobili of this species were receptive for about 30 days. P. uliginosa released pollen intensively for 8 days and its megastrobili were receptive for about 25 days (Table 3). The differences between the starting dates of phenological phases of pollen release and development of megastrobili of P. sylvestris and P. uliginosa were much smaller in 2000, which was characterized by an extremely early, warm and dry spring. Intensive release of pollen by P. sylvestris from Szczeliniec Wielki Mt took place when P. uliginosa megastrobili were receptive (Fig. 7), whereas pollen release by P. uliginosa occurred at the end of pollen reception by megastrobili of P. sylvestris (Fig. 8) in all three years of observation, just as occurred at We˛gliniec. Discussion Phenological phases typically occur earlier on the lowlands than in the mountains. According to this rule, the spring development of vegetation on the Silesian Lowland, the region of Węgliniec, should take place about 10 days earlier than in the Stołowe Mts (Molga 1974). The phenological observations on pollen release and FLORA (2003) 198 381 Fig. 4. Differences in development of reproductive organs of Pinus uliginosa at a Silesian Lowland (We ˛ gliniec) and Stołowe Mts (Batorów) site reception of P. sylvestris and P. uliginosa confirmed this pattern only in 1999 and 2001. By contrast, in 2000, when spring was early and warm, earlier pollen release and reception was observed in both, P. sylvestris and P. uliginosa in the Stołowe Mts. This can probably be explained by the influence of deep inversions of temperatures on the Silesian Lowland at the northern foothills of Sudethians (Schmuck 1969). The specific response of the populations of P. sylvestris and P. uliginosa from the Stołowe Mts to the atypical springtime temperature regime in this year possibly results from this inversion. The smaller differences between cold and warm years in dates of phenophases at We˛gliniec than at Batorów are difficult to explain without the analysis of previous weather conditions in the years of observations (Sarvas 1967 ; Boydak 1977; ChaŁupka 1993). We have not sufficient climatic data, but a comparison of the air tem382 FLORA (2003) 198 peratures and relative humidity of April and May of 2000 from We˛gliniec and Batorów (Figs. 1 and 2) shows that the delay of development of micro- and megastrobili of both species observed at We˛gliniec in 2000 can be interpreted as a result of the influence of greater oscillations between day and night temperatures and higher average relative air humidity of this site. Observations of the phenological phases of P. sylvestris pollen release and pollen reception in seed-orchards showed that differences in the dates of them typically amount to one to three days on average (Jonsson et al. 1976 ; Boydak 1977; Koski 1991; ChapŁuka 1993 ; Dengler, after ChaŁupka 1993). In our data these differences were greater, especially in the years with cold and humid springs, but comparable and even shorter in the year with an extremely dry and warm spring (Table 4 and 5). Table 4. Delay of starting dates of pollen release as compared with pollen reception in Pinus sylvestris Tree no. Delay of pollen release [days] Silesian Lowland 1 2 3 4 5 6 7 8 9 10 11 12 Stołowe Mts 1999 2000 2001 Average 1999 2000 2001 Average –3 7 4 15 3 4 10 10 15 3 10 14 0 3 0 0 3 0 3 3 0 0 5 0 5 5 5 5 5 5 11 6 9 11 11 9 1 5 3 6.7 3.7 3 8 6.3 8 4.7 8.7 7.7 9 7 9 12 7 7 10 8 8 8 10 3 3 6 0 10 6 11 10 11 15 13 22 13 15 15 13 17 14 15 11.3 7.7 11.3 10.3 7.3 10.7 9.7 12 10.7 11.3 15.2 10.2 Average 7.7 1.4 7.3 5.4 8.5 7.0 Standard deviation 5.58 1.76 2.67 2.50 1.50 3.77 2.56 1.450 Table 5. Delay of starting dates of pollen release as compared with pollen reception in Pinus uliginosa Tree no. Delay of pollen release [days] Silesian Lowland 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Stołowe Mts 1999 2000 2001 Average 1999 2000 2001 Average 7 0 0 7 4 4 7 7 7 – –7 14 0 15 4 – 0 0 0 0 0 0 0 – 0 0 2 0 – – 3 – 11 9 0 7 0 – –2 9 9 12 –5.5 –0 –1.0 –3.5 –5.0 –4.3 –2.3 –4.7 –2.3 – –3.0 –7.7 –3.7 –9.0 – 0 6 – 6 6 – 6 0 3 – 7 11 – 7 14 7 – 11 13 – 15 15 – 13 10 13 13 10 13 – 7.33 10.67 – 8.67 10.0 10.0 9.50 7.0 9.67 10.0 12.8 9.1 Average 5.0 0.5 9.9 –3.4 3.9 Standard deviation 5.70 1.19 4.83 –3.00 2.64 Intensive pollen release by both species lasted about 6 –12 days, which is comparable with literature data (Chalupa 1964 ; Jonsson et al. 1976; WesoŁy 1982 ; Burczyk & ChaŁupka 1997). The megastrobili were receptive for a longer time (Table 3). 2.78 1.79 1.24 Pollen perception by Pinus uliginosa took place at the moment of full pollen release by P. sylvestris every year. Thus, the probability of pollination of P. uliginosa megastrobili with P. sylvestris pollen seems to be high. It is also strongly greater at We˛gliniec, in particular if we FLORA (2003) 198 383 Fig. 5. Development of megastrobili of Pinus uliginosa (- - - - ) and microstrobili of P. sylvestris ( ) at the Silesian Lowland site take into consideration the rather small number of individuals of P. uliginosa there and large number of individuals of P. sylvestris in the immediate vicinity. The possibility of pollination of individuals of P. sylvestris by P. uliginosa pollen appears much lower. The difference between possibilities of pollination P. uliginosa by P. sylvestris pollen can be even greater if we accept the rule “first come – first served” (Sarvas 1972). According to that rule, the first pollen grain that enters the pollen chamber takes part in fertilization. There is also some possibility of pollination of P. uliginosa megastrobili in the Stołowe Mts. by pollen of P. sylvestris transported from lowlands. The time differences between pollen release by P. sylvestris on the Table 6. Average duration of pollen release and pollen reception in Pinus uliginosa and P. sylvestris Year Site Average duration [days] Pinus uliginosa 1999 2000 2001 384 Silesian Lowland Stołowe Mts Silesian Lowland Stołowe Mts Silesian Lowland Stołowe Mts FLORA (2003) 198 Pinus sylvestris Reception Pollen release Reception Pollen release 15.2 14.3 8.8 11.3 20.6 24.8 9.1 10.4 8.3 10.9 11.3 12.9 16.2 20.1 10.7 9.9 14.3 15.0 8.3 14.0 8.0 9.4 10.8 12.3 Fig. 6. Development of megastrobili of Pinus sylvestris (- - - - ) and microstrobili of P. uliginosa ( ) at the Silesian Lowland site lowland and pollen reception by P. uliginosa in the mountains would permit this process. Additionally, P. sylvestris produces great amounts of pollen grains per hectare of the adult forests (Sarvas 1962 ; ChaŁupka & Fober 1977; Koski 1991; ChaŁupka 1993). The species predominates in the forests on the lowland of Central Europe and the pollen grains are normally trans- ported for a hundreds and sometimes thousands of kilometers by convecting air flows (Tikhomirov 1950). By contrast, the pollination of P. sylvestris by pollen of P. uliginosa, and also of P. mugo, seems to be rather unlikely, because pollen release in the mountains is considerably delayed in relation to the period of pollen reception in the lowland locations. Table 7. Delay of pollen release by microstrobili and pollen reception by megastrobili in Pinus uliginosa as compared to Pinus sylvestris Year Site Average delay [days] Pollen release 1999 2000 2001 Silesian Lowland Stołowe Mts Silesian Lowland Stołowe Mts Silesian Lowland Stołowe Mts Pollen reception start intensive end start intensive end 6 5 8 3 5 2 6 8 8 5 4 5 5 6 8 6 9 10 2 13 9 4 1 3 8 9 8 5 13 7 7 5 7 4 7 9 FLORA (2003) 198 385 Fig. 7. Development of megastrobili of Pinus uliginosa (- - - - ) and microstrobili of P. sylvestris ( ) at the Stołowe Mts site Conclusions Our three-year record of phenological observations shows that periods of development of megastrobili of P. uliginosa and microstrobili of P. sylvestris overlap, enabling cross-pollination (Figs. 5 and 7) at both, a mountain and a lowland site. Development of P. uliginosa microstrobili is delayed as compared to P. sylvestris megastrobili, but does not strictly rule out crosspollination, but such an event will have a much lower probability (Figs. 6 and 8). The differences in the dates of development of microand megastrobili of both species varied significantly during three years of observation, but in each year crosspollination was principally possible. The possibility of pollination of P. uliginosa by P. sylvestris may be strongly increased if this follows the rule “first come – first served”. A substantially greater possibility of pollination of P. uliginosa megastrobili by P. sylvestris pollen creates the potential danger of genetic erosion in the first spe386 FLORA (2003) 198 cies, especially in the small population of the species which is surrounded by P. sylvestris at We˛gliniec. In 2000, characterized by an early, warm and dry spring, the populations of P. sylvestris and P. uliginosa in the mountain site of the Stołowe Mts developed reproductive organs earlier than those at We˛gliniec (Silesian Lowland). This was probably due to the effects of a temperature inversion, but a specific response of the mountain populations to the anomalous temperature climate cannot be excluded. The megastrobili of P. uliginosa developed earlier than microstrobili in any case, as it is the rule and even more pronounced in P. sylvestris too. Acknowledgements The study was sponsored by the Polish Committee for Scientific Research Grant 6P04G 060 16 with support of Institute of Dendrology. 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