J Ornithol (2011) 152:37–44 DOI 10.1007/s10336-010-0543-7
ORIGINAL ARTICLE
Genetic variation in Black Grouse populations with different lekking systems in the Czech Republic Jana Svobodova´ • Gernot Segelbacher Jacob Ho¨glund
•
Received: 22 December 2009 / Revised: 16 May 2010 / Accepted: 8 June 2010 / Published online: 26 June 2010 Ó Dt. Ornithologen-Gesellschaft e.V. 2010
Abstract Here, we describe genetic diversity of lekking and non-lekking populations of Black Grouse Tetrao tetrix in the mountains bordering the Czech Republic. In total, 250 individuals from eight lekking and eight non-lekking study sites were genotyped at 11 microsatellite loci. Significant pair-wise FST values between the different regions indicated low dispersal between localities, and birds could be assigned to three main mountain ranges (Sˇumava Mts, Krkonosˇe Mts and Krusˇne´ Mts). We found lower genetic diversity at study sites with solitary displaying males compared to sites where birds aggregate at leks. Additionally, genetic diversity was significantly more strongly associated with type of display than with spatial location of the sites. Given that lekking behaviour may be related to population density, we suggest that a shift from lekking to
Communicated by M. Wink. J. Svobodova´ (&) Department of Ecology, Faculty of Environmental Science, Czech University of Life Sciences, Kamy´cka´ 1176, 165 21 Prague 6, Czech Republic e-mail:
[email protected] J. Svobodova´ Department of Population Biology, Institute of Vertebrate Biology v.v.i., Academy of Sciences of the Czech Republic, Kveˇtna´ 8, 603 65 Brno, Czech Republic G. Segelbacher Department of Wildlife Ecology and Management, University Freiburg, Tennenbacher Str. 4, 79106 Freiburg, Germany G. Segelbacher J. Ho¨glund Population Biology and Conservation Biology, Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Norbyv. 18D, 752 36 Uppsala, Sweden
solitary displaying males is an alarming sign for conservationists indicating decreasing effective population sizes and declining populations. Keywords Black Grouse Conservation Lekking Microsatellites Tetrao tetrix
Introduction Genetic variability of species can be significantly influenced by their mating system (Frankham et al. 2002). In lekking species, for example, only a few males reproduce. Thus, the effective population size is smaller than the actual census size in a comparable non-lekking population (e.g. Bellinger et al. 2003; Bouzat and Johnson 2004; Johnson et al. 2004). Loss of genetic diversity is increased in small populations (Frankham 2005; Ho¨glund 2009). Thus, the risk of extinction is higher for lekking populations compared to equally sized non-lekking populations where matings are more evenly distributed (Ho¨glund 1996). The Black Grouse Tetrao tetrix is generally described as a lekking species throughout its distribution range, i.e. groups of males display together in arenas to attract females during the breeding season. Thus, matings within one population are biased towards fewer males than predicted by chance (Ho¨glund and Alatalo 1990, 1995). However, non-lekking Black Grouse populations (Kruijt and Hogan 1967; Kruijt and de Vos 1988; Ho¨glund and Sto¨hr 1996) and observations of copulations of single displaying males (Kruijt et al. 1972; de Vos 1983) have also been reported, although these instances appear to occur less frequently. The occurrence of displaying single males has been interpreted as a result of them being of
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lower quality and/or juvenile males that fail to access a lekking arena (Kruijt and Hogan 1967). However, Ho¨glund and Sto¨hr (1996) identified all solitarily displaying males in a non-lekking population as adults and suggested low population density and lack of suitable habitats as the cause for the absence of leks. In the Czech Republic, Black Grouse populations have declined since the beginning of the twentieth century, increasingly rapidly during the last five decades. A temporary increase in numbers was noticed in the 1970s, but only in populations in the north of the country where largescale forest destruction has occurred due to industrial emissions. As Black Grouse are adapted to open forest landscapes, this artificially created more suitable habitat for Black Grouse (Sˇˇtastny´ et al. 1997). Today, total population size of Black Grouse in the Czech Republic is estimated to be in the order of 800–1,000 displaying males, and the birds are limited to the mountains bordering the country (Sˇˇtastny´ et al. 2006). The aim of this study was to evaluate genetic diversity of Black Grouse in the Czech Republic and compare the genetic diversity (1) of different mountain regions, and (2) between lekking and non-lekking populations. If solitary display is associated with low population density, genetic diversity of non-lekking populations should be lower than in lekking populations, as low population size generally correlates with low genetic variability (Ho¨glund et al. 2006; Larson et al. 2008). We further predicted that genetic diversity of Black Grouse populations in the Czech Republic should be lower than in more continuous Black Grouse populations as, for example, in Scandinavia. Black Grouse populations in the Czech Republic are isolated from other populations in Central Europe and the population sizes are relatively small. On the other hand, the period of population isolation has been short and a temporary increase of population numbers during the 1980s could have maintained genetic diversity. Thus, to study the extent of fragmentation of the Czech Republic Black Grouse population, we investigated the level of population divergence among and within mountain ranges using analyses both with predefined population structure and analyses which were based on no a priori assumptions of structure.
Methods Sampling and genotyping We used moulted feathers or feathers from carcasses found in the field as sources for genomic DNA. Samples were classified as male or unidentified sex according to the shape and coloration of the feathers. Feathers were put individ-
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ually in envelopes and were stored at room temperature from 1 to 20 months. DNA was extracted from feather material ca. 0.5 cm from the root end and from the umbilicus (Horva´th et al. 2004) using the DNeasy Tissue Kit (Qiagen) following the manufacturer’s protocol with slight modifications. All samples were genotyped at 11 tetra nucleotide microsatellite loci (Tut1, Tut3, Tut4, Bg4, Bg6, Bg10, Bg12, Bg14, Bg15, Bg16, Bg18, Bg19; Pirtney and Ho¨glund 2001; Segelbacher et al. 2000). Two tetraplex (1.Tut1, Bg4, Bg6, Bg12; 2.Tut3, Bg4, Bg10, Bg19), one triplex (Bg14, Bg16, Bg18) and one single (Bg15) PCR, was carried out for PCR amplification. All multiplex reactions were amplified in a total reaction volume of 20 ll, containing 4.5 ll of the DNA with 0.1–0.4 lM of each primer and 59 Multiplex PCR Master Mix (Qiagen) and 0.59 Q-solution. PCR amplification was conducted using the following conditions: an initial denaturation step of 15 min at 95°C followed by 40 cycles of 30 s at 94°C, 90 s at 55°C (at 51°C for triplex and duplex), 60 s at 72°C, and final extension of 30 min at 60°C. PCR products were resolved by capillary electrophoresis on ABI Prism 3130 and analysed using GeneMapper 3.7 software. PCR amplification of feather DNA was repeated up to four times depending on the quality of extracted DNA. In the Czech Republic, surveys of Black Grouse numbers and distribution are based on numbers of displaying males (Ma´lkova´ 2000). However, population estimates should be considered with caution as most of the areas were visited only once during any lekking period (Table 1). Black Grouse occur in four mountain regions at the country’s borders: Sˇumava Mts in the south, Krusˇne´ Mts in the north-west, and Jizerske´ Mts and Krkonosˇe Mts in the north, respectively (Fig. 1). In these mountain areas (at 16 study sites), feathers were collected during the summer months in 2004–2007, but most localities were visited in 2005 (Table 1). The study sites were classified as separate locations when separated by at least 20 km distance, based on the average dispersal distance in females (Caizergues and Ellison 2002; Ho¨glund et al. 1999; Willebrandt 1988). The sites were classified as lekking or non-lekking according to the location and behaviour of displaying males within the site. If males were displaying solitarily more than 0.5 km from one another, the site was considered non-lekking. Except for two populations in the Krusˇne´ Mts, all lekking sites were found in the south, in the Sˇumava Mts, where the habitat is predominantly peat bogs and moorland. All nonlekking sites were located in the northern mountains (Fig. 1) within maturating forest stands (e.g. Bartosˇ and Soucˇek 2004). These forests died in the 1970s due to the impact of industrial air pollution, and were subsequently replanted.
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Table 1 Genetic diversity of Black Grouse Tetrao tetrix populations in the Czech Republic Mountains (no. of Popn name Population displaying males)a abbreviation (density/km2)
Display
n (n males)b HE
HO
AR
FIS
Sˇumava Mts (80)
c
Lek
25 (18)
0.66
0.74
3.81
-0.13
Lek
23 (11)
0.69
0.63
4.19
0.09
SR
Pasecka´ slatˇ Stra´zˇenska´ slatˇ Pra´sˇily
Lek
11 (10)
0.67
0.63
3.53
0.06
SV
Sveˇtlı´k
Lek
7 (0)
0.72
0.56
4.25
0.24
SZ
Zvonkova´ Dobra´
Lek
9 (1)
0.72
0.61
4.27
0.16
Lek
9 (1)
0.73
0.57
4.44
0.22
Alfre´dka (4) Non-lekking 18 (8) Pomeznı´ hrˇeben (7) Non-lekking 18 (8) Tetrˇevı´ boudy (6) Non-lekking 14 (8) Zeleny´ vrch (5) Non-lekking 9 (7)
0.65 0.63
0.61 0.64
3.84 3.33
0.06 -0.02
SP SS
SD Krkonosˇe Mts (128)
KA KP KT
Jizerske´ Mts Mts
JI
(64) Krusˇne´ Mts
GU
(253)
CB NA HA CI
Mean ± SD
Gru¨nwald (10) Cˇervene´ blato Nakle´rˇov (10) Harbartice Cı´novec (4)
Lek
0.63
0.55
4.03
0.14
0.63
0.46
3.37
0.28
9 (8)
0.71
0.71
4.55
-0.01
Non-lekking 18 (6)
0.62
0.57
3.85
0.09
Lek
27 (10)
0.65
0.60
3.97
0.07
Non-lekking 17 (13)
0.64
0.68
3.83
-0.07
Non-lekking 18 (4)
0.65
0.56
3.91
Lek
0.70 ± 0.25 0.64 ± 0.23 4.15 ± 1.47
0.09 ± 0.03
0.63 ± 0.22 0.58 ± 0.21 3.73 ± 1.32
0.08 ± 0.03
Non-lekking
0.13
2
Mountain range and population names with number of displaying males/km in parenthesis where data were available, n number of analysed individuals, number of analysed males in parenthesis HE Expected heterozygosity, HO observed heterozygosity, AR allelic richness, FIS deviations from Hardy–Weinberg expectations. In 2005, 80 males were displaying in the Sˇumava Mts, 128 in the Krkonosˇe Mts, 64 in the Jizerske´ Mts and 253 in the Krusˇne´ Mts
a
b
n Number of analysed individuals, number of analysed males in parentheses
c
Incomplete data
Fig. 1 Map of the geographical locations of the sampled populations of Black Grouse Tetrao tetrix in the Czech Republic. White squares are lekking populations and white spots are non-lekking populations. For abbreviations of populations, see Table 1
Statistical analyses Linkage disequilibrium between all pairs of loci within each population, and between all populations, was analysed
using Genepop (Raymond and Rousset 1995). All loci were further checked for allelic dropout and the presence of null alleles using Microchecker (van Oosterhout et al. 2004). To control for (and exclude) repeated analyses of samples
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from the same individual, the data were screened using the genotypic match function in the Excel add-in Microsatellite Toolkit (Park 2001). We genotyped 444 samples, but all 11 microsatellite loci were successfully amplified in just 250 samples. In addition, samples which did not differ in more than two alleles were considered to be from the same individual and were excluded from all analyses (n = 13). In total, 250 individuals (121 males, 129 unidentified sex) were included in the final dataset for further analyses. The probability that relatedness within local population is high due to re-sampling of individuals from one family group is low. Genetic clustering within all individuals was investigated with the admixture model in Structure 2.1 (Pritchard et al. 2000). Five replicates were used for each simulation of 1–10 independent genetic clusters (k), with 500,000 permutations and an initial burn-in of 100,000 generations. K was estimated by Evanno’s calculation (Evanno et al. 2005), which is based on the second order rate of change in the log probability of the data between successive values of k (Dk). Genetic variation within each population was characterised by allelic richness (AR), observed heterozygosity HO, expected heterozygosity HE and FIS (deviations from Hardy–Weinberg expectations). Genetic differentiation between populations was determined by computing pair wise FST. Isolation-by-distance was tested by calculating the correlation between genetic distance (Nei 1978) and geographic distance among pairs of populations, and the significance was determined with a Mantel test. All calculations of genetic variation was obtained after 10,000 permutations in FSTAT (Goudet 2001). Effect of display type (lekking vs non-lekking) on genetic variability of Black Grouse was evaluated using generalised linear models with mixed effects (GLMM) with AR, FIS, HE and HO as dependent variables. In addition to display type, the effect of mountain region was also included as covariate in the model. Display type and mountain region were included as fixed effects. We controlled for differences in numbers of samples per population by including sample size as random effect. The same GLMM model was also used for the restricted dataset including only samples that were reliably assigned to males (n = 119) from 13 populations. Three populations had to be excluded from these latter analyses (i.e. SD, SV, SZ) due to small sample sizes (Table 1). In all analyses, the significance of any particular explanatory variable was selected by change of deviance between full and reduced model (deletion tests; Crawley 2002). All GLMMs were performed using the software R (R Development Core Team 2008).
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Simulations To examine whether the loss of genetic variation in Black Grouse could be influenced by genetic drift and mating system, we used the program BottleSim (Kuo and Janzen 2003). We assumed a starting population size of 9,000 males declining to a size of 10 males during the simulation. We assumed one lek for every ten Black Grouse males, and that either an average of two males per lek would reproduce, or as alternative scenario, all males within the lek would mate. The female numbers were considered equal to the number of males per lek. Simulation parameters were set to random mating (with either two or all males), generation overlap with one year to maturity, and an average lifespan of 3 years (Hjorth 1970).
Results No linkage disequilibrium was found between any pairwise loci combination (P [ 0.05). No allelic dropout was found in any of the loci, but we detected potential null alleles for loci Bg10 (22.7%) and Bg14 (29.6%). Both loci Bg10 and Bg14 were included in all subsequent analyses, because comparisons of AR, HE, HO and FIS values including and excluding both loci, and data from consecutive analyses, revealed similar results. Pair-wise FST comparisons revealed significant differences for study sites among mountain ranges, and in most cases also within mountains (Table 2). The admixture model with an a priori undefined genetic population differentiation in Structure (both with and without Evanno’s calculations) suggested three independent clusters that best explained the data (Fig. 2). Thus, we assigned birds from the Jizerske´ Mts to the neighbouring populations in the Krkonosˇe Mts. The three main clusters correspond to the main mountain ranges in the Czech Republic. The average genetic diversity in lekking populations was higher than in non-lekking populations (Table 1). Significant differences were revealed for HE and AR (Table 3). Although non-lekking populations mostly occurred in the northern mountains, the mountain effect was only significant for HE (Table 3), indicating that display type was more strongly associated with the genetic makeup of populations than the effect of mountain (region). The lowest level of heterozygosity and allelic richness was detected in the non-lekking population in the Jizerske´ Mts. We found no evidence for significant deficit of heterozygosity (FIS) in any population (Table 1). Interestingly, genetic diversity of the lekking population (GU) from the northern mountains was more similar to lekking
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Table 2 Pair-wise FST between Black Grouse populations in the Czech Republic KA KA KP
KP
KT
SP
SS
SR
SV
SZ
SD
CB
NA
HA
CI
CH
GU
JI
0.11
0.13
0.20
0.13
0.20
0.09
0.09
0.13
0.17
0.18
0.15
0.12
0.22
0.14
0.05
0.18
0.19
0.19
0.19
0.12
0.16
0.19
0.21
0.17
0.20
0.14
0.23
0.15
0.15
0.23
0.11
0.17
0.09
0.12
0.14
0.14
0.13
0.12
0.09
0.17
0.13
0.13
KT SP
0.16
SS
0.17
0.14
0.13
0.12
0.22
0.19
0.20
0.15
0.23
0.14
0.19
0.14
0.04
0.03
0.05
0.15
0.12
0.09
0.07
0.14
0.06
0.13
0.10
0.13
0.13
0.18
0.20
0.19
0.16
0.23
0.16
0.17
0.00
0.07
0.10
0.11
0.11
0.07
0.16
0.07
0.08
0.02
0.13
0.13
0.12
0.10
0.19
0.06
0.10
0.16
0.11
0.11
0.08
0.15
0.06
0.10
SR SV SZ SD CB
0.16
NA HA CI CH GU
0.16
0.11
0.16
0.12
0.16
0.05
0.06 0.04
0.14 0.09
0.07 0.06
0.18 0.16
0.07
0.05
0.11
0.10
0.19 0.14
JI The adjusted nominal level (5%) for multiple comparisons is 0.000417. Significant values are marked in bold (P values obtained after 2,400 permutations). All values are Bonferroni corrected. For abbreviations of populations, see Table 1
Fig. 2 Bayesian assignment probabilities for k = 3. Each vertical bar corresponds to one individual. Individuals from the Jizerske´ Mts have high assignment probabilities to cluster 1. The figure was generated by the programme STRUCTURE
Table 3 Results from GLMM model analyses for factors (displaying type and mountain as fixed effects, sample size as random effect) influencing genetic diversity of lekking and non-lekking populations of Black Grouse in the Czech Republic (n = 250, 16 populations) Ddf
Variable Dependent AR FIS HO HE
v2
P
Explanatory Display type
1
6.76
Mountain
2
4.07
0.009 0.13
Display type
1
0.06
0.806
Mountain
2
1.56
0.459
Display type
1
2.8
0.094
Mountain
2
2.25
0.325
Display type Mountain
1 2
12.71 9.05
[0.001 0.011
populations from the Sˇumava Mts in the south, than to nonlekking populations from the north. This results further strengthens the observation that genetic variability is stronger associated with display type rather than environmental parameters in the different regions. A Mantel test revealed a correlation between genetic distance and geographic distance for both the entire Czech Republic (r = 0.497, R2 = 0.25, P \ 0.001), and within the Krusˇne´ Mts (r = 0.884, R2 = 0.78, P \ 0.001). However, Mantel tests within any other mountain region did not reveal significant results (Sˇumava Mts: r = 0.082, R2 = 0.007, P [ 0.762; Krkonosˇe Mts and Jizerske´ Mts: r = 0.074, R2 = 0.006, P [ 0.882). Mating system clearly affected levels of genetic variation in our stochastic simulations (Fig. 3). Both number of alleles and observed heterozygosity were lower in the
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Fig. 3 Simulated number of alleles and observed heterozygosity under two demographic scenarios. a The effect on number of alleles when all males are allowed to mate (black), and when two males per lek are allowed to reproduce (grey). b The effect on observed heterozygosity (color codes as in a). See text for details. Values given are means and vertical bars indicate standard errors obtained from 1,000 simulations under each scenario
scenario where only two males per lek were allowed to reproduce, compared to the alternative scenario with all males reproducing. Not surprisingly, the effect on number of alleles was more pronounced compared to the effect on observed heterozygosity (see also Cornuet and Luikart 1996; Bellinger et al. 2003).
Discussion In comparison to other Black Grouse populations, the populations in the Czech Republic display levels of genetic diversity that are similar to other fragmented populations in Europe (Caizergues et al. 2003; Ho¨glund et al. 2006). Thus, the Czech Republic Black Grouse appear to have lost genetic variation in comparison to the larger populations in Scandinavia. This loss of genetic diversity may be explained by the recent population decline and range contraction (Sˇˇtastny´ et al. 2006), which has been resulted from greater isolation and smaller population size of the remaining subpopulations. A temporary increase in the
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number of populations residing in planted forests, mountains and military areas with first forest stage regeneration, and thus providing favourable habitat for Black Grouse (e.g. Børset 1973), does not seem to have mitigated this loss of genetic variation. In some, but not all, isolated European populations, there are significant signs of increased FIS (Ho¨glund et al. 2006). However, no significant FIS were found for any population in the Czech Republic, suggesting that contemporary populations in the Czech Republic are currently in migration drift equilibrium. This could also explain the significant correlation between geographical and genetic distance within the entire Czech Republic. Previous studies have shown that Black Grouse display genetic differences at very fine spatial scale (i.e. between leks; Ho¨glund et al. 1999; Lebigre et al. 2008; Segelbacher et al. 2006). This genetic differentiation is likely to be caused by an almost complete lack of male natal dispersal. While females disperse approximately 20 km in their first year, males are often recruited to the lek where their male parent displayed (Caizergues and Ellison 2002; Warren and Baines 2002; Willebrandt 1988). Negative changes of environmental conditions are expected to lower population fitness including male survival and lead to a decrease of numbers of displaying males in leks. In the end, most leks in an area may hold only a single male. It seems that Black Grouse abandon lekking when population density becomes too low (Ho¨glund and Sto¨hr 1996). This phenomenon was also observed in Capercaillie Tetrao urogallus (personal observation) and lekking ungulates (e.g. Langbein and Thirgood 1989). Therefore, non-lekking populations are likely to be smaller than lekking populations, and are expected to exhibit significantly lower genetic diversity, which is supported by the results of this study. This scenario probably occurs in the northern mountains in the Czech Republic where maturing forest stands are currently causing a decline in Black Grouse numbers and which today are mostly occupied by non-lekking populations, a phenomenon also observed in a study area in Scotland (Pearce-Higgins et al. 2007). Although data on population density are not available for all localities included in this study, we conclude that the average population density of two lekking populations (mean = 10.0 birds/km2) in the northern mountains was higher than that of six non-lekking populations (mean = 5.2 birds/km2; Table 1). In addition, the simulated effect of display type on observed heterozygosity and allele numbers also supports this proposed scenario. Low population density and non-lekking behaviour in the northern mountains could also be due to the absence of suitable lekking arenas such as meadows, clearcuts and open bogs, as new-planted trees on the air-polluted clearcuts are now maturating. However, in many areas in the
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Table 4 The GLMMs results for factors (displaying type and mountain as fixed effects, sample size as random effect) influencing genetic diversity of lekking and non-lekking populations of Black Grouse males in the Czech Republic (n = 119, 13 populations) Ddf
Variable
v2
Explanatory
AR
Display type
1
3.76
Mountain
2
0.78
0.675
Display type
1
0.10
0.757
Mountain
2
2.80
0.246
Display type
1
2.41
0.121
Mountain
2
2.59
0.275
Display type
1
3.41
0.121
Mountain
2
2.65
0.265
HO HE
Genetische Variabilita¨t in Birkuhnpopulationen mit unterschiedlichem Balzsystem in Tschechien
P
Dependent
FIS
Zusammenfassung
0.052
northern mountains, it is possible to find open habitats suitable for lekking arenas, particularly pastures. We further found that the factor of mountain region, which is linked with habitat type (i.e. absence and presence of lekking arenas), did not affect genetic variability of Black Grouse in most cases (except for HE as the explanatory variable). For these reasons, we suggest that display type rather than mountain region alone is a predictor of population size, and could be the ultimate driver of the observed correlations. To test the possible impact of a biased sex ratio on our results, we restricted our analysis to males (n = 119, 13 populations) only (Table 4). Results from the males-only dataset were similar to analyses including all individuals, only HE remained non-significant in the restricted dataset, which is likely to be due to lower sample sizes per region. Further, pairwise FST values were correlated between the full and restricted male dataset (Mantel test: r [ 0.304, P = 0.061). We thus conclude that there is no reason to assume that genetic variation among the sampled Black Grouse populations could be influenced by potential sex biased sampling. Despite an ongoing population decline of Black Grouse in the Czech Republic, we did not observe low heterozygosity levels for any of the studied populations (Sˇˇtastny´ et al. 2006). However, we found significant pair-wise FST values within regions indicating low or absence of dispersal between different locations in this study. This result is further supported by the clustering analysis. Thus, the mountain ranges can be considered as holding isolated populations with relatively low population size. A shift from lekking to solitary displaying in the studied populations is an alarming signal for conservationists, as it probably indicates low population density, which in turn is a signal of low population size.
Wir beschreiben hier die genetische Vielfalt in Birkhuhnpopulationen Tetrao tetrix der tschechischen Bergregionen. Dort gibt es sowohl Vorkommen mit einzeln balzenden Birkha¨hnen, als auch Balzpla¨tze an denen sich mehrere Ha¨hne zur Balz einfinden. Insgesamt untersuchten wir 250 Individuen von 8 unterschiedlichen Vorkommen mittels 11 genetischer Marker (Mikrosatelliten). Zwischen den einzelnen Gebieten fanden wir signifikante paarweise FST Werte, was auf einen geringen genetischen Austausch zwischen den einzelnen Gebieten hindeutet. Die Birkhu¨hner konnten den drei Hauptgebirgszu¨gen (Sˇumava, Krkonosˇe, Krusˇne´) zugeordnet werden. In Gebieten mit einzeln balzenden Birkha¨hnen fanden wir eine geringere genetische Vielfalt im Vergleich zu Gebieten mit gro¨ßeren Balzpla¨tzen. Die genetische Vielfalt war dabei sta¨rker mit der Art des Balzplatzes (einzeln balzend oder mehrere Ha¨hne) als mit dem ra¨umlichen Vorkommen der Population in den verschiedenen Bergzu¨gen korreliert. Unter der Annahme das die Ha¨ufigkeit mehrerer Ha¨hne an einem Balzplatz von der Populationsdichte abha¨ngen, stellt das Auftreten von einzeln balzenden Ha¨hnen ein alarmierendes Signal fu¨r den Naturschutz dar, da es eine reduzierte effektive Populationsgro¨ße und abnehmende Besta¨nde widerspiegelt. Acknowledgments We thank D. Resˇl, T. Lorenc, M. Trˇesˇnˇa´k and D. Fertsˇa´k for their help with feather collections in the field. We also thank M. Vyskocˇilova´ for her help with the laboratory work and two anonymous referees for valuable comments on previous versions of the manuscript. The data on Black Grouse numbers in the Czech Republic were provided by Agency for Nature Conservations and Landscape Protection of the CR (AOPK CˇR). The research was supported by the Grant Agency of the Czech Republic (GACˇR 206/ 06/P302), Internal Grant Agency of the faculty of Environmental Sciences (IGA 48/2006).
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