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Spatial and microgeographical genetic differentiation of black alder (Alnus glutinosa Gaertn.) populations
Forest Ecology and Management 160 (2002) 3–9
Spatial and microgeographical genetic differentiation of black alder (Alnus glutinosa Gaertn.) populations Dusˇan Go¨mo¨ry*, Ladislav Paule Faculty of Forestry, Technical University in Zvolen, T.G. Masaryka 24, SK-960 53 Zvolen, Slovak Republic Received 22 June 2000; received in revised form 16 November 2000; accepted 16 November 2000
Abstract Three Alnus glutinosa populations from central Slovakia were investigated by means of isozyme analysis. The average expected heterozygosity ranged from 0.151 to 0.173. Despite significant differences in allele frequencies among populations, genetic differentiation was low (F ST ¼ 0:022). Intrapopulation fixation indices indicate no or very slight deviation from panmictic expectations towards the heterozygote deficiency. No indications were found that the investigated populations have experienced recent bottlenecks in population size. A spatial autocorrelation analysis based on Moran’s I indicates the existence of a spatial genetic structuring in the population Kra´l’ova´. Patch size was estimated to be 70–100 m, which is more than observed in other broadleaved tree species. This difference is explained by the linear shape of alder populations and a higher mobility of seeds. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Alnus glutinosa; Isozymes; Genetic differentiation; Spatial autocorrelation
1. Introduction Black alder (Alnus glutinosa [L.] Gaertn.) is a tree species with a pan-European range. In the north, it reaches the Polar circle, whereas the southernmost occurrences are located in North Africa. In the east, it grows behind the Ural mountains under the continental climate of the western Siberia. Within this vast range, it mounts from lowlands up to 1800 m a.s.l. in the Alps and 1300 m a.s.l. in the Carpathians. It means that it is a species able to adapt to very variable climatic conditions. At the same time, it requires specific soil conditions, mainly concerning moisture.
*
Corresponding author. Tel.: þ421-855-5206-226; fax: þ421-855-5332-654. E-mail address: [email protected] (D. Go¨mo¨ry).
It occupies primarily sites with flowing ground water along streams, but also sites with stagnating ground water like peat bogs. However, in contrast to most hygrophilous tree species, it survives also on drier sites, where it behaves as a pioneer species (PancerKotejowa and Zarzycki, 1980). Site requirements lead to a considerable fragmentation of the distribution range. Black alder regenerates generatively as well as vegetatively, mainly by sprouts, rarely by root suckers. Sprouts are mostly very vigorous and lead to the formation of polykormic individuals. Rooting of lateral branches of fallen trees was also observed, after the maternal stem decayed, the sprouts form clonal groups. In central Europe, large alder stands, which were occurring on the river terraces, in dead arms of rivers and lakes, swamps, etc. have mostly been destroyed by
0378-1127/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 2 7 ( 0 1 ) 0 0 4 6 5 - 0
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D. Go¨mo¨ry, L. Paule / Forest Ecology and Management 160 (2002) 3–9
human activities. However, alder still occurs along practically all streams, even very small ones. Therefore, typical alder populations differ from other tree species by a specific shape, since they are extended mainly in one dimension. This might affect considerably the gene flow within populations and subsequently the genetic structure. Prat et al. (1992) investigated the genetic structure of alder populations within the major part of its European range. They did not find clear geographical trends, but a considerable local and regional differentiation. Similar patterns were found in the North American alder species Alnus rugosa and Alnus crispa (Bousquet et al., 1987a, 1988). The aim of this study was to find out how the fragmentation of the range and the subsequent isolation of populations reflected by genetic differentiation, and to what extent the specific bionomy of this species (mainly the linear shape of populations) leads to spatial genetic structuring.
2. Materials and methods Three black alder (A. glutinosa Gaertn.) populations were sampled in the close vicinity of the town Zvolen in central Slovakia (198080 E/488350 N). Their exact position is given in Fig. 1. In spring 1999, at the time of ripening of male catkins, twigs with dormant buds
Fig. 1. Location of the investigated black alder populations: (1) Bienˇ ; (2) Neresnica; (3) Kra´ l’ova´ .
and male catkins were taken from all available adult trees (fructifying trees with BHD over 7 cm) in spatially continuous sections of the respective populations 39, 48 and 93 trees in populations Neresnica, Bienˇ and Kra´ l’ova´ , respectively. In the population Kra´ l’ova´ , the sampled trees were labeled and their exact position was identified from an aerial photograph. Vegetative tissues and pollen released from catkins were analyzed separately. Pollen was stored at 4 8C up to 4 weeks until the analysis. Buds together with cambium were homogenized in a Tris–HCl extraction buffer pH 7.3 with the addition of PVP 40, PVP 360, EDTA II, Tween 80, PEG, 2-mercaptoethanol (1% each), 0.025% DTT, and 0.5% Na-ascorbate. Pollen was homogenized manually in the same extraction buffer. Enzyme separation was performed electrophoretically in 12% (w/v) starch gels using three buffer systems (lithium borate–Tris citrate pH 8.1; sodium borate pH 8.0–Tris citrate pH 8.7, and Tris histidine–Tris citrate buffer pH 7.0). Staining procedures followed Cheliak and Pitel (1984). Where possible, the interpretation of zymograms followed Prat et al. (1992) and Bousquet et al. (1987b, 1988). A parallel analysis of diploid (buds) and haploid (pollen) tissues was performed to facilitate reading of zymograms. Alleles were designated by their relative migration rates as related to the most frequent one. Allelic frequencies at each locus were calculated based on diploid genotypes. Differences in allelic frequencies between populations were tested using the probability test (Raymond and Rousset, 1995). Genetic diversity was characterized by the mean number of alleles per locus, proportion of polymorphic loci, observed and expected heterozygosities. Deviations of the genotypic structures from the Hardy–Weinberg equilibrium were tested following Rousset and Raymond (1995). Sign test and Wilcoxon sign-rank test (Cornuet and Luikart, 1996) were used to detect recent population bottlenecks. To assess the spatial genetic structure in the population Kra´ l’ova´ , spatial autocorrelation method was used. Moran’s I (Sokal and Oden, 1978) was estimated for each of 10 distance classes with equal ranges of 70 m. The overall significance of correlograms was tested using Sˇ ida´ k’s criterion (Geburek, 1993).
D. Go¨ mo¨ ry, L. Paule / Forest Ecology and Management 160 (2002) 3–9
3. Results 3.1. Inheritance of isozyme systems In total, 10 isozyme systems controlled by at least 15 loci (putative in some cases) were analyzed. The inference about the genetic control of isozyme variants was based on the knowledge of the subunit number in multimeric enzymes and the comparison of isozyme phenotypes in pollen an vegetative tissues. For several enzyme systems, scoring was much easier and much more reliable in pollen than in buds (shikimate dehydrogenase, isocitrate dehydrogenase). Glutamate dehydrogenase even exhibited activity only in pollen. However, in 6-phosphogluconate dehydrogenase, isozyme phenotypes in the more rapidly migrating zone in pollen have not shown any correspondence with those in vegetative tissues. In buds, this zone is apparently controlled by a single locus with two allelic variants, whereby the enzyme is dimeric (heterozygous individuals exhibit a triple-banded phenotype), but we have not succeeded in finding any genetic interpretation for the pattern found in pollen. It seems that in pollen, different loci are expressed than in buds. In malate dehydrogenase two zones could be found in both tissue types, whereby the second one was variable with double-banded phenotypes in pollen and triplebanded phenotypes in buds (controlled by the locus Mdh-B). In pollen, an additional zone with two bands was observed. According to our interpretation, this zone is controlled by another locus (Mdh-C) forming an interlocus heterodimer with the product of Mdh-B (Fig. 2). In the remaining cases, there was a good concordance between isozyme phenotypes in pollen and buds, whereby for dimeric or tetrameric enzymes, intralocus hybrid bands were absent in pollen. Four zones have not shown any variation, and after comparing with the results of Bousquet et al. (1987b, 1988,
Fig. 2. Observed MDH phenotypes and their genetic interpretation.
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1990), they were interpreted as controlled by monomorphic loci (Pgi-A, Mdh-A, Mdh-C, Got-A). Two zones were highly variable (Fest-A, Idh-B) but no reliable scoring was possible. 3.2. Microgeographical differentiation Out of 16 scored loci, 12 were found to be polymorphic in at least one population. Despite very small geographic distances between populations, significant heterogeneity of allelic frequencies was found in eight loci (Table 1). The levels of the genetic multiplicity and diversity differed among populations as well (Table 2). The population Kra´ l’ova´ , even when represented by the biggest sample, appears to posses least alleles and least polymorphic loci. Like in the other broadleaved species, there is no concordance between the levels of the genetic diversity and multiplicity. The population Neresnica, which is the most rich one in alleles, does not exhibit the highest diversity levels at the same time. Like in most tree species, the level of genetic differentiation as revealed on the basis of nuclear genetic markers, is very low. The values of FST ranged from 0.001 to 0.082, that means that only a small portion of the total variation is due to interpopulation differences (Table 3). The locus with the highest differentiation among populations is Got-B, what probably results from a high frequency of the Got-B/ 28 allele in the population Kra´ l’ova´ . The average intrapopulation fixation index FIS is near zero (0.003). However, there is a considerable variation among loci as well as among populations. In the population Neresnica, there was no significant deviation from Hardy–Weinberg equilibrium found. On the other hand, three significant deviations towards the heterozygote deficiency were found in the population Bienˇ (Fest-B, Mdh-B, 6pgdh-A). In Kra´ l’ova´ , heterozygote deficiency was at the significance limit (P ¼ 0:0606) in Fest-B. Since these deviations are not consistent among loci within individual populations, it is questionable if the positive fixation indices are due to inbreeding. Selection or pure chance is a more plausible explanation. Neither the sign test nor the Wilcoxon sign-rank test proved a significant gene diversity excess over the equilibrium value, which indicates a recent population bottleneck, in any of the studied populations
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Table 1 Allelic frequencies of polymorphic loci in the investigated black alder populations Locus
Allele
Population
Pa
Population
N
na
nab
PP
HO
HE
Bienˇ Neresnica Kra´ l’ova´
36.3 41.8 86.5
1.9 2.0 1.6
2.5 2.7 2.1
62.5 75.0 56.3
0.154 0.171 0.153
0.173 0.158 0.151
Bienˇ
Neresnica
Kra´ l’ova´
117 100 83 80
0.141 0.782 0.064 0.013
0.143 0.806 0.051 0.000
0.074 0.881 0.045 0.000
0.1622
Gdh-A
125 100
0.435 0.565
0.389 0.611
0.410 0.590
0.8958
Pgm-A
105 100 95
0.013 0.936 0.051
0.043 0.915 0.043
0.048 0.952 0.000
0.0169
Pgm-B
67 59
0.974 0.026
0.957 0.043
1.000 0.000
0.0111
Idh-A
105 100
0.224 0.776
0.121 0.879
0.077 0.923
0.0189
Mdh-B
77 67 57
0.090 0.897 0.013
0.049 0.951 0.000
0.000 1.000 0.000
0
6pgd-A
100 87
0.526 0.474
0.671 0.329
0.653 0.347
0.1038
6pgd-B
53 40
1.000 0.000
0.988 0.012
1.000 0.000
0.4664
Skdh-A
105 100
0.375 0.625
0.222 0.778
0.442 0.558
0.0058
Got-B
60 44 28
0.000 1.000 0.000
0.050 0.925 0.025
0.000 0.813 0.187
0
Pgi-B
89 73 57
0.000 0.962 0.038
0.051 0.949 0.000
0.005 0.995 0.000
0.0007
Mnr-A
134 100 87
0.064 0.936 0.000
0.031 0.949 0.020
0.114 0.886 0.000
0.0187
Fest-B
a
Table 2 Characteristics of the genetic multiplicity a diversity of the investigated black alder populationsa
Probability test (Raymond and Rousset, 1995).
(probabilities for sign test were 0.135, 0.176, and 0.507 for Neresnica, Bienˇ , and Kra´ l’ova´ , respectively). 3.3. Spatial structure We compared genotypes of neighboring trees by means of a cluster analysis. No groups of spatially proximate identical genotypes, indicating natural cloning, were identified.
a N: average sample size; na: mean number of alleles per locus; PP: proportion of polymorphic loci; HO: observed heterozygosity; HE: expected heterozygosity. b For polymorphic loci (loci monomorphic in all populations excluded).
Spatial autocorrelation analysis was performed for nine independent alleles. The resulting correlograms are presented in Fig. 3a–c (distance classes are characterized by centers, not ranges). Even if the courses of the lines for individual alleles are not identical, there seems to exist a general sinusoidal pattern, which can be identified in the correlograms, and which is reflected by average values of Moran’s index over alleles. As expected, the autocorrelations in the first distance class are generally positive, with two significant values. Then autocorrelations decrease, and at the distance of 170–250 m they become significantly negative. Within the distance range of 300–450 m, positive values prevail again. Interestingly, in the distance class of 525 m, almost all Moran’s indices are negative, whereby for six out of nine loci, the deviation from the expected value is significant. Five out of nine correlograms proved to be Table 3 Single-locus F-statistics in the investigated black alder populations Locus
FIS
FIT
FST
GST
Fest-B Gdh-A Pgm-A Pgm-B Idh-A Mdh-B 6pgd-A 6pgd-B Skdh-A Got-B Pgi-B Mnr-A
0.107 0.075 0.060 0.038 0.201 0.191 0.131 0.012 0.016 0.116 0.045 0.091
0.116 0.073 0.052 0.023 0.164 0.218 0.146 0.004 0.053 0.025 0.025 0.076
0.009 0.001 0.008 0.014 0.031 0.033 0.018 0.008 0.037 0.082 0.020 0.014
0.010 0.001 0.011 0.032 0.033 0.054 0.013 0.035 0.038 0.056 0.019 0.013
Average
0.003
0.020
0.022
0.022
D. Go¨ mo¨ ry, L. Paule / Forest Ecology and Management 160 (2002) 3–9
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Fig. 3. (a) Correlograms for alleles Idh-A/105, 6pgdh-A/100, Fest-B/117 and Fest-B/83. (b) Correlograms for alleles Gdh-A/125, Got-B/28, Mnr-A/134, Pgm-A/105 and Skdh-A/105. (c) Mean correlogram and numbers of significant positive (shaded column) and negative (blank column) Moran’s I indices in individual distance classes.
significant (for 6pgd-A/100, Fest-B/83, Got-B/28, Mnr-A/134, and Skdh-A/105). These fluctuations indicate a patchy genetic structure within the population, with a patch size of approximately 70–100 m. This course is mainly shaped by Got-B/28, Fest-B/ 83, and Idh-A/105, but for most alleles, the correlogram is at least partially concordant with the described
trend. In only one distance class, both significant positive and negative were found (class 455 m, two and one positive and negative values, respectively). The most deviating allele is Mnr-A/134, where all indices up to the distance of 315 m are above the expected value, and for all higher distance classes they become negative.
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4. Discussion Despite its economical importance, black alder does not belong to intensively studied tree species, at least regarding genetic structure. Therefore, the information about the inheritance of isozyme systems is very scarce. Linares-Bensimo´ n (1984) tested the Mendelian inheritance of aminopeptidases and glutamate–oxaloacetate transaminases. Prat et al. (1992) analyzed 16 isozyme systems controlled by 19 polymorphic loci. They did not present any results of inheritance testing, but they state that they observed Mendelian segregation in open-pollinated progenies for all the proposed loci. However, for several isozyme loci that we used, we had no reference in A. glutinosa, therefore we used the results published for the American alder species A. crispa, A. rugosa, and Alnus sinuata (Bousquet et al., 1987b, 1988, 1990). Another basis for the inference about the genetic control was a parallel analysis of buds and pollen. Our results are in a good concordance with the cited studies. The only exception is the Fest-B locus. Prat et al. (1992), who used polyacrylamide gel electrophoresis, report about monomeric structure of the enzyme produced by the most rapidly migrating locus. Similarly, Bousquet et al. (1987b) found monomeric structure in b-esterases of A. crispa. Phenotypes that we found in the slowly migrating zone of fluorescent esterase, indicate a dimeric structure (triple-banded pattern in diploid tissues, double-banded pattern with missing hybrid band in pollen). Since there was a perfect concordance between the phenotypic expression in diploid and haploid tissues, we adopted the hypothesis of the genetic control of this zone by a single locus. The levels of the genetic multiplicity, characterized by the mean number of alleles and the proportion of polymorphic loci, correspond to those found by Prat et al. (1992), however, we found slightly lower diversity. It is very interesting that there is a similarity in the levels of diversity between black alder and the American alder species (A. crispa, A. rugosa, A. sinuata). Although the distribution of the expected heterozygosities over loci is not homogeneous among species, the average values are very close to each other (cf. Bousquet et al., 1987a,b, 1988, 1990). Only 2.2% of the total variation is due to interpopulation differences. This is much less than in the study of Prat et al. (1992), who found F ST ¼ 0:204.
This difference was expected, since in the cited study, populations from almost the whole Europe were included, whereby in our case the air distances between populations range from 5 to 10 km. Prat et al. (1992) observed a strong small-scale differentiation and absence of any clear large-scale geographical trends. They suppose that this pattern is due to isolation of populations and life-history traits of black alder, which undergoes successive colonization/retreat cycles accompanied by severe population bottlenecks. However, there is no indication that our populations have experienced any recent bottlenecks. In fact, alder behaves as a pioneer species on wet meadows, which it colonizes but it is successively replaced by other tree species. However, along the streams, where the site conditions are permanently favorable for alder, it is a very competitive species, which can be considered a climax one. These sites have continuously been occupied by alder populations, counting several tens or hundreds of individuals, so that at least in these cases, bottleneck effects are improbable. Alder is quite a common species in all regions where the natural character of landscape has not been severely changed. It does not form large continuous stands, but individuals or small groups are dispersed along all streams, so that its distribution over the landscape is quite regular. These individuals may serve as stepping stones for gene flow among larger populations mediated by pollen. Nevertheless, gene flow does not seem to be as effective as in case of predominant forest tree species with continuous ranges like beech or oaks, since significant differences on a very small geographical scale occur. Luka´ cˇ ik (2000) observed considerable differences in the representation of morphological traits among alder populations in different regions within Slovakia. Although environmental effects contribute considerably to this morphological variation, the magnitude of differences in traits where a higher heritability can be expected (like bark color and form) indicates also a genetic differentiation. Since most of the genetic variation in black alder proved to reside within populations, the question how is it spatially organized appears to be relevant. A relatively good concordance among the correlograms of different alleles, indicating a common mechanism in forming of genetic substructuring, is not a common phenomenon in forest tree species. In general, very weak spatial genetic structures have been reported
D. Go¨ mo¨ ry, L. Paule / Forest Ecology and Management 160 (2002) 3–9
(Leonardi and Menozzi, 1996; Merzeau, 1991; Streiff et al., 1998; an overview for forest tree species is also given in Geburek (1993)). A distinct spatial structure is mostly associated with a severe human interference (Knowles et al., 1992). It must be emphasized that in most studies, much smaller ranges of distance classes are used, allowing to identify a finer genetic structure. Smaller patch sizes of approximately 30 m were observed in Quercus laevis and Fagus sylvatica (Berg and Hamrick, 1995; Leonardi and Menozzi, 1996; Merzeau, 1991). We cannot exclude, that there exists a finer fluctuation of Moran’s I in black alder as well, but due to the linear shape of the investigated population (which is typical for this species), smaller distance classes could not be chosen, since they would lead to very small sample sizes within them. Microhabitat selection and isolation by distance due to a limited pollen and seed flow are generally considered the main causes of spatial genetic structuring. In our case, the similar course of correlogram curves for different alleles indicates that the latter mechanism is the more probable one. Except for polykormic individuals, we have not observed any clonal groups. However, there may exist groups of relatives within the population—not only half-sib families, but also parent–offspring groups, since the stand is readily uneven-aged. Alder is a self-sterile wind-pollinated tree species, with small and light seeds which are easily transported by wind and water. Maybe these factors contribute to a larger patch size than in self-compatible tree species with heavy seeds like beech or Turkey oak. Acknowledgements The authors are indebted to Mrs. Zuzana Slancˇ ´ıkova´ for the technical assistance with sampling and laboratory analyses. Aerial photographs were kindly provided by the Institute of Forest Management, Zvolen. This study was supported by Grant nos. 1/7056/20 and 1/ 4036/97 from the Slovak Grant Agency for Science. References Berg, E.E., Hamrick, J.L., 1995. Fine-scale genetic structure of a Turkey oak forest. Evolution 49, 110–120.
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Bousquet, J., Cheliak, W.M., Lalonde, M., 1987a. Genetic differentiation among 22 mature populations of green alder (Alnus crispa) in central Quebec. Can. J. For. Res. 17, 219–227. Bousquet, J., Cheliak, W.M., Lalonde, M., 1987b. Allozyme variability in natural populations of green alder (Alnus crispa) in Quebec. Genome 29, 345–352. Bousquet, J., Cheliak, W.M., Lalonde, M., 1988. Allozyme variation within and among mature populations of speckled alder (Alnus rugosa) and relationships with green alder (A. crispa). Am. J. Bot. 75, 1678–1686. Bousquet, J., Cheliak, W.M., Wang, J., Lalonde, M., 1990. Genetic divergence and introgressive hybridization between Alnus sinuata and A. crispa (Betulaceae). Plant Syst. Evol. 170, 107–124. Cheliak, W.M., Pitel, J.A., 1984. Techniques for starch gel electrophoresis of enzymes from forest tree species. Information Report PI-X-42, Petawawa National Forest Institute, Chalk River. Cornuet, J.M., Luikart, G., 1996. Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144, 2001–2014. Geburek, T., 1993. Zur Analyse der genetischen Architektur von Waldbesta¨ nden anhand ra¨ umlicher Autokorrelationen. Allg. Forst- u. Jagdztg. 164, 137–144 (in German). Knowles, P., Perry, D.J., Foster, H.A., 1992. Spatial genetic structure in two tamarack [Larix laricina (Du Roi) K. Koch] populations with differing establishment histories. Evolution 46, 572–576. Leonardi, S., Menozzi, P., 1996. Spatial structure of genetic variability in natural stands of Fagus sylvatica L. (beech) in Italy. Heredity 77, 359–368. Linares-Bensimo´ n, C., 1984. Versuche zur Viabilita¨ tsselektion and Enzym-Genloci bei Alnus glutinosa (L.) Gaertn. Go¨ ttingen Research Notes in Forest Genetics, Georg-August-Universita¨ t, Go¨ ttingen (in German). Luka´ cˇ ik, I., 2000. Premenlivost’, rastova´ charakteristika a ekolo´ gia drevı´n Alnus glutinosa L. (Gaertn.), Pinus mugo Turra a Taxus baccata L. v lesny´ ch porastoch Slovenska. Habilitation thesis, Technical University in Zvolen, Zvolen (in Slovak). Merzeau, D., 1991. Estimation des parame`tres du mode de reproduction et des structures ge´ ne´ tiques du heˆ tre (Fagus sylvatica L.). PhD thesis, Universite´ de Bordeaux I, Bordeaux (in French). Pancer-Kotejowa, E., Zarzycki, K., 1980. Zarys ekologii. In: Białobok, S. (Comp.), Olsze-Alnus Mill. Nasze drzewa les´ne, Tom 8, Pan´stwowe Wydawnictvo Naukowe, Warszawa-Poznan´, pp. 230–257 (in Polish). Prat, D., Leger, C., Bojovic, S., 1992. Genetic diversity among Alnus glutinosa (L.) Gaertn. populations. Acta cologica 13, 469–477. Raymond, M., Rousset, F., 1995. An exact test of population differentiation. Evolution 49, 1280–1283. Rousset, F., Raymond, M., 1995. Testing heterozygote excess and deficiency. Genetics 140, 1413–1419. Streiff, R., Ducousso, A., Kremer, A., 1998. Organisation spatiale de la diversite´ ge´ne´tique et flux polliniques dans une cheˆnaie mixte. Gen. Sel. Evol. 30 (Suppl. 1), 137–152 (in French).
Spatial and microgeographical genetic differentiation of black alder (Alnus glutinosa Gaertn.) populations Dusˇan Go¨mo¨ry*, Ladislav Paule Faculty of Forestry, Technical University in Zvolen, T.G. Masaryka 24, SK-960 53 Zvolen, Slovak Republic Received 22 June 2000; received in revised form 16 November 2000; accepted 16 November 2000
Abstract Three Alnus glutinosa populations from central Slovakia were investigated by means of isozyme analysis. The average expected heterozygosity ranged from 0.151 to 0.173. Despite significant differences in allele frequencies among populations, genetic differentiation was low (F ST ¼ 0:022). Intrapopulation fixation indices indicate no or very slight deviation from panmictic expectations towards the heterozygote deficiency. No indications were found that the investigated populations have experienced recent bottlenecks in population size. A spatial autocorrelation analysis based on Moran’s I indicates the existence of a spatial genetic structuring in the population Kra´l’ova´. Patch size was estimated to be 70–100 m, which is more than observed in other broadleaved tree species. This difference is explained by the linear shape of alder populations and a higher mobility of seeds. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Alnus glutinosa; Isozymes; Genetic differentiation; Spatial autocorrelation
1. Introduction Black alder (Alnus glutinosa [L.] Gaertn.) is a tree species with a pan-European range. In the north, it reaches the Polar circle, whereas the southernmost occurrences are located in North Africa. In the east, it grows behind the Ural mountains under the continental climate of the western Siberia. Within this vast range, it mounts from lowlands up to 1800 m a.s.l. in the Alps and 1300 m a.s.l. in the Carpathians. It means that it is a species able to adapt to very variable climatic conditions. At the same time, it requires specific soil conditions, mainly concerning moisture.
*
Corresponding author. Tel.: þ421-855-5206-226; fax: þ421-855-5332-654. E-mail address: [email protected] (D. Go¨mo¨ry).
It occupies primarily sites with flowing ground water along streams, but also sites with stagnating ground water like peat bogs. However, in contrast to most hygrophilous tree species, it survives also on drier sites, where it behaves as a pioneer species (PancerKotejowa and Zarzycki, 1980). Site requirements lead to a considerable fragmentation of the distribution range. Black alder regenerates generatively as well as vegetatively, mainly by sprouts, rarely by root suckers. Sprouts are mostly very vigorous and lead to the formation of polykormic individuals. Rooting of lateral branches of fallen trees was also observed, after the maternal stem decayed, the sprouts form clonal groups. In central Europe, large alder stands, which were occurring on the river terraces, in dead arms of rivers and lakes, swamps, etc. have mostly been destroyed by
0378-1127/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 2 7 ( 0 1 ) 0 0 4 6 5 - 0
4
D. Go¨mo¨ry, L. Paule / Forest Ecology and Management 160 (2002) 3–9
human activities. However, alder still occurs along practically all streams, even very small ones. Therefore, typical alder populations differ from other tree species by a specific shape, since they are extended mainly in one dimension. This might affect considerably the gene flow within populations and subsequently the genetic structure. Prat et al. (1992) investigated the genetic structure of alder populations within the major part of its European range. They did not find clear geographical trends, but a considerable local and regional differentiation. Similar patterns were found in the North American alder species Alnus rugosa and Alnus crispa (Bousquet et al., 1987a, 1988). The aim of this study was to find out how the fragmentation of the range and the subsequent isolation of populations reflected by genetic differentiation, and to what extent the specific bionomy of this species (mainly the linear shape of populations) leads to spatial genetic structuring.
2. Materials and methods Three black alder (A. glutinosa Gaertn.) populations were sampled in the close vicinity of the town Zvolen in central Slovakia (198080 E/488350 N). Their exact position is given in Fig. 1. In spring 1999, at the time of ripening of male catkins, twigs with dormant buds
Fig. 1. Location of the investigated black alder populations: (1) Bienˇ ; (2) Neresnica; (3) Kra´ l’ova´ .
and male catkins were taken from all available adult trees (fructifying trees with BHD over 7 cm) in spatially continuous sections of the respective populations 39, 48 and 93 trees in populations Neresnica, Bienˇ and Kra´ l’ova´ , respectively. In the population Kra´ l’ova´ , the sampled trees were labeled and their exact position was identified from an aerial photograph. Vegetative tissues and pollen released from catkins were analyzed separately. Pollen was stored at 4 8C up to 4 weeks until the analysis. Buds together with cambium were homogenized in a Tris–HCl extraction buffer pH 7.3 with the addition of PVP 40, PVP 360, EDTA II, Tween 80, PEG, 2-mercaptoethanol (1% each), 0.025% DTT, and 0.5% Na-ascorbate. Pollen was homogenized manually in the same extraction buffer. Enzyme separation was performed electrophoretically in 12% (w/v) starch gels using three buffer systems (lithium borate–Tris citrate pH 8.1; sodium borate pH 8.0–Tris citrate pH 8.7, and Tris histidine–Tris citrate buffer pH 7.0). Staining procedures followed Cheliak and Pitel (1984). Where possible, the interpretation of zymograms followed Prat et al. (1992) and Bousquet et al. (1987b, 1988). A parallel analysis of diploid (buds) and haploid (pollen) tissues was performed to facilitate reading of zymograms. Alleles were designated by their relative migration rates as related to the most frequent one. Allelic frequencies at each locus were calculated based on diploid genotypes. Differences in allelic frequencies between populations were tested using the probability test (Raymond and Rousset, 1995). Genetic diversity was characterized by the mean number of alleles per locus, proportion of polymorphic loci, observed and expected heterozygosities. Deviations of the genotypic structures from the Hardy–Weinberg equilibrium were tested following Rousset and Raymond (1995). Sign test and Wilcoxon sign-rank test (Cornuet and Luikart, 1996) were used to detect recent population bottlenecks. To assess the spatial genetic structure in the population Kra´ l’ova´ , spatial autocorrelation method was used. Moran’s I (Sokal and Oden, 1978) was estimated for each of 10 distance classes with equal ranges of 70 m. The overall significance of correlograms was tested using Sˇ ida´ k’s criterion (Geburek, 1993).
D. Go¨ mo¨ ry, L. Paule / Forest Ecology and Management 160 (2002) 3–9
3. Results 3.1. Inheritance of isozyme systems In total, 10 isozyme systems controlled by at least 15 loci (putative in some cases) were analyzed. The inference about the genetic control of isozyme variants was based on the knowledge of the subunit number in multimeric enzymes and the comparison of isozyme phenotypes in pollen an vegetative tissues. For several enzyme systems, scoring was much easier and much more reliable in pollen than in buds (shikimate dehydrogenase, isocitrate dehydrogenase). Glutamate dehydrogenase even exhibited activity only in pollen. However, in 6-phosphogluconate dehydrogenase, isozyme phenotypes in the more rapidly migrating zone in pollen have not shown any correspondence with those in vegetative tissues. In buds, this zone is apparently controlled by a single locus with two allelic variants, whereby the enzyme is dimeric (heterozygous individuals exhibit a triple-banded phenotype), but we have not succeeded in finding any genetic interpretation for the pattern found in pollen. It seems that in pollen, different loci are expressed than in buds. In malate dehydrogenase two zones could be found in both tissue types, whereby the second one was variable with double-banded phenotypes in pollen and triplebanded phenotypes in buds (controlled by the locus Mdh-B). In pollen, an additional zone with two bands was observed. According to our interpretation, this zone is controlled by another locus (Mdh-C) forming an interlocus heterodimer with the product of Mdh-B (Fig. 2). In the remaining cases, there was a good concordance between isozyme phenotypes in pollen and buds, whereby for dimeric or tetrameric enzymes, intralocus hybrid bands were absent in pollen. Four zones have not shown any variation, and after comparing with the results of Bousquet et al. (1987b, 1988,
Fig. 2. Observed MDH phenotypes and their genetic interpretation.
5
1990), they were interpreted as controlled by monomorphic loci (Pgi-A, Mdh-A, Mdh-C, Got-A). Two zones were highly variable (Fest-A, Idh-B) but no reliable scoring was possible. 3.2. Microgeographical differentiation Out of 16 scored loci, 12 were found to be polymorphic in at least one population. Despite very small geographic distances between populations, significant heterogeneity of allelic frequencies was found in eight loci (Table 1). The levels of the genetic multiplicity and diversity differed among populations as well (Table 2). The population Kra´ l’ova´ , even when represented by the biggest sample, appears to posses least alleles and least polymorphic loci. Like in the other broadleaved species, there is no concordance between the levels of the genetic diversity and multiplicity. The population Neresnica, which is the most rich one in alleles, does not exhibit the highest diversity levels at the same time. Like in most tree species, the level of genetic differentiation as revealed on the basis of nuclear genetic markers, is very low. The values of FST ranged from 0.001 to 0.082, that means that only a small portion of the total variation is due to interpopulation differences (Table 3). The locus with the highest differentiation among populations is Got-B, what probably results from a high frequency of the Got-B/ 28 allele in the population Kra´ l’ova´ . The average intrapopulation fixation index FIS is near zero (0.003). However, there is a considerable variation among loci as well as among populations. In the population Neresnica, there was no significant deviation from Hardy–Weinberg equilibrium found. On the other hand, three significant deviations towards the heterozygote deficiency were found in the population Bienˇ (Fest-B, Mdh-B, 6pgdh-A). In Kra´ l’ova´ , heterozygote deficiency was at the significance limit (P ¼ 0:0606) in Fest-B. Since these deviations are not consistent among loci within individual populations, it is questionable if the positive fixation indices are due to inbreeding. Selection or pure chance is a more plausible explanation. Neither the sign test nor the Wilcoxon sign-rank test proved a significant gene diversity excess over the equilibrium value, which indicates a recent population bottleneck, in any of the studied populations
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Table 1 Allelic frequencies of polymorphic loci in the investigated black alder populations Locus
Allele
Population
Pa
Population
N
na
nab
PP
HO
HE
Bienˇ Neresnica Kra´ l’ova´
36.3 41.8 86.5
1.9 2.0 1.6
2.5 2.7 2.1
62.5 75.0 56.3
0.154 0.171 0.153
0.173 0.158 0.151
Bienˇ
Neresnica
Kra´ l’ova´
117 100 83 80
0.141 0.782 0.064 0.013
0.143 0.806 0.051 0.000
0.074 0.881 0.045 0.000
0.1622
Gdh-A
125 100
0.435 0.565
0.389 0.611
0.410 0.590
0.8958
Pgm-A
105 100 95
0.013 0.936 0.051
0.043 0.915 0.043
0.048 0.952 0.000
0.0169
Pgm-B
67 59
0.974 0.026
0.957 0.043
1.000 0.000
0.0111
Idh-A
105 100
0.224 0.776
0.121 0.879
0.077 0.923
0.0189
Mdh-B
77 67 57
0.090 0.897 0.013
0.049 0.951 0.000
0.000 1.000 0.000
0
6pgd-A
100 87
0.526 0.474
0.671 0.329
0.653 0.347
0.1038
6pgd-B
53 40
1.000 0.000
0.988 0.012
1.000 0.000
0.4664
Skdh-A
105 100
0.375 0.625
0.222 0.778
0.442 0.558
0.0058
Got-B
60 44 28
0.000 1.000 0.000
0.050 0.925 0.025
0.000 0.813 0.187
0
Pgi-B
89 73 57
0.000 0.962 0.038
0.051 0.949 0.000
0.005 0.995 0.000
0.0007
Mnr-A
134 100 87
0.064 0.936 0.000
0.031 0.949 0.020
0.114 0.886 0.000
0.0187
Fest-B
a
Table 2 Characteristics of the genetic multiplicity a diversity of the investigated black alder populationsa
Probability test (Raymond and Rousset, 1995).
(probabilities for sign test were 0.135, 0.176, and 0.507 for Neresnica, Bienˇ , and Kra´ l’ova´ , respectively). 3.3. Spatial structure We compared genotypes of neighboring trees by means of a cluster analysis. No groups of spatially proximate identical genotypes, indicating natural cloning, were identified.
a N: average sample size; na: mean number of alleles per locus; PP: proportion of polymorphic loci; HO: observed heterozygosity; HE: expected heterozygosity. b For polymorphic loci (loci monomorphic in all populations excluded).
Spatial autocorrelation analysis was performed for nine independent alleles. The resulting correlograms are presented in Fig. 3a–c (distance classes are characterized by centers, not ranges). Even if the courses of the lines for individual alleles are not identical, there seems to exist a general sinusoidal pattern, which can be identified in the correlograms, and which is reflected by average values of Moran’s index over alleles. As expected, the autocorrelations in the first distance class are generally positive, with two significant values. Then autocorrelations decrease, and at the distance of 170–250 m they become significantly negative. Within the distance range of 300–450 m, positive values prevail again. Interestingly, in the distance class of 525 m, almost all Moran’s indices are negative, whereby for six out of nine loci, the deviation from the expected value is significant. Five out of nine correlograms proved to be Table 3 Single-locus F-statistics in the investigated black alder populations Locus
FIS
FIT
FST
GST
Fest-B Gdh-A Pgm-A Pgm-B Idh-A Mdh-B 6pgd-A 6pgd-B Skdh-A Got-B Pgi-B Mnr-A
0.107 0.075 0.060 0.038 0.201 0.191 0.131 0.012 0.016 0.116 0.045 0.091
0.116 0.073 0.052 0.023 0.164 0.218 0.146 0.004 0.053 0.025 0.025 0.076
0.009 0.001 0.008 0.014 0.031 0.033 0.018 0.008 0.037 0.082 0.020 0.014
0.010 0.001 0.011 0.032 0.033 0.054 0.013 0.035 0.038 0.056 0.019 0.013
Average
0.003
0.020
0.022
0.022
D. Go¨ mo¨ ry, L. Paule / Forest Ecology and Management 160 (2002) 3–9
7
Fig. 3. (a) Correlograms for alleles Idh-A/105, 6pgdh-A/100, Fest-B/117 and Fest-B/83. (b) Correlograms for alleles Gdh-A/125, Got-B/28, Mnr-A/134, Pgm-A/105 and Skdh-A/105. (c) Mean correlogram and numbers of significant positive (shaded column) and negative (blank column) Moran’s I indices in individual distance classes.
significant (for 6pgd-A/100, Fest-B/83, Got-B/28, Mnr-A/134, and Skdh-A/105). These fluctuations indicate a patchy genetic structure within the population, with a patch size of approximately 70–100 m. This course is mainly shaped by Got-B/28, Fest-B/ 83, and Idh-A/105, but for most alleles, the correlogram is at least partially concordant with the described
trend. In only one distance class, both significant positive and negative were found (class 455 m, two and one positive and negative values, respectively). The most deviating allele is Mnr-A/134, where all indices up to the distance of 315 m are above the expected value, and for all higher distance classes they become negative.
8
D. Go¨ mo¨ ry, L. Paule / Forest Ecology and Management 160 (2002) 3–9
4. Discussion Despite its economical importance, black alder does not belong to intensively studied tree species, at least regarding genetic structure. Therefore, the information about the inheritance of isozyme systems is very scarce. Linares-Bensimo´ n (1984) tested the Mendelian inheritance of aminopeptidases and glutamate–oxaloacetate transaminases. Prat et al. (1992) analyzed 16 isozyme systems controlled by 19 polymorphic loci. They did not present any results of inheritance testing, but they state that they observed Mendelian segregation in open-pollinated progenies for all the proposed loci. However, for several isozyme loci that we used, we had no reference in A. glutinosa, therefore we used the results published for the American alder species A. crispa, A. rugosa, and Alnus sinuata (Bousquet et al., 1987b, 1988, 1990). Another basis for the inference about the genetic control was a parallel analysis of buds and pollen. Our results are in a good concordance with the cited studies. The only exception is the Fest-B locus. Prat et al. (1992), who used polyacrylamide gel electrophoresis, report about monomeric structure of the enzyme produced by the most rapidly migrating locus. Similarly, Bousquet et al. (1987b) found monomeric structure in b-esterases of A. crispa. Phenotypes that we found in the slowly migrating zone of fluorescent esterase, indicate a dimeric structure (triple-banded pattern in diploid tissues, double-banded pattern with missing hybrid band in pollen). Since there was a perfect concordance between the phenotypic expression in diploid and haploid tissues, we adopted the hypothesis of the genetic control of this zone by a single locus. The levels of the genetic multiplicity, characterized by the mean number of alleles and the proportion of polymorphic loci, correspond to those found by Prat et al. (1992), however, we found slightly lower diversity. It is very interesting that there is a similarity in the levels of diversity between black alder and the American alder species (A. crispa, A. rugosa, A. sinuata). Although the distribution of the expected heterozygosities over loci is not homogeneous among species, the average values are very close to each other (cf. Bousquet et al., 1987a,b, 1988, 1990). Only 2.2% of the total variation is due to interpopulation differences. This is much less than in the study of Prat et al. (1992), who found F ST ¼ 0:204.
This difference was expected, since in the cited study, populations from almost the whole Europe were included, whereby in our case the air distances between populations range from 5 to 10 km. Prat et al. (1992) observed a strong small-scale differentiation and absence of any clear large-scale geographical trends. They suppose that this pattern is due to isolation of populations and life-history traits of black alder, which undergoes successive colonization/retreat cycles accompanied by severe population bottlenecks. However, there is no indication that our populations have experienced any recent bottlenecks. In fact, alder behaves as a pioneer species on wet meadows, which it colonizes but it is successively replaced by other tree species. However, along the streams, where the site conditions are permanently favorable for alder, it is a very competitive species, which can be considered a climax one. These sites have continuously been occupied by alder populations, counting several tens or hundreds of individuals, so that at least in these cases, bottleneck effects are improbable. Alder is quite a common species in all regions where the natural character of landscape has not been severely changed. It does not form large continuous stands, but individuals or small groups are dispersed along all streams, so that its distribution over the landscape is quite regular. These individuals may serve as stepping stones for gene flow among larger populations mediated by pollen. Nevertheless, gene flow does not seem to be as effective as in case of predominant forest tree species with continuous ranges like beech or oaks, since significant differences on a very small geographical scale occur. Luka´ cˇ ik (2000) observed considerable differences in the representation of morphological traits among alder populations in different regions within Slovakia. Although environmental effects contribute considerably to this morphological variation, the magnitude of differences in traits where a higher heritability can be expected (like bark color and form) indicates also a genetic differentiation. Since most of the genetic variation in black alder proved to reside within populations, the question how is it spatially organized appears to be relevant. A relatively good concordance among the correlograms of different alleles, indicating a common mechanism in forming of genetic substructuring, is not a common phenomenon in forest tree species. In general, very weak spatial genetic structures have been reported
D. Go¨ mo¨ ry, L. Paule / Forest Ecology and Management 160 (2002) 3–9
(Leonardi and Menozzi, 1996; Merzeau, 1991; Streiff et al., 1998; an overview for forest tree species is also given in Geburek (1993)). A distinct spatial structure is mostly associated with a severe human interference (Knowles et al., 1992). It must be emphasized that in most studies, much smaller ranges of distance classes are used, allowing to identify a finer genetic structure. Smaller patch sizes of approximately 30 m were observed in Quercus laevis and Fagus sylvatica (Berg and Hamrick, 1995; Leonardi and Menozzi, 1996; Merzeau, 1991). We cannot exclude, that there exists a finer fluctuation of Moran’s I in black alder as well, but due to the linear shape of the investigated population (which is typical for this species), smaller distance classes could not be chosen, since they would lead to very small sample sizes within them. Microhabitat selection and isolation by distance due to a limited pollen and seed flow are generally considered the main causes of spatial genetic structuring. In our case, the similar course of correlogram curves for different alleles indicates that the latter mechanism is the more probable one. Except for polykormic individuals, we have not observed any clonal groups. However, there may exist groups of relatives within the population—not only half-sib families, but also parent–offspring groups, since the stand is readily uneven-aged. Alder is a self-sterile wind-pollinated tree species, with small and light seeds which are easily transported by wind and water. Maybe these factors contribute to a larger patch size than in self-compatible tree species with heavy seeds like beech or Turkey oak. Acknowledgements The authors are indebted to Mrs. Zuzana Slancˇ ´ıkova´ for the technical assistance with sampling and laboratory analyses. Aerial photographs were kindly provided by the Institute of Forest Management, Zvolen. This study was supported by Grant nos. 1/7056/20 and 1/ 4036/97 from the Slovak Grant Agency for Science. References Berg, E.E., Hamrick, J.L., 1995. Fine-scale genetic structure of a Turkey oak forest. Evolution 49, 110–120.
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