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ISBN 952-10-2817-3 (pdf) ISSN 1235-4449 4.1. Conservation of genetic biodiversity in a changing landscape, with special reference to capercaillie (Tetrao urogallus) in Finland Hannaleena Mäki-Petäys, Markku Orell University of Oulu, Department of Biology, P.O.Box 3000, FI-90014 Oulu, Finland. [email protected] () CONSERVATION GENETICS Conservation genetics is the theory and practice of genetics in the conservation of species as dynamic entities, which are capable of evolving to cope with environmental changes to minimise the risk of extinction (Frankham et al. 2002). Conservation genetics is a relatively young discipline and it is developing rapidly due to the new molecular techniques available (Smith & Wayne 1996). Although ecological, economical, and political factors may be of primary concern for avoiding the extinction of most endangered species, long-term persistence, genetics and related considerations have also become the focus of conservation efforts. This has only recently become possible because new molecular techniques have facilitated genetic studies of endangered species (Hedrick 2001). As a discipline, conservation genetics also includes the resolution of taxonomic uncertainties and the delineation of management units. It may additionally involve the genetic management of small populations to ensure that as much genetic diversity is maintained as possible, as well as the forensic use of molecular genetic analyses to increase our understanding of a species’ biology. Conservation genetics focuses on the processes within small, fragmented populations, and on practical approaches designed to minimise any harmful trends. Small population size is typically the main reason for the loss of genetic diversity, whereas restricted gene flow can prevent the exchange of alleles between fragmented populations (Frankham et al. 2002). Genetic diversity Genetic diversity encompasses the whole variety of alleles and genotypes present in a population or species. It is typically described by measuring polymorphism, average heterozygosity and allelic diversity. Genetic diversity can be measured for many different traits, including quantitative characters, deleterious alleles, proteins, nuclear DNA loci, mitochondrial DNA, chloroplast DNA and chromosomes (Hedrick 1999). The maintenance of genetic diversity is a primary objective in the management of wild and captive populations of threatened species. The loss of genetic diversity, i.e. genetic erosion, is often associated with reduced reproductive fitness (Crnokrak and Roff 1999). Further, genetic diversity is the raw material for adaptive evolutionary Anneli Jalkanen & Pekka Nygren (eds.) 2005. Sustainable use of renewable natural resources — from principles to practices. University of Helsinki Department of Forest Ecology Publications 34. http://www.helsinki.fi/mmtdk/mmeko/sunare Mäkipetäys & Orell Fig. 1. Male capercaillie on tree top is an impressive sight that has become rare in Finnish forests. Maintenance of sufficient genetic variation in the population is essential to ensure the viability of the species in the future. Photo: Pekka Nygren change, and thus a prerequisite for the evolution of populations to cope with environmental changes (Lande 1995). The risk of genetic erosion is highest in small, isolated populations because of increased random genetic drift, elevated inbreeding and reduced interpopulation gene exchange (Gilpin 1991, Raijmann et al. 1994, Hedrick 2001). This can particularly be the case where a single continuous population has been fragmented into smaller subpopulations. Habitat fragmentation Habitat loss and fragmentation due to human land use are considered to be the most important deterministic threats to many species. Commercial logging has resulted in large-scale changes in forest structure, which in turn affect forest species at the population level. The viability of a species may additionally be jeopardised by demographic, genetic or environmental stochasticity – factors which are amplified in small populations (Holsinger 2000, Keller and Weller 2002). From a genetic point of view, habitat fragmentation has several impacts on population genetic structure and diversity. Restricted gene flow together with small population size has been shown to lead to the genetic differentiation of populations and to loss of heterozygosity and allelic richness, causing inbreeding depression. Inbreeding and loss of genetic diversity can reduce reproduction in the short term and diminish the capacity of populations to 2 Conservation of genetic biodiversity in a changing landscape respond to environmental change in the long term, leading to an increased risk of extinction (Keller and Weller 2002). The effects of fragmentation depend critically upon the level of gene flow among the fragments, which is in turn dependent on factors related to environmental and population patterns, such as the number and spatial structure of populations. The effects of fragmentation are also time-dependent, as well as being influenced by the dispersal ability of the species and associated migration rates between the remaining habitat fragments (e.g. Frankham et al. 2002). In this paper we concentrate on the effects of fragmentation on the genetic population structure of the capercaillie (Tetrao urogallus) in Finland, including genetic diversity and gene flow. It is assumed that the capercaillie suffers from forest fragmentation, and it is suggested to be an ‘umbrella species’ of old-growth forest (Suter et al. 2002). More information on the capercaillie is thus urgently needed to facilitate conservation. DECLINED POPULATIONS OF THE CAPERCAILLIE The capercaillie is protected by law in several European countries and it is included in the respective national Red Books (Hilton-Taylor 2000). In Finland, the relative density of the species has declined rapidly since the 1940s due to various human activities (Lindén and Rajala 1981). The current population size is on the average 300,000 breeding individuals (Helle et al. 1999b). This is about one third of the species’ population in the 1960s. In addition to this overall decline in population size, the species’ sex-ratios have also changed over recent decades (Helle et al. 1999a). Previously the Finnish population was female-biased (60% females, 40% males), but in Southern Finland females currently only make up around 50% of the population, although they still constitute around 60-65% of total populations in the north (Helle et al. 1999a). Several reasons have been put forward to explain the population decline of the capercaillie. Increased predation pressure associated with changes in the structure of forest landscape has been proposed as a factor (Storaas et al. 2001). Nowadays game hunting extends over wider areas as the intensifying network of forest roads has reduced the numbers of remote natural reserves for the capercaillie. Although hunting may have led to decline on a local scale, the most seriously affected populations are in Southern Finland where hunting pressure is the lowest. This would indicate that the major threats to the capercaillie are forest fragmentation and changes in the age composition of forests due to human land use (Helle 1999a, b). Capercaillie mating occurs at leks that consist of groups of males visited by females. Distribution of mating is highly distorted so that only a few males manage to reproduce (Höglund and Alatalo 1995). This type of breeding behaviour means that the effective population size of the capercaillie is considerably smaller than the actual population size, because of the high number of non-breeding males. This makes the species even more vulnerable to environmental disturbances. The lekking behaviour of the capercaillie affects dispersal behaviour, which in turn, has an effect on the gene flow between breeding areas, i.e. leks. Males tend to be devoted to their leks, while females can visit several leks during their lifetime (Storch 1997), even though most females tend to return to the same lek and mate with the same male year after year (Hjorth 1970, Wegge and Larsen 1987). It has been suggested that 3 Mäkipetäys & Orell the capercaillie has sex-biased natal dispersal with males being the philopatric sex (Koivisto 1963, Lindén 2002). Earlier studies are based on ecological data and did not include the use of molecular markers to address the question of differential dispersal between the sexes. There are many difficulties in determining the relative contributions of males and females to gene flow using ecological methods, since the sample sizes needed are relatively large. Furthermore, dispersal rates are often unreliable, as dispersers do not necessarily breed (Frankham et al. 2002). New molecular methods have great potential for examining sex-specific gene flow. Many other subjects can more easily be investigated by using molecular, rather than ecological methods, including estimates on how closely individual birds are inter-related. An other important issue, in addition to dispersal behaviour, is the uncertainty regarding the existence of different sub-species of the capercailllie in Finland. Previous work has indicated that three different subspecies – Tetrao urogallus urogallus, T. u. major, and T. u. uralensis – occur in Finland (Johansen 1957, Lindén and Teeri 1985). This conclusion has mainly been based on observed morphological differences (Helminen 1963, Lindén and Väisänen 1986) and behavioural differences (Jaakola 1999) between sub-species. However, there is no information that indicates whether these subspecies should be treated as separate conservation units or a single unit in the planning of management programmes. When planning land use and forestry at the landscape level, it is important that the level of gene flow between leks is known. Protected forests in eastern Finland, such as those adjoining the “Green Belt” forests of Russian Karelia, may ultimately serve as important source areas for maintaining the genetic diversity of capercaillie subpopulations in western Finland, and even more widely for the long-term persistence of the species in the whole Fennoscandia. THE GENETIC STRUCTURE OF THE CAPERCAILLIE IN FINLAND We began the studies on the population genetics of the capercaillie in Finland in spring 2001 with the collection of information on the genetic diversity of the capercaillie population. At first, we focused on the spatial structure of the population, and the relatedness of individuals within leks. The data have a good coverage over the country (Fig. 2), which makes it possible to analyse dispersal patterns of the capercaillie at different spatial scales more reliably than previously. Information on genetic diversity and gene flow can be linked with the landscape data in GIS (Geographical Information Systems) to estimate the effects of forest fragmentation on these patterns. Analysis at the landscape level is particularly important in Finnish forests near Russian Karelia, becuase the forests on Russian side of the border have been considered as a potential source of immigrant birds that could maintain the genetic diversity of the Finnish capercaillie population in the future. We have also designed our study to resolve taxonomic uncertainties and to define conservation and management units for declining populations. The ultimate goal of the study is to determine suitable management options for the species and to provide information for helping to ensure that land use and forestry are planned in an ecologically sustainable way. 4 Conservation of genetic biodiversity in a changing landscape Fig. 2. Locations of sampled leks (indicated by ) in Finland. Subpopulations (max. distance between samples 50 km) are circulated by black line, whereas different colours (blue, green, yellow and red) indicate four groups obtained by genetic analyses. Zonation is based on the subspecies distribution. Genetic analyses Samples for genetic analyses were collected from the lekking sites of the capercaillie. Samples were fresh faeces collected under the trees, in which males have stayed over several nights. The minimum distance between the samples was 60 meters, which should be adequate to separate the sleeping sites of different males in territories. Females lack territories, thus only one female sample per lek-site was collected, with the exception that faeces were less than one day old. The sex of the sample in the field was estimated from the size of the faeces. Later the sex of all samples was confirmed by DNA-based sex identification (Griffiths et al. 1998). The study involved the examination of the degree of genetic variability in the Finnish capercaillie population using co-dominant nuclear microsatellites and single stranded conformation polymorphism (SSCP) from mitochondrial DNA (mtDNA), as molecular markers. The mtDNA differs from nuclear DNA as it is inherited only from the mother, whereas nuclear DNA is inherited from both parents. Using these two different markers allowed us to examine the factors such as sex-specific gene flow. Microsatellite loci are segments of DNA with very short sequences repeated in tandem at one or more places in the genome (Queller et al. 1993). The large number of microsatellite loci, and their high variability, makes them potentially important tools for examining the genetic structure of the populations. We used nine species-specific microsatellite loci developed by Segelbacher et al. (2000) for revealing the nuclear variation. In total, we genotyped about 500 individuals collected from 200 leks (Fig. 2). All loci used were independent and highly variable. 5 Mäkipetäys & Orell Table1. Number of individuals (Nind) in subpopulations, number of microsatellite alleles (Nall) and gene diversity (HE) calculated over loci by software FSTAT. Nind Nall HE Sodankylä 15 Meltaus 24 Rovaniemi 24 Palokas 18 Simo 22 Litokaira 32 Posio 14 P-Kuusamo 47 E-Kuusamo 48 Syöte 54 Pudasjärvi 20 Pohjanmaa 9 K-Suomi 27 E-Suomi 31 56 74 60 65 56 64 61 74 71 87 67 21 71 69 0,76 0,77 0,76 0,82 0,72 0,70 0,73 0,75 0,73 0,75 0,76 0,88 0,76 0,75 Table 2. Number of individuals (Nind) and alleles (Nall.), expected (HE) and observed (HO) heterotsygosity, and FST values with standard errors. All values are estimated over subpopulations and loci by using software FSTAT. Nind. Nall. HE HO FST Microsatellites Females Males Total 105 280 385 107 128 137 0.74 0.75 0.76 0.54 0.56 0.55 0.035 ± 0.012 0.025 ± 0.003 0.025 ± 0.004 SSCP 187 15 0.030 SSCP is based on 3-dimensional conformation, as determined by a single-stranded nucleotide sequence, and rate of its migration in polyaclylamide gel (Hayashi 1991). Even a single nucleotide difference may change the conformation of the single strands enough to change their mobility in the gel. Thus SSCP is a useful method for detecting single nucleotide polymorphism without having to sequence the homologous DNA fragments isolated from a large number of individuals. To examine the genetic variation in mtDNA, we genotyped ca. 200 samples from 120 leks by using SSCP. The segment used in this study was originally designed for the grey partridge (Perdix perdix) by Liukkonen-Anttila et al. (2002). All genotypes were verified by sequencing 40 samples for both directions. 6 Conservation of genetic biodiversity in a changing landscape Results Genetic population structure and gene flow We have already obtained preliminary results on the genetic diversity and population structure of the capercaillie in Finland. Relatively high genetic diversity, measured as the number of alleles and gene diversity, was found in all studied subpopulations (Table 1). There was no genetically differentiated subpopulations when microsatellite data was analysed by using Bayesian clustering (BAPS; see Corander et al. 2003, 2004). Moreover, the genetic differentiation of subpopulations estimated by FST was relatively low (Table 2). The FST indicates the probability that two alleles drawn randomly from a population fragment are identical by descent. Additionally, studies using microsatellites have as yet shown no clear signs at the genetic level of the existence of subspecies of the capercaillie. However, more structure was found for mtDNA (Table 2) by Bayesian analyses, but it did not match the suggested distribution of subspecies (Fig. 2). Most genetically differentiated and geographically constant group consisted of leks located in north-eastern Finland (Syöte, Pohjois-Kuusamo and Etelä-Kuusamo). This supports the results of Liukkonen-Anttila et al. (2004), with no mtDNA sequence structuring related to suggested capercaillie subspecies’ ranges in Finland. However, some rare haplotypes and high genetic diversity in north-eastern Finland was also found in this study. According to our preliminary results, the genetic differentiation at the lek level was relatively strong (FST = 0.094 ± 0.01), indicating partly restricted gene flow between leks. Also the level of inbreeding coefficients, or the probability that two alleles in an individual are identical by descent (FIS), were quite high (FIS = 0.182 ± 0.031). Similar and even higher inbreeding values in lek level (0.19–0.31) has been recorded for other grouse species (lesser prairie chicken Tympanuchus pallidicinctus; Bouzat and Johnson 2004). The partly restricted gene flow together with high level of inbreeding could be a consequence of kin selection in leks, as described for the black grouse (Tetrao tetrix) (Höglund et al. 1999). Kin selection theory predicts that by joining a lek where a relative is likely to reproduce, the attractiveness of the lek is raised and males unsuccessful in obtaining copulations may gain via inclusive fitness. Sex-Biased dispersal The preliminary result based on comparison of genetic population structure between sexes and between differently inherited markers indicate that males have a stronger impact upon gene flow than earlier assumed. The result of roughly equal gene flow of sexes do not agree with the previously held assumption that females are the dispersing sex, whereas males are highly devoted to their leks. This is particularly interesting because earlier studies have mainly concentrated on the average dispersal distances of the sexes, but not on the maximum distances. Yet long dispersal distances can have an important role in the level of gene flow, and in mixing the different gene pools of populations. Further, earlier dispersal estimates are based on ecological studies, which poorly measure the effective dispersal affected by factors like polygamy, i.e the distortion of mating, of the species. Although the female would be the more often dispersing sex, the rare long dispersing males can have strong influence on the genetic structure of the leks. The dispersal of males with high fitness further magnifies strength of the male gene flow as only one or few males in a lek can reproduce. It is also possible that the dispersing male is not superior itself, but its offspring will have heterosis advantage in a new lek site. In the black grouse, males that never obtained a lek territory 7 Mäkipetäys & Orell had lower level of heterozygosity than males that were observed in a territory, possibly showing the effects of heterosis (Höglund et al. 2002). The unexpected results also imply that more information will be needed on the structure of leks and the lekking behaviour of the capercaillie for designing conservation practices that ensure genetic diversity and facilitate efficient dispersal and gene flow between leks in the future. Implications for conservation and management The preliminary results of our study support the importance of the connectivity of forests in the management of capercaillie populations. In forest management, it is important to maintain a sufficient network of both current well-functioning and potential yet unoccupied lek sites for sustaining efficient natal dispersal of the capercaillie. Moreover, the importance of male dispersal may earlier have been underestimated. This should be taken into account when planning hunting and game management. When considering the conservation and/or management unit of the capercaillie, our results support the importance of populations in NE Finland. More detailed analyses in landscape level are needed to obtain information for local management. Even though we found high level of genetic diversity in all subpopulations in Finland, it does not necessarily imply that the capercaillie is not genetically vulnerable. Changes in allelic distributions and reduction of genetic diversity can take quite a long time unless the population goes through a severe bottleneck. The forest fragmentation in Finland may be too recent to be detected in genetic analyses, and the observed level of genetic diversity may reflect the past situation rather than the current one. High level of inbreeding and partly restricted gene flow between leks indicate that limited dispersal caused by, for example, forest fragmentation can have severe consequences in genetic diversity of the species in the long term. Male contribution in gene flow may be stronger than earlier thought. Male dispersal may be connected to the long distance dispersal for new habitat patches, including establishment of new leks. Therefore, the habitat availability may strongly affect the genetic diversity of the populations. If there is a factor, like the mating system, that increases the level of inbreeding in populations, it magnifies still the importance of rare dispersal events. The strength of male dispersal is also closely related to the polygamy of the species. Changes in landscape structure tend to decrease the size of the leks, which in turn may decrease the level of polygyny. It has been observed that larger leks with many displaying males attract more females than smaller leks (Alatalo et al. 1992). Therefore, we assume that the size of the leks also has an important influence on maintaining genetic diversity in population. Dispersal can be linked to mortality of the sexes. Forest fragmentation can increase the predation especially of the dispersing sex. Hunting may also increase mortality of dispersing individual. To be able to discuss on the relation between dispersal, mortality and fragmentation, local analyses of dispersal behaviour in different habitats are needed. In general, there may be differences in dispersal behaviour between geographical areas in Finland. The dispersal behaviour may vary between different environments depending, for example, on the availability of habitats in that area. It is too early to say discuss the local results as the analyses are still in process. There was no clear evidence for the existence of subspecies in Finland, confirming the results of Liukkonen-Anttila et al. (2004). Thus, the subspecies would not be the best management units of the capercaillie. However, some structuring was 8 Conservation of genetic biodiversity in a changing landscape found, and especially NE Finland differed from the other areas in terms of mtDNA haplotype distribution. This result supports the idea that gene flow from the east can be important in the maintenance of the genetic diversity in western Finland. ACKNOWLEDGEMENTS We want to thank the staff of Arctic Centre (University of Lapland), Finnish game and Fisheries institute, Metsähallitus, and the Oulanka research station for their contributions to work in the field, and Joanna Aalto and Laura Törmälä for work in the lab. Many thanks also to David Hughes, Tuija Liukkonen and Pekka Pamilo giving helpful comments on the manuscript. The study was funded by the Academy of Finland (52772), the Finnish Cultural foundation, North Ostrobothnian Fund of the Culture Foundation, Finnish Game Foundation and European Social Fund (Naturpolis Kuusamo). REFERENCES Alatalo, R., Höglund, J., Lundberg, A. & Sutherland, W.J. 1992. Evolution of black grouse leks: female preference benefit males in larger leks. Behav. Ecol. 3: 53-59. Berg, L.M., Lascoux, M. & Pamilo, P. 1998. The infinite island model with sexdifferentiated gene flow. Heredity 81: 63-68. Bouzat, J.L. & Johnson, K. 2004. Genetic structure among closely spaced leks in a peripheral population of lesser prairie-chickens. Mol. Ecol. 13: 499-505. Corander, J., Waldmann, P. & Sillanpää, M.J. 2003. Bayesian analysis of genetic differentiation between populations. Genetics 163: 367-374. —, Waldmann, P., Marttinen, P. & Sillanpää, M.J. 2004. 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