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). We thank Eva, Tajana, Herwig, and Wolfgang
for technical assistance in collecting the samples as well as
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