Genetic Variation of Pinus cembra Along an Elevational Transect in Austria Raphael Thomas Klumpp Marcus Stefsky Abstract—The genetic variation of Pinus cembra was analyzed by means of isozyme gene markers along an elevational transect at Koetschach Valley (Salzburg State/Austria). Mature stands and their natural recruitment were studied at three elevation levels: subalpine zone, high montane zone, and middle montane zone. Sample included juvenile and mature individuals. Thirteen enzyme systems encoding for 22 gene loci were scored. The results showed increasing allelic multiplicity with increasing altitude in mature stands and decreasing polymorphism with increasing altitude in juvenile populations. Surprisingly high values of allelic multiplicity and hypothetic gametic multilocus diversity were found in middle elevation populations, which have potential for generating genetically diverse gametes in future generations. Seed dispersal by a nutcracker bird species, as well as gene flow by local wind systems, may be the reason for this phenomenon, which is obviously strengthened by strong selection forces. Key words: Pinus cembra, isozyme, elevation, population, genetic diversity, seed dispersal, Nucifraga caryocatactes. Introduction ____________________ Pinus cembra L., a locally important species in Europe, has had multiple uses over centuries. The species has been used for timber, when indoor use for country style furniture and wall ornaments were in fashion, and the large seeds (nuts) were used to improve the diets of farmers living in alpine pastures. Presently, the blue cones are harvested for preparing liquor, which leads to crown damage and problems for species that depend on the seeds for food like nutcracker birds (Nucifraga caryocatactes L.). It is well known that P. cembra was eliminated in lower elevations by competition. It is less commonly known that virgin forests at the timberline in alpine mountains were harvested in order to extend alpine pastures, even up to the 20th century (Fromme 1957). Furthermore, large amounts of timber from these forests were needed and utilized. In: Sniezko, Richard A.; Samman, Safiya; Schlarbaum, Scott E.; Kriebel, Howard B., eds. 2004. Breeding and genetic resources of five-needle pines: growth, adaptability and pest resistance; 2001 July 23–27; Medford, OR, USA. IUFRO Working Party 2.02.15. Proceedings RMRS-P-32. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station. Raphael Thomas Klumpp and Marcus Stefsky are with the Institute of Silviculture, University of Natural Resources and Applied Life Sciences, Peter Jordan Str. 70 A-1190, Vienna, Austria. Contact Klumpp: Phone 0043 1 47654 4063. Fax 0043 147654 4092. Email: raphael.klumpp@boku.ac.at 136 Hence, distortion of genetic architecture is anticipated in all natural populations. In spite of these facts, we hypothesize that there is still a relatively high level of genetic variation in high elevations, as those forests have not been regularly managed. Furthermore, the competitiveness of P. cembra, as well as the mutualism with nutcracker birds at these elevations, may lead to more effective preservation of the gene pool. In contrast, populations at lower elevation may have suffered a reduction of genetic diversity due to regular management activities and limited activity of nutcrackers in such dense forests. Isoenzyme studies in P. cembra and related species have been conducted by different authors. Heterozygosity is low in comparison to other pine species (Szmidt 1982), and species can be easily differentiated (Goncharenko and others 1992, Politov and Krutovskii 1994). Different numbers of gene loci have been found to be encoding the same enzyme systems of different pine species (Bergmann and Hattemer 1995), but P. cembra and its relatives from subgenus Strobus Lemm. section Strobus exhibit the same number of gene loci controlling the enzyme system of 6PGDH (Bergmann and Gillet 1997). Genetic architecture in conifer populations can be affected by the age of the population. Investigations on the natural recruitment in P. sylvestris L. revealed that there is an excess of homozygotes in the embryo stage, which decrease as age advances according to the species’ life cycle (Muona and others 1987, Yazdani and others 1985). Similar results have been found in other coniferous species, such as P. radiata D. Don. (Plessas and Strauss 1986), Pseudotsuga menziesii [Mirb.] Franco (Shaw and Allard 1982), and Abies alba Mill. (Hussendoerfer 1998). Variation along elevational transects has been observed by means of isozyme gene markers in several species. Mitton and others (1980) found clinal variation at two gene loci in ponderosa pine (P. ponderosa Laws.). Ruetz and Bergmann (1989) found variation of allelic structures along one elevational transect in Norway spruce (Picea abies (L.) Karst.). However, other studies, for example Neale and Adams (1985) in balsam fir (Abies balsamea (L.) Mill.), could not confirm this finding. Similarly, some publications on Norway spruce (Konnert 1991) and European beech (Fagus sylvatica L.) (Loechelt and Franke 1995) did not show clinal variation along elevational gradients. These studies were conducted on populations in southwestern Germany, where silvicultural activities by humans over centuries may have lead to distortion of natural variation. The reproductive potential of this species is currently in danger, as the local forest authority is unable to control the harvesting of cones in Austria. As this tree species is less competitive than other species, we initiated an investigation on genetic diversity along 11 elevational transects in the USDA Forest Service Proceedings RMRS-P-32. 2004 Genetic Variation of Pinus cembra Along an Elevational Transect in Austria Austrian Alps (Alpine Mountains). This study will provide information to guide gene conservation/restoration efforts and can be used as well to indicate changes in genetic diversity due to global warming over time. In this paper, initial results will be reported using isozyme gene markers to study diversity along a single Austrian transect. Materials and Methods ___________ The Koetschach Valley in the State of Salzburg, Austria, is found in the eastern part of the central Alps, which Klumpp and Stefsky exhibits a subcontinental, cold winter climate. Sampling areas were designated on a single slope with northern exposition at three different elevation levels (table 1). The transect number is AT1, and the sampling area was designated as “U” for the subalpine zone, “M” for the high montane zone, and “L” for the middle-montane zone. Samples were taken from adult and juvenile trees (51 to 149 individuals per population) and recorded by age class. Isoenzyme analyses were conducted following the methods described by Cheliak and Pitel (1984) and Hertl (1997). The analyses used 13 enzyme systems, which encode for 22 gene loci (table 2). Data were scored using the GSED pro- Table 1—Characteristics of the sampling area. Transect AT 1 Altitudinal zone Elevation Forest community Subalpine (U) High montane (M) Middle montane (L) 1900 – 2100 m 1600 – 1700 m 1200 – 1300 m P. cembra – larch Larch – Norway Spruce forest with forest with Pinus spruce forest with some P. cembra and mugo Turra P. cembra larch Semipodsol Soil Precipitation 900 – 1100 mm Total number of individuals: 149 148 Adult population 99 100 34 Juvenile population 49 48 17 51 Table 2—Analysed enzyme systems and gene loci. Nomenclature Enzyme system Analysed loci Alaninaminopeptidase (E.C. 3.4.11.2) AAP AAP-A AAP-B Aspartataminatransferase (E. C. 2.6.1.1) AAT AAT-A AAT-B AAT-C Aconitase (E. C. 4.2.1.3) ACO ACO-A Diaphorase (E.C. 1.6.4.3) DIA DIA-A DIA-B Glutamatdehydrogenase (E. C. 1.4.1.2) GDH GDH-A Isocitrat-Dehydrogenase (E. C. 1.1.1.42) IDH IDH-B Leucin-Aminopeptidase (E. C. 3.4.11.1) LAP LAP-A LAP-B Malat-Dehydrogenase (E. C. 1.1.1.37) MDH MDH-A MDH-B MDH-C Menadion-Reduktase (E. C. 1.6.99.2) MNR MNR-A 6-Phosphogluconic-Dehydrogenase (E. C. 1.1.1.44) 6-PGDH 6-PGDH-A 6-PGDH-C Phosphoglucose-Isomerase (E. C. 5.3.1.9) PGI PGI-A PGI-B Phosphoglucomutase (E. C. 2.7.5.1) PGM PGM-A Shikimat-Dehydrogenase (E. C. 1.1.1.25) SKDH SKDH-A USDA Forest Service Proceedings RMRS-P-32. 2004 137 Genetic Variation of Pinus cembra Along an Elevational Transect in Austria Klumpp and Stefsky gram (Gillet 1994). We used allelic multiplicity (number of alleles M), relative allelic multiplicity (M/Mmax) and the average number of alleles per locus (A/L) for describing allelic variation (Hattemer and others 1993). Genetic diversity was described using average (observed) heterozygosity (Ho), diversity of the gene pool (V) and hypothetic gametic multilocus diversity (Vgam) (Gregorius 1978, 1987). The genetic variation within each population was quantified by calculating the percentage of polymorphic loci (P95), where the frequency of the most common allele does not exceed 95 percent. Results and Discussion __________ The P. cembra subpopulation from the middle montane zone on the valley floor is scattered in a spruce-dominated forest that also includes larch. Correspondingly, only 34 adult and 17 young individuals were found, which represented approximately 80 percent of the whole population in this area. This bias in sampling was considered when drawing some conclusions from this study. The number of observed alleles was found to obviously increase with elevation in the adult populations. However, the highest value (M=36) was found at medium elevation in juvenile populations (table 3). In the high montane zone, 75 percent of the known variants were represented in the juvenile population, as a total of 48 alleles over 22 gene loci, were found in this valley. Closer inspection revealed that variants with allele frequencies of more than 5 percent were found to comprise between 58.3 and 64.6 percent of the total known variants. Adult populations obviously possessed more rare variants with an allele frequency of 1 percent, such as AT1-U: M/Mmax=83.3 and M(f>1)/Mmax=77.1, than in young populations, such as AT1-U: 70.8: 70.8 (table 3). Surprisingly, the populations of the middle elevation exhibited not only the highest values of observed alleles, but also high values of relative allelic multiplicity (M/Max), which was 75 percent of the known variants (table 3). Stone pine appears to be different from other species, where clinal variation was not found in allozyme gene markers (see for example Moran and Adams 1989), or was only found only in certain loci (for example Mitton and others 1980). This may be due to strong selective forces such as the harsh climate at the timberline, or competition with spruce in the valley floor may influence the gene pool composition of this species. Moreover, trees resulting from movement of seed by birds (compare Marzluff and Balda 1992) may have enhanced the existing gene flow (primarily by pollen transport up and down the slopes), which leads to high level of genetic multiplicity. A comparison of selected parameters of genetic variation in mature stands from different elevations shows a slight increase of multiplicity (M) with elevation but nearly no trends in the other parameters (fig. 1). In contrast, no trend was found in the heterozygosity in the juvenile stands, and polymorphism decreased with elevation. Multiplicity as well as hypothetic gametic multilocus diversity (the potential for generating genetically diverse gametes in future generations) is highest in the middle elevation (fig. 2). This indicates that the genetic architecture in the juvenile populations at the middle elevation has been preserved better than at other locations/age classes (fig. 3; table 3). Conclusions ____________________ The higher allelic multiplicity in juvenile populations at the mid-montane zone is due to a combination of birds and wind. Nutcracker birds transport seed to the middle slopes from higher and lower elevations, as the middle slopes provide less snow cover and easy access. Snow cover at the timberline and the dense forest in the valley are not as attractive for habitats for the bird, and allelic multiplicity is correspondingly lower. Pollen transport by local wind systems cause gene flow among populations up and down slopes, thereby the mid-montane zone has an influx of genes from populations at both higher and lower elevations. Selection forces are obviously active, which to a certain extent keeps the gene pool of the timberline population different a Table 3—Genetic variation at transect AT1 in the Koetschach valley, Salzburg State / Austria AT1 - U Number of individuals Number of loci Number of observed alleles (M) M/Mmax (%) A/L A/L ≥ 5 % A/L ≥ 1 % P95 in % Ho (observed) Gene pool diversity (V) Hyp. gam. diversity (Vgam) AT1 – M AT1 - L Ab Jc A J A J Average 100 22 40 83.3 1.82 1.27 (58.3) 1.68 (77.1) 23 0.095 1.10 28.3 49 22 34 70.8 1.54 1.27 (58.3) 1.55 (70.8) 18 0.081 1.09 24.6 100 22 38 79.2 1.73 1.32 (60.4) 1.64 (75.0) 18 0.083 1.12 27.3 48 22 36 75.0 1.64 1.32 (60.4) 1.64 (75.0) 27 0.083 1.13 37.1 34 22 34 70.8 1.54 1.36 (62.5) 1.55 (70.8) 32 0.083 1.13 26.4 17 22 32 66.7 1.45 1.41 (64.6) 1.41 (64.6) 32 0.086 1.12 24.8 — 22 35.7 74.3 1.62 1.33 (60.6) 1.58 (72.2) 25 0.085 1.12 28.1 a Where U refers to the subalpine zone, M refers to the high montane zone, and L refers to the middle montane zone of transect AT1. A - Adult population J - Juvenile population b c 138 USDA Forest Service Proceedings RMRS-P-32. 2004 Genetic Variation of Pinus cembra Along an Elevational Transect in Austria 50 40 AT1 - U 30 AT1 - M 20 Klumpp and Stefsky for laboratory assistance. The advice and many helpful comments by Prof. Gerhard Mueller-Starck (Munich/Germany) is greatly appreciated. Furthermore, we thank Scott Schlarbaum and Richard Sniezko for helpful comments on an earlier draft. AT1 - L 10 References _____________________ 0 M P95 Ho Vgam Figure 1—Average genetic variation for selected parameters in mature stands from different elevation. 40 35 30 25 20 15 10 5 0 AT1 - U AT1 - M AT1 - L M P95 Ho Vgam Figure 2—Average genetic variation for selected parameters in the juvenile population from different elevation. 40 35 30 25 20 15 10 5 0 adult juvenile M P95 Ho Vgam Figure 3—Average genetic variation for selected parameters in adult and juvenile populations at sample area AT1- M (high montane zone). from that of the valley population. Strong competition with the dominating spruce at the ground of the valley reduces P. cembra populations to the extent that the opportunity for rare variants in the gene pool is limited. Acknowledgments ______________ This study was funded by the European Union as part of the project on “biodiversity of alpine forest ecosystems” (EU CT96-1949). 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