Environmental factors and intrinsic processes affecting habitat use of European ground ( citellus): putting science into conservation practice

Ph.D. Thesis

Csongor Gedeon Department of Ethology, Biological Institute, Faculty of Science, Eötvös Loránd University,

Supervisors

Dr. Vilmos Altbäcker (Department of Ethology, Eötvös Loránd University, Budapest, Hungary)

Regents’ Prof. Lee C. Drickamer (Department of Biological Sciences, Northern Arizona University, Flagstaff, USA)

Ph.D. School of Biology Head of PhD School: Prof. Dr. Anna Erdei

Ph.D. Program of Ethology Head of Ph.D. Program: Dr. Ádám Miklósi

- Budapest, 2011 -

1 I. Acknowledgements

Following the general guideline, I will try to keep this section short. Not because it is less important than the other parts, but because I truly believe that all people who contributed to this dissertation know exactly how grateful I have always been for their work or encouraging words or for their mere existence supporting me along the road for several years. I do not think I have been a regular or typical Ph.D. student. First of all, I have been working for a living in a regular workplace while I was doing my scientific work. Secondly, as I have a beautiful family, whom I love so much, they sometimes distracted my attention. I think it happens frequently and normally. Finally, I consider myself a real generalist in every aspect of life. Consequently, believing in me or in my abilities to succeed sooner or later, or believing that finally I would be able to produce something worthwhile might have been a challenge.

Eventually, herebelow come the acknowledgements, which is rather a list of good, encouraging, motivating, enthusiastic, open-minded, flexible, helpful, and friendly people.

First and foremost I want to thank my local and remote supervisors Vilmos Altbäcker and Lee C. Drickamer. I am particulary grateful to Lee who took everything I asked from him voluntarily. I also want to thank a few other scientists who had major impact on this thesis via common publications, scientific cooperation and/ or fellowship (in alphabetical order): Miklós Dombos, Ilse Hoffmann, Gábor Markó Eva Millesi, István Németh, Attila Sándor, Franz Suchentrunk, and Olivér Váczi. I am also grateful for several anonymous reviewers for criticising but at the same time encouraging me to improve the manuscripts that I submitted during my Ph.D. studies. At last but not least, l am grateful to my ’small’ family Réka, Blanka, Rozi, Flóra, and my parents, brothers who always encouraged me to finish this route. My research work has been funded by the Hungarian-American Fulbright Commission for Educational Exchange, by the Austrian-Hungarian Action Programme for Scientific Cooperation, by the grant of the Austrian Science Fund to Ilse Hoffmann, by the Mecenatura Conference Grant, and by the European Science Foundation’s short term Conservation Genetics grant, only to mention the most significant ones. 2 II. Table of Contents

I. Acknowledgements ______2

II. Table of Contents______3

III. Introduction ______5 1. Soil ______7 2. Vegetation ______8 3. Human activities ______9 4. Mating system ______10 5. Genetic diversity in neutral genes ______10

IV. Study species ______12 1. Ground dwelling squirrels ______12 2. European ground (Spermophilus citellus) ______12 3. The Gunnison’s (Cynomys gunnisoni) ______14

V. Objectives – Questions ______16 1. Background ______16 2. Objectives ______16

VI. Nest material selection affects nest insulation quality for the European ground squirrel (Spermophilus citellus) ______18 Abstract ______18 Introduction ______19 Material and Methods ______20 Results ______24 Discussion ______26 Acknowledgments ______28

VII. Morning release into artificial burrows with retention caps facilitates success of European ground squirrel (Spermophilus citellus) translocations ______29 Abstract ______29 Introduction ______30 Materials and Methods ______31 Results ______34 Discussion ______35 Acknowledgments ______36

VIII. Medium-height grass and angled artificial burrow entrance to favour European ground squirrel translocations ______37 Abstract ______37 Introduction ______38 Material and methods ______40 Results ______44 Discussion ______47

3 IX. Importance of burrow-entrance mounds of Gunnison’s prairie dogs (Cynomys gunnisoni) for vigilance and mixing of soil ______51 Abstract ______51 Introduction ______52 Discussion ______56

X. Dwindling genetic diversity in European ground squirrels______58 Abstract ______58 Introduction ______59 Materials and methods ______60 Results ______63 Discussion ______67 Conclusions ______73 Acknowledgements ______73

XI. Does ecological isolation or population size influence genetic diversity in the European ground squirrel (Spermophilus citellus)? ______79 Abstract ______79 Introduction ______80 Material and methods ______83 Results ______87 Discussion ______89

XII. Putting science into conservation action: Notes on the guideline for reintroduction of European ground squirrels ______93

XIII. Future challenges: Captive breeding programme of European ground squirrels ______97

XIV. Literature review ______99

XV. Final remarks ______120

XVI. Summary ______125

XVII. Záró megjegyzések ______126

XVIII. Összefoglalás ______132

XIX. Publications ______133

4 III. Introduction

In principle, scientific research is driven and motivated by curiosity (Lorenz 1998). Curiosity always raises more and more and newer questions in the mind of the researcher. The sequence from the moment of asking until answering finally determines the scientific discipline, which is distinguishable fom other disciplines by this process. Recently, finite knowledge, in unlimited quantity, is available for people, thus curiosity motivates us to ask more and more special questions. However, in spite of the high specialisation there are yet fundamental questions to answer. All disciplines build on these and return to them when they acquire novel findings. From the principle of Biology, as “Nothing in biology makes sense except in the light of evolution (Dobzhansky 1973)”, to the four main questions of Ethology, or to the bottomline and theory of Ecology scientific investigation includes an infinite number of questions or theories. Ecology askes what kind of population compositions (including the abiotic environmental factors) can be observed, what is the quantity of different populations and in what spatial and temporal or enviroenmental pattern they co-exist, and eventually how can we explain these population compositions and patterns (Jordán, Scheuring, and Vida 2002). From a methodological viewpoint, to answer the fundamental questions in biological sciences is a complicated and hard task, because these are complex questions and they can be divided into multiple subquestions. Consequently, to answer basic questions would require the knowledge of the methodology or scientific approach of other subdisciplines.

In my dissertation, I will inverstigate and approach the fundamental question of Ecology from the viewpoint of factors that influence the habitat use of the European ground squirrel and the Gunnison’s prairie dog, two ecologically similar ground dwelling sciurids. I attempted to learn and understand the importance of a few mechanisms by which the association between the habitat and ground squirrels is maintained. In other words, to see if a few extrinsic or intrinsic factors affect the habitat use of ground squirrels substantially. During the research I was driven by my curiosity, by previous findings, and by the limited financial resources. Consequently, the coherence between the studied mechanisms that are supposed to affect habitat use of ground squirrels is subjective and arbitrary. Secondly, I had to apply both molecular and supra-individual, field and laboratory tests. My general, scientific approach and questioning is best reflected by the methodology used by naturalists: „[a naturalist] is

5 basically an explorer and tester of evolutionary and ecological ideas that are developed to reveal and explain regularities in nature (Grant 2000).”, and (Schmidly 2005) wherever they are to be found (macroecology, population genetics, and so on), by whatever means are available (field experimentation, analysis of DNA, and so on).” In habitat use models, "When are associated with a particular (micro)habitat, it is often presumed that they “prefer” to be there or that they have actively “selected” that habitat and rejected the others available to them. However, there are three broad classes of model that could explain such a pattern of distribution. The first is settlement/ recruitment: juvenile individuals could recruit in greater numbers to that microhabitat than to others and not subsequently leave. The second alternative is that the population distributes itself randomly throughrout the available habitat, but differential mortality leads to the reduction of numbers in the “unfavourable” microhabitats. The last possibility is that, after widespread recruitment, adults exhibit behaviour that establishes and maintains their position in the “favoured” microhabitat. These models are equivalent to the terms “birth rate”, “death rate”, and “migration” in demographic equations and such processes can act alone or in combination to give rise to observed associations” (Crowe TP 1998).

6 1. Soil

Soil properties are major factors that govern ecosystem processes (Chapin, Matson, and Mooney 2002) and are of paramount importance for ground dwelling animals. The characteristics of soil within the habitat could be the main determinants of habitat use (Parer 1985; Vitale, Zoppe, and Bollini 1989; McCarley 1966; Scharff and Burda 1998).For burrowing , the physical property of the soil determines basically if digging is feasible. However, soil properties and morphology are also influenced by several factors e.g. the nature of the parent material, climate, topography, time and the biota. Soil fauna (micro, meso, macro and megafauna) contributes significantly to habitat heterogeneity at the landscape scale (Kinlaw 1999; Whitford and Kay 1999). In particular, vertebrate biopedturbation, the disturbance of soil by vertebrate animals, has been implicated in structuring ecosystems and ecosystem function (Butler 1995; Whitford and Kay 1999). For example, the burrows or diggings of a range of mammals have been shown to change soil bulk density, water infiltration, pH, organic matter or nutrient content, which has led to changes in surrounding plant communities (Reichman 1990; Kinlaw 1999; Whitford and Kay 1999)). This interaction between the soil and its mammalian dwellers is well illustrated by e.g. Oryctolagus cuniculus which digs its burrow system under the roots of Juniperus communis (Parer 1985; Altbäcker, Nyéki, and Kertész 1997)). These shelters and resting places are more durable than burrows in the seahore sand dunes (Vitale, Zoppe, and Bollini 1989). Another pictorial example of soil disturbance by vertebrate animals (biopedturbation) is the burrows and mounds of many subterranean, fossorial, and semi-fossorial mammals. The mounds associated with ground-dwelling sciurid burrows serve as protection against burrow flooding, as vantage points to scan for predators or conspecifics (King 1959), as burrow ventilation aids (Bailey 1931; Scheffer 1947; King 1955, 1959; Vogel and Bretz 1972; Vogel, Ellington, and Kilgrove 1973; Hoogland 1995; Hoogland 2003). Burrows can be flooded from the underground water base, consequently ground dwelling squirrels should excavate their burrow systems in safe distance from the water table. As a result of this, animals usually prefer elevations in habitats. Additionally, during burrowing subsoil is mixed and deposited at the entrance, and results in bare, loose mounds that increase heterogeneity of habitats while providing patches for dicot plants (Whitford and Kay 1999).

7 2. Vegetation

Habitat quality is considered one of the most important elements of a species’ critical needs (IUCN 1998, 1998) to be addressed in habitat management or reintroduction. For ground dwelling sciurids, the habitat encompasses both the above-ground and underground environments. For natural colonies of ground squirrels, one of the most important factors of above-ground environment to favour survival of colonies is grass height (Kis et al. 1998). Ground squirrels abandon sites where the grass grows tall, and prefer to colonize areas where the vegetation is already low (Koford 1958; Clark 1979; Snell 1985; Knowles 1986; Foster 1990; Kis et al. 1998). Vegetation usually differentiates ground dwelling sciurid colonies from surrounding areas. The height of vegetation is markedly shorter within colonies. Shorter vegetation results from foraging or from clipping tall plants at the base without consuming (King 1955; Hoogland 1995) or simply because of climatic factors (i.e. low precipitation) that limit growing of grasses on steppes or prairies. Short vegetation can facilitate the detection of predators or conspecifics, but on the other hand, it can also facilitate ariel predators in detecting their prey. Moreover, composition of the plant community within the territory of the colony can also be radically different from other uncolonized areas (Koford 1958; Foster 1990; Whicker 1988). Dietary studies of ground squirrels (Váczi, Koósz, and Altbäcker 2006; Koósz 2002) and European rabbits (Oryctolagus cuniculus) and hares (Lepus europaeus) (Katona et al. 2004) have shown the importance of xeromorph fescues (i.e. Festuca pseudovina, F. vaginata); however, they emphasized only the importance of these plants in the diet. F. pseudovina, a typical plant species of short grasslands in Europe, is a common, but not the most frequent element in the diet of squirrels (Gedeon et al. 2010). Its use as nest material (at least in case of burrowing, nesting animals) was indicated by our results, as it resists either dry or wet conditions without dramatic morphological changes. In addition, it may serve as a food item at the end of before emergence from the burrows. To survive unpleasant environmental periods the quality of nesting material can be as important as food items for ground dwelling sciurids, and fescue and plants alike might serve both functions. Experimental evaluation of specific abiotic (Váczi, Koósz, and Altbäcker 2006; Wagner and Drickamer 2004) and biotic (Kis et al. 1998) components could reveal characteristics of the habitat that are key to survival and thus provide valuable information for conservation efforts.

8

3. Human activities

Human activity affects populations in general (Channell and Lomolino 2000) and ground dwelling sciurids in particular (Lomolino 2001). For the European ground squirrel, barriers to migration between colonies and habitat fragmentation, intensive agriculture, and aforestation or lack of management of primary or secondary steppes are the main effects that influence populations negatively. Although anthropogenic influences are usually considered disadvantageous, there are examples (e.g. additional food by human visitors on recreational areas, regular mowing or grazing, protection from predators, etc.) which demonstrate their positive effect on ground dwelling squirrel survival and increasing colony census density (Dobson and Kjelgaard 1985; Dobson, Smith, and Xue Gao 2000; Hubbs and Boonstra 1997; Michener 1989; Hoffmann, Millesi, Huber, et al. 2003). For the Gunnison’s or other prairie dog species, there are additional anthropogenic actions that have affected their survival negatively, such as poisoning, shooting, intensive livestock grazing (Seglund 2004).

9 Intrinsic factors that affect habitat use

4. Mating system

The majority of mammals have polygynous or promiscuous mating systems (Zeisset and Beebee 2008; Dobson, Smith, and Xue Gao 2000). The mating system of a species is genetically determined but yet is also under the influence of several environmental factors, i.e. the habitat quality (Begon 1990; Davis 1985). Theoretically the maximal carrying capacity of a habitat (habitat quality) is the ultimate factor that would prohibit unlimited increase of census colony density and size. In other words, habitat quality determines the minimal, necessary area for an individual to maintain itself and perform its species specific behaviours. As the habitat quality changes its carrying capacity changes too, and this can result in lower or higher census densities or sizes. The density dependence of social behaviour is evident (Goss- Custard and Sutherland 1997) and certain behaviours cannot be performed below a certain threshold of available partners (at very low densities) (Anthony 2000; Allee 1931). However, social reproductive suppression of potentially reproductive males or females in a group is also a density dependent phenomenon -albeit in high density groups- which reduces the number of breeding individuals and may decrease a population's rate of increase (Anthony 2000; Wade, Gong, and Arnold 1997; Wasser 1983). For ground squirrels, it means that the mating system is certainly under the influence of habitat quality via census density. As census density may change annually in ground dwelling squirrels, the mating system can also vary between populations (subpopulations) or years.

5. Genetic diversity in neutral genes

Genetic diversity is considered of paramount importance to maintain evolutionary potential or individual fitness (Frankham 1996; Paetkau, Shields, and Strobeck 1998). In natural populations of mammals the generally accepted factors to influence genetic diversity of populations via genetic drift or natural selection are i.e. population size or/ and density, ecological (effective) and/ or genetic isolation, mating system (polygynous in particular) and social structure, metapopulation and spatial structure, and population history (Amos and

10 Harwood 1998; Booy 2000). All other things being equal, small populations carry less selectively neutral genetic diversity than equivalent larger ones (Amos and Harwood 1998) in natural populations. Although this relationship is supported by Kimura’s theory (Kimura 1984) of neutral molecular evolution, and by several studies of wild populations (Frankham 1996; Young, Boyle, and Brown 1996; Amos and Harwood 1998) set out several alternative explanations (e.g., recent bottleneck, inbreeding, metapopulation structure, breeding system) when the positive correlation of census population size and genetic diversity is weakened or even destroyed. Ground dwelling squirrels recently inhabit fragmented landscapes, where the former metapopulation structure (Hanski and Gilpin 1997) has disintegrated. On the other hand, these fragmented landscapes frequently provide good-quality habitat, which in turn can maintain high census population densities and sizes. All in all, habitat quality, mating system, census density and size, genetic diversity are strongly interrelated mechanisms.

11 IV. Study species

1. Ground dwelling squirrels

Ground-dwelling squirrels (Spermophilus spp.) and prairie dogs (Cynomys spp.) and their relatives are an abundant and frequently studied group of burrowing mammals in the Holarctic ecozone, making their long burrow systems and building their nests at various depths in the soil (Hut and Scharff 1998; Michener 2000, 2002, 2004; Mrosovsky 1968; Ružić 1978). Ground-dwelling sciurids vary in demographic characteristics and in social organization from solitary Spermophilus tridecemlineatus to colonial Cynomys ludovicanus (Armitage 1981; Michener 1983; Barash 1989; Schwagmeyer 1990; Armitage 1999). They occupy a broad range of habitats, including tropical deciduous forests, hot and cold deserts, prairie and steppe, tundra, and woodlands (Howell 1938; Nadler et al. 1982; Yensen 1999; Ruff 1999). Two subfamilies of squirrels are currently recognized: Pteromyinae, the flying squirrels; and Sciurinae, consisting of nine tribes (i.e. Marmotini including genus Cynomys, as prairie dogs, Marmota, as , Spermophilus, as ground squirrels, and Ammospermophilus, as antelope squirrels genus) (McKenna 1997). The Spermophilus genus is represented by the European and spotted (S. suslicus) ground squirrels or s(o)usliks in Europe, and the Cynomys genus, represented by C. leucurus, C. gunnisoni, C. parvidens, C. mexicanus, C. ludovicanus, occurs only in North America.

2. European ground squirrel (Spermophilus citellus)

Scientific and Common name

European ground squirrel appeared first as Mus citellus in the Systema naturae (Linnaeus 1766). Then, it was named as Citellus citellus until the first half of the twentieth century(Oken 1815-1816). Finally, in 1956, the International Commission on Zoological Nomenclature (Opinion 417) concurred and validated the next oldest appropriate name, Spermophilus (Cuvier 1825) for the genus. Since then it has been called Spermophilus citellus, however

12 some authors from Central and Eastern Europe, and especially from the former Soviet Union, continued to use Citellus until as recently as 1995 (Gromov and Erbaeva 1995). The common name squirrel comes from the Latin word sciurus which was itself borrowed from Ancient Greek word σκίουρος, skiouros. Skiouros means shadow-tailed, referring to the bushy appendage possessed by many of its members. Ground comes from Old English grund which means bottom, foundation, surface of the earth.

Natural history

European ground squirrels are obligate hibernators and diurnal with bimodal activity patterns, and whose reproduction is limited by a relatively short active season (Katona, Váczi, and Altbäcker 2002; Mrosovsky 1968; Kryštufek 1999). They remain continuously underground from the end of August to March (Millesi et al. 1999) European ground squirrels have long and elaborate burrow systems (Ružić 1978), which provides a stable and safe environment against predators and adverse environmental conditions (Hut and Scharff 1998). Burrow entrances can be divided into two kinds: there are vertical burrows (pointing straight down at an angle of 90 degrees to a horizontal surface) and angled ones (sloping in one direction at an angle of 30-35 degrees to a horizontal surface) (Hut and Scharff 1998; Ružić 1978). The average number of entrances per burrow system and individual is four (Altbäcker 2005; Hut and Scharff 1998). Although they live in colonies, it appears that every individual makes its own burrow system and consequently they cannot be regarded as truly social . Being diurnal, they spend the night in the burrow system and forage for food during the day.

Conservation status

Due to the decrease of extensive animal husbandry that had maintained short grass ground- squirrel habitat for centuries, the species has recently become endangered in Europe. According to the 2008 IUCN Red List vers. 3.1, the species is considered vulnerable, (A2bc), with a decreasing population trend (Coroiu et al. 2008). It is also listed in Appendix II of the Bern Convention and the Annexes II and IV of the EU Habitats and Species Directive. Its important status in the short grass steppe habitat is underlined by several facts e.g., it is one of the main prey of several protected raptors (i.e., Falco cherrug, Aquila heliaca) (Kovács 2008); its burrows serve as a refuge for the protected Bufo viridis and Vipera berus (Váczi 13 2005). In addition to its natural habitat, the short grass steppe, ground squirrels have adapted well to live on grassy airports (Váczi, Koósz, and Altbäcker 2006) or recreational parks (Hoffmann, Turrini, and Brenner 2008). Initiated by the Hungarian Birdlife partner, MME in 1990s and later on the Department of Ethology, Eötvös Univeristy, translocation of European ground squirrels has taken place at several sites in Hungary.

3. The Gunnison’s prairie dog (Cynomys gunnisoni)

Scientific and Common Name

The name Cynomys first appeared in the XIXth century (Rafinesque 1817), which derives from the Greek for "mouse dog." “Prior to (Hollister 1916) careful taxonomic revision, different biologists had assigned prairie dogs to the following genera: Arctomys, Monax, Cynomis, Cynomomus, Mamcynomiscus, and Spermophilus (Hoogland 1995). C. gunnisoni was first mentioned and described as a distinct species by Baird (1855). Earlier it was assigned into the Spermophilus genus (Baird 1855). Prairie dogs are named for their habitat and warning call, which sounds similar to a dog's bark. The common name prairie dog is attested to from at least 1774. The 1804 journals of the Lewis and Clark Expedition note that in September 1804, they "discovered a Village of an annamale the French Call the Prairie Dog which burrow in the grown" (Woodger and Toropov 2004).

Natural history

Gunnison’s prairie dog is a large (675-1350 g), colonial and social (Slobodchikoff 1988; Rayor 1988; Hoffmeister 1986; Fitzgerald 1974), facultatively hibernating, ground dwelling sciurid, which constructs long and complex burrow systems (Longhurst 1944). Hibernation lasts for 4-5 months in Arizona from around October until March (Hoogland 1998). Gunnison’s prairie dogs reproduce only once annually and survival of offspring in the first few years is below 60 %. Both their burrow systems and social organization are really similar to those of ground squirrels (Pizzimenti and Hoffmann 1973). Burrow entrances are usually located on slopes or small hummocks rather than in depressions, which protects the burrows from flooding. The older burrow systems are deeper, have more entrances at the surface, and 14 more bifurcations below (Pizzimenti and Hoffmann 1973).They modify the surface of the landscape through grazing activities and digging for seeds (Shalaway and Slobodchikoff 1988), which increases the patchiness of grasses and shrubs on prairie dog colonies (Bangert and Slobodchikoff 2000). Consequently, as they influence soil properties and vegetation fundamentally (Carlson and White 1987; Whicker 1988; Munn 1993), they are classified as keystone species (Kotliar et al. 1999; Miller et al. 2000) and physical ecosystem engineers (Ceballos, Pacheco, and List 1999; Kotliar, Lange, and Rozenberg 2000; Gallie and Drickamer 2008; Bangert and Slobodchikoff 2000) in grassland ecosystems in western North America.

Conservation status

According to the 2008 IUCN Red List vers. 3.1, the species is considered as ‘least concern’ because it still occupies most of its historic range, although it is much reduced in colony size and distribution within that range, and because its populations are not declining fast enough to qualify for listing in a more threatened category. It occurs from central Colorado to central Arizona, including southeastern Utah and much of the northwestern half of New Mexico in the United States. It occurs from 1,830-3,660 m above sea level. Nevertheless, populations in all states within the range have declined significantly compared to historical levels (U.S. Fish and Wildlife Service 2006) and its population trend is decreasing. Apart from habitat destruction and shooting by humans, a major threat to the survival of prairie dogs comes from bubonic plague, caused by the non-native bacterium, Yersinia pestis.

15 V. Objectives – Questions

1. Background There are intrinsic and extrinsic factors that affect habitat use by ground squirrels and their survival, including the species’ specific requirements of hibernation and reproduction. Intrinsic factors include physiological, behavioural, and genetic patterns. Extrinsic factors encompass ecological processes and environmental conditions. Both mechanisms act synergistically, primarily at the population level, and affect life-history traits.

Since 1995 a few Ph.D. and M.Sc. research studies have been carried out on the Department of Ethology to investigate the direct relationship between extrinsic factors (incidentally resources, such as temperature, food material) and microhabitat usage, hibernation, temporal and spatial activity. These studies are the starting point for my study in which we attempted to investigate the effect of additional extrinsic factors (i.e. nest material, burrow, ecological isolation, census population density and size, etc.) and intrinsic processes, such as behaviour or genetic diversity, on habitat use.

The primary subject animals of this study were two semi-fossorial sciurid species: the European ground squirrel (Spermophilus citellus; EGS(s) hereafter) and the Gunnison’s prairie dog (Cynomys gunnisoni; GPD(s) hereafter). These diurnal, hibernating animals provide good examples for studying population dynamics of burrowing mammals.

The specific results for my questions gave answers to key questions in reintroduction biology: Under what circumstances are behavioural or genetic considerations important to reintroduction success? What restoration or management is required for the population to survive?

My study added some notes on the guideline for a more evidence-based management of European ground squirrels.

2. Objectives Our main purposes were: (I) In relation with behavioural biology: To understand the factors contributing to 16 the nest material choice of semi-fossorial burrowing mammals (Spermophilus citellus) under natural and laboratory conditions. To test the consequences of preferring fresh green nest material in nest building as insulation. We asked the following questions: Is there a specific preference for fescue in nest construction? Does the moisture content of the plant affect choice? To what extent does the moisture content of the nest material affect nest insulation? (Chapter IX.) To describe and analyze the characteristics and functions of GPD mounds and to analyze the behaviour of GPDs in relation to their mounds. We investigated mound and burrow structure with the following questions: Are GPD geomorphic agents? Our questions regarding GPD behaviour in relation to their mounds included: Does the frequency of the on-mound and off-mound behaviour differ significantly? Does the frequency of the vigilance behaviour change with vegetation height? Is the direction of the vigilance behaviour different between individuals at the perimeter and centre of the colony? (Chapter XII.)

(II) In relation to standard population genetics: To estimate genetic diversity at eleven polymorphic microsatellite loci of seven EGS populations. To compare the data particularly to data received in genetically depleted populations. (Chapter XIII.)

(III) In relation with ecological genetics: to clarify whether variations in census population density and size or ecological isolation are detectable in genetic diversity indexes for the polygynous EGSs. (Chapter XIV.)

(IV) In relation with conservation ecology: To test and evaluate how habitat manipulation (grass height, man-made burrow entrance angle) and different release methods (time: morning vs. afternoon, retention of animals) can influence the ‘success’ of wild-to-wild translocations of ground squirrels. Moreover, to observe the dynamics of new burrow constructions in the translocated animals during the active season until hibernation. (Chapters X. and XI.)

17 VI. Nest material selection affects nest insulation quality for the European ground squirrel (Spermophilus citellus)

Csongor I. Gedeon, Gábor Markó, István Németh, Viktor Nyitrai, and Vilmos Altbäcker Published in the Journal of Mammalogy, 91(3): 636-641, 2010

Abstract Natural nests of the European ground squirrel, Spermophilus citellus, are constructed almost exclusively of fresh fescue (Festuca pseudovina: Poaceae). We performed laboratory experiments to understand the functional significance of preference of wild squirrels for nest material. We examined the factors contributing to nest quality by monitoring the construction and analyzing the composition of squirrel nests. As in the wild, squirrels showed strong preference for fescue during the laboratory tests and preferred fresh material to dry material. Because preference for fresh material was not expected, as high water content reduces insulation, we manipulated the moisture content of nests made from either fresh or dry fescue to determine how nest quality and moisture level contribute to insulation. We found that the insulation property of fresh grass nests was superior to nests constructed from dry grass only. Intracellular water in the nest material did not affect thermal conductance and insulation significantly. We concluded that fresh fescue provided a more flexible material that allows squirrels to construct nest with better insulation under both field and laboratory conditions.

Key words: insulation, moisture content, nest, nest material, Spermophilus citellus

18 Introduction Nest-building has evolved in many taxa but is used mainly by endothermic animals that can maintain body temperature above ambient conditions. Although nests share common features within a taxon, their properties are determined by their function. The insulation quality of nests is dependent on several factors, such as nest structure (McGowan 2004; Redman, Selman, and Speakman 1999), height, thickness and volume (Grubbauer and Hoi 1996; Szentirmai 2005), nest material quality (Mertens 1977), and moisture content (Pinowski 2006).

Ground-dwelling sciurids (Spermophilus spp.) are an abundant group of burrowing mammals, making their long burrow systems and building their nests at various depths in the soil (Hut and Scharff 1998; Michener 2000, 2002, 2004; Mrosovsky 1968; Ružić 1978). Squirrel burrows and nests serve a number of functions, such as providing shelter during resting, sleeping, or hibernation and protection against unpleasant environmental periods (Hut and Scharff 1998)), defense from predators, as a place to store food, and for reproduction (Bethge 2004; Long, Martin, and Barnes 2005; Meadows 1991); and (Pizzimenti and Hoffmann 1973). In general, nests of burrowing small mammals insulate them from fluctuating ambient temperatures (Casey 1981). As a result, the nest can affect the magnitude of energy conservation and consequently is considered crucial (Bethge 2004; Geiser 1988; Houston and McNamara 1993; Lovegrove 2001; McCafferty, Moncrieff, and Taylor 2003; Redman, Selman, and Speakman 1999) or even critical to survival (Barclay 2001; Pinowski 2006) or reproduction (Lamprecht 2004).

Squirrels spend about 7 months underground during the hibernation period (Millesi et al. 1999) when even small differences in insulation can be of functional significance as breeding success is closely linked to weight at vernal emergence (Millesi et al. 1999). Nest insulation hardly influences nest temperature during torpor because the very low metabolic rate during torpor results in a minimal temperature gradient between the torpid animal and its surrounding. Therefore energy savings from the nest is usually marginal during torpor itself. However, a good nest will help an individual save energy during arousal and normothermia when energy expenditure is high during hibernation season. In addition, should a hibernator be forced to thermoregulate during torpor because ambient temperature falls

19 below the optimum (Buck 2000; Geiser 1988), the temperature gradient between animal and surrounding will increase, and good nest insulation will reduce energy expenditure. Nest quality also can contribute to survival in the active season. The relationship of insulation and loss of body mass change in the active season (i.e., during the mating period), when better insulation quality of the nest can help reduce loss in body mass. Mating or rearing offspring is energetically costly primarily due to decreased foraging time (Millesi et al. 1999); therefore a good nest can save energy, contributing to reproductive success. Our manipulative study on the European ground squirrel (Spermophilus citellus) has shown that to avoid high temperatures (Váczi 2005) ground squirrels hide underground in the humid nest chamber at midday during summer drought periods, possibly to enhance cooling (Váczi, Koósz, and Altbäcker 2006). In a field study focusing on plant use for dietary and nest-building purposes we found that squirrel nests contained fescue (Festuca pseudovina) in quantities well above chance level, but contrary to our expectations relative to insulation needs, nests contained fresh green material not the dry hay known to be preferred by similarly sized Mongolian gerbils and pikas (Millar 1972).

The aim of the study was to (1) understand factors contributing to the nest material choice of the European ground squirrel, and (2) test the consequences of preference for fresh green nest material in nest building as insulation. We asked the following questions. Do European ground squirrels have a specific preference for fescue in nest construction? Does moisture content of the plant affect choice? To what extent does moisture content of the nest material affect nest insulation?

Material and Methods Animals and housing. — Our field and laboratory study procedures were part of an approved research permission issued by the Authority of Nature Conservation in charge. In addition, our laboratory procedure also was approved by the Ethical Committee for Animal Experiments at Eötvös University, and followed the rules detailed in guidelines of the American Society of Mammalogists (Gannon and Sikes 2007). After the experiment, in the spring of 2008, study animals were released into their original habitat.

20 Eight wild male European ground squirrels were trapped on the grassy airport of Dunakeszi, Duna-Ipoly National Park (47°36'51.7"N; 19° 8'32.5"E), Hungary, in September 2007 and were transferred to a climate-controlled room at Eötvös Loránd University, Budapest. Squirrels were housed individually in plastic containers of 15 x 21 x 36-cm size. Containers were open at the top but were covered with a removable wire mesh. Barrel-shaped (16-cm diameter × 8-cm height), opaque plastic boxes were situated in every container as next boxes with removable tops. The squirrels could enter the next box through an elbow-shaped tunnel (6 cm in diameter) attached to the side of the box. Room temperature was measured continuously and maintained constant (approximately 25 ºC). Illumination (L:D = 12:12, with light on at 0600 h) was constant, and both food (rabbit chow; Galgavit Ltd., Gödöllő, Hungary) and water were given ad libitum during experiments (Németh, Nyitrai, and Altbäcker 2009).

Nest material choice.—To determine whether squirrels selectively choose certain materials for nest building, 2-way nest material choice tests were conducted where we tested (1) the species preference and (2) the moisture content preference in nest building. For these experiments Festuca pseudovina (pseudovina fescue) and Bothriochloa ischaemum ( yellow bluestem) plant tussocks were collected on the grassy airport of Dunakeszi, as these were the dominant species of the grassland and the main constituents of natural squirrel nests. Consequently, animals were familiar with the plants provided for nest construction. Nest-building behavior of the animals was recorded in their home cages by a continuous and computerized digital video system (Cameras PTC-150S; Elmo Ltd., Nagoya, Japan) positioned 2.5 m above the cages. In both experiments two grass tussocks of equal volume (2 dm3) and known composition (dried Festuca versus dried Bothriochloa in experiment 1, and fresh, humid Festuca versus dried Festuca in experiment 2) were presented to the animals for nest building in the early morning, and their behavior was recorded continuously. When one-half of the volume of the grass was used the test was terminated. At that point possible difference in grass usage should have been maximum because if only fresh or dry grass had been used for nest building, squirrels would have switched to the other tussock if they had required more grass for nest construction. Consequently, the fresh: dry grass ratio would have been biased due to limited volume of grass resources. The position of the two grass tussocks was assigned randomly to one of the opposite corners of the cages, with a distance of 25-35 cm between the tussocks. From the video recording we quantified the building behavior between 0900-1200 h (three hours), which is considered to 21 be the main period for surface activity of these squirrels in the wild (Váczi, Koósz, and Altbäcker 2006). The time duration (min) spent within 10 cm from the two grass tussocks was quantified from the video material as a measure of nest material collection. Subsequently, assembled nests were removed from the next boxes. From each nest three independent samples were picked with forceps, each containing 25-40 pieces of grass, and these samples were analyzed with a microscope using 40X magnification (Petróczi and Altbäcker 1994). In the analyses we determined the grass species (experiment 1) and, based on the color of the grass leaves, whether they were fresh (humid, dark-green) or dry (light-green) material (experiment 2). The average of the three samples was subjected to statistical analysis.

Nest quality manipulation.—The basic arrangement for manipulation of the nest insulation was similar to nest material choice tests described above, but with the difference that only one grass tussock of either fresh (Group Fresh) or dried fescue (Group Dry) was presented to the animals for nest building. In this experiment we investigated how nest insulation depends on the (1) moisture content and (2) nest properties (wall thickness, nest mass) of the assembled nests. On the basis of the previous two way choice tests and on our earlier observations, one grass tussock of known volume, mass, and composition (humid Festuca or dried Festuca) was presented to the animals for nest building between 0800-0700 h (23 hours). Eleven hours light was sufficient for the animals to complete a nest of either material. Each animal was allowed to construct one “dry” and one “fresh” nest. Upon completion of the nests the following morning we removed the next boxes with the nests and provided new material and a new next box. To determine the insulation value in relation to grass condition and moisture content we manipulated the moisture content of the nests in a sequence of four phases. In each phase the quality of nest insulation was measured. The four phases correspond to different level of moisture content of the nest material on a dry weight basis. First, we determined the difference in weight between fresh and completely dried nest material in a pilot study (not included in the present data set). When fresh fescue was dried in a laboratory drier until mass remained constant (i.e., essentially 100 percent solid), weight half of the original weight indicating that fresh material contained about 50% water. For Group Fresh nests (n = 8) were built from fresh, humid Festuca, and the insulation was measured in all four phases. In each phase the moisture content of the otherwise intact nests was set to a certain level. In Phase 1 the nest contained 50% water (water content is 100% on a dry weight basis). In Phase 2 the nest contained 25 % water (water content is 50% on a dry 22 weight basis). In Phase 3 the nest contained 0% water (water content is 0% on a dry weight basis), and in Phase 4 the nest contained again 25% water (water content is 50% on a dry weight basis) by adding the appropriate amount of water with a water-sprayer to the dried nest. Group Dried nests (n = 8) were built from dried Festuca, and the insulation was measured only twice corresponding to Phase 3 and 4 of Group Fresh. Finally, nests were removed from the boxes, and nest mass (g) was measured on a dry weight basis. The thickness of the nest wall and height of the nest also were measured (mm) at five randomly chosen points with a ruler, and the averaged value for each nest was used for further analysis. Additionally, lengths and widths of the slightly elliptical nests also were measured.

The design of the experimental device followed the method of Redman et al. (1999). Glass bottles (150 ml filled) with hot (50 ± 1°C) water were inserted into each squirrel nest kept at room temperature (25 ± 2°C). These bottles represented the animals within the nest, and the slower their temperature decreased the better the insulation was because thermal insulation refers to the decrease in rate of heat transfer. Each bottle was closed with a rubber lid through which a temperature-sensitive resistor (thermistor) was inserted. The temperature of the water bottle within the nest (T b) and room temperature (T a) were measured by thermistors. The sensors were previously calibrated to the nearest 0.1 °C against a precision quicksilver thermometer in a water bath between 0 and 40 °C. The signals from the thermistors were received with a model multimeter (M-4640A; Metex Instruments, Seoul, South Korea) via a channel multiplexer controlled and recorded by a personal computer. Finally, the multimeter was interfaced to a computerized system of data acquisition that recorded at 2-min intervals. Data recording lasted 5 h, enough for the bottles to cool to room temperature. The cooling constant, a (h-1), and insulation, I (ºC Watt-1), were calculated following a recent approach (Nicol and Andersen 2007). The relationship between time and cooling rate is best expressed by a negative exponential equation, where a was estimated by an iterative nonlinear regression procedure:

−Time⋅a Tb −Ta = b⋅e + ε

Tb - Ta is the difference between the temperature of water in the bottle (Tb) and the room temperature (Ta); b is the initial value of temperature difference and ε refers to the residual variability (error) which has standard normal distribution with zero expected value. Based on the

23 specific heat capacity of water (4.18 J g-1 ºC-1) and bottle (0.84 J g-1 ºC-1; mean [+ SD] mass: 102.66 ± 0.26) the thermal energy content of the 150 ml of heated water (50 ºC) equaled 35.65 kJ. For example, a one degree drop in temperature over 1 min would equal 713 J ºC-1, equivalent to 11.88 J s-1 or 11.88 W. At an initial temperature gradient of 25 ºC between water bottle and ambient, the thermal conductance (C) would be 0.475 W ºC-1. Statistical analyses. —All statistics were performed using Statistica version 8.0 (StatSoft, Inc. 2008). For both the Bothriochloa / Festuca species preference experiment and the fresh / dry nest material choice experiment either a parametric paired t-test or a nonparametric Wilcoxon matched-pair test (Z) were used, depending on the distribution (normality) of data. Violance of normality assumption was tested with Shapiro-Wilk test and Q-Q plots. In experiment 3 a mixed-model ANOVA was used to test the effects of a) moisture content in Festuca grass and b) nest material used for construction on nest insulation index. To separate the effect of nest mass, nest height, nest length, nest width, and nest-wall thickness on nest insulation a best-subset regression model was used. The variance inflation factor was used to determine whether predictor variables were correlated (Hill 2006). To compare the insulation of nests made either from dry grass (Phase 3-4; two cohorts) or fresh grass (Phase 1-4; four cohorts), we used a nested ANOVA. The Bonferroni post hoc test was used for multiple comparisons following a significant ANOVA outcome. To examine relationships between insulation and both dry mass of nest and wall thickness Pearson correlation (r) was used. Summary data are reported as mean + SD, unless stated otherwise. The criterion for statistical significance was P .05.

Results Nest material preference. —Squirrels (mean body mass: 172.5 ± 31.1) showed a preference for Festuca for nest material, both in terms of the composition of finished nests and time spent collecting nest material. The final nest contained significantly more Festuca (it comprised average 90 % ± 5 of the nest material) than Bothriochloa (t4 = 18.82, P < 0.001). Squirrels also spent significantly more time in the vicinity of the Festuca (median 82 %, range 73-85, of time spent at fescue) than at the Bothriochloa tussock ( Z = 2.02, n = 5, P < 0.05).

24 Quality of nest material. —When subjects were given a choice between fresh and dry

Festuca, they showed a preference for fresh Festuca ( t 7= 4.24, P < 0.001; 97 % ± 4 of nest material was fresh fescue). In addition, the animals spent more time in the vicinity of fresh tussocks (median 76 %, range 67-85, of time spent at fresh fescue) compared to dry ones ( Z = 2.52, n = 8, P < 0.05).

Moisture content manipulation.— Both the quality of raw material (fresh vs. dried: F135 = 119.91, P < 0.0001) and moisture content (untreated, half-dried, dried, half humid, remoisturized:

F4,35 = 21.54, P < 0.0001 ) influenced insulation significantly in a mixed-model ANOVA (Fig. 1).

F igure 1 Mean (± SD) thermal insulation of nests constructed by European ground squirrels (Spermophilus citellus). Phases 1-4 refer to different moisture content (see text for details). Gray bars represent fresh nest materials (fescue) and white bars dried nest materials (fescue). Different letters above bars indicate significant differences (P < 0.01) between or among means. The difference in insulation (I ) between untreated nests built from fresh, humid Festuca (Phase 1) and post-treated nests built from fresh, humid Festuca (Phase 2 and 3) were not significant (P > 0.05). However, I of Phase 4 was significantly different (P < 0.0001) from Phase 1, 2, and 3 built from fresh, humid Festuca. Mean thermal conductance values (in Watt ºC-1) of nests of cohort “fresh” were: CPhase_1 = 2.53 ± 0.19, CPhase_2 = 2.36 ± 0.15, CPhase_3 = 2.36 ± 0.13, and CPhase_4 = 2.94 ± 0.14. In the case of nests built from dried Festuca the difference in the 25 insulation between untreated nests and half humid, remoisturized nests was significant (P < 0.01; -1 Fig. 1). Mean thermal conductance values (in Watt ºC ) of nests of cohort “dry” were: CPhase_3 =

2.95 ± 0.21 and CPhase_4 = 3.31 ± 0.26.

Walls of the assembled nests made from fresh grass were significantly thicker (t14 = 4.36, P < 0.001) than the walls of the nests made from dried grass (Group Fresh = 4.28 ± 0.39 mm, Group Dried = 3.14 ± 0.63 mm; Table 1). Wall Nest thickness Height (cm) Length (cm) Width (cm) Dry Weight (g) material (cm) Fresh 4.28 ± 0.39 3.86 ± 0.41 19.63 ± 0.52 14.56 ± 0.82 19.25 ± 3.54 Dry 3.14 ± 0.63 3.48 ± 1.12 18.00 ± 2.14 14.06 ± 1.15 13.75 ± 2.71

Table 1 Descriptive data (means ± SDs) for composition of fresh (n = 8) and dry (n = 8) nests constructed by European ground squirrels (Spermophilus citellus).

Both the dry mass of the nest (r = 0.56, n = 16, P < 0.05) and the wall thickness (r = 0.80, n = 16, P < 0.0001) correlated with the insulation significantly. According to a best-possible- subset regression model, where nest mass, wall thickness, nest height, nest length, and nest width were entered as independent continuous predictors, and nest material (fresh or dry) as a categorical predictor, wall thickness and nest mass (F2,13 = 14.87, P < 0.001) explained 65 % of the variation in nest insulation. Wall thickness by itself explained 61 % of the variation in nest insulation (wall thickness: F1,16 = 16.16, P < 0.01; nest weight: F1,13 = 2.64, P >0.05). The variance inflation factor was 0.8; therefore wall thickness and nest mass are considered uncorrelated, reliable predictors.

Discussion European ground squirrels showed a specific preference for fescue (Festuca pseudovina), one of the dominant grass species of grasslands inhabited by squirrels in Hungary. The preference was evident both in the behavior of animals during construction and in the composition of resulting nests in the laboratory choice tests. Preference for fresh material was not expected. Thermal insulation refers to the capacity of materials to reduce heat transfer. Heat energy can be transferred by four main mechanisms: conduction, convection, radiation, and evaporation-condensation. According to thermal conductivity and evaporative heat transfer, higher water content of a material increases thermal conductivity and evaporation rate, and as a result reduces insulation. 26 However, conductance was not affected by intracellular moisture content; i.e., the insulation of nests made of fresh, humid Festuca did not increase with drying. Insulation is dependent on the thermophysical properties (Bozikova 2005) and condition of the material. The insulation properties of feathers rank best, followed by grasses and then mosses (Hilton 2004; Reid et al. 2002), but grasses are less affected by moisture than are feathers (Cramp 1977; Limona 1992). In contrast, the sprayed-on water on the surface of the nest material decreased insulation significantly. The reduced negative effect on thermal conductivity of intracellular water within the leaves of xeromorph fescues (Festuca pseudovina, F. rupicola, and F. valesiaca) can be attributed to the external and internal morphological structures of their leaves (high ratio of air-filled sclerenchyma, constantly-curled leaf blades, thick cuticles, pilosity, epistomic epidermis with a low stomata index, and recessed stomata under the epidermis). These characteristics help them resist dry and wet conditions (Kovács-Láng 1995) and hence buffer the fluctuations in temperature and humidity within underground burrows (Meadows 1991). The negative effect of surface water on insulation probably was due to evaporative cooling. Squirrels could build better insulating nests from fresh grass possibly because it provided a more flexible and treatable material, and they did not have to make a trade-off between fresh, humid and old, dry fescue. Finally, it resulted in better structure and thicker walls, which reduces air permeability of the nest and prevents the penetration of cold air to the animal (Banks-Lee, Mohammadi, and Ghadimi 2004; Epps 1988; Van de Weerd et al. 1997). The importance of nest architecture was supported by the result that wall thickness was positively correlated with insulation level, an effect that also was observed for nests of Microtus agrestis (Redman, Selman, and Speakman 1999). Nest insulation is not necessarily correlated with nest size (Ellison 1995), but our results agreed with those of Redman (1999) that wall thickness appears to be a better predictor for the insulation properties of a nest. Nests should provide insulation for animals during stressful climatic periods. European ground squirrels construct underground nests below the freezing line, at a depth between 0.5- 1.0 m (Hut and Scharff 1998; Ružić 1978). The preference of fresh fescue grass in nest construction can be explained by the good insulation quality, which is a valuable feature in the active season when the temperature gradient between body and environment is great, but also during the normothermic periods of hibernation when the temperature gradient between body and environment is greatest (particularly prior to final emergence). Dietary studies of squirrels (Koósz 2002), and European rabbits (Oryctolagus cuniculus) and hares (Lepus europaeus) (Katona, Váczi, and Altbäcker 2002) have shown the 27 importance of xeromorph fescues; however, they emphasized only the importance of these plants in the diet. F. pseudovina is a common, but not the most frequent, element in the diet of squirrels. Its use also as nest material (at least in case of burrowing, nesting animals) was indicated by our results, but because it resists either dry or wet conditions, it might serve as a proper food item at the end of hibernation before emergence from burrows. To survive unpleasant environmental periods the quality of nesting material can be as important as food items, and fescue might serve both functions. This notion requires additional field and laboratory study. Experimental evaluation of specific abiotic (Váczi, Koósz, and Altbäcker 2006; Wagner and Drickamer 2004) and biotic (Kis et al. 1998) components could reveal characteristics of the habitat that are key to survival and thus provide valuable information for conservation efforts. Our results suggest that squirrels may rely on dominant, abundant, and readily available xeromorph fescues of dry meadows because of their importance to nest building.

Acknowledgments We thank Lajos Porupcsanszki, Head of the Dunakeszi Airport, and the Duna-Ipoly National Park for permitting us to trap protected animals in the airport area. We thank Miklós Dombos, András Horváth, Olivér Váczi, Barbara Koósz, Peter Sandy, and Alexandra Kövér for suggestions regarding the manuscript. In addition, we express our thanks to the anonymous reviewers for their useful and helpful comments, and in particular to Lee C. Drickamer for thoroughly editing the text.

28 VII. Morning release into artificial burrows with retention caps facilitates success of European ground squirrel (Spermophilus citellus) translocations

Cs. I. Gedeon, O. Váczi, B. Koósz,V. Altbäcker Published in the European Journal of Wildlife Research pp. 1-5. doi:10.1007/s10344-011- 0504-3

Abstract Relocating ground squirrels within their natural distribution range is a popular tool in wildlife management in Central-Eastern Europe. Nevertheless, wildlife management lacks both a carefully developed and tested translocation guide and methods. We evaluated conditions of release method (time of release and retention of animals) that affect short-term settlement of translocated ground squirrels in the central region of Hungary. In a field experiment we translocated 117 individuals from an international airport to a protected site in 2000. We found that release time should precede the animals’ natural, daily activity peak. The use of retention caps combined with artificial burrows instead of complex acclimation cages works successfully to prevent animals from dispersing from the release site.

Keywords: retention cap, timing of release, translocation, ground squirrel

29 Introduction Relocating animals within their natural distribution range is a popular tool in wildlife management and conservation biology (Bajomi 2010). The technique is used to solve human- animal conflicts, to restock game populations for hunting, and to re-establish new populations of endangered species (Fischer and Lindenmayer 2000; IUCN 1987, 1998; Seddon, Armstrong, and Maloney 2007). In general, a reintroduction is considered successful if it results in a self-sustaining population (Calvete 2004; Seddon 1999). Texeira (Teixeira 2007) consider the three main objectives of a reintroduction to be: (1) survival of the animals after release, (2) settlement of animals in the release area, and (3) successful reproduction in the release area. The first and second objectives deal with the days or weeks immediately after release of the animals. Consequently, long-term survival at the release site strongly depends on this critical period (Seddon 1999). There are a lot of factors that influence the outcome of this critical period including e.g., suitable habitat, predator exclusion and confinement of the animals to the release site. Skilled reintroduction specialists (Roe AK 2004), and use of artificial burrows and acclimation cages are considered fundamental in preventing ground dwelling sciurids from escaping quickly from the release site (Hoogland 2005; Simms 2009).

Acclimation cages consist of several parts (e.g, nest chamber, retention basket, etc.) and serve to help acclimation to the new location (Simms 2009). Nevertheless, they have substantial costs to prepare and later, in the critical period usually, require additional work to remove from the release site. Substitution of acclimation cages with artificial burrows combined with retention caps seems a cost efficient, easily applicable, and non-invasive tool.

An additional, important but less investigated factor is timing of release both on daily and annual scales (Poole 2009; Pinter-Wollman 2009). Bimodal activity pattern occur in many diurnal rodents (Váczi, Koósz, and Altbäcker 2006), and the first activity peak is frequently followed by a midday pause and then by a lower activity peak in the afternoon (Katona, Váczi, and Altbäcker 2002). Bimodality of daily activity pattern is regulated by the interaction of endogenous and environmental factors (Aschoff 1966), but ambient temperature is particularly important (Váczi, Koósz, and Altbäcker 2006). Semifossorial burrowing animals, which have continuous access to a relatively stable environment underground, forage only on and perform the most of their behaviours on the surface, but during unpleasant environmental 30 conditions they return to their burrows (Hut and Scharff 1998). Consequently, the aboveground daily activity peak of these animals reflects convenient environmental conditions and success of colonization might depend on release timing relative to activity peak.

The European ground squirrel (Spermophilus citellus) is an endangered species in decline. It is an obligate hibernator and diurnal with a bimodal daily activity pattern. Reproduction is limited by a relatively short active season. The former metapopulation structure (Hanski 1997) of the species has disintegrated, and increasing loss of suitable habitat threatens population persistence (Spitzenberger 2001; Cepáková 2002; Kryštufek 1999; IUCN 2008). According to the 2008 IUCN Red List vers. 3.1, the species is considered vulnerable (A2bc), with a decreasing population trend (Coroiu et al. 2008). Due to its importance in the food chain, translocation of ground squirrels seems to be necessary to re-establish former populations, but critical skills and techniques are lacking.

In our study we attempted to develop and test easily applicable and low cost techniques to increase ground squirrel translocation success, and to contribute for a future translocation guide based on scientific results. From the abovementioned factors that influence the critical period after translocations (i.e., mortality and survival), both timing of release and retention of the animals in the artificial burrows require careful preparations. Therefore, we undertook a combined experiment to test and evaluate how two factors, morning against afternoon release and retention against no-retention of animals, influence the settlement and success of translocated ground squirrels during the critical period after release.

Materials and Methods Study area In April 18-20, 2000, 117 ground squirrels were reintroduced to a 160 x 120 m site (Fig. 1) in a protected grassland (~40 ha) north from the village of Tahitótfalu at Szentendrei Island, Hungary, belonging to Duna Ipoly National Park (N47° 47’ 13”; E19° 05’ 28”).

31

Figure 1 Map of the release site at Szentendrei island.

Ground squirrels became extirpated more than 10 years before the translocation and extensive grazing was terminated, resulting in vegetation transformation. To provide short grass habitat for ground squirrels (Kis et al. 1998) grazing with sheep was restarted in advance of the ground squirrel release. Additionally, we employed predator exclusion by keeping the release site under surveillance from hides and by fences (Rouco 2008). Capture and transport All founder animals were wild-born, reproductively active individuals trapped with snares (Hulová 2008) at the grassland of the Budaörs grassy airport (N47°27'7"; E18°59'9"). During trapping we found about a 1:1 sex ratio that we maintained during the release. Snaring is carried out by laying a thin, spiral copper snare around the burrow entrance. A piece of copper string is joined to a metal rod by tying a knot. The rod with the snare is stuck into the soil close to the entrance. The snare catches the animals when they enter or leave the burrow. The string is long enough to let the animal hide its head in the burrow. Usually, captured animals stay calm in this position until release. The animals stay in the traps about 15-45 minutes (our checking interval), depending on weather conditions. Temporary injuries occurred if the

32 checking interval exceeded 75 minutes due to bad weather or the snare was too strong and could not be torn. The injured animals were kept under surveillance until they recovered and then they were released on the capturing site.

Until release into artificial burrows, captured animals were housed in separated wire cages (10 x 10 x 40 cm) in a climatized trailer. We provided the animals with slices of fresh apple as an overnight water and food source. At the release site, there were neither remaining ground squirrels nor burrow remnants. Release method Release occurred at the end of April, which we considered the safest period for translocating ground squirrels. All individuals had emerged from hibernation, but pregnancies would all still be in the first trimester (Katona, Váczi, and Altbäcker 2002). In preparation for translocation, captured animals were arbitrarily divided into four groups. Each group was marked on the side of the body with neutral, animal livestock dyes of different colours (Dalton Ultra Spraymark, Livestock linemarker, Dalton Supplies, Oxfordshire, England). We released the individuals of two groups in the late afternoon of the day of capture. One group (plugged group, “A”; 30 individuals) was released into burrows plugged with wood caps after the animals entered the burrows, to retain animals in the burrows. The other group (unplugged group, “B”; 29 individuals) was released into burrows that remained unplugged. We released the individuals of the other two groups in the morning on the day after capture. Within these groups we applied the same treatments: plugged group (30 individuals) as “C”, and unplugged group (28 individuals), as “D”. In all group s, we released the animals into artificial, vertical burrows (50 cm long with 4.5 cm diameter) that served as seminatural places for shelter. We drilled the burrows with an electric device (Makita BBA520, Japan). The burrows were spaced 4.5 m apart in all directions. The animals in the two plugged groups were able to leave the artificial burrow only if they dug themselves out of the burrow. The animals in the two unplugged groups could leave or enter their burrows without any digging effort. We determined release locations of individuals belonging to different groups arbitrarily in the four 40 by 40 m grid cells. Care was taken to ensure a distribution of one individual per one artificial burrow. Releases were done for three consecutive days from 22 to 24 of April (1st day 39, 2nd day 46, 3rd day 32 individuals). Beginning from 28th April we began recapturing ground squirrels in the release grid; this continued for 5 consecutive days. Both recapturing and capturing were done during the activity peaks (0900 - 1100; 1400 - 1600) with the same 33 snaring method. Unfortunately, on the 11th day after release heavy rainfalls were coming and lasted for a few weeks, which made it impossible to continue recapturing and evaluate further the result of the translocation. Statistical analysis In our experiment, we compared the frequency of occurrence of recaptured animals in the four groups (morning release with retention cap “C”, afternoon release with retention cap “A”, morning release without retention cap “D”, and afternoon release without retention cap “B”) by Chi-square statistics. We used STATISTICA (data analysis software system), version 9.0 for the analysis.

Results During the 5-day recapture period we caught 25 different individuals. We recaptured significantly more individuals from the groups C and D released in the morning (18 individuals) than from the groups A and B released in the afternoon (7 individuals; χ2 = 5.8, df = 1, p < 0.05). When we compared the total number of recaptured animals from the “plugged” groups A and C (19 individuals) with the “unplugged” groups B and D (6 individuals) the difference was also significant (χ2 = 6.77, df = 1, p < 0.01). When we compared the number of recaptured animals in the group C (15 individuals) with the other three groups (A, B, and D; 4, 3, 3 recaptured individuals respectively) we found significant differences (χ2 = 8.39, df = 1, p < 0.005; χ2 = 11.43, df = 1, p < 0.01; χ2 = 10.94, df = 1, p < 0.001, respectively; Fig. 2).

Figure 2 Relative number of recaptured animals (recapturing percentage) after translocation in the 4 different groups. Y axis shows the relative percent of recaptured animals in each experimental group (number of recaptured individuals in one group/ number of all recaptured animals × 100).

Mortality at capture and handling procedure was under 4%. We did not find dead animals in the area or in any of the burrows after the translocation (the bottom of the artificial burrow 34 was visible with the help of a torch), which shows that plugging permitted the animals to dig out of the burrows and did not result in extreme cooling or loss of the animals within the burrows.

Discussion We tested if release time and retention of reintroduced animals in the release site affect initial colonization. Both factors should be considered as important determinants of reintroduction success. We recaptured the largest proportion of those animals that were released before their activity period, and were retained in the artificial burrows by plugging the entrance.

The daily activity peak of ground squirrels falls in the morning (between 0900-1100) (Katona, Váczi, and Altbäcker 2002). We recaptured more individuals from the group released in the morning than from the group released in the afternoon, consequently release time before the natural activity peak of the animal favours settlement on the release site in the critical period after translocation. In natural conditions, ground squirrels spend the most time aboveground at the morning activity peak. During the natural activity pause starting at noon, ground squirrels stay in the burrows (Váczi, Koósz, and Altbäcker 2006; Koshev 2008). Therefore release in or directly before the “midday siesta” (Katona, Váczi, and Altbäcker 2002) seems particularly disadvantageous for the animals in the critical period because their natural activity level is depressed. Release in the morning is advantageous as it provides more time for ground squirrels to acclimate to the new environment. This extended daily acclimation period is similar to the longer confinement period that results in short-term survival in rabbit reintroductions (Rouco 2008). Finally, we believe this technique is a valuable option to reintroduction specialists working with animals whose activity peaks occur during daytime hours (Moorhouse, Gelling, and Macdonald 2009). Releasing the animals without providing at least temporal shelter, results in panic reactions and erratic movements of ground squirrels. Our method of plugging, which forced them to stay an hour or two underground prevented such panic reactions and enhanced colonization if the release also coincided with their activity pattern. The squirrels started to establish their own burrows either by extending the original short burrows, or making new burrows in the unoccupied habitat surrounding the release grid. As we recaptured animals only in the release grid, the recapture rate (see Fig. 2) underestimates colonization success. Larger scale

35 monitoring and individual marking of animals would be necessary to test the exact movements. Even when predrilled artificial burrows are provided, the animals can be especially vulnerable in the first few days. The artificial burrow with retention cap provides shelter for the animals until they excavate their own burrow system and animals can shuttle between them and aboveground foraging sites. They work similarly to natural burrows, which provide a thermally stable environment underground and an option to avoid hypothermia and hyperthermia (Long, Martin, and Barnes 2005). From the practical point of view, our results show that the use of retention caps in combination with the artificial burrows could work as effectively as complicated and expensive acclimation cages (Simms 2009). Our techniques help ground squirrels to settle down at the release area and they can be applied for reasonable cost and effort (Fischer and Lindenmayer 2000).

Acknowledgments Duna-Ipoly National Park Directorate and Alsó-Tisza-vidéki Environment, Nature Protection and Water Authority permitted us the scientific work on the protected species (permission number: 1363/2/2000). In addition, we express our thanks to the anonymous reviewers for their useful and helpful comments, and in particular to Lee C. Drickamer for thoroughly editing the text. The experiments comply with the current laws of the Republic of Hungary.

36 VIII. Medium-height grass and angled artificial burrow entrance to favour European ground squirrel translocations

Subtitle: Translocation and habitat manipulation

Cs. I. Gedeon, G. Boross, A. Németh, V. Altbäcker Accepted to Wildlife Biology on 25 July 2011

Abstract Relocating ground squirrels has become a popular tool in Central Europe for conservation purposes; however few techniques have been carefully evaluated. Therefore wildlife managers lack tested and justified methods in reintroductions. To contribute to a translocation guide for ground squirrels, we evaluated conditions of habitat manipulation (grass height, artificial burrow entrance angle) that we expected to affect settlement of translocated ground squirrels during the critical period after release. In a field experiment we translocated 173 individuals from an international airport to a protected area in south-eastern Hungary in 2007. We released the animals into angled or vertical artificial burrows, in a design of a 300 × 300 m grid. We manipulated the grass height by creating medium- or short-height grass in the grid cells. We found that animals preferred angled (~ 30 º) artificial burrows and medium-height grass (18 cm ± 1.51). The angled slope of the burrow entrance facilitates digging it from the ground surface. Moreover, although ground squirrels choose the short grass habitat in undisturbed situations, overhead protection by grasses is valuable after a translocation. This result implies that in order to accomplish a successful translocation action it is not sufficient to know the habitat preference of a species in undisturbed situations, but tendentious experiments are needed to learn how and to what extent habitat characteristics should be manipulated. Keywords: burrow entrance angle, grass height, translocation, Spermophilus citellus

37 Introduction Relocating animals within their natural distribution range is a popular tool in wildlife management and conservation biology (Bajomi 2010). The technique is used to solve human- animal conflicts, to restock game populations for hunting, and to re-establish new populations of endangered species (IUCN 1987, 1998; Fischer and Lindenmayer 2000; Seddon, Armstrong, and Maloney 2007). According to Texeira (Teixeira 2007) the three main objectives of a reintroduction are: (1) survival of the animals after release, (2) settlement of animals in the release area, and (3) successful reproduction in the release area. Two of these objectives deal with the critical period (which is estimated two weeks post-release for the European ground squirrel (Spemophilus citellus = ground squirrel) because mortality rate proved to be the highest during this period in previous translocations), and the third aim talks about a self-sustaining population (Seddon 1999; Calvete 2004). Although, there is no general agreement on the ‘success’ of translocations (Seddon 1999), survival at the release site strongly depends on what happens in the critical period. Initial loss due to panic responses influences the fate of the animals or the success of the release in the critical period (Griffith 1989; Miller 1999; Gerber 2003; Cheyne 2006; Moorhouse, Gelling, and Macdonald 2009). Habitat quality is considered one of the most important elements of a species’ critical needs (IUCN 1998) to be addressed in a reintroduction. For ground dwelling sciurids, the habitat encompasses both the above-ground and underground environments. For natural colonies of ground squirrels, one of the most important factors of above-ground environment to favour survival of colonies is grass height (Kis et al. 1998). Animals abandon sites where the grass grows tall, and settle where short grassland is established.

For the underground habitat, ground squirrels have long and elaborated burrow systems (Ružić 1978), which provides a stable and safe environment against predators and unpleasant environmental conditions (Hut and Scharff 1998). Burrow entrances can be divided into two kinds: there are vertical burrows (pointing straight down at an angle of 90 degrees to a horizontal surface) and angled ones (sloping in one direction at an angle of 30-35 degrees to a horizontal surface) (Ružić 1978; Hut and Scharff 1998). As ground dwelling sciurids strongly depend on their burrow systems perpetually, the use of artificial burrows and acclimation cages is recognized as an essential tool in the reintroduction of ground dwelling rodents (Anstee and Armstrong 2001; Truett 2001; Simms 2009). During 38 reintroductions of ground squirrels, wildlife managers of national parks in Hungary usually applied vertical artificial burrows, because it is easier to drill in the soil and there has been no data if angled or vertical artificial burrows facilitate more the settlement in the critical period after reintroductions.

The European ground squirrel is an obligate hibernator and diurnal rodent with bimodal activity pattern whose reproduction is limited by a relatively short active season. According to the 2008 IUCN Red List vers. 3.1, the species is considered vulnerable, (A2bc), with a decreasing population trend (Coroiu et al. 2008). It is also listed in Appendix II of the Bern Convention and the Annexes II and IV of the EU Habitats and Species Directive. Its importance in the short grass steppe habitat is underlined by several facts e.g., it is one of the main preys of several protected raptors (e.g., Falco cherrug, Aquila heliaca) (Kovács 2008); its burrows serve as a refuge for the protected Bufo viridis and Vipera berus (Váczi 2005). In addition, as the ground squirrel has adapted well to live on grassy airports (Váczi, Koósz, and Altbäcker 2006) or recreational parks (Hoffmann, Turrini, and Brenner 2008), its high density can create damage and safety concerns in airports through raptors that predate them and through burrow mounds that are potentially hazardous to small airplanes during landing and taking off. Thus regulating its abundance by translocations is sometimes a remedy to solve the human-animal conflict (Massei 2010).

As a result of the decline in ground squirrel populations, reintroduction as a management tool has been used frequently in Hungary (e.g., in 2007 and 2008 more than 2000 animals were released in Natura 2000 habitats according to the Interim Report (LIFE06 NAT/H/000096), and about 200 animals were reintroduced to from Hungary between 2005 and 2007 (IUCN 2010). Nevertheless, only a few studies provided well-documented information that can be linked to the success or failure of these reintroductions. Consequently, nature conservation managers lack an evidence-based translocation guide for ground squirrel translocations.

In our study we attempted to test important determinants of pre-release habitat manipulation on the reintroduction success. We undertook a combined experiment to test and evaluate how (1) grass height on the release site and (2) entrance angle of artificially drilled burrows can influence the successful settlement during the critical period after translocation of wild-caught 39 ground squirrels. Both features require only careful design during planned translocations without additional high costs.

Material and methods 173 reproductively active (about 1:1 sex ratio) ground squirrels were captured at Ferihegy International Airport (N47°25'8.88"; E 19°15'7.20") and then reintroduced to Szeri-puszta (N46°31’ 19"; E20°04'44"), a protected grassland (~ 150 ha) in the Kiskunság National Park, in the Duna-Tisza Köze region, Hungary, on 16 July, 2007. Because of human safety concerns at the airport we were required to implement capturing as quickly and smoothly as possible. Consequently, capturing involved several persons and required very careful preparation and management of capturing, transport, and release. These circumstances combined with high animal density at the airport allowed us to capture 173 animals during less than two days (14 and 15 July), and finally to release them on 16 July at Szeri-puszta. Ground squirrels became extinct more than 10 years before the translocation on the release site, because extensive grazing by sheep was terminated and resulted in vegetation transformation. Consequently, there were neither remaining ground squirrels nor burrow remnants on the release site. To provide short grass habitat for ground squirrels (Kis et al. 1998) grazing with sheep (Hungarian Merino and Racka) was restarted in advance of the ground squirrel release. Additionally, after and during the translocation, we employed predator exclusion on the release site by guarding continuously the animals from hides for two weeks (Rouco 2008). During this period volunteers were observing and guarding the area from the close-by tent under the supervision of an expert. After the animals entered into daily rest (including all night), guards applied spotlight for checking the area.

Ground squirrel trapping was carried out by snaring (Hulová 2008; Gedeon, Váczi, et al. 2011). Our snare was a piece of 0.2 mm copper string joined to a metal rod by tying a knot, and placed around the burrow entrance. The rod with the snare is stuck into the soil close to the entrance. The string is long enough to let the animal hiding its head in the burrow. The snare catches the animals when they enter or leave the burrow. A captured animal usually stays calm in this position until release, but if they move frantically, the wire brakes and the animal is not hurt. Due to regular checking, the animals stay in the traps for a maximum of 45 minutes or shorter, depending on actual weather conditions. Temporary injuries occurred in

40 less than 1% of animals if the checking period had exceeded 75 minutes due to bad weather and the snare had been too strong and could not be torn. The injured animals were kept under surveillance until they recovered and then they were released on the site of capture. Until release into artificial burrows, captured animals were housed in separated wire cages (10 × 10 × 40 cm). We provided the animals with slices of fresh apple as an overnight water and food source. All animals were released in the morning period, on the day after capture, and we applied retention bottles to keep animals in the burrows and eventually to increase translocation success in the critical period. Retention bottles are poorly transparent glass bottles (0.5 Litre). The practical advantages of them combined with artificial burrows that they work as effectively (Altbäcker 2005) as complicated and expensive acclimation cages (Simms 2009) but they are even more cost efficient (Fischer and Lindenmayer 2000), easily applicable, and non-invasive than retention caps (Gedeon, Boross, et al. 2011). These bottles were removed from the burrows next day when we checked that animals were able to dig out themselves from the burrows (Fig 1).

Figure 1 Picture on a retention bottle at the release site.

The release site (a 6 × 6 cell grid; 50 × 50 m) included the 200 × 200 m inner area, where the animals were released, and the 50 × 50 m buffer zone around the inner area (Fig 2).

41

Figure 2 Map of the release site and the experimental design in Szeri-puszta.

The release site was signed with signposts (49) in each grid cell corner, and within the inner area (200 × 200 m; 4 × 4 cell grid), 50 cm long and 5 cm wide artificial burrows were drilled with an electric device (Makita BBA520, Japan). We drilled two kinds of artificial burrows: angled and vertical burrows, similarly to the natural burrow types (see above). The order of artificial burrow types (angled or vertical) within a grid cell was tessellated and burrows were spaced 12.5 m in all directions. It resulted in a total of 256 artificial burrows in the inner area (16 grid cells), 128 angled and 128 vertical burrows. In other words we had equal number of burrows in grid cells either with medium-height (unmown) grass or with short (mown) grass (see below for the description of unmown and mown grass). The animals were able to leave the artificial burrow only if they dug themselves out of the burrow as we plugged the animals in the burrow with retention bottles (Fig 1). In order to prevent introducing ground squirrels to an already occupied burrow due to movement of previously released animals, all artificial burrows were plugged in with bottles before the operation. Plugs were removed immediately before the release to that particular burrow and placed back again. This method guaranteed one individual per one artificial burrow.

The grass height of the grid cells was alternately mown (short grass; mean = 6 cm ± 0.55; range: 0 – 14 cm) or unmown (medium-height grass; mean = 18 cm ± 1.51; range: 2 – 44 cm) by a tractor before the release (Fig 1). Grass height was measured during the experiment at

42 each data record occasion by measuring the height of the longest blade of grass at 9 randomly chosen locations within each grid cell (Váczi and Altbäcker 2005). We recorded on a map of the release site if a burrow had been occupied by an animal on each data recording day. We defined a burrow as occupied if fresh soil or fresh faecal pellets were at the burrow entrances. After the release, we counted the number of inhabited man-made (angled, vertical) burrows as well as burrows dug by the animals in each cell on the release site, 6 times (1, 2, 3, 7, 37, 72 days after release; 17, 18, 19, 23 July, 22 August, and 26 September) after translocation. After each data collection we removed the indicators of occupancy (fresh soil or faecal pellets), so that at the next data collection we did not record the burrow as occupied based on older signs.

Before we counted the number of inhabited burrows, we always did visual census (observation and counting) of the ground squirrels in eight 50 × 50 m grid cells from the same hide close-by (Váczi, Koósz, and Altbäcker 2006) by one observer. Basically, it applies a scanning procedure (Altmann 1974). The visual census was done during the activity peak between 0900 – 1100. Each grid cell was scanned for 5 minutes with a regular binocular. It resulted three visual censuses from each grid cell during the two hour long observation sessions (one observation session lasted for 40 minutes). From the 5-minute long observation of one grid cell, we used the maximum number of distinct animals seen at the same time within each grid as data. By this method we avoided double counting of some animals. Observations began one hour after arriving at the colony to allow for habituation to the observer.

Statistical analysis

In the analysis we applied a multivariate test for repeated measures on data of occupancy of burrows or number of observed animals, since normality assumptions were violated (Mauchley Sphericity Test and Shapiro-Wilk Test), and because grouping variables (grass height, as medium-height and short; burrow form, as new or artificial; burrow type, as slanted or vertical) were interacted with each other. The main ‘between-effect factors’ in the analysis correspond to the main questions or grouping variables in the experiment (primarily grass height and burrow type, secondarily burrow form), whilst ‘within-effect’ was ‘Days after release’, analysed as repeated measures. 43 For the X values of ‘Days after release’, the time interval between consecutive data recording occasions was not even (data recording in days after the release: 1, 2, 3, 7, 37, and 72). To equalise the differences, we linearized data recording days (X values) by applying a log10 transformation of days. It resulted in a linear trend between occasions (1 – 6 on X axis) and log10days (Y axis; correlation coefficient, r = 0.96). Basic statistical results are given as mean ± SE. We used STATISTICA (data analysis software system), version 9.0 for the analysis.

For Figures 3-6 ‘Medium-height grass’ was shown with grey colour and ‘Short grass’ was shown with black colour. X axis always shows the linearized data recording days (1, 2, 3, 7, 37, 72 days after release) which corresponds to 17, 18, 19, 23 July, 22 August, and 26

September by applying a log10 transformation of days after release. Y axis always shows the mean number of inhabited burrows per grid cell and its standard error (mean ± SE).

Results Significant grass height difference was maintained between short (6 cm ± 0.55) and medium-height (18 cm ± 1.51) grass grid cells during the whole experiment (Grass height: F = 81.64, df = 1, 14, p < 0.001; Days after release: F = 11.71, df = 1, 70, P < 0.001; Interactions: F = 406.24, df = 1, 14, p < 0.001; Fig. 3).

Figure 3 Change of ‘Grass height’ in the grid cells of the release site.

However, plant height on ‘medium-height grass’ grid cells decreased significantly between 37 and 72 days after release because of extreme drought during the experiment. The results 44 showed that the number of inhabited burrows changed significantly from the 1st day after the release until the 72nd day, by the time all animals disappeared from the surface, and most probably hibernated (Days after release effect; Wilks lambda = 0.65; F = 5.84, df = 5, 53, p < 0.001. Burrow occupancy changed from the 1st day until the 72nd day, with a significant increase in burrow occupancy on the 7th day after the release compared to the first three days (p < 0.01). Then the number of inhabited burrows did not change significantly until the 72nd day (Fig. 4).

Figure 4 Change of the number of inhabited burrows in relation with ‘Days after release’ and ‘Grass height’.

Comparing the change in the number of inhabited new vs. artificial burrows, the number of inhabited artificial burrows descended while the number of inhabited new burrows ascended during the experiment (Days after release * Burrow type (new or artificial) effect; Wilks lambda = 0.35, F = 19.08, df = 5, 53, p < 0.001; Bonferroni post-hoc test: p < 0.001; Fig. 5).

45

Figure 5 Change of the number of inhabited burrows in relation with ‘Burrow type (New or Artificial)’ and ‘Grass height’.

The change in numbers of inhabited angled burrows paralleled the pattern for all artificial burrows: numbers first increased then decreased (after the 7th day), while the number of inhabited vertical burrows increased steadily. (Days after release * Burrow form (Slanted or Vertical) effect; Wilks lambda = 0.54; F = 9.06, df = 5, 53, p < 0.001). Nevertheless, the overall difference between angled and vertical artificial burrows was not significant (Bonferroni post-hoc test: p = 0.31; Fig. 6).

Figure 6 Change of the number of inhabited burrows in relation with ‘Burrow form (Angled or Vertical)’ and ‘Grass height’.

Although the number of inhabited burrows was always higher in the medium-height grass than in the short grass (Fig. 4), the difference was not significant (Days after release * Grass 46 height effect; Wilks lambda = 0.88, F = 1.51, df = 5, 53, p = 0.20). Regarding the number of observed animals in medium-height or short grass grid cells, we observed more animals in the short than in the medium-height grass (Wilks lambda = 0.32, F = 4.29, df = 5, 10, p < 0.05). However, it should be borne in mind that the average number of animals observed in short or medium-height grass was equal to one or even less. Therefore, if we took into account the interaction of ‘Days after release’ and ‘Grass height’ effects the difference in the number of observed animals disappeared (Days after release * Grass height effect; Wilks lambda = 0.57, F = 1.5, df = 5, 10, p = 0.27).

Mortality at capture and handling procedure was less than 1 %. We did not find dead animals on the release site neither in the burrows nor near the burrows after the translocation (the bottom of artificial burrows seemed empty with a torch). It shows that plugging with bottles permitted the animals to dig out of the burrows and did not result in extreme cooling or loss of the animals within the burrows.

Discussion Our results suggested that angled artificial burrows in comparison with vertical artificial burrows and medium-height grass in comparison with short grass favour translocation success in the critical period after release. The artificial burrows, as expected from earlier findings (Truett 2001; Simms 2009), provided shelter and refugia for the animals in the critical period before they excavated their own burrow system. Translocated animals could use them to shuttle between the burrows and the above-ground foraging sites according to their needs. This result is in accordance with the finding of (Ružić 1978), whose study implied that angled burrows are dug from the outside in contrast to vertical ones. From a logical point of view we can also add that scratch digging is expected to be very hard in a vertical burrow because the excavated soil would fall back on the digger.

As the environmental conditions were stable in the summer of the translocation (i.e., summer drought; no precipitation from the day of the translocation until the end of September), we can say that the end of the critical period can be more closely determined in the light of the results regarding burrow occupancy. The increasing trend of artificial burrow usage (both vertical

47 and angled burrows) changed to decreasing between the 7th (23 July; highest number of inhabited artificial burrows) and 37th day (22 August) after the release. In parallel with this trend, the number of new burrows dug by the animals began to increase around the 7th day after release. We might conclude that the critical period ended between 7th and 37th days after the release. It means that the relocated animals got over the stress being caused by the disruption of their normal way of life in the original habitat (Teixeira 2007) within this period. This finding is in accordance with the result of (Calvete 2004) who determined the critical period for translocated rabbits (Oryctolagus cuniculus) about 7-10 days after release. Nevertheless, as the trend changed within this approximately 30-day long period and we did not have additional sampling events during this term we are not able to specify more precisely the length of the critical period after the release. Additionally, there must be other elements that could be influencing the burrow use of the animals. Consequently, any more inferences on the length of the critical period would be speculative.

The discrepancy between the counted number of inhabited burrows and the number of observed animals after release should also require some explanation. First, microrelief of the release site was unfavourable for visual observations in the area; second, the size of the animals’ home range (Turrini et al. 2008) and the grid cell area per capita (between 208 and 227 m2) is comparable in magnitude. Therefore the animals may have left the home grid cell for feeding and exploration, resulting in positions different from home grid during the observation periods. However, each individual uses one burrow system only in nature (Hut and Scharff 1998; Millesi 1998). Additionally, since surplus (256 burrows were drilled but only 173 ground squirrels were captured and released), artificial burrows were plugged before the animals were released, all burrow occupancy represented one ground squirrel (double counting was avoided by this method; animals were not able to use these extra burrows). Consequently, we were better able to detect the presence and location of the animals in the grid by counting occupied burrows than by visual scanning or census of animals. Burrow occupancy records seemed a more reliable and conservative technique than visual census.

Our results also suggest that the effect of vegetation height on settlement of released ground squirrels at release site cannot be left out of consideration. In relocating water voles, Moorhouse and co-workers (Moorhouse, Gelling, and Macdonald 2009) also concluded that the proper habitat characteristics were vital for the long-term persistence. Kis and co-workers’ 48 (Kis et al. 1998) results suggested that ground squirrels prefer short vegetation to tall grass. There are several hypotheses for the preference of short grass in the diurnal ground squirrel within natural conditions: (1) it enables predator detection, (2) it helps in social communication between conspecifics, and (3) moving and navigation may require less energy in shorter than in taller grass. Newly translocated individuals, however, may have different preference for settlement than individuals in a natural, “undisturbed” population of the same species. Our results showed that ground squirrels initially preferred the area where the grass was medium-height. It seems probable that grass tussocks provided a natural hide for the translocated individuals in the first days like shrubs provide overhead protection for Octodon degus (Ebensperger and Hurtado 2005; Hardman 2006). Our experience of having difficulties in observing the animals in those grid cells where the grass was higher confirms this explanation. In general, this suggests that it is not enough to know the habitat preference of a species in undisturbed situations, but tendentious experiments are needed to learn how and how much we should manipulate the habitat characteristics to accomplish a successful translocation action.

The success of a translocation can and should be evaluated on multiple temporal scales (Seddon 1999; Simms 2009). Post release monitoring of the animals occurred via observation until end of September, when ground squirrels usually go to hibernation (Millesi et al. 1999) in Hungary. Although long-term success was not studied and recorded with the same method, monitoring took place until 2009, and the national park directorate in charge of the protected area will continue to monitor in the following 5 years, too. In 2008, after the first hibernation and on the national monitoring day of the ground squirrel programme of the Hungarian Biodiversity Monitoring System (Váczi and Altbäcker 2005), about 30-40 individuals were observed in an adjacent area to the release site. In 2009, this number increased to 60-80 individuals, occupying a larger area than in the preceding year. Following the generally used visual census technique (Simms 2009) and burrow counting method (Váczi and Altbäcker 2005; Koshev 2008) to monitor ground dwelling sciurids, the national park directorate monitoring coordinator (Tamás Nagy, pers. comm.) estimated the number to be 100-120 individuals. The increasing number of the animals in these two years seems to fulfil the requirement of a ‘successful translocation’ in the long-term too (Seddon 1999). Finally, our experiments confirm the findings that long-time persistence, which primarily depends on longevity and reproductive success, is strongly connected to the first (critical) 49 period after release of the translocated animals, and habitat quality (grass height and artificial burrow entrance angle similar to natural ones with mounds) at release site has a large effect on this critical period (Letty, Marchandeau, and Aubineau 2007; Moorhouse, Gelling, and Macdonald 2009).

Acknowledgments-this study was accomplished in the framework of the Falco cherrug LIFE programme (LIFE06 NAT/H/000096). We are extremely grateful to Tamás Nagy, State Ranger Service, Kiskunság National Park Directorate, for his still ongoing collaboration and enthusiasm to help the study. In addition, we express our thanks to the anonymous reviewers for their useful and helpful comments, and in particular to Lee C. Drickamer for thoroughly editing the text. Duna-Ipoly National Park Directorate and Alsó-Tisza-vidéki Environment, Nature Protection and Water Authority permitted us the scientific work on the protected species (permission numbers: 1231/1/2007). The experiments comply with the current laws of the Republic of Hungary.

50 IX. Importance of burrow-entrance mounds of Gunnison’s prairie dogs (Cynomys gunnisoni) for vigilance and mixing of soil

Csongor I. Gedeon, Lee C. Drickamer, and Andrew J. Sanchez-Meador

Accepted to The Southwestern Naturalist on 1 November 2010

Abstract Aboveground mounds and underground burrows are multifunctional and influence behavior and habitat of Gunnison’s prairie dogs (Cynomys gunnisoni). Four colonies were studied June- September 2004 to examine function of mounds with respect to vigilance, and to estimate magnitude of soil mixed by these prairie dogs. Frequency of vigilance atop mounds increased in taller vegetation and individuals at perimeters of colonies oriented toward the outside more frequently than to the interior of colonies. Mounds accounted for an average of 10,374 kg of soil/ha that was excavated from the burrow. This mass of subsoil moved to the surface and the 7- 17 m3 of air in the burrow make the geomorphic effect of prairie dogs potentially significant.

Resumen Montículos de tierra construidos en la superficie y tuneles construidos bajo tierra tienen muchas funciones y afectan el comportamiento y el hábitat de los perritos de las praderas de Gunnison (Cynomys gunnisoni). Cuatro colonias de esta especie fueron estudiadas de Junio a Septiembre del 2004 para tratar de determinar la función de los montículos con relación a la vigilancia contra depredadores, y también para calcular la cantidad de tierra mezclada por estos animales. La frecuencia de los comportamientos de vigilancia cuando los animales se encontraban sobre los montículos aumento cuando la vegetación estaba mas crecida (mas alta); además, los animales en las márgenes de las colonias se encontraban con más frecuencia orientados hacia afuera en vez de hacia el interior de la colonias. Los montículos contenian un promedio de 10.374 kg de tierra por hectareo excavados del tunel. Esta cantidad de tierra transportada a la superficie, y los 7-17 m3 de aire en el tunel, indican que el efecto geomórfico de los perritos de las praderas es considerable.

51 Introduction Prairie dogs (Cynomys) dig burrows that serve numerous functions, e.g., protection from the environment, defense from predators, as a place to store food, hibernation, and reproduction (Pizzimenti and Hoffmann 1973; Meadows 1991). During burrowing, subsoil is mixed and deposited at the opening, which results in bare, loose mounds that increase heterogeneity of habitats (Whitford and Kay 1999). Mounds also protect against flood ing. Black-tailed prairie dogs (C. ludovicianus) use mounds as vantage points to scan for predators or conspecifics (King 1959, 1955). Vigilance behavior comprises a significant part of aboveground behavior of prairie dogs (Longhurst 1944); its frequency varies between pre-juvenile and post-juvenile periods (Verdolin and Slobodchikoff 2002) and by location within the colony (Armitage 1962; Svendsen 1974; Hoogland 1979). Relatively high mounds and low vegetation facilitate better detection of predators and receiving visual signals from conspecifics. Whether taller vegetation is protective or obstructive for prairie dogs is not clear. In constructing burrows, Gunnison’s prairie dog (C. gunnisoni) leaves excavated dirt at the opening to burrows without further modification, which results in asymmetric mounds (Pizzimenti and Hoffmann 1973; Fitzgerald 1974). Mounds usually are neither disproportionately downhill on slopes (Longhurst 1944), nor do they have specific orientation on flat areas (Bailey 1931; Scheffer 1947). For burrowing mammals, size (i.e., surface area and volume) of mound varies (Vogel, Ellington, and Kilgrove 1973) and whether the mound at the opening to burrows correlates with size of burrow system is questionable (Butler 2006; Schulz 1978). Each burrow system of C. ludovicianus mixes ca. 200-225 kg of soil (Whicker 1988). With 50-300 openings and 25-300 systems/ha (Butler 2006) this translates to 5,000-67,500 kg of mixing of soil/ha. The extent of mixing of soil by prairie dogs is considerable in terms of processes in soil and modification of habitat (Hansell 1993). We examined use of mounds at openings of burrows for vigilance and mixing of soil from construction of burrows. Gunnison’s prairie dogs inhabit the Four Corners region of the Southwest (Hoogland 2003). Main goals of our study were to analyze vigilance behavior by Gunnison’s prairie dog in relation to their mounds and to evaluate the magnitude of mixing of soil via building mounds. We collected field data on four colonies June-September 2004 within Flagstaff, Coconino County, Arizona. Colonies were separated by uninhabitable urban areas. We designated study areas as ball field (35°13'N, 111°35'W), old cemetery (35°12'N, 111°34'W), 52 golf course (35°12'N, 111°34'W), and police station (35°11'N, 111°39'W). Colonies were similar in population density, habitat (secondary growth, high-desert grasses and forbs), and exposure to humans and vehicular traffic. Behavioral observations followed methods of (Verdolin and Slobodchikoff 2002). We observed each colony once every 7 days for 2 h. Two colonies were observed in the morning (0800-1100 h) and the other two colonies were observed in the afternoon (1500-1800 h). These periods correspond to bimodal activity peaks of Gunnison’s prairie dog (Longhurst 1944; Rayor 1988). We switched morning and afternoon observations so that colonies were monitored in morning or afternoon every other day. We repeated behavioral measures for 4 weeks, providing 8 h of observation/colony. Observations were made over areas 10,000 m2 in size within each colony from natural elevations and blinds. Prairie dogs were not marked for identification during this study to avoid artificially increased vigilance and altered behavior. Because emergence of juveniles significantly modifies magnitude of vigilance behavior, we studied colonies after juveniles emerged from burrows (Verdolin and Slobodchikoff 2002). To obtain behavioral information in relation to mounds, we used binoculars and instantaneous-scan sampling (Altmann 1974). A 30-min observation session was divided into 10 3-min sampling intervals. We classified behavior into five categories, i.e., resting-lying (lying or four feet on the ground), vigilance (Verdolin and Slobodchikoff 2002), burrowing (digging activity at the burrow), social interaction (aggressive or amiable behavior), and other (behavior not in the four named categories and most often meant off-mound feeding). We determined whether prairie dogs were on or off the mound at each record of their behavior. We further assessed locations as high or low on the mound, or inside the opening of the burrow. We measured height of vegetation as tall (>1 m), medium (1-0.5 m), or short (<0.5 m) on each site by using four transects across the colony. Orientation of vigilance behavior at the periphery and interior areas of colonies was recorded at two locations (golf course and old cemetery) using four compass directions (N, E, S, and W). We designated four subareas on each site to record vigilance behavior. We used 30-min observation sessions, divided into five 6-min intervals; this provided 2 h of observation/subarea. We defined the periphery as a 10-m-wide space at the edge of the colony. Using a map of the study area, we defined whether animals at the periphery looked outward from the colony, looked inward, or direction could not be determined. Examination of data for prairie dogs in the center of the colony revealed no directional bias, and data were not analyzed further. We estimated density of burrows (openings/ha) by recording exact locations of all 53 inhabited burrows for the 10,000-m2 observation areas at each site. These areas encompassed 3- 25% of total area of colonies (police station 25%, old cemetery 20%, ball field 6%, and golf course 3%). A burrow was occupied if fresh soil or fresh fecal pellets were at the opening. We tallied 824 active openings across all four locations (ball field, 161 openings/ha; old cemetery, 190 openings/ha; golf course, 215 openings/ha; police station, 258 openings/ha). To record locations of openings we used a Trimble GPS R4 GPS Receiver with a post-processed differential GPS for correction of data. We estimated density of mounds and number of prairie dogs by dividing the count of inhabited burrows by three, because we assumed that on average three openings belong to one burrow system (Butler 2006; Verdolin, Lewis, and Slobodchikoff 2008). We estimated size of population by multiplying number of prairie dogs in our 10,000-m2 observation areas by area of the entire colony. For estimating size of mounds, we first measured≥15 independent, randomly chosen mounds on each study area (total n = 68 mounds). Each mound was divided into eight equal- sized pie-sections. Volume of each pie-section was determined based on the polar-grid system, which made it possible to determine surface area of a pie-section from the pie-angle (α = 45°) and radius of pie-section. Knowing surface area of a pie-section, we were able to calculate volume by using the formula:

2 2 n  d   d   45 id +  − id −  πh = ∑  i V pie , i=1  2   2   360 where n is number of measurements of height, d is radius of pie-section, and h is height of that section. Because the circular mound was divided into eight sections, the polar angle was always 45°. Because height of mound changed from the opening to the edge of the mound, it was necessary to measure height of mound at several intervals. We placed a metal rod vertically into the ground at each opening. By means of a level, a string, and a ruler, we measured height of mound (from surface of the ground) every 10 cm from the opening to the outer edge of the mound in each pie-section. To validate calculations, we directly measured volume of pie-sections with a 1-L cylinder (accurate to10 ml) for 64 pie-segments from eight mounds. We compared measured and calculated volumes of pie-segments using linear regression. From these calculations, we estimated volume and weight of a mound. We used these findings, combined with our estimates

54 of density of burrows, to assess magnitude of mixing of soil. We used the calculation of Vleck (1981) to determine how much soil was moved to the surface during burrowing. To determine if mound and burrow were of equal size, we compared mass of a mound with the theoretical, total mass of soil removed from a burrow system of average length. All statistics were performed using Statistica (version 8.0, www.statsoft.com). To calculate percentage of vigilant individuals, we followed (Verdolin and Slobodchikoff 2002), where vigilance, P, was equal to Va/Na; Va was number of prairie dogs vigilant for each observation session and Na was number of prairie dogs observed during each session. These values were arcsine-transformed to meet assumptions of normality. In analysis of behavioral data, repeated-measures analysis of variance was used. We used a binomial test to analyze data concerning differences in vigilance directed to the periphery versus inward toward the colony. Procedures were approved (protocol 2002-31) by the Northern Arizona University Institutional Animal Care and Use Committee.

Vigilance was significantly related to position on the mound (F7,16 = 81.72, P < 0.001, 2 Partial η = 0.97) and height of vegetation (F11,70 = 14.16, P < 0.001, Partial η2 = 0.91), but not to time of day (Fig. 1).

Figure 2 Average percentage of behavior (vigilant and other) by Gunnison’s prairie dogs (Cynomys gunnisoni) as a function of height of vegetation (tall, medium, short), time of day (a, morning; b, afternoon), and location of animal relative to the mound at the opening of the burrow. 55 Vigilance was performed most frequently on top of mounds (P < 0.001). Proportion of individuals being vigilant increased from short to medium and tall vegetation; vigilance was greater in tall vegetation versus short vegetation (P < 0.001) and greater in medium vegetation versus short vegetation (P < 0.01). Animals on the perimeter of the colony looked outward significantly more frequently than expected by chance when performing the vigilance posture (binomial test: n = 271, P < 0.001). Neither volume nor surface area of mound were significantly different on the four sites 2 (F6,130 = 1.46, P = 0.200, Partial η = 0.06). Consequently, we used the composite mean (data from all four sites) of volume and surface area of mound in all calculations related to mounds. Average density of mounds was 68.7/ha (± 6.86 SE), with an average area of 2.092 m2 (± 0.13 SE) and volume of 0.15 m3 (± 0.02 SE), which is equal to ca. 151-242 kg of soil if density of soil is 1-1.6 g/cm3. Taking into account only the direct effect of construction, this translates into 143.72 m2 and 10,374-16,625 kg of disturbance to soil on average/ha. Average diameter of burrow was 13.8 cm (± 1.3 SD). Gunnison’s prairie dogs mix ca. 15-24 kg of soil during construction of 1 m of burrow. If we extrapolate from mean length of burrow in (Verdolin, Lewis, and Slobodchikoff 2008), we assume that these prairie dogs mix ca. 210-335 kg of soil/burrow system (14,427-23,015 kg/ ha). There was a significant positive 2 relationship between maximum diameter and volume of mounds (R = 0.73, F1,66 = 181.28, P < 0.001).

Discussion We draw four conclusions. 1) Gunnison’s prairie dogs use mounds for vigilance; vigilance increases with taller vegetation. The higher frequency of vigilance in situations with medium and tall vegetation indicates the ability to adapt behavior to changing natural conditions. As vegetation grows during summer, changing the rate of vigilance provides greater security. This is similar to shifts in vigilance with changes in vegetation observed in degus (Octodon degus; (Ebensperger and Hurtado 2005). 2) Vigilance is more frequent by individuals on the periphery than those in the interior of the colony, and vigilance activities by those on the periphery are directed outward from the colony (Verdolin and Slobodchikoff 2002). It is adaptive to the colony to have an early warning system that is focused to potential danger from outside the colony. Ariel predators can come from any direction, but ground predators will approach the periphery. Greater vigilance by individuals living in perimeter

56 areas that is directed outside the colony provides warnings of potential ground predators. 3) It is possible to estimate volume of burrows by measuring amount of subsoil displaced on the mound. This discovery should permit better estimation of volume of burrows and it provides a method, using the pie-shaped sections and computational formula, to achieve that end. Information on volume of burrows can be applied to situations where prairie dogs are being translocated and established in artificial burrows. 4) During construction of burrowsystems, Gunnison’s prairie dogs move and mix 10,000-16,500 kg of soil/ha, which is within the range excavated by black-tailed prairie dogs (Butler 2006). Effects of burrowing animals as ecosystem engineers has a significant geomorphological impact on many landscapes (Gabet, Reichman, and Seabloom 2003), but measuring magnitude of bioturbation by a population, colony, or individual is difficult (Nevo 1999). North American pocket gophers (Geomyidae), acknowledged engineers of ecosystems, excavate ca. 18 m3/ha/year on average (Smallwood 1999). For Gunnison’s prairie dogs, we determined that excavation of soil was 6.5-16.6 m3/ha, taking into account only the volume of mounds as excavated soil. This indicates that Gunnison’s prairie dogs are having an ecological impact equal to effects of pocket gophers, which provides support for statements made by (Detling 1987) and fits the ecosystem-engineer concept (Jones 1994; Gutiérrez 2006).

We acknowledge B. Birch, M. Keindl, and N. Sieben for help and advice on statistical analyses, E. Volodina, I. Volodin, A. Zareva, J. Hoogland, and D. Wagner kindly provided helpful comments on early versions of the manuscript, and A. Novák and M. Sandy translated the abstract into Spanish. Two anonymous reviewers and J. Frey provided valuable assistance in completing the final version of the manuscript. This research was supported, in part, by a Hungarian-American Fulbright Commission Scholarship to C. I. Gedeon and by the Department of Biological Sciences at Northern Arizona University.

57 X. Dwindling genetic diversity in European ground squirrels

Hichem Ben Slimen*, Csongor I. Gedeon*, Ilse E. Hoffmann, and Franz Suchentrunk * contributed equally to this paper

Submitted to Mammalian Biology

Abstract Populations of the European ground squirrel (Spermophilus citellus) are increasingly fragmented, and only a coordinated conservation effort at the European level may guarantee long time survival of this species. To evaluate the suggestion of an earlier study that Czech populations were genetically depleted, inbred, and strongly genetically isolated, and to obtain a more general population genetic picture on a larger geographic scale, we screened 116 individuals from seven local populations in Hungary, , and for allelic variability at 14 microsatellite loci. Of those, eleven were not monomorphic and could be used for population genetic calculations. We found a high (23.4%) proportion of private alleles, but only 12.4% of genetic diversity was partitioned among populations. Genetic variability was significantly higher than in the earlier studied Czech populations, but significantly less than in undisturbed populations of S. suslicus and S. brunneus, which have a similar biology. It was also lower than in most presumably undisturbed populations of other Sciurid species. This, together with the observed degree and pattern of genetic differentiation among populations, such as pairwise FST values across geographic distances, suggested disintegration of metapopulation structures. A Bayesian STRUCTURE analysis suggested anthropogenic translocations among Hungarian populations. Indeed, our data on genetic diversity and its distribution do not object to such conservation measures; actually, we advise such conservation measures in addition to others for local and isolated populations to increase chances of survival and to avoid inbreeding at low densities.

Keywords genetic differentiation, genetic diversity, Marmotini, population genetics, Spermophilus citellus

58 Introduction The European ground squirrel (Spermophilus citellus (L., 1766)) is an endangered species in decline. Populations are increasingly fragmented, and only a coordinated conservation effort at the European level may sustain their viability. According to the 2008 IUCN Red List vers. 3.1, the species is considered vulnerable, (A2bc), with a decreasing population trend (Coroiu et al. 2008). It is also listed in Appendix II of the Bern Convention and the Annexes II and IV of the EU Habitats and Species Directive. The species’ distribution is split into two major ranges in central and south-eastern Europe and several additional isolated patches (Fig. 1; (IUCN 2008; Ružić 1978).

VOR DUN PÉL

VIE

SIÓ

PEC DET

PEC Black VIE Sea PEL500 km SIÓ DUN DET VOR

Figure 3 Geographic range of S. citellus (hatched areas, IUCN 2008) and populations studied: DET – Denis Tepe (Romania), n = 20, DUN – Dunakeszi (Hungary), n =19, PÉL – Pécel (Hungary), n = 8, PEC – Pecs (Hungary), n = 16, SIÓ – Siófok (Hungary), n = 16, VOR – Vöröskővár (Hungary), n = 15, VIE – Vienna (Austria), n = 23. Inset: Three-dimensional plot of FCA-coordinates for populations.

Increasing loss of suitable habitat is considered one of the major threats for population persistence (IUCN 2008; Cepáková 2002; Kryštufek 1999; Spitzenberger 2001). 59 Fragmentation and deterioration of available habitat, along with the possibility of stochastic processes at relatively low effective population sizes, are adverse maintaining local genetic diversity. This in turn might result in local short-term viability problems due to inbreeding depression. In the longer term, loss of genetic diversity in small isolated populations reduces their capability to adapt to future environmental challenges. Any conservation measures to be undertaken for local colonies or regional populations should consider levels and patterns of genetic diversity. First data on Czech and Slovak S. citellus populations showed low genetic variability, high levels of inbreeding, and strong genetic differentiation among populations (Hulová 2008). In this study, we estimate genetic diversity at eleven polymorphic microsatellite loci of seven S. citellus populations from Hungary, Romania, and Austria, and compare the data particularly to those of the Czech populations (Hulová 2008), to 1) obtain a more general picture of genetic diversity of local populations, 2) evaluate the conclusion of (Hulová 2008) that all so far analyzed Czech populations are genetically depleted, and to 3) evaluate levels of genetic differentiation among populations from areas of supposedly less fragmented and deteriorated habitats, as emphasized by (Hulová 2008). In addition, by screening populations from both major parts of the distribution range, we also check for marked genetic divergence between the two ranges due to possibly independent evolutionary histories since the late Pleistocene, as indicated by mitochondrial DNA (Kryštufek, Bryja, and Bužan 2009).

Materials and methods Samples and molecular analysis

A total of 117 European ground squirrels were live-trapped at seven localities, subsequently termed populations, in Austria, Hungary and Romania (Fig. 1). From these animals we obtained tissue by small clips of the tail tip and hair samples (Hoffmann, Turrini, and Brenner 2008; Hoffmann, Millesi, Huber, et al. 2003). Live trapping and sampling was approved by the Austrian Advisory Committee for Animal Experiments (BMBWK-66.006/0012- BrGT/2006), the Municipal Department for Environmental Protection (MA22-20/3143/05), and by the Hungarian and Romanian authorities for nature conservation (Ethical Committee: 48/OP/2002; DOPog–4201-04-73/03/jr, DOPog–4201-04-22/04/jr). Total DNA was extracted using the GenEluteTM Mammalian Genomic DNA Miniprep kit. Fourteen microsatellite loci

60 were selected: Ssu1, Ssu5, Ssu13, Ssu15, Ssu16 (Gondek 2006), IGS-1, IGS-6, IGS-110b, IGS-BP1, IGS-CK1 (May 1997), MS53, MS56, ST10 and SX (Hanslik 2000). Amplification of locus-specific DNA was performed as in (Gondek 2006; May 1997), and (Hanslik 2000). The PCR products were electrophoresed on a LI-COR 4200 automated sequencer along with a fluorescently labelled size standard (50–350bp sizing standard; LI- COR ® Biotechnology Division). Allele lengths were determined using Gene ImageIR 3.52. (LI-COR, Inc., © 1990–1998).

Data analysis Genetic variation Among all loci analyzed, two (IGS-CK1, ST10) were monomorphic (hence, overall rate of polymorphism: 85.7%) and were excluded from all further analyses. The IGS-6 locus was also excluded from further analyses, as the MICRO-CHECKER 2.2 program (Van Oosterhout 2004) showed heterozygote deficiency and a significant presence of null alleles in all analyzed populations. The remaining 11 loci were tested for deviation from Hardy-Weinberg equilibrium and genotypic linkage disequilibrium using the Markov chain method with default parameter settings in GENEPOP 3.4 (Raymond 1995). Allele frequencies, mean number of alleles (A), observed (Ho), and expected (He) heterozygosity were calculated for each locus and population with GENETIX 4.05.2 (Belkhir 2004). The same program was used to calculate overall and population-specific inbreeding coefficients (FIS, (Weir 1984) and associated significance levels for deviation from zero by permutation tests (10 000 permutations). FSTAT 2.9.3.2. (Goudet 1995, 2001) was used to calculate locus-specific values of allelic richness (Rsl) based on a rarefaction approach. Normally distributed population-specific allelic richness (Rs) values were obtained by averaging Rsl values over all respective loci separately for each population. We tested Rs for variation across populations with a General

Linear Model (GLM) based on Rsl values, and concordance of Rsl among populations with Kendall’s coefficient W using SPSS 10.0.1.

Genetic differentiation The population genetic structure was quantified using (Weir 1984) estimators (θ) of Wright’s

FST, as calculated by the MSA software (Dieringer 2002). Significance levels were adjusted after 10 000 permutations. To quantify overall differentiation of a single population from all 61 others, we calculated the “mean FST “ (mFST) as the population-specific arithmetic mean of all pairwise FST values involving a particular population. Population-specific variation of FST values (represented by mFST) was tested by a Generalized Least Squares Regression model

(GLS) of randomized FST values with a restricted maximum likelihood approach (REML), to account for different variances, using S-PLUS 6.2®. Using randomized values was necessary to obtain statistically independent values, which is a precondition for the test (the raw data are not statistically independent). Prior to the GLS we used a Levene´s test to check for variance homogeneity of the randomized values (which was not the case; see “Results”). The amount of partitioning of microsatellite diversity among populations was calculated by an AMOVA model considering all populations equally using ARLEQUIN 1.1 (Schneider 2000). To test for the amount of gene flow, we calculated coalescence theory derived maximum likelihood estimates for the current migration rates (Beerli 2001) among the populations with a Markov Chain Monte Carlo (MCMC) approach as implemented in Migrate (Beerli 2001). The GENETIX program was used to run a Factorial Correspondence Analysis (FCA) on individual genotypes and population differentiation. Resultant individual FCA scores were tested for significant variation across populations separately for each of the first ten FCA factors by a GLS model. We further used a Bayesian approach to estimate the likelihood of an individual’s genotype to be assigned to the population from which it was sampled (Cornuet 1999; Rannala 1997). Specifically, we applied the Bayesian method of (Rannala 1997) and computed probability following the resampling algorithm of (Paetkau 2004) implemented in GENECLASS 2 (Piry 2004). Furthermore, we examined individually based spatial genetic structuring of the whole data set with STRUCTURE (Pritchard 2000; Falush 2003). Thereby we assessed the most likely number of population groupings compatible with the observed genotypic distribution. The likelihood (L) when assuming different numbers of populations (K) was calculated under an “admixture model” and “correlated allele frequencies among populations” with burn-in of 200 000 and the number of MCMC repetitions after burn-in of 500 000. We calculated L for ten runs per K = 1–8. Additionally, we calculated ΔK for each K = 2–7 following (Evanno 2005).

Isolation by distance We assessed the association between genetic similarity and geographic distance by regressing

FST/(1-FST) on the logarithm of geographical distance, which is expected to be linear under isolation by distance in two dimensions (Rousset 1997). For any two populations, the 62 geographical distance was measured as the shortest straight line. To test for a significant concordance between the genetic and geographic distance matrices, the Mantel correlation coefficient (Mantel 1967), r, was calculated using GENETIX with 10 000 matrix permutations.

Recent bottlenecks To detect possible strong reductions of effective population sizes we tested for significantly higher Ho than expected from observed allele frequencies using BOTTLENECK 1.2.02 (16 II. 99) (Cornuet 1996). Specifically, we performed one-tailed Wilcoxon sign rank tests separately for each population both for the “Infinite Allele Model” (IAM) of evolution of loci and a “Two-Phased Model of Mutation” (TPM); for the TPM we used the default settings in the program (for theoretical aspects see also (Di Rienzo 1994). Possible influences of sample sizes on our results were examined with Pearson’s correlations, and significance decision was based on the B-Y approach, which is less stringent than the Bonferroni approach (Narum 2006). In addition, for further hints of a bottleneck history, we calculated population-specific M-ratios, which are the mean ratio of the number of alleles to the range in allele size (Garza 2001).

Results Genetic variation and Hardy-Weinberg Equilibrium A total of 64 alleles (for allele frequencies see supporting information) were detected for all eleven loci considered, with an average of 5.82 alleles per locus. Numbers of alleles per locus ranged from 3 (Ssu15, IGS-BP1) to 9 (IGS-1, MS56), and total numbers of alleles per population ranged from 27 (SIÓ) to 47 (DET). Among all alleles, 15 (23.4%) were private and 49 were shared by at least two populations. He ranged from 0.423 in SIÓ to 0.524 in DET. All pairs of loci were found in genotypic linkage equilibrium. Only three populations (VOR, DUN, PÉL) were in Hardy-Weinberg equilibrium. However, only DET showed a significant heterozygote deficit as indicated by FIS. Rs did not vary significantly across populations (F6,76 = 1.128, p = 0.355, GLM; p = 0.734 for Levene’s test), and there was no concordance of Rsl values across populations (Kendall’s W = 0.149, df = 6, p = 0.133).

Population-specific indices of genetic diversity and FIS values are listed in Table 1.

63 SIÓ VOR DUN DET PEC PÉL VIE

Ho 0.348 0.399 0.381 0.384 0.413 0.457 0.463

(s.e.) (0.200) (0.270) (0.207) (0.240) (0.219) (0.243) (0.230)

He 0.423 0.424 0.433 0.524 0.453 0.492 0.4780

(s.e.) (0.206) (0.235) (0.202) (0.243) (0.208) (0.202) (0.210)

A 2.727 3.182 3.727 4.455 3.000 3.182 3.727

Rs 2.410 2.672 2.686 3.462 2.621 2.983 2.835

FIS 0.209 0.095 0.147 0.291* 0.121 0.139 0.056

mFST 0.138 0.119 0.107 0.078 0.131 0.060 0.097

(s.d.) (0.028) (0.086) (0.043) (0.047) (0.061) (0.03) (0.055)

Table 2 Population-specific indices of genetic diversity and genetic structure, based on eleven polymorphic microsatellite loci. Observed heterozygosity (Ho); expected heterozygosity (He); mean number of alleles per locus (A); allelic richness (Rs); inbreeding coefficient (Weir 1984), and mean FST (mFST). Dispersion measures in parentheses. See Appendix A for population-specific allele frequencies. Asterix denote significantly higher value than zero.

Population structure

Pairwise FST values between populations ranged from 0.046 to 0.219, and only three (14.3%) did not differ significantly from zero (Table 2).

SIÓ VOR DUN DET PEC PÉL VIE SIÓ 0.137 0.155 0.151 0.164 0.086 ns 0.139 0.774 0.475 1.047 1.585 1.265 3.509 VOR 0.115 0.150 0.219 0.120 0.177 1.177 1.179 1.206 1.305 0.365 1.830 DUN 0.057 0.147 0.054 ns 0.112 1.972 4.059 1.340 1.892 0.159 1.855 DET 0.109 0.065 0.072 2.606 0.753 1.775 3.829 0.416 2.486 PEC 0.107 0.038 1.924 0.668 1.442 0.599 0.950 3.234 PÉL 0.046 ns 0.902 6.626 1.474 3.090 2.749 4.522 VIE 0.752 3.114 2.068 3.340 3.649 3.473

Table 3 Estimators of Wright’s FST (θ, upper values above diagonal) and estimated numbers of migrants per generation (see 2.2.2; in italics and grey), source populations in columns, receiving populations in rows. Bold values indicate minimum and maximum of >0 migrants (ns denote values not significantly higher values than zero).

64 Pairwise population-specific FST values varied among populations (F6,35 = 13.017, p < 0.001), with mFST ranging between 0.06 (PÉL) and 0.138 (SIÓ) (Table 1). Variances of pairwise FST values varied across populations too (F6,35 = 4.12, p = 0.003). But there was no significant correspondence of the genetic and geographic distance matrices (r = -0.089, p = 0.549; see Fig. 2). 0.3 Mantel test r = -0.089 p = 0.549

ST 0.2 F - /1 ST F

0.1 pairwise

0 2 3 4 5 6 7

geographic distance [ln km] Figure 4 Spatial pattern of pairwise genetic differentiation: moderate to large dispersion of values of pairwise genetic differentiation does not increase with geographical distance.

Most of the allelic variation was found within populations (87.5%, p < 0.001), whereas variation due to differentiation among populations was relatively low (12.5%, p < 0.001 AMOVA). The estimated numbers of migrants between pairs of populations (Table 2) were not homogeneously distributed and ranged between 0.159 (from PÉL to DUN) and 6.626 (from VOR to PÉL). The individual FCA scores varied significantly across populations for seven out of the first ten factors, which altogether conveyed 43.8% of allelic variation (for descriptive and test statistics see supporting information).

65 Based on individual genotypes, 94 (80.3%) out of all ground squirrels were correctly assigned to their population of origin using the Bayesian approach as described in “Materials and methods”. Percentages of correct population assignment ranged from 52.6% (DUN) to 100.0% (DET, PÉL) and did not depend on population sample size (r = -0.456, p = 0.152, n = 7). Following the standard procedure (Pritchard 2000, 2004) to infer the most likely number of populations inherent in our data set, we concluded that K = 5 populations. However, the ΔK values for each K between 2 and 7 (K = 2: 1.21; K = 3: 15.24; K = 4: 25.84; K = 5: 13.49; K = 6: 1.83; K = 7: 2.13) suggested that K = 4 was the most likely number of populations (see Fig. 3 for individual population proportions (Q values) per population).

100%

Q4

0.0% 100%

Q5

0.0%

SIÓ VOR DUN DET PEC PÉL VIE

Figure 5 Bayesian STRUCTURE results for S.-citellus genotypes assigned to four (Q4) and five (Q5) populations, respectively. Stacked bars represent individuals; grayscale corresponds to percentages allocated to STRUCTURE populations computed from our data set.

Bottleneck histories

When testing for higher Ho than expected from observed allele frequencies, we found significant results for the IAM in PEC (p = 0.01) and VIE (p = 0.007), but only a tendency for the TPM in PEC (p = 0.08). Mean population-specific M-ratios ranged from 0.782 (PÉL) to 0.886 (DUN and SIÓ); i.e., they did neither exhibit much variation nor reveal hints for episodes of low effective population sizes in the more remote past. M-ratios were not significantly correlated with population sample sizes (r = 0.519, p = 0.116, n = 7 populations; r = 0.496, p = 0.159, n = 6 populations excluding DET, as this population had an FIS significantly higher than zero, which could have effected the M-ratio).

66

Discussion Genetic diversity Genetic diversity of the presently studied local populations of S citellus from Hungary, Romania, and Austria is higher than for the Czech populations studied by (Hulová 2008). Numbers of alleles per locus (A) are currently for all but one (SIÓ) populations greater than the maximum observed in the Czech populations. Also, our data on heterozygosity (Ho, He) exceed the maxima reported for the Czech populations. The A and Ho values of the Slovak

(Bratisilava) population (Hulová 2008), however, lie within the range of our results; it´s He is even markedly above the maximum He we found. These comparisons support the conclusion of the above authors that the Czech populations are genetically depleted. However, our comparison rests on different numbers of loci, with more than double the number of loci than the former study. Increased numbers of loci scored appear to give better estimates of genetic diversity than increased numbers of individuals (Nei 1978); (Shriver 1993). Only three loci (IGS-1, IGS-110b, MS56) scored are identical in both studies, and this might blur our conclusions. But even when based on these three loci only, the present A values are higher than those of the Czech populations (mean population-specific A = 2.0; range = 1.67–2.33). Also, locus-specific heterozygosities for two of those loci are significantly lower in the Czech populations than both in our study and the Bratislava population; means of population-specific Ho, He, and p-values for the Czech vs. the presently studied populations together with the Bratislava population are as follows: IGS1: 0.000/0.000 vs. 0.583/0.669 (p = 0.001/0.001*); IGS110b: 0.414/0.253 vs. 0.498/0.364 (p = 0.181/0.081); MS56: 0.312/0.101 vs. 0.668/0.542 (p = 0.001*/p = 0.001*). In line with these results, no private alleles were found in any of the Czech populations, whereas four were present in the Bratislava population (see Table 2 in (Hulová 2008)), and we found an average of two, despite slightly lower sample sizes compared to the Czech populations. Private alleles that occur usually at low frequencies in one population may be eliminated by stochastic processes (genetic drift), particularly at low effective population sizes over many generations (e.g., (Halliburton 2004). All these comparisons corroborate the conclusion that the Czech populations have indeed lost genetic diversity in the recent past.

67 The only population we examined from the south-eastern major distribution of S. citellus (DET) had tentatively higher genetic diversity than those from central Europe. This is paralleled by a similar trend for increased mitochondrial gene pool diversity in the south- eastern range (Kryštufek, Bryja, and Bužan 2009), and consistent with decreasing landscape fragmentation in Europe with increasing geographic latitude (Anděl 2009; Jones 2009).

Values of He and A are considered to sufficiently reflect levels of genetic diversity at microsatellite loci, and are commonly used as surrogate for overall nuclear genetic diversity of populations (Garner 2005; Hedrick 1999). In a meta-analysis (Reed 2003) found a positive relationship between fitness components and genetic diversity in plants, , and vertebrates. They suggested that heterozygosity measured by molecular markers, via its link with population size, can give some indication of population fitness. In mammals, He appears to sensibly indicate demographic challenges. According to (Reed 2003) placental populations having incurred demographic threats in the recent past (“challenged populations”) exhibited consistently lower heterozygosity (mean 0.502) than unchallenged (“healthy”) populations (mean 0.677). In comparison, not only the “challenged” Czech S. citellus populations, but also all populations of our study as well as the Bratislava population studied by (Hulová 2008) have relatively low heterozygosity (see Fig. 2 and 3 in (Garner 2005). Moreover, most investigations and reviews on Sciuridae indicate higher genetic diversity than that found presently: (Stevens 1997) for Columbian ground squirrels (S. columbianus), (Da Silva 2006; Cohas 2009, 2007), (Hanslik 2000) for alpine marmots (Marmota marmota), (Jones 2005; Roach 2001) for prairie dogs (Cynomys ludovicianus), (Schulte-Hoestedde 2001) for yellow-pine chipmunks (Tamias amoenus), (Fike 2009) (in press)(Lane 2007; Shibata 2003; Trizio 2005) for tree squirrels (Sciurus niger, S. vulgaris, S. carolinensis, Tamiasciurus hudsonicus, S. lis), (Selonen 2005) for Siberian flying squirrels (Pteromys volans). Compared to our study, only one island population of S. vulgaris in Pentraeth, Anglesey, U.K. (Ogden 2005), the endangered Delmarva squirrel (S. niger cinereus, see (Lance 2003), and the endangered Vancouver Island (M. vancouverensis, (Kruckenhauser in press) had similar or even lower levels of genetic diversity. To help interpret these findings, we compared studies, which report indices of genetic diversity of North American (Garner 2005) and eastern European (Biedrzycka 2008) ground- squirrel populations: Based on eight microsatellite loci, (Garner 2005) found that in Idaho ground squirrels (S. brunneus), all populations of the northern subspecies S. b. brunneus had significantly higher genetic diversity than those of the southern subspecies S. b. endemicus. 68 According to Biedrzycka and Konopiński (Biedrzycka 2008) spotted-souslik (S. suslicus) populations from the southern had higher genetic diversity than those in Poland and west Ukraine. Both studies concluded that degradation and isolation of populations in the respective areas had caused the drop of genetic diversity. These latter two studies report data in a way that allows direct statistical treatment together with our data, despite some of the screened loci were different. Hence, we statistically compared our population-specific He, A, and Rs values and those of (Hulová 2008) with those of (Garner 2005) and (Biedrzycka 2008), employing three GLS models (Table 3).

S. citellus S. brunneus S. suslicus Genetic This study & Czechia1 S. b. brunneus2 S. b. endemicus2 S Ukraine3 Poland, W Interspecific

diversity Slovakia1 (8) (6) (7) (8) (4) Ukraine3 variation

(10)

He 0.48 (0.06) 0.33 0.58 (0.03) 0.43 (0.09) 0.71 (0.01) 0.43 (0.11) p = 0.002*

p < 0.001* 0.42–0.61 (0.07) 0.54–0.62 0.24–0.50 0.70–0.72 0.20–0.57

0.24–0.43

A 3.45 (0.54) 2.43 4.02 (0.36) 3.45 (0.83) – – p = 0.005*

p < 0.001* 2.72–4.45 (0.27) 3.38–4.50 2.00–4.88

2.20–2.80

Rs 2.81 (0.34) – 3.61 (0.13) 3.45 (0.83) 5.51 (0.32) 2.56 (0.54) p < 0.001*

– 2.41–3.46 3.38–3.81 2.0–4.88 5.09–5.88 1.45–3.07

Table 4 Comparison of genetic diversity in S.-citellus, S.-brunneus and S.-suslicus populations (numbers of populations in parentheses). Means (standard deviation in parentheses) and ranges are given for population-specific values of He (expected heterozygosity), A (mean number of alleles per locus), and Rs (mean locus-specific allelic richness). (1calculated from (Hulová 2008); 2 calculated from (Garner 2005); 3 (Biedrzycka 2008); * significant differences between populations (intraspecific effect; p values in first column below respective index of genetic diversity) and between species (p values in last column) as resulting from three GLS models, that accounted for different variances by REML and corrected for multiple testing by the B-Y approach. GLS model for He: arcsin He 0.5 ~ sample size + species * pop history (“unchallenged” vs. “challenged”); GLS model for A: A ~ sample size + species * pop history (for S. citellus and S. brunneus only); GLS model for Rs: Rs ~ sample size + species).

Interestingly, we found significant variation of genetic diversity among the three species independent of population history and sample size per population, with the lowest values for the S. citellus populations. Thus, the apparently typical level of genetic diversity of S. citellus as calculated in our study is lower than in the two closely related (Helgen 2009) and ecologically similar species.

69 S. citellus most probably colonized Europe during interglacial periods 220 –185 ka ago (Bridgland 2004). Assuming that the species originates from a glacial refuge in Anatolia (Gündüz 2007), we might expect relatively low genetic diversity. In addition, some loss of genetic diversity might have occurred during the later expansion of S. citellus in Europe. Spitzenberger (Spitzenberger 2001) found evidence for a reduction of its distribution range towards the end of the last ice age. According to the latter authors, S. citellus has at that time most likely also disappeared from the Carpathian Basin and persisted only in a refuge in the Balkans. The lack of subfossil records during large parts of the Holocene may indicate local population decreases in central Europe and spread to the current western edge of its distribution range (i.e., Austria, ) only in historical times. Based on subfossil finds, even in the Carpathian Basin a substantial expansion of the distribution range only occurred within the last millennium. Presumably, the species has re-colonized the Pannonian Region during the deforestation of large parts of the landscape in Neolithic times (Spitzenberger 2001) and perhaps later on in medieval times. Such a recent history might have contributed to a generally lowered level of genetic diversity for S. citellus, which presumably has further declined in the course of increased habitat loss and deterioration, and ensuing population fluctuations in the past 50 years (Hoffmann, Millesi, Pieta, et al. 2003) Extensive fluctuations in effective population size combined with increasing isolation of local populations in the cultural landscape may cause a decrease in genetic diversity already over a few generations. For instance, (Srikawan 2000) demonstrated erosion of genetic diversity in three small-mammal populations in Thailand occurring after rainforest fragmentation; and their findings accorded to what is expected by theory, namely that allelic diversity is more sensitive than heterozygosity to detect genetic erosion. However, although genetic diversity is expected to decrease more severely at low effective population sizes in mitochondrial DNA than at microsatellite loci, (Gündüz 2007) and (Kryštufek, Bryja, and Bužan 2009) did not find a particularly low level of mtDNA diversity in S. citellus.

Genetic differentiation Genetic population differentiation in our study was low to moderate as demonstrated by the

AMOVA and FST values, but by-and-large within the range of the genetically depleted Czech populations (Hulová 2008). The tendency for both a higher level (mFST = 0.162) and a higher variance (0.008) of pairwise genetic differentiation among the Czech populations relative to the present study (mFST = 0.115, variance = 0.002) was, however, not significant (F1,34 = 70 1.297, p = 0.263, GLS of randomized arcsin square root-transformed pairwise FST values;

Levene’s test: F1,34 = 0.956, p = 0.335). An increase both in genetic differentiation and in its variance in pairwise population comparisons might have been expected under different levels of genetic drift, with increasing drift leading to increased population differentiation (Halliburton 2004). Thus, the above results suggest that, despite generally higher genetic diversity, the presently studied populations have experienced genetic drift compatible to that of the Czech populations. This interpretation is supported by the lack of any geographic pattern of genetic relatedness among the populations, despite significant differentiation, and by the absence of genetic isolation by geographic distance. Moreover, the variance of pairwise

FST values does not increase with geographic distance (Fig. 2). This together with the somewhat elevated level of genetic differentiation in our study indicates absence of a migration-drift equilibrium. Rather, genetic drift within local populations would dominate over gene flow among populations. The extent of such drift effects is likely population- specific, and this might have produced the observed range of pairwise population differentiation. A pronounced short-term effect is probable only in very small populations, whereas long-term drift effects do accumulate also in larger populations. Generally, the magnitude of drift may also depend on fluctuations in effective population size, which could also reflect periods of high variance in female reproductive output or varying proportions of males participating in reproduction. The significant signal for a recent drop in effective population size in the PEC and VIE populations may be due to such changes in reproductive structure. According to a field study on S. citellus (Hoffmann, Millesi, Huber, et al. 2003), such changes in reproductive patterns may occur within less than a decade, along with significant changes in population density. In a free-ranging population, median litter size varied from four in the first three to six in the last four years, and the percentage of reproductively active yearling males increased with decreasing population density (Hoffmann, Millesi, Huber, et al. 2003). Ground squirrels are sensitive to changes in ecological factors (Koshev 2008), facilitating fluctuations in population density. Instable population dynamics, interacting with “scramble-competition polygyny” (Millesi 1998), can decrease effective population size in the long and short term. The lack of dispersal corridors together with an increased mortality risk for ground squirrels leaving their familiar area (Hoffmann, Muck, and Millesi 2004; Hoffmann, Millesi, Pieta, et al. 2003; Turrini et al. 2008) inhibits gene flow between our currently studied populations. Even considering the two geographically closest populations in the environs of 71 Budapest, the unsuitable habitat surrounding the PÉL population impedes any natural gene exchange with the DUN population since at least 20 years. Hence, the relatively high migration estimates and the low FST values for several of our study populations may be due to stochastic allele similarities rather than ongoing gene exchange: for instance, 2.5 and 3.3 migrants between VIE (Austria) and DET (Romania) per generation are certainly overestimated, given the geographic distance (>1000 km) and the distribution gap between those populations (see Fig. 1). To our knowledge, neither did anthropogenic translocations of animals occur between these two populations. For the Hungarian populations, however, translocations in the course of mitigation and compensation measures and restocking programs are common. Particularly the VOR population has been used as a source population for translocations to the DUN and perhaps also to the PÉL populations (Authority of Nature Conservation of Hungary, pers. comm., Gedeon, pers. observ.). Additionally, Birdlife International Hungary has performed several, sometimes undocumented, reintroductions in the last 20 years, attempting to provide sustainable prey populations for endangered raptors. The population PÉL has been isolated and small over the last 20–30 generations, consisting of no more than ca. 50–100 animals (Gedeon, pers. observ.). Therefore, we had expected a considerable extent of genetic drift, which is obviously not the case, as mFST for PÉL is the lowest value of all studied populations (Table 1). Consistent with the low genetic population differentiation, but contrary to what can be expected under strong drift over many generations at small effective population size, genetic diversity for the PÉL population is not particularly low. The theoretical amount of immigration from the VOR population exceeds all other estimated numbers of dispersers per generation (Table 2), suggesting anthropogenic translocations to the PÉL population in the recent past. The low FST between the DUN and PÉL populations might, however, partly reflect a former cohesion of ground-squirrel colonies in this area. As the STRUCTURE results, the FCA did not facilitate the interpretation of the geographic distribution of genetic diversity: the first ten factors explained less than 50 percent of the allelic diversity, suggesting a complex composition of allelic variability adherent to the data set and partitioned among individuals. The significant variation of individual FCA scores for most of the first ten factors, nevertheless, indicates that this complex diversity varies substantially across the local populations studied. In addition to effects of local drift and

72 anthropogenic translocations it may reflect the presence of residuals of earlier geographic patterns of genetic differentiation.

Conclusions Altogether, the pattern of genetic differentiation as revealed in our study suggests the action of genetic drift in local populations that have been disconnected by anthropogenic obstruction of dispersal corridors, and, on a larger geographic scale, the ongoing genetic disintegration of metapopulation structures. Population fragmentation, isolation and local extinction are indeed occurring (Spitzenberger 2001; Kryštufek 1999; IUCN 2008). In Hungary at least ten out of 63 populations have disappeared since 2000, when the Hungarian national ground-squirrel monitoring began. Overall, our results together with previous findings on S. citellus denote a relatively low heterozygosity with local genetic depletion (Hulová 2008), and suggest anthropogenic impact on local populations, maladapted dispersal behaviour and disintegration of metapopulation structures. In our opinion, the fragmentation, and ultimately, disappearance of local colonies implies a more serious threat to the persistence of the species in its current distribution than possible negative effects (e.g., outbreeding depression) of reintroduction or translocation programs. Carefully planned and evaluated translocations can be an option to counterbalance local extinctions and substitute natural recolonisations, which maintained the metapopulation structure in historical times. In general, our data do not suggest serious drawbacks for potential anthropogenic translocation and restocking programs for local populations as regards genetic aspects. Actually, from our genetic findings we are inclined to support such measures for local populations at low densities, should other (e.g., ecological) circumstances not discourage them. However, it remains open to what degree genes possibly adapted to regional environmental situations (e.g., MHC genes) may reduce the future success of restocking and introduction programs. To minimize possible negative genetic effects, we recommend using neighbouring source populations for translocations whenever feasible.

Acknowledgements The FWF (Austrian Science Funds, P18108-B03 to Ilse E. Hoffmann) and the AÖU (Stiftung Aktion Österreich-Ungarn grant to Csongor I. Gedeon) provided financial support for field work, and laboratory and data analyses. These institutions did neither intervene in the study

73 design, collection, analysis, and interpretation of the data nor in the writing of the report and in the decision to submit the paper for publication. We thank István Németh and Éva Szabó for their assistance in the field, and Attila Sándor in particular, who provided local know-how in remote areas of Romania. We are thankful to Vilmos Altbäcker for his theoretical and practical comments.

74 Appendix A Population-specific allele frequencies at eleven polymorphic microsatellite loci. SIÓ VOR DUN DET PEC PÉL VIE Ssu1 204 0.0227 206 0.0938 210 1.0000 0.9667 0.7632 0.7895 0.9063 0.9375 0.7955 212 0.0263 0.0263 214 0.0263 0.0526 216 0.0333 0.1842 0.1316 0.0625 0.1818 Ssu13 175 0.7188 0.9333 0.9444 0.9118 0.6667 0.8750 1.0000 177 0.0333 0.0278 0.1333 179 0.2500 0.0333 0.0588 0.2000 181 0.0313 0.0278 0.1250 183 0.0294 Ssu5 141 0.0313 143 0.0667 0.0278 0.5556 0.1250 0.2500 145 0.2813 0.6667 0.3056 0.2500 0.1875 0.1250 0.0227 147 0.1875 0.2222 0.0556 0.6250 0.1875 0.5227 149 0.5313 0.2667 0.4444 0.1389 0.0313 0.6875 0.2045 Ssu15 185 0.0263 189 0.3750 0.6000 0.2368 0.2500 0.3333 0.5000 0.5909 191 0.0938 0.1333 0.1842 0.2000 0.5000 0.0625 0.1364 193 0.5313 0.2667 0.5526 0.4750 0.1667 0.4375 0.2727 195 0.0750 Ssu16 190 0.8571 0.8000 0.9375 0.8846 1.0000 0.8333 0.8500 194 0.1429 0.0500 0.0313 0.1154 0.1667 0.1500 196 0.1500 0.0313

75 IGS-1 SIÓ VOR DUN DET PEC PÉL VIE 85 0.0333 0.0625 87 0.1333 0.0455 89 0.5313 0.2667 0.3947 0.2895 0.6250 0.5000 0.5000 91 0.2667 0.0227 93 0.0938 0.1000 0.0263 0.0263 0.0313 95 0.1333 97 0.0526 0.0526 105 0.3750 0.0667 0.5263 0.5789 0.3438 0.4375 0.4318 107 0.0526 IGS- 110b 133 0.1053 0.1000 0.2000 0.1875 0.0952 136 0.0263 0.1000 139 0.5000 0.4667 0.2895 0.5000 0.2667 0.2500 0.3810 142 0.5000 0.5333 0.5789 0.2750 0.5333 0.4375 0.5000 148 0.0250 0.1250 0.0238 IGS-BP1 87 0.0625 0.0227 90 0.6563 0.6333 0.1579 0.1250 0.2667 0.3750 0.2727 93 0.3438 0.3667 0.8421 0.8750 0.7333 0.5625 0.7045 ST10 121 0.0227 123 0.0313 0.0263 0.1750 0.2500 0.1875 0.1136 125 0.0625 0.0667 0.0526 0.1500 0.4688 0.4375 0.5000 127 0.7188 0.9000 0.6842 0.3250 0.1250 0.2500 0.1818 129 0.0333 0.0263 0.1000 0.1563 0.0625 0.1591 131 0.1875 0.2105 0.2000 0.0625 0.0227 133 0.0500 SX

76 140 0.1429 0.0227 142 0.9063 0.3000 0.2353 0.2500 0.7500 0.2857 0.6136 SIÓ VOR DUN DET PEC PÉL VIE 144 0.0625 0.7000 0.7647 0.3250 0.2500 0.5714 0.2955 146 0.0313 0.0750 0.0682 148 0.1250 150 0.0750 152 0.0750 154 0.0500 156 0.0250 MS56 111 0.2143 0.0263 0.0750 0.0625 0.1136 113 0.4688 0.0714 0.0789 0.1250 0.0313 0.4375 0.0909 115 0.1563 0.2143 0.5000 0.4000 0.5313 0.2500 0.5000 117 0.1563 0.3929 0.2895 0.2750 0.3125 0.1875 0.1591 119 0.2188 0.1071 0.0789 0.0750 0.0625 0.0625 0.0455 121 0.0263 0.0625 0.0227 123 0.0500 0.0682

77 Appendix B Summary of descriptive and test statistics (GLS) of the factorial correspondence analysis (FCA) for the first ten factors. Fpop and associated p values (as resulting from the GLS) for variation of individual scores across populations are given for each factor. Asterisks denote significant variation of individual scores across populations under the B-Y criterion for multiple testing (see 2.2.5) % allelic cumulative FCA Factor variation % of allelic GLS

explained variation Fpop p explained 1 6.03 6.03 16.49 <0.001* 2 5.60 11.63 21.63 <0.001* 3 5.36 16.98 10.79 <0.001* 4 4.40 21.39 9.96 <0.001* 5 4.05 25.43 3.03 0.009* 6 4.00 29.44 12.85 <0.001* 7 3.84 33.29 2.67 0.019 8 3.71 36.99 0.88 0.512 9 3.47 40.46 1.81 0.102 10 3.38 43.84 7.01 <0.001*

78 XI. Does ecological isolation or population size influence genetic diversity in the European ground squirrel (Spermophilus citellus)?

Csongor I. Gedeon, Ilse E. Hoffmann, Olivér Váczi, Franz Knauer, and Franz Suchentrunk

Manuscript

Abstract Genetic diversity is considered of paramount importance to maintain evolutionary potential in populations or individual fitness. Particularly for conservation issues it is crucial to know how genetically diverse an endangered species is, and what factors may explain genetic variation among populations. In this study we investigated whether genetic diversity of local populations (four in Hungary and five in Austria) of the declining European ground squirrel was influenced by census population size and ecological isolation of the populations. We genotyped 144 individuals at eleven polymorphic microsatellite loci to assess population- specific allelic richness by a rarefaction approach to account for different sample sizes. We based estimates of population size on standardized counts of numbers of entrances of burrows, and we used the isolation index of Rodríguez and Delibes to obtain population- specific values of ecological isolation; they were calculated using grid-based presence/ absence data of ground squirrels around each local population. Using a generalized least squares fit by maximum likekihood approach and following AICc-based best fit model selection and model averaging, we found higher allelic richness in the Austrian populations than in the Hungarian populations (relative variable importance: 0.98), but no significant influence of either ecological isolation (rel. variable improtance: 0.40) or census population size (rel. variable importance: 0.03). Increased levels of ecological isolation seem to reduce allelic richness of populations as well, but data of more populations are needed to cearify that. The unexpected region effect might partly result from generally higher and more fluctuating population densities in Hungary, which might reduce effective population sizes because of relative high proportions of non-reproducing yearling males (corresponding to the ”scramble- competition polygyny” and yearling male supression hypothesis).

79 Introduction Genetic diversity is considered of paramount importance to maintain evolutionary potential or individual fitness (Paetkau 2004; Frankham 1996). In reintroduction and re-enforcement programs the increase of genetic diversity in small, isolated populations is one of the key issues (IUCN 1998). Therefore it is important to know how genetically diverse a species is, and what factors may explain the variation or the loss of genetic variation within and among populations. In natural populations of mammals the generally accepted factors acting at group level to influence genetic diversity of populations via genetic drift or natural selection are i.e. population size or/ and density, ecological (effective) and/ or genetic isolation (disrupt of gene flow between local populations), mating system (polygynous in particular) and social structure, metapopulation and spatial structure, and population history (Amos and Harwood 1998; Booy 2000). For the negative effect of genetic drift, i.e., the loss of genetic diversity, it is primarily determined by the census population size of the smallest known population, and the number of generations it has been held at around that level (Amos and Balmford 2001).

According to neutral genetic theory, census population size is positively correlated with genetic diversity (Allentoft 2009; Frankham 1996), in natural populations: If ecological attributes of populations are identical, and they only differ in census population size, smaller populations carry less genetic diversity than larger ones (Amos and Balmford 2001). While this relationship is supported by Kimura’s theory (Kimura 1984) of neutral molecular evolution, and by several studies of natural, wild populations (Frankham 1996; Young, Boyle, and Brown 1996) a significant variation in the ratio of effective to census population size ratio

(Ne/ Nc) can affect it seriously. (Amos and Harwood 1998), set out several alternative explanations (e.g., recent bottleneck, inbreeding, metapopulation structure, breeding system) when the positive correlation of census population size and genetic diversity is weakened or even destroyed. However, if this scenario is not modified essentially by other factors we can expect genetic diversity to increase with population size.

Ecologically and consequently genetically isolated populations with low probability of immigration are more prone to the disadvantageous consequences of genetic drift (Young, Boyle, and Brown 1996; Amos and Harwood 1998), or to the negative effect of inbreeding, because ecological isolation could potentially disrupt gene flow among colonies and adversely modify their genetic variability. Features, such as limited dispersal ability, special habitat 80 requirements, or high degrees of sociality, further increase the likelihood of becoming genetically isolated. Nevertheless, dispersal of vagile species and thus immigration can also be impeded if barriers in the intervening habitat matrix are effective (Ricketts 2001). However, there is no simple relationship between the magnitude of ecological isolation and genetic diversity, and how seriously isolation impacts genetic variation within and among populations (Saunders 1991) remains a question. If strong ecological isolation coincides with ‘good’ or ‘high-quality’ habitat (Davis 1985; Begon 1990) then population density can reach high values. Theoretically the maximal carrying capacity of the habitat is the ultimate factor that would prohibit further increase of population density of a colony. Either in high or low population densities social behaviour can be affected severely (Goss-Custard and Sutherland 1997). For low population density, interactions cannot be performed below a certain threshold of available partners (at very low densities) but for high population density, the frequency of agonistic interactions to exclude potential competitors from reproduction increases with population density (Allee 1931; Anthony 2000). The latter, social reproductive suppression reduces the number of breeding individuals and may decrease a population's rate of increase (Wasser 1983; Arnold 1997; Anthony 2000). Nevertheless, it is expected that extremely low or high densities have only severe consequences on the population (Creel 1995). The European ground squirrel (Spermophilus citellus) gives a typical example for the density dependence of reproductive suppression: In a long term study of a ground-squirrel colony in Austria, most yearling males were sexually immature with strong intra-sexual competition acting on the few reproductive ones at high density. At low density, however, all yearling males in the same area were reproductively active and able to mate (Hoffmann, Millesi, Huber, et al. 2003; Millesi 2004).

S. citellus colonies occur mainly in isolated habitat fragments (Leitner 1986; Ružić 1978; Mitchell- Jones 1999; Spitzenberger 2001), which is characteristic for relic populations (Begon 1990). Such residues of various colony sizes and densities likely constituted metapopulations (Hanski 1997), which formerly occupied continuous areas north and south of the river Danube (Hoffmann, Millesi, Pieta, et al. 2003). The species’ critical state (Coroiu et al. 2008) is mainly due to development and construction, intensive agriculture, and afforestation (Hoffmann, Millesi, Pieta, et al. 2003). These activities lead to habitat loss and fragmentation of available habitats, resulting in the ecological isolation of population fragments that may consist of as few as 3 reproductive individuals in Austria (Hoffmann 81 2005) or less than 50 ground squirrels in more than 50 % of all Czech colonies (Matějů 2008). An additional constraint is the fact that it is an obligate hibernator that produces maximum one litter per year. Millesi and co-workers (Millesi 1998) termed them as marginally social and their mating system as ‘scramble-competition polygyny’, which can be considered as a special ‘non-defence polygyny’(Davis 1985).

These life-history and population-ecological characteristics make this endangered, polygynous sciurid a good subject to examine genetic diversity (allelic richness and expected heterozygosity), and its relation to population-ecological attributes (census colony size, population density, ecological isolation, regional effect). Our objective was to clarify whether variations in these population-ecological characteristics are detectable in genetic diversity indices. Our hypotheses were tested with several genetic methods based on microsatellite data. We estimated genetic diversity within populations representing two geographic regions of the species’ distribution range, with Hungary as the core region and Austria as the northwestern region.

We expected the least isolated colonies to be genetically most diverse, and vice versa. In this regard we asked if ecological isolation of ground-squirrel colonies affects genetic diversity significantly. On the basis of historical data, we presume that all these colonies were part of a large, undisturbed metapopulation (Ben Slimen 2011). We expected census population size to be positively correlated with genetic diversity. In accordance with the expectations of neutral genetic theory, larger colonies of S. citellus should have higher genetic diversity than smaller colonies. In this regard we asked if census colony size affects genetic diversity of ground squirrels significantly. As census size was estimated from population density data, it was recommended to investigate the effect of census colony density on genetic diversity. We expected no significant correlation, because however with increasing population density the census colony size also increases but at the same time the denser the colony the more yearling males are reproductively suppressed. It finally can decrease effective population size. In this regard we asked if census colony density affects genetic diversity of S. citellus-colonies significantly. We expected that there were no significant difference in genetic diversity index between the two geographic regions or that genetic diversity is larger in Hungary that in Austria. In this regard we asked if ther were distinct regional differences in genetic diversity. 82

Additionally, we quantified differentiation among colonies and regions, estimated rates of contemporary gene flow and tested for a pattern of isolation by-distance. Taking into account the effect of bottlenecks on the rapid loss of genetic diversity, which is considered essential in conservation (Allentoft 2009) and if genetic bottlenecks occurred it would modify the explanatory power of our model to explain variation in genetic diversity indices among colonies, therefore we also investigated the signs of bottlenecks in the colonies. Quantification of genetic differentiation among colonies (regions) and inbreeding coefficients, estimation of rates of contemporary gene flow, tests for a pattern of genetic isolation by- distance, and investigation of signs of recent bottleneck are not yet evaluated factors that may subvert the expected relationship between population census size or density, ecological isolation, regional effect, and genetic diversity.

Material and methods Sampling and Study sites In 2006 and 2007, a total of 144 European ground squirrels was trapped by snaring and Tomahawk live traps, respectively. Detailed description of the capture technique appears in (Gedeon, Váczi, et al. 2011) and in (Hoffmann, Millesi, Pieta, et al. 2003). Tip-tail tissue and hair-bulb samples were collected from each individual on ten different sites (termed colonies hereafter). We sampled five colonies from Hungary and five from Austria (Table 1). In Hungary, three are on the right side of or near the River Danube, as Pécs, Hármashatárhegy- Vöröskő and Siófok, and two on the left side of or near the River Danube, as Dunakeszi and Pécel. From the Austrian colonies, three are on the left side of the River Danube in Vienna- Stammersdorf, Vienna-Falkenberg and in Gerasdorf, and two on the right side of the River Danube in Vienna-Radio Station and near Trausdorf west of Lake Neusiedl. The study sites are inhabited by ground-squirrel colonies of different census-colony sizes and densities, and exposed to varying degrees of ecological isolation. Tissue samples were collected once from each individual for DNA extraction, and after sampling, each animal was released at its capture site. For later DNA extraction the tail tips were frozen at -18 Celsius or stored in 96% ethanol. The traps were spread evenly throughout the area to avoid non-random sampling of related individuals. Detailed information on the localities, number of analysed individuals per locality, as well as

83 the size of colonies estimated from census colony density and maximum area of each colony is given in Table 1. Colony census Estimated Number of WGS84 WGS84 CS CC density colony sampled X Y (ind/ size (ind) individuals ha) FB AUS 23.913 120 17 48.1850 16.2258 ST AUS 17.526 4381 16 48.1837 16.2455 GB AUS 11.186 134 20 48.1860 16.2733 RS AUS 7.801 390 11 48.9120 16.2440 TD AUS 4.082 408 14 47.4820 16.3324 SIO HUN 114.167 4875 16 46.5124 18.6461 VOR HUN 70.833 1300 15 47.3318 18.5837 DUN HUN 43.333 3969 19 47.3652 19.8325 PEC HUN 60.000 4867 16 45.5926 18.1416

colony symbol (CS), country code (CC), global position (WGS84)

Table 1 Estimates of colony size and density, and central coordinates of sampled colonies.

Live trapping and sampling was approved by the Austrian Advisory Committee for Animal Experiments (BMBWK-66.006/0012-BrGT/2006) and by the local authorities for nature conservation. Field procedures were carried out according to the Ethical Committee for Animal Experiments at Eötvös University, and followed the rules detailed in ASM guidelines (Gannon and Sikes 2007).

DNA extraction and microsatellite typing Total DNA was extracted from tissue samples using the GenEluteTM Mammalian Genomic DNA Miniprep kit. On the basis of a recent population-genetic study (Ben Slimen 2011) et al., submitted), the following 11 microsatellite loci with different levels of polymorphism were selected for this study: Ssu1, Ssu5, Ssu13, Ssu15, and Ssu16 (Gondek 2006), IGS-1, IGS-110b, IGS-BP1 (May 1997), MS53, MS56, SX (Hanslik 2000). Amplification of DNA was performed as in Gondek et al. (Gondek 2006), May et al. (May 1997), and Hanslik et al. (Hanslik 2000). The PCR products were electrophoresed on a LI-COR 4200 automated sequencer along with a fluorescently labelled size standard (50-350bp sizing standard; LI-

84 COR ® Biotechnology Division). Allele lengths were determined using Gene ImageIR (LI- COR, Inc., © 1990-1998).

Genetic variation, test for HW proportions and genotypic linkage equilibrium Microsatellite loci were tested for deviation from Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium using the Markov chain method in GENEPOP vers. 3.4 (Raymond 1995). Default parameter settings of 1000 dememorizations, 100 batches, and 1000 iterations per batch were used for Markov estimations. Significance levels were adjusted using the sequential Bonferroni correction for multiple comparisons (Rice 1989). Allele frequencies, mean number of alleles (A), observed (Ho) and expected (H e) heterozygosity were calculated for each locus and for each population with GENETIX (Belkhir 2004). The same program was used to calculate overall and population-specific (Weir 1984) estimators of Fis and respective significance levels for deviation from zero by permutation tests (10000 permutations). The FSTAT vers. 2.9.3.2. programme (Goudet 2001, 1995) was used to calculate locus-specific values of allelic richness (Rsl) based on a rarefaction approach to account for different sample sizes. Colony-specific allelic richness (Rs) was obtained by averaging Rsl values over all loci, respectively.

Census colony density and size Census population density and size of each colony were estimated using and following the S. citellus protocol of the Hungarian Biodiversity Monitoring System (Váczi 2005), combined with capture-mark-recapture and observations (Millesi et al. 1999). Counting of ground- squirrel burrow entrances shortly after hibernation (22 April) is a reliable method of estimating and comparing differences in population density (Katona, Váczi, and Altbäcker 2002; Koshev 2008). We estimated colony size via dividing the number of ground-squirrel burrow entrances per hectare (density data) by six (Hut and Scharff 1998; Katona 1997) and then multiplied by the estimated size of the maximal colony area (ha). Maximal colony area was estimated by determining the colony perimeter with GPS receivers on the field and then by calculating surface area within the perimeter.

Ecological isolation Ecological isolation was quantified with an isolation index (ISOL INDEX hereafter) after to × (Rodríguez 2003), with ISOLATION INDEX = 퐷푖 푘 푤푖 푒 85 ∑푖=1 푆푡 where k = number of neighbouring populations, D = distance to the nearest neighbour, and St is the sum of all the neighbours’ sizes. This formula for isolation assumes that (1) all neighbours contribute immigrants, (2) the contribution of each neighbour decreases geometrically with distance (estimated by the exponential function eDi), and increases linearly with neighbour size, (3) immigrant contribution is additive, and (4) the effect of distance is weighted (w) by neighbour size, where wi = colony sizei/ St. This way of modelling isolation is conceptually similar to that used by Hanski (Hanski 1994; Hanski and Gilpin 1997). For the ISOL INDEX a colony was determined as a continuum of occupied adjacent grid cells connected either by sides or corners. The freely available 1-km UTM reference grid for Europe (Van Liedekerke 2009), combined with Google Earth® satellite map of the nine colonies provided the tool for calculating ISOL INDEX. In addition to the geographical information system (grid and satellite map) and perhaps more importantly, our a priori knowledge about the sampled colonies and its environs gave us the opportunity to have a reliable and updated picture on the connectivity of the sampled colonies to other colonies or populations. Based on data indicating short dispersal distances of S. citellus (< 1 km) (Hoffmann, Muck, and Millesi 2004; Sutherland 2000; Turrini et al. 2008), an unoccupied, adjacent 1-km grid cell was considered enough to separate a colony from another one in the neighbourhood. The higher the ISOL INDEX, the more isolated the colony was. The sampled colonies differed in degree of ecological isolation, and we expected that the genetic diversity indices (Rs and He) decrease with increasing isolation (Milne and Forman 1986).

Statistical analysis To explain variation in genetic diversity index (population specific overall allelic richness Ar) by two ratio (census colony size or census colony density, ecological isolation) and one nominal variable (region), we applied a generalized linear model (GLZ) approach (McCullagh 1989). Before model selection, as a preliminary analysis of the data, descriptive statistics and Pearson correlation analysis were done to show important relationships among the predictors and between the predictors and genetic diversity indices (Pearson 1896). Before calculating correlation coefficients and when necessary, data were log-transformed to ensure normality. All general analyses were performed with the software STATISTICA v.9 (StatSoft 2009) or R programme (R 2008). During selection of the best-subset model, we included ecological isolation, region and population size or population density as predictors and allelic richness as 86 response variable and (Global model: lm (formula = alrich ~ (ecological isolation + population size/ population density + region)^2). For model selection, we employed Akaike’s information criterion for restricted data set (AICc hereafter) (Akaike 1973) as it is recommended to use AICc if sample size is smaller than 40. We permitted second order effects of predictors in the final model, as the number of predictors (3) was relatively low compared to the number of cases (9). Otherwise the interpretation or understanding of the results would have been difficult and because we might have found spurious but yet significant interactions between predictors. The best-subset model was then chosen corresponding to the smallest value of AICc.

Results Correlating genetic diversity indices Census colony density and ecological isolation index were negatively correlated s with Rs (r = -0.8, p = 0.005; r = -0.83; p < 0.005; r = -0.67, p < 0.05, respectively). In other words, allelic richness significantly decreased with increasing density or isolation. Expected heterozygosity significantly increased with Rs (r = 0.84, p < 0.005). When data from each country were analysed separately, the correlation was not significant for Austria (r = 0.54, p = 0.35), and marginally significant for Hungary (r = 0.84, p = 0.07). Other ecological predictors did not show significant correlations with He.

Regional comparisons Census colony density was significantly higher in Hungary than in Austria (Austria: 13 ± 8; Hungary: 63 ± 34; t-test: t = -4.03 d.f. = 8; p < 0.005), whereas census colony size was similar

(p = 0.48). Allelic richness and H e were higher in Austria than in Hungary. The differences were significant for Rs (Austria: 3.00 ± 0.1; Hungary: 2.68 ± 0.2; t-test: t = 3.19; d.f. = 8; p <

0.05), and marginally significant for He (Austria: 0.52 ± 0.04; Hungary: 0.46 ± 0.04; t-test: t = 10.23; d.f. = 8; p > 0.05). Hungarian and Austrian colony-groups did not differ significantly in

Ho and Fis. Linear distance (km) between colonies in Hungary was significantly higher than in Austria (F = 12.19, d.f. = 1, 19, p < 0.005; Table 2).

87 FB ST GB RS TD SIO VOR DUN PEC PEL

FB 2.43 5.76 17.81 58.5 206 211.4 219.7 293.6 241.9

ST 3.4 17.29 57.5 204.8 208.8 217.6 292.2 239.5

GB 16.7 56 202.3 205.3 214 289.2 236

RS 40.62 190.9 203.1 212.4 276.4 233.7

TD 155.7 183.2 194.5 239.3 214.1

SIO 101.6 115.1 96.83 117.9

VOR 14.43 181.6 30.83

DUN 193.7 24.43

PEC 186

Table 2 Pairwise linear distances (km) between sampled colonies.

Explaining genetic diversity indices The best-subset model for Rs included only region (Model 1 in Table 3, AIC−4.95). = The second best-subset model for Rs included ecological isolation and region. This model is just a bit less fitting our data (delta is the difference in AICc to the best fitting number one model, and the difference is only minimal, but nevertheless it fits only second best). There was no significant effect of census colony density or size on Rs in the best-subset model.

Table 3 Model selection values (Akaike’s Information Criterion) for generalized least squares linear models with maximum likelihood estimates fit to encounter allelic richness index value.

Population genetic statistics: genetic variation, tests for Hardy–Weinberg proportions, and genotypic linkage equilibrium Allelic richness (Rs), based on a minimum sample size of 11 individuals per site, ranged from

2.56 (Siófok) to 3.45 (Stammersdorf and Radio Station). Expected heterozygosity (He) values 88 were ranging from 0.44 (Hármashatárhegy-Vöröskő) to 0.58 (Stammersdorf). Observed values for the coefficient of inbreeding (Fis) were significant for four colonies from Austria and two colonies from Hungary. Lower values (only significant in six colonies) were found in Austria. No statistically significant linkage disequilibrium was observed in the 11 loci tested.

Discussion The natural metapopulation structure and continuous grassy, steppe-like habitat of S. citellus have been recently fragmented by anthropogenic activitites (Hoffmann, Turrini, and Brenner 2008) and colonies are prone to different degrees of ecological and environmental constraints. Consequently S. citellus provided a good opportunity to examine which ecological population characteristics how much affect genetic diversity of natural colonies. We aimed to explore the population-ecological mechanisms, in particular ecological isolation, that are closely associated with genetic diversity index. Significant effects were detected for allelic richness (Rs) by region (Hungary as core area and Austria as edge of the species’ distribution range) and although not statistically important (relative variable importance) ecological isolation (isolation hereafter) also appered to affect genetic diversity. Both factors are extrinsic mechanisms therefore we must address these factors one-by-one to interpret the results of our models.

Regional effect There likely are differences in habitat quality between Hungary and Austria, which can result in different carrying capacities of S.citellus habitats. Hungary represents the core area of the species’ distribution range, whereas Austria is located at the westernmost edge. Compared to Austria, there are yet more grassy fields dominated by fescue species (Gedeon et al. 2010) all over Hungary, which are considered the natural, optimal habitat of S. citellus (IUCN 2010). Therefore it is reasonable to assume that habitat quality is lower in Austria than in Hungary. On the one hand, in accordance with this idea, we observed higher average census colony density in Hungarian colonies, indicating that the habitats in Hungary can maintain more individuals. On the other hand the data from the Hungarian ground squirrel monitoring showed that census colony density varied more between colonies and between years (Váczi O., unpub.data).. If the fluctuation of census colony density had been dramatic it could have resulted in decreasing Ne in the long run, and decreasing Ne would have eventually lead to

89 lower genetic diversity. The census colony density data of these colonies however provided substantial but not dramatic variation between years since 2000 (Váczi O., unpub.data). Also we found that Hungarian colonies made up rather distinct genetic units, while Austrian populations were more dispersed or scattered according to the genetic assignment test. This may indicate that dispersal and corresponding gene flow occurred more frequently between colonies in Austria than in Hungary (Anthony 2000). For the ‘regional effect’ it means that habitats of colonies are more interconnected in Austria than in Hungary, which is yet able to maintain a level of dispersal and gene flow in a degree that can decrease genetic differentiation (see assignment test results) of colonies and counteract the effects of genetic drift. In conclusion, habitat difference between Hungary and Austria can be the result of complex processes. The regional effect in our best-subset model does not carry a simple, underlying mechanism.

Ecological isolation Ecological isolation did not have significant effect on Rs (Model 1 in best-subset models) but the second best fitting model including isolation was almost as good as the foirst one. It indicated that the effect of isolation cannot be ignored and further data would be required to clearify its importance. However, the picture appears complex and requires explanation and recapture of the specific results and their interrelation. Briefly, we found that average census colony density is larger in Hungary. Habitat quality determines the carrying capacity of a habitat, which predicts that habitat level is higher in Hungary than in Austria. In other words (optimal) core habitats in Hungary can maintain higher intrinsic rates of increase than (suboptimal) edge habitats in Austria (Thomas, Harper, and Morgan 2001). But ecological isolation was larger in the Hungarian colonies, and unexpectedly both genetic diversity indices were lower in the Hungarian colonies. This does not support the general prediction telling that within-species genetic diversity declines from the centre of the geographical range to the periphery (Eckert, Samis, and Lougheed 2008). Moreover Ho was always less than He in all colonies independently from the region, indicating that all colonies have been ecologically isolated in different degrees and have received less external gene flow recently. The genetic assignment test suggests a higher extent of dispersal between colonies in Austria than in Hungary. We did not find evidence on recent genetic bottleneck in the colonies nor was the effect of isolation by distance substantial and significant. All populations were found to be in Hardy-Weinberg 90 equilibrium based on values of Fis and He, suggesting no significant inbreeding. This result is not in agreement with previous findings on S. citellus colonies in Czech Republic (Hulová 2008). Taking into account all the abovementioned, ecological isolation is expected to be a principal mechanism that can explain variation in genetic diversity (lower in Hungarian colonies) and exceeds the counter effect of colony size or density. Moreover, taking into account the reproductive suppression in S. citellus, isolation and density may act synergistically, strengthening each others’ negative effect on genetic diversity. If high census colony density coincides with high isolation, they may comprise a ‘polygyny impact’ via increased yearling male suppression, and eventually further decreasing genetic diversity. On the other hand, low census colony density can be advantageous for mature yearling males in terms of reproductive success. But we cannot exclude that low census density may be associated with disadvantageous metabolic and immune changes on the basis of adrenal activity (Millesi 1998; Millesi et al. 1999; Millesi 2004) and higher individual predation risk (Hoffmann, Millesi, Pieta, et al. 2003).

Sample size and He We applied only Rs as a surrogate for gentic diversity. This approach is in accordance with the result of Pruett and Winker (Pruett 2008), who concluded that H e performs better (more sensitive to) in populations with high genetic diversity, and Rs performs better in populations with low genetic diversity at small sample sizes. We found that neighbouring populations characterized by higher allelic richness have also higher He, whereas the strong correlation does not hold for populations further apart (Comps et al. 2001), which measn a distance dependent relationship. The distance between colonies ranged 2 and 59 km for the Austrian colonies, whereas this ranged between 14 and 186 km for the Hungarian ones. We can conclude that that genetic diversity indices may behave differently in varying ecological and genetic conditions (Widmer and Lexer 2001) and it was recommended to use only Rs for genetic diversity. Inn addition, Rs is more sensitive to isolation and fragmentation than He because rare alleles do not contribute to He but to Ar (Nei 1978; Cornuet 1996).

Conclusions for conservation We found higher allelic richness in the Austrian populations than in the Hungarian populations, but only low support for either ecological isolation or census population size. However, increased levels of ecological isolation seem to reduce allelic richness of 91 populations as well, but data of more populations are needed to clearify that. The unexpected region effect might partly result from generally higher population densities in Hungary, possibly reducing effective population sizes because of relative high proportions of non- reproducing yearling males (corresponding to the ”scramble-competition polygyny hypothesis”). It might also result from longer existing isolation of individual colonies in Hungary than in Austria; according to field data, the latter have been longer in contact with each other and several are still not very much isolated. To restore or maintain genetic diversity and reduce inbreeding the barriers for dispersal should be identified. If recent anthropogenic barriers cannot be eliminated or corridors cannot be re-established to recover dispersal between colonies then relocations can provide a tool to increase or at least to maintain a certain level of genetic diversity that finally maintains evolutionary potential and increased individual fitness. Taking into account the potential negative effect of high census densities on genetic diversity, source colonies for translocations can be identified if density is monitored systematically and at least annually.

92 XII. Putting science into conservation action: Notes on the guideline for reintroduction of European ground squirrels

Motto: If you measure and monitor it, you can manage it!

Relocations have occurred for 30 years at least in Hungary. The first, pioneer relocations were done by the regional groups of Birdlife International of Hungary. Then the Department of Ethology of Eötvös University developed a protocol for translocations. On the basis of the experiences and international recommendations of IUCN on reintroductions, we determined critical points that should be addressed in ground squirrel translocations. Some points were tested experimentally, but a few others yet remained untested. Nevertheless, as relocations are inevitable because of land use change of ground squirrel habitats, grassy airport renewal programmes, motorway constructions through habitats, reintroduction specialists are inspired or perhaps as it can be expected from a committed conservationist, forced to compose a common guideline to minimize the damage of colonies and mortality of wild-caught ground squirrels.

The following list includes our recommendations which we consider the critical points to be addressed during a translocation or reintroduction of ground squirrels. I would not consider it a fully comprehensive translocation guide we only address the most important issues herebelow.

Source population: How should one select the source colony? How many individuals should one translocate?

Annual and continuous monitoring of census size and density of ground squirrel populations provide valuable information on choosing wild populations for translocation. Within high census density yearling males are suppressed, therefore a certain density range can be determined as satisfactory and below or above that threshold translocation is encouraged. Number of translocated individuals should be about 200 if a new colony is established.

Capturing: When and how should one trap ground squirrels (Chapter VII. and VIII.)?

93

Either gentle snaring or pouring appears a less expensive and feasible trapping technique. Although gentle snaring does not endanger animal survival as much as pouring and its harmless application is supported by experiments, it requires more personnel (10-20 people/ hectare and time (12 working hours/ day) to apply. Consequently, in summer when the daily temperature is above 25 °C and there is low chance of precipitation, pouring can be an alternative to gentle snaring, as it is faster and requires only a few persons. Most importantly, we can say that within optimal circumstances gentle snaring is for rescuing all individuals from an area and pouring is for catching a certain number of individuals as quick as possible.

Before capturing one should do a census survey by visual census of ground squirrels in the source colony in the first week of April or last week of July. Visual census of the ground squirrels means observation and counting of individuals from a hide by at least one observer. Basically, it applies a scanning procedure (Altmann 1974). The visual census should be done during the activity peak between 09:00 – 11:00. The total area is split into 1-ha areas where the observer scans one area for 5 minutes with a regular binocular at least 5 times. One should use the maximum number of distinct animals seen at the same time within the area as data. By this method one can avoid double counting of some animals. Observation should begin one hour after arriving at the colony to allow for habituation to the observer. After the survey of ground squirrels, catching should be done in spring or in later summer. In spring, catching is considered safe when all individuals emerged from hibernation, but pregnancies would all still be in the first trimester (Katona, Váczi, and Altbäcker 2002) (12-20 April). In summer, one should do catching after weaning when young ground squirrels left the nest burrows, from about 10th July until 10th August.

Transport: How should one transport the ground squirrels?

Captured animals should be housed in separate wire cages (10 x 10 x 40 cm) in a climatized trailer or room. One should provide food and water for the animals kept temporarily in the wire cages. Slices of fresh apple are a good food and water source in captivity.

Release site: How to choose the release area? How big should the release area be? How should we prepare the release area and site for the translocation? When and how should we 94 release ground squirrels? (Chapter VII. and VIII.)

The release area should be similar (i.e. vegetation, soil texture) to the original habitat of translocated ground squirrels (usually dominated by fescue grass, water table depth is one or more meter, soil depth is minimally 50 cm, soil texture is soft, and the grass is medium height). Its minimal size should reach at least 5 ha if we take into account that the average density of ground squirrels is 60 individuals/ ha (Katona 1997) and 200-500 individuals are considered necessary to maintain a colony. Vegetation height should not exceed 15 cm, which can be maintained by grazing (sheep, cattle) if possible. One should release ground squirrels in the morning as it provides more time for them to acclimate to the new environment. By morning release, the daily acclimation period is extended similarly to the longer confinement period in rabbit reintroductions (Rouco et al. 2010), which supports short term survival. During release, one should let one individual loose in a separate, angled (~ 30 °) artificial burrow combined with a retention bottle (it works as successfully as expensive acclimation cages).

Post-release activities: How and how long should we carry on guarding or monitoring? How can we decide if a translocation is successful?

After and during the translocation one should employ predator exclusion on the release site by guarding continuously the animals from hides for at least two weeks (Rouco 2008). After the animals enter into daily rest (including all night), guards should apply a spotlight for checking the area. The success of a translocation can and should be evaluated on multiple temporal scales (Seddon 1999; Simms 2009). Post release monitoring of the animals should occur via observation for 5 years. One should do visual census (Simms 2009) of the ground squirrels on the release site for at least three days during the activity peak(s). Then in every month from the release until the first hibernation used-burrow counting is recommended (Katona, Váczi, and Altbäcker 2002; Váczi and Altbäcker 2005; Koshev 2008) to monitor ground dwelling sciurids. From year after the release ground squirrel monitoring is recommended on the national monitoring day (22 April) of the ground squirrel programme of the Hungarian Biodiversity Monitoring System (Váczi and Altbäcker 2005). Data on ground squirrel census numbers at the release area are required to decide if the 95 translocation was ‘successful‘ in the long-term (Seddon 1999).

Release site treatment: What are the priorities in a ground squirrel habitat to maintain the colony? First of all, vegetation height should be maintained at a maximum of 15 cm. Secondly, regular monitoring is necessary to efficiently manage the translocated animals. Thirdly, disturbance of the colony should be minimized as translocated individuals require more than 5 years to become fully established in the new habitat.

96 XIII. Future challenges: Captive breeding programme of European ground squirrels

“Habitat protection alone is not sufficient if the expressed goal of the World Conservation Strategy, the maintenance of biotic diversity, is to be achieved. Establishment of self- sustaining captive populations and other supportive intervention will be needed to avoid the loss of many species, especially those at high risk in greatly reduced, highly fragmented, and disturbed habitats. Captive breeding programmes need to be established before species are reduced to critically low numbers, and thereafter need to be co-ordinated internationally according to the sound biological principles, with a view to the maintaining or re- establishment of viable populations in the wild.” (IUCN 1987)

The message of the IUCN policy statement is absolutely clear, and if we assess the conservation status of the European ground squirrel on the basis of the recommendations in the IUCN policy statement and in the light of the results of various research studies on the ecology of ground squirrels we should come to the conclusion that the establishment of ex situ population(s) or to start captive breeding programmes are fully recommended. The Hungarian national biodiversity monitoring programme since 2000 has provided reliable data to conclude that its habitat has been reduced recently, and is more and more fragmented and disturbed by anthropogenic influences. Although there is no additional, long term monitoring in other regions of its distribution range where there are yet large populations (>500 individuals), we argue that in general, its short-grass habitat (primary or secondary steppe) is not as stable as it was historically and its sustainable usage is not guaranteed. Ground squirrel colonies are more and more isolated from each other, the good-quality habitats are less and gets smaller, the metapopulation structure has been disintegrated, and the colony census densities and sizes oscillate. All in one, a captive breeding programme in a region where there are yet thousands of ground squirrels with sufficient genetic diversity is most recommended.

With the support of LIFE funding there have been several captive breeding programmes of endangered mammals (i.e. Mustela lutreola, Lynx pardinus, Monachus monachus, Rupicapra pyrenaica ornate, Ovis gmelini musimon varietas corsicana), therefore its financial background is also available at the moment. From a practical point of view, I would mention

97 two examples of captive breeding programmes of ground squirrels. Between 2005 and 2007 about 200 European ground squirrels were reintroduced to Poland (to Kamien Śląski, to Jakubowie Lubińskim, and to the vicinity of the village Głębowice). The reintroductions were successful (number of animals born there is increasing), therefore there is certain knowledge and experience in breeding ground squirrels ex situ. The other example includes a captive breeding programme of thirteen-line ground squirrels (S. tridecemlineatus) (Vaughan et al. 2006) for research on hibernation.

To summarize, there is readily available information on the breeding of ground squirrels and only the protocol and long-term management facilities need to be developed. On the basis of earlier experiences in keeping ground squirrels in laboratory conditions, they adapt well to captivity as they are not truly social animals, long-term husbandry proved to be feasible, natural diet can be substituted by rabbit or dog chow food, and management of hibernation, mating, and successful reproduction also seem workable. In addition, as Hungary has several colonies with high census densities and sizes the management of census density in natural colonies is recommended because of the negative effects of high density on the reproduction of yearling males. Taking into account the recent conservation status of the European ground squirrel and from all these experiences and scientific results we can conclude that time has come to develope a species protection plan including a captive breeding programme and protocol. It may provide an effective remedy for the continous loss of ground squirrel populations, or at least it can severly reduce the currently experienced negative (meta)population demographic trend.

98 XIV. Literature review

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119 XV. Final remarks In this thesis I have investigated the relationship between a few environmental factors (i.e. fescue grass, soil, burrow, vegetation height, region) or intrinsic processes (i.e. genetic variability, population density or size) and habitat usage for ground dwelling sciurids. In addition, I have attempted to give a guideline for ground squirrel reintroductions.

My studies indicate that European ground squirrels show a strong and significant preference for fresh, green Festuca pseudovina in laboratory and natural conditions. Insulation property of the squirrels’ fresh grass nests was superior to nests constructed from dry grass only. Intra- cellular water in the nest material did not affect insulation significantly. We concluded that fresh fescue provided a more flexible material that allows squirrels to construct nest with better insulation under field and laboratory conditions as well. Experimental evaluation of specific abiotic (Váczi et al. 2006; Wagner and Drickamer 2004) and biotic (Kis et al. 1998) components could reveal characteristics of the habitat that are keys to survival and thus provide valuable information for conservation efforts. Our results suggest that squirrels may rely on dominant and abundant xeromorph fescues of dry meadows because of their importance to nest building. Although we did not test experimentally the hypothesis if ground squirrels prefer and actively choose those habitat patches (“birth rate” and “migration” models to explain the distribution of animals) where fescue dominates but we can conclude that ground squirrels cannot spare fescues for resting in the active season or for hibernation. Consequently, it can be assumed that populations will expand to unoccupied areas and survive for longer-time and reintroductions will be successful if the habitat is characterized by fescues.

We draw four conclusions about burrow mounds of Gunnison’s prairie dogs, which have similar ecology to European ground squirrels. 1) Gunnison’s prairie dogs use mounds for vigilance; vigilance increases with taller vegetation. The higher frequency of vigilance in situations with medium and tall vegetation indicates the ability to adapt behaviour to changing natural conditions. As vegetation grows during summer, changing the rate of vigilance provides greater security. This is similar to shifts in vigilance with changes in vegetation observed in degus (Octodon degus; Ebensperger 2005). 2) Vigilance is more frequent by individuals on the periphery than those in the interior of the colony, and vigilance activities by 120 those on the periphery are directed outward from the colony (Verdolin 2002). It is adaptive to the colony to have an early warning system that is focused on potential danger from outside the colony. Ariel predators can come from any direction, but ground predators will approach the periphery. Greater vigilance by individuals living in perimeter areas that is directed outside the colony provides warnings of potential ground predators. 3) It is possible to estimate volume of burrows by measuring the amount of subsoil displaced in the mound. This discovery should permit better estimation of volume of burrows and it provides a method, using the pie-shaped sections and computational formula, to achieve that end. Information on volume of burrows can be applied to situations where prairie dogs are being translocated and established in artificial burrows. 4) During construction of burrow systems, Gunnison’s prairie dogs move and mix 10,000-16,500 kg of soil/ha, which is within the range excavated by black-tailed prairie dogs (Butler 2006). Effects of burrowing animals as ecosystem engineers have a significant geomorphological impact on many landscapes (Gabet, Reicman, and Seabloom 2003), but measuring magnitude of bioturbation by a population, colony, or individual is difficult (Nevo 1999). North American pocket gophers (Geomyidae), acknowledged engineers of ecosystems, excavate ca. 18 m3/ha/year on average (Smallwood 1999). For Gunnison’s prairie dogs, we determined that excavation of soil was 6.5-16.6 m3/ ha, taking into account only the volume of mounds as excavated soil. This indicates that Gunnison’s prairie dogs are having an ecological impact equal to the effects of pocket gophers, which provides support for statements made by Detling and Whicker (1987) and fits the ecosystem-engineer concept (Jones et al. 1994; Gutierrez and Jones 2006). All these four conclusions show that vegetation influences habitat quality and has consequences on the behaviour of prairie dogs, but on the other hand prairie dogs also have noticeable effect on the vegetation. Different models act in combination (Crowe and Underwood 1998) to explain associations of animals and certain habitats or habitat characteristics but these results call the attention to the fact that ground dwelling sciurids are also active, key players in this interactive game of the habitat and its users. Either the “birth rate”, “death rate” or “migration” model explains their habitat use, ground dwelling sciurids modify dramatically the habitat (both the surface and the topsoil) and its quality.

Topics addressed in Chapters X. and XI. support the hypothesis (Seddon 1999) that long-time persistence of ground squirrel colonies, which primarily depends on longevity and reproductive success, is strongly connected to the first (critical) period after release of the 121 translocated animals, and habitat quality (grass height and artificial burrow entrance angle with mounds similar to natural ones) at release site has a large effect on this critical period (Letty, Marchandeau, and Aubineau 2007; Moorhouse et al. 2009). For the critical period after release and habitat quality we draw four conclusions that seriously influence the survival of ground squirrels. 1) Release time should correspond to the natural activity peak of the translocated animals, for European ground squirrels the morning period. Release in the morning is advantageous as it provides more time for ground squirrels to acclimate to the new environment. This extended daily acclimation period is similar to the longer confinement period that results in short-term survival in rabbit reintroductions (Rouco et al. 2010). 2) Plugging the burrow entrance after release can increase the chance to hold more animals at the releasing area (burrow fidelity or settlement) while to avoid panic escape. Releasing the animals without providing at least temporal shelter, results in panic reactions and erratic movements of ground squirrels. Our method of plugging, which forced them to stay an hour or two underground prevented such panic reactions and enhanced colonization if the release also coincided with their activity pattern. The artificial burrow with retention cap provides shelter for the animals until they excavate their own burrow system and animals can shuttle between them and aboveground foraging sites. They work similarly to natural burrows, which provide a thermally stable environment underground and an option to avoid hypothermia and hyperthermia (Long et al. 2005). From the practical point of view, our results show that the use of retention caps in combination with the artificial burrows could work as effectively as complicated and expensive acclimation cages (Simms 2009). 3) Our results suggested that angled artificial burrows in comparison with vertical artificial burrows and medium-height grass in comparison with short grass favour translocation success in the critical period after release. 4) We can conclude that the critical period after translocation ended between the 7th and 37th days after the release. It means that the relocated animals got over the stress caused by the disruption of their normal way of life in the original habitat (Teixeira et al. 2007) within this period. This finding is in accordance with the result of Calvete and Estrada (2004) who determined the critical period for translocated rabbits (Oryctolagus cuniculus) about 7-10 days after release. 5) The translocated animals prefer medium-height grass (18 cm ± 12 SD) to short (6 cm ± 3SD) grass after translocation. Our results showed that ground squirrels initially preferred the area where the grass was medium-height. It seems probable that grass tussocks provided a natural hide for the translocated individuals in the first days like shrubs provide overhead protection for Octodon degus (Ebensperger and Hurtado 2005; Hardman 122 and Moro 2006). In general, these results suggest that it is not enough to know the habitat preference of a species in undisturbed, “natural” situations, but manipulative experiments are needed to learn how and how much we should manipulate the habitat characteristics to accomplish a successful translocation action.

All habitat usage models (“birth rate”, “death rate”, and “migration”) takes into account the quality of the habitat which determines if the animals’ reproduction is or is not successful or if they move to the best place in the mosaic of habitat spots with different quality. From the viewpoint of fitness attributes, the quality of the habitat provides the base on which the population will increase if fitness attributes are beneficent in that environment. Although the magnitude of correlation between fitness attributes and genetic diversity at neutral marker loci is not clear (neutral molecular markers are not reliable indicators of adaptive variation), it is expected that neutral markers provide estimates of genetic diversity that may reflect the genetic health of wild populations (Beebee and Rowe 2008). Consequently, genetic diversity (genetic health) estimates the populations’ capacity to adapt and survive on new habitats via adapting their life history traits (behaviour, reproduction) to the novel or continuously changing environment. This work intended to study the genetic diversity of ground squirrels in general, and how much ecological characteristics can be linked to genetic diversity.

Generally speaking, genetic diversity of S. citellus populations from Hungary, Romania and Austria was higher than that of the Czech populations studied by Hulová and Sedlácek (2008). However, all our populations were located within the range of a “Northern” mtDNA phylogroup, that exhibited relatively little subdifferentiation despite its wide geographic distribution (Kryštufek et al. 2009); this common phylogeographic background might help to explain why we did not find a pronounced variation of genetic diversity across our local populations. In a metaanalysis Reed and Frankham (2003) found a positive relationship between fitness components and genetic diversity in plants, invertebrates, and vertebrates. They suggested that heterozygosity measured by molecular markers, via its link with population size, can give some indication of population fitness. According to Reed and Frankham (2003) placental mammal populations having incurred demographic threats in the recent past (“challenged populations”) exhibited consistently lower heterozygosity (mean 0.502) than unchallenged (“healthy”) populations (mean 0.677). In comparison, not only the “challenged” Czech S. citellus populations, but also all populations 123 of our study as well as the Bratislava population studied by Hulová and Sedlácek (2008) have relatively low heterozygosity. Moreover, most investigations and reviews on Sciuridae indicate higher genetic diversity than that found presently. In a free-ranging population, median litter size varied from four in the first three to six in the last four years, and the percentage of reproductively active yearling males increased with decreasing population density (Hoffmann et al. 2003). Ground squirrels are sensitive to changes in ecological factors (Koshev and Kocheva 2007), facilitating fluctuations in population density. Instable population dynamics, interacting with “scramble-competition polygyny” (Millesi et al. 1998), can decrease effective population size in the long and short term.

Population fragmentation, isolation, and local extinction are indeed occurring (IUCN 2008; Kryštufek 1999; Spitzenberger ger and Bauer 2001). In our opinion, the ongoing fragmentation, isolation and ultimately, disappearance of local colonies imply a more serious threat to the persistence of the species in wide ranges of its current distribution than possible negative effects (e.g., outbreeding depression) of reintroduction or translocation programs. Carefully planned and evaluated translocations can be an option to counterbalance local extinctions and substitute natural recolonisations. Overall, our data do not suggest serious drawbacks for potential anthropogenic translocation and restocking programs for local populations as regards genetic aspects. Actually, we are inclined to support such measures for local populations at low densities, as target population for translocated individuals, or for extremely high densities, as source population for translocation, should other (e.g., ecological) circumstances not discourage them. However, it remains open to what degree genes possibly adapted to certain regional environmental situations (e.g., MHC genes) may affect the success of restocking and introduction programs (under changed environmental conditions). To minimize possible negative genetic effects, we recommend using neighbouring source populations for translocations whenever feasible.

124 XVI. Summary In my Ph.D. thesis I investigated a few factors that influence the habitat use of the European ground squirrel (Spermophilus citellus; as EGS hereafter) and the Gunnison’s prairie dog (Cynomys gunnisoni; as GPD hereafter), two ecologically similar ground dwelling sciurids. I attempted to show the importance of a few extrinsic and intrinsic mechanisms by which the association between the habitat and ground squirrels is maintained. EGS show a strong preference for fresh Festuca pseudovina. Insulation property of the squirrels’ fresh grass nests was superior to nests constructed from dry grass only. Our results suggest that squirrels may rely on dominant xeromorphic fescues of dry meadows because of their importance to nest building. Consequently, it can be assumed that populations will expand to unoccupied areas and survive for longer-time and reintroductions will be successful if the habitat is characterized by fescues. GPD use mounds for vigilance and it increases with taller vegetation. Vigilance is more frequent and directed outward by individuals on the periphery than those in the interior of the colony. GPD we excavates about 6.5-16.6 m3/ ha/ year, so they can be considered as ecosystem-engineers indeed. It is possible to estimate volume of burrows with our calculation method by measuring the amount of subsoil displaced in the mound. Information on volume of burrows can be applied to monitor how the size of natural or artificial burrows changes. Results of translocation experiments showed that release time should correspond to the morning activity peak of EGS, the artificial burrow with retention cap prevents panic reactions, and angled artificial burrows and medium-height grass (18 cm ± 12 SD) favour translocation success in the critical period after release. Genetic diversity of EGS populations from Hungary, Romania and Austria was higher than that of the Czech populations but all studied populations have relatively low heterozygosity in comparison with other sciurids. Nevertheless, and perhaps as a result of simultaneously acting factors the genetic diversity of populations of neighbouring regions can differ significantly. To summarize, ground squirrels are sensitive to changes in environmental factors, facilitating fluctuations in population size. Ecological sensitivity, interacting with “scramble- competition polygyny” imply a more serious threat to the persistence of the species than possible negative effects of translocations as regards the genetic aspects. This notion is illustrated by the ongoing, rising isolation and disappearance of colonies.

125 XVII. Záró megjegyzések Dolgozatomban néhány környezeti tényező (ú.m.: növényzet, különös tekintettel a csenkeszre; talaj; járat; növényzet magassága) vagy populáció szintű belső folyamat (ú.m.: genetikai változatosság, populáció denzitása és mérete) és az élőhelyhasználat közötti összefüggést vizsgáltam az ürgék esetében. Továbbá, az eredmények tükrében, az ürgetelepítésekhez kísérelek meg útmutatót nyújtani.

Eredményeim azt mutatják, hogy a közönséges ürge (Spermophilus citellus) előnyben részesíti a friss, zöld sovány csenkeszt (Festuca pseudovina) mind laboratóriumi, mind természetes körülmények között. A friss csenkeszből készült fészkek szigetelő képessége meghaladta a száraz csenkeszből épült fészkek szigetelési tulajdonságát. A növényi intracelluláris nedvességtartalom nem befolyásolta szignifikánsan a szigetelőképességét. Az eredményekből arra következtettünk, hogy a friss csenkesz rugalmasabb nyersanyagot nyújt a fészeképítéshez akár laboratóriumi, akár természetes környezetben, ezáltal az abból épült fészek jobb szigetelőképességgel rendelkezik. Általánosságban elmondható, hogy az élőhely bizonyos abiotikus (Váczi et al. 2006; Wagner 2004) és biotikus (Kis et al. 1998) komponenseinek a vizsgálata rámutathat az élőhely azon tényezőire, amelyek a túlélés szempontjából kulcsfontosságúak. Ezen a tényezők ismerete értékes információval szolgál az ürge védelméhez, különös tekintettel az áttelepítésekre. Habár kísérletesen nem igazoltuk, hogy az ürgék aktívan keresik az olyan élőhelyfoltokat, ahol a csenkesz nagy tömegben fordul elő (“születési ráta” és “migrációs” élőhely modellek az ürgék eloszlásának magyarázatára) de elmondható, hogy az ürgék nem nélkülözhetik a csenkeszt, mint alapanyagot a pihenő- fészkekhez az aktív időszakban, tavasztól őszig, valamint a hibernáció során, ősztől tavaszig. Következésképpen feltételezhető, hogy a lokális populációk térbeli kiterjedése akkor növekszik, illetve azok hosszú távú fennmaradása úgy biztosított, amennyiben az élőhely karakterisztikus faja a csenkesz.

A közönséges ürgéhez hasonló ökológiájú Gunnison prérikutyák (Cynomys gunnisoni) járataihoz tartozó kupacokról az alábbi négy következtetést vonhatjuk le: 1) A prérikutyák a kupacokat használják megfigyelésre, valamint a megfigyelés napi aránya a más viselkedés kategóriák rovására a növényzet magasságának növekedésével emelkedik. A közepesen magas és magas növényzetben mutatott megfigyelés gyakorisága arra enged következtetni,

126 hogy a prérikutyák képesek viselkedésüket a növényzet magasságához, mint változó környezeti tényezőhöz igazítani. Ahogy a növényzet magassága növekszik, úgy emelkedik a megfigyelés mértéke, ezáltal biztosítva nagyobb biztonságot a csökkent látótávolság miatt fokozódó veszélyekkel szemben. A jelenség hasonló a degu (Octogon degus) esetében tapasztaltakhoz (Ebensperger and Hurtado 2005). 2) A kolónia peremén élő egyedeknél magasabb a megfigyelés mértéke, valamint az a kolóniához képest kifelé irányuló (Verdolin and Slobodchikoff 2002). A kolónia peremén élő egyedek fokozottabb figyelése, azaz a korai figyelmeztető rendszer, adaptív értékű, hiszen a szárazföldi ragadozók a kolónia perifériája felől közelíthetnek így azok észlelése ezáltal könnyebb. 3) A járatok térfogatát a kupacok térfogatának mérésével megbecsülhetjük. A kupacok térfogatának mérésére kidolgozott eljárás segítségével pontosabban becsülhető a járatok térfogata (mérete). Ez az információ jól hasznosítható az áttelepítések során, illetve a mesterséges vagy új járatok méretének változása jobban nyomon követhető így az áttelepítést követően. 4) A Gunnison prérikutyák által megmozgatott mintegy 10 – 16,5 t talaj összevethető a fekete-farkú prérikutyák (Cynomys ludovicanus) talajmozgatásával (Butler 2006). Az ökoszisztéma mérnök talajlakó állatok geomorfológiai hatása jelentős (Gabet et al. 2003), azonban annak meghatározása akár egyed, kolónia, vagy populáció szinten rendkívül nehéz (Nevo, 1999). A közismerten ökoszisztéma- mérnök Észak-Amerikai hörcsögfélék (Geomidae) évente kb. 18 m3 talajt mozgatnak meg, míg a Gunnison prérikutyák, ha csak a kupacok térfogatát vesszük figylembe, kb. 6,5 – 16,6 m3 talajt. Ez a “megművelt” talajmennyiség alátámasztja Detling and Whicker (1987) állítását és összhangban van az ökoszisztéma mérnök fajokról alkotott meghatározással, azaz a Gunnison prérikutyák igazi ökoszisztéma mérnöknek tekinthetőek (Jones et al. 1994; Gutierrez and Jones 2006). A prérikutyákra megfogalmazott négy következtetés azt mutatja, hogy a növényzet és a talaj befolyásolja az élőhely minőségét és a prérikutyák viselkedését, illetve a prérikutyák maguk is észrevehető hatással bírnak a növényzetre és talajra egyaránt. Különféle modellek (mechanizmusok) egyszerre működése képes csak megmagyarázni az élőlények és az adott élőhely összefüggéseit (Crowe and Underwood 1998), azonban a jelen eredmények arra is rámutatnak, hogy a talajlakó ürgék és rokonaik ugyancsak aktív, kulcsfontosságú elemei az élőhely és használói között játszódó “interaktív játéknak”. Akár a “születési ráta”, akár a “halálozási ráta”, akár a “migrációs” modellek magyarázzák helyesen az élőhely használatát az ürgéknek és rokonaiknak, mindenképpen jelentősen módosítják az ürgék és rokonaik az élőhelyet (mind a felszínt, mind a felső talajszintet). 127

A X. és XI. fejezetekben tárgyaltak megerősítik azt az elképzelést, hogy az ürge kolóniák hosszú távú fennmaradása (állatok túlélése és sikeres szaporodása; Seddon, 1999) erősen kötődik az áttelepítése utáni első, ún. kritikus időszakhoz. Továbbá, az élőhely minősége (a növényzet magassága és a természetes kupacos lyukakhoz hasonló mesterséges, ferde lyukak) az elengedés helyszínén jelentősen befolyásolja a kritikus időszakot (Letty, Marchandeau, and Aubineau 2007; Moorhouse et al. 2009). Az áttelepítés utáni kritikus időszakra és élőhely minőségre vonatkozólag négy következtetést fogalmaztunk meg, amely komolyan befolyásolja az áttelepített egyedek fennmaradását. 1) Az elengedés ideje a természetes körülmények között tapasztalt aktivitási csúcshoz kell, hogy illeszkedjen, ami a közönséges ürge esetében reggel van (de. 9:00 – 11:00). A reggeli elengedés hosszabb időszakot biztosít az állatoknak az első napon az új területhez való alkalmazkodáshoz. A hozzászoktatási időszak növelése az üregi nyúl áttelepítések esetében hasonló eredményt mutatott (Rouco et al. 2010). 2) Az áttelepített és lyukakba helyezett állatok pánikszerű menekülésének megakadályozása a mesterséges járatok elzárásával hozzásegít, hogy minél több állat maradjon az elengedés helyszínén. Amennyiben nem biztosítunk átmeneti búvóhelyet és nem kényszerítjük az állatokat az új élőhelyhez a kezdeti kritikus időszakban, akkor az állatok pánikszerűen elhagyják az elengedési helyszínt. Járatdugózási eljárásunk, amennyiben az elengedés illeszkedett a természetes napi aktivitási ritmushoz, elősegítette a kolonizációt és megelőzte a pánikreakciókat az első órákban. A mesterséges járatok átmeneti búvóhelyet szolgáltatnak, amíg az állatok elkészítik saját járataikat. Hasonlóan működnek a természetes járatokhoz, azaz kiegyenlítettebb hőmérsékletet és páratartalmat biztosítanak a talajban, ami lehetővé teszi pl. a hypo-, és hyperthermia elkerülését (Long et al. 2005). Gyakorlati szempontból tekintve az üvegpalackkal ledugózott mesterséges lyukakra elmondható, hogy hatékonyan működnek, azaz a drága és komplikált akklimatizációs ketrecek (Simms 2009) pótolhatók velük. 3) Eredményeink azt sugallták, hogy a ferde (30°-os a vízszinteshez képest) mesterséges lyukak a függőlegessel szemben kedvezően hatnak az áttelepítési sikerre a kritikus időszakban. 4) Megállapíthatjuk, hogy a kritikus időszak vége az áttelepítés utáni 7. és 37. nap közöttire tehető. Ez azt jeleni, hogy az állatok az áttelepítés miatti stresszt ennyi idő alatt heverték ki (Teixeira et al. 2007). Az eredmény összhangban van Calvete and Estrada (2004) adataival, akik üregi nyulakra ezt az időszakot 7 – 10 napra becsülték. 5) Az áttelepített állatok a közepesen magas füvet (18 cm ± 12 SD) preferálták az alacsonyabbal szemben (6 cm ± 3SD). Valószínű, hogy a közepesen magas fűcsomók természetes, felszíni 128 rejtőzködést biztosítottak az első napokban, hasonlóan a deguknál (Octodon degus) tapasztaltakkal (Ebensperger és Hurtado 2005; Hardman és Moro 2006). Általánosságban azt a következtetést vonhatjuk le, hogy az áttelepítések során módosulhatnak az állatok preferenciaigényei a természetes, zavartalan állapothoz képest. Ezért manipulációs kísérletekkel szükséges megállapítani, hogy milyen kezelést, mekkora mértékben kell végeznünk az új élőhelyen ahhoz, hogy sikeres áttelepítést hajthassunk végbe.

A „születési” és „halálozási” ráta, valamint „migrációs” élőhelyhasználati modellek az élőhely minőségét veszik figyelembe az élőhelyen belüli elterjedés magyarázatára, amely meghatározza, hogy az állatok szaporodása sikeres vagy sem, illetve vajon aktívan keresik-e a kedvező, jobb minőségű foltokat az élőhely mozaikban. Az élőhely minősége határozza meg, hogy a fitneszt kedvezően befolyásoló tulajdonságok végső soron a populáció méretének növekedését okozzák. Habár a fitneszt befolyásoló tulajdonságok és a neutrális genetikai markereknél mért változatosság közötti korreláció nem egyértelmű (neutrális molekuláris markerekben mért genetikai változatossága nem jelzi megbízhatóan az adaptív genetikai értéket és változatosságot), azt mondhatjuk, hogy a neutrális markereknél mért genetikai változatosság jól tükrözi a genetikai egészségét a vadonélő populációknak (Beebee és Rowe 2008). Következésképpen a genetikai változatosság (úm. egészség) mértéke becslést nyújt a populáció azon képességéről, hogy mennyire képesek az egyedek adaptálódni és fennmaradni új élőhelyen, azaz mennyire képesek életmenet stratégiájukat (viselkedés, szaporodás) egy új, folytonosan változó környezethez alakítani. Reed and Frankham (2003) pozitív összefüggést talált a fitneszt befolyásoló tulajdonságok és a genetikai változatosság között egy átfogó metaanalízisben mind növények, gerinctelenek és gerincesek esetében. Véleményük szerint a neutrális molekuláris markereknél mért heterozigócia foka a populáció mérettel (egyedszám) való kapcsolaton keresztül bizonyos fokig hűen jelzi a populáció fitneszét. Dolgozatomban a közönséges ürge genetikai változatosságát vizsgáltam, valamint azt, hogy a genetikai diverzitás mértéke mennyiben kapcsolható ökológiai folyamatokhoz.

Vizsgálataink alapján a magyarországi, ausztriai, és romániai ürgepopulációk genetikai diverzitása magasabb, mint Hulová and Sedlácek (2008) által vizsgált csehországi, genetikailag elszegényedett populációké. Az általunk vizsgált populációk az északi mtDNS törzscsoportba tartoznak, amely aránylag csekély genetikai eltérést mutat a tág földrajzi elterjedés ellenére (Kryštufek et al. 2009). Ez a phylogeográfiai háttér állhat a hátterében, 129 hogy miért nem találtunk jelentős különbséget a genetikai diverzitásban a vizsgált populációk között. Reed and Frankham (2003) vizsgálata szerint azok a méhlepényes emlőspopulációknál, amelyek a közelmúltban demográfiai krízisen mentek át (ú.n. “challenged populations”) a heterozigócia foka alacsonyabb, mint azoknál, amelyeket ilyen hatás nem ért (ú.m. „unchallenged populations”, azaz egészséges). Összehasonlítva eredményünket Hulová és Sedlácek (2008) vizsgálatával azt találjuk, hogy nemcsak a „challenged” cseh populációk, de mind a hazai, mind az ausztriai, mind a szlovák (Pozsony) populációk genetiaki változatosága viszonylag viszonylag alalcsony. A Sciuridae családba tartozó fajokon végzett genetikai tanulmányok általában magasabb genetikai változatosságot mutatnak, mint a jelenlegi vagy a cseh vagy szlovák populációkon végzett vizsgálatok. A viszonylag alacsony genetikai változatosság hátterében állhat egyrészt, hogy az ürgék érzékenyek az ökológiai tényezőkben bekövetkező változásokra (Koshev és Kocheva 2007). A változó populációdenzitás kedvezőtlen hatását erősítő a „scramble competition polygyny” sajátos rendszere. Hoffmann és mtsai. (2003) vizsgálata alapján, egy vadon élő populációban az alom mérete az első három évben tapasztalt négyről hatra változott a következő négy évben, valamint a reproduktívan aktív fiatal hímek aránya emelkedett a populáció denzitás csökkenésével. Összességében elmondható, hogy az ürgepopulációk dinamikai tényezőinek változékonysága, a „scramble competition polygyny” szaporodási rendszerrel a háttérben csökkentheti az effektív populáció méretet mind rövid, mind hosszú távon (Millesi et al., 1998), amely a genetikai változatosság csökkenéséhez vezet. Az ürge hosszú távú fennmaradását ugyancsak veszélyeztető tényező, hogy a (meta)populációk fragmentálódása, ökológiai elszigetelődése, illetve lokális kolóniák kipusztulása jelenleg is folyik az elterjedési területen (IUCN 2008; Kryštufek 1999; Spitzenberger and Bauer 2001). Meggyőződésünk, hogy ezek a folyamatok sokkal nagyobb veszélyt rejtenek magukban a faj fennmaradása szempontjából, mint az áttelepítésekben rejlő kockázatok (pl.: kültenyésztési hatás). A gondosan megtervezett és kivitelezett áttelepítések lehetőséget teremtenek a lokális kihalások ellensúlyozására, illetve a természetes rekolonizáció (immigráció) helyettesítésére. Eredményeink alapján, genetikai aspektusokat figyelembe véve, az áttelepítések nemcsak, hogy nem rejtenek magukban veszélyeket a lokális populációkra, hanem a magas denzitású populációk, mint forrás-, a tartósan alacsony denzitásúak, pedig mint célpopulációk jöhetnek számításba áttelepítésekhez, amennyiben más ökológiai szempontok ezt nem módosítják (pl.: parazitáltság). Mindazonáltal egyelőre nem ismert, hogy a szelekciós nyomás alatt álló gének szintjén (pl.: MHC gének) milyen mértékű 130 az adaptáció a környezeti állapothoz, amely befolyásolhatja az áttelepítések sikerét, amennyiben a forrás és célterültek eltérőek. Következésképpen ajánlott a szomszédos populációk közötti mesterséges géncsere, azaz az áttelepítés, amennyiben kivitelezhető.

131 XVIII. Összefoglalás Disszertációmban megismertetek néhány fontos külső (környezeti) vagy belső folyamatot, amelyek meghatározóak a közönséges ürge (Spermophilus citellus; továbbiakban ürge) vagy Gunnison préri kutya (Cynomys gunnisoni; továbbiakban préri kutya) élőhelyhasználatában. Az ürge előnyben részesíti a friss sovány csenkeszt (Festuca pseudovina) a fészeképítésben, a magasabb mértékű szigetelőképessége miatt. Eredményeim azt mutatják, hogy az ürgék nem nélkülözhetik a rövid füves pusztákon karakterisztikus szárazságtűrő csenkeszt, mint fészek alapanyagot. Feltételezhető, hogy a populációk térbeli kiterjedése akkor növekszik, illetve azok hosszú távú fennmaradása akkor biztosított, amennyiben az élőhely karakterisztikus faja a csenkesz. A préri kutyák a kupacokat használják ragadozó elkerülő megfigyelésre, és a megfigyelés napi aránya a növényzet magasságának növekedésével emelkedik. A kolónia peremén élő egyedek megfigyelés mértéke magasabb, és az a kolóniához képest kifelé irányuló. A kupacok térfogatának mérésére kidolgozott számítási eljárásunkkal jól becsülhető a járatok térfogata. Segítségével a mesterséges vagy új járatok méretének változása jobban nyomon követhető. A préri kutyák által “megművelt” talajmennyiség (min. 6,5 – 16,6 m3) összhangban van az ökoszisztéma mérnök fajokról alkotott meghatározással. Az ürge áttelepítések során azt tapasztaltuk, hogy az új élőhelyen való megtelepedést elősegíti, ha az elengedési időpontja a reggeli aktivitási csúcshoz illeszkedik, továbbá a mesterséges járatok elzárása megakadályozza az áttelepített állatok pánikszerű menekülését, így hozzásegít a terület kolonizációjához. A ferde mesterséges lyukak közepesen magas füves élőhellyel párosítva (18 cm ± 12 SD) kedvezően hatnak az áttelepítési sikerre. A magyarországi, ausztriai, és romániai ürgepopulációk genetikai diverzitása viszonylag magasnak mondható, mint a genetikailag elszegényedett cseh ürge populációké. Azonban a Sciuridae családba tartozó más fajok magasabb genetikai változatosságot mutatnak, mint akár a hazaiak akár az ausztriaiak. Az eredményeink alapján elmondható, hogy az ürgék érzékenyek a környezeti tényezőkben bekövetkező változásokra, ezért a populációk mérete erőteljesebben ingadozik térben és időben egyaránt. Ez az ökológiai érzékenység a „scramble competition polygyny” szaporodási rendszerrel párosulva sokkal nagyobb veszélyt rejtenek magukban a faj fennmaradása szempontjából, mint az áttelepítésekben rejlő genetikai kockázatok. Ezt támasztja alá az ürge populációk eltűnése, elszigetelődése az ürge elterjedési területén.

132 XIX. Publications

Published papers and manuscripts included in the thesis

Published or accepted papers in international scientific journals

Gedeon, Cs., Markó, G., Németh, I., and Altbäcker, V. 2010. Nest material selection affects nest insulation quality in the European ground squirrel (Spermophilus citellus). Journal of Mammalogy 91(3):636-641.

Gedeon, C., Váczi, O., Koósz, B., & Altbäcker, V. 2011. Morning release into artificial burrows with retention caps facilitates success of European ground squirrel (Spermophilus citellus) translocations. European Journal of Wildlife Research, 1-5. doi: 10.1007/s10344- 011-0504-3

Gedeon, Cs., Drickamer, L.C., Meador, A. S. 2011. Importance of Gunnison’s prairie dog (Cynomys gunnisoni) burrow entrance mounds for vigilance and soil mixing. Manuscript is accepted to The Southwestern Naturalist.

Gedeon, Cs., Boross, G., Németh, A., and Altbäcker, V. 2011. Release site manipulation to favour European ground squirrel (Spermophilus citellus) translocations. Accepted to Wildlife Biology.

Manuscripts in revision and to be submitted Gedeon, Cs., Hoffmann, I., Vaczi, O., Suchentrunk, F. XXXX. Does population size or ecological isolation affect genetic diversity in the polygynous European ground squirrels (Spermophilus citellus)?

Ben Slimen, H., Gedeon, Cs., Hoffmann, I., and Suchentrunk, F. XXXX. Dwindling genetic diversity in European ground squirrels (Spermophilus citellus). Manuscript is accepted to Mammalian Biology; Abstract published in Mamm.Biol. 73 (2008)).

133 Conference presentations related to the thesis Gedeon, C., Hoffmann, I., Váczi, O., Knauer, F., and Suchentrunk, F. 2011. Does ecological isolation or population size influence genetic diversity in the European ground squirrel, Spermophilus citellus? poster; 6th European Congress of Mammalogy, Paris, France

Gedeon, C., Hoffmann, I., Váczi, O., and Suchentrunk, F. 2011. Population characteristics and genetic diversity in European ground squirrels. oral pres.; Molecular Phylogeny and Microevolution in rodents, Vienna, Austria;

Gedeon, Cs., Drickamer, L.C., Meador, A. S. 2010. Estimating significance of the mounds at the entrances to burrows of Gunnison’s prairie dogs for vigilance and soil mixing. oral pres.; 3rd European Ground Squirrel Meeting, Ordu, Turkey;

Gedeon, Cs., Váczi, O., Boross, G., Németh, A., Koósz, B., and Altbäcker, V. 2009. Burrow entrance angle and grass height influence translocation success in the Spermophilus citellus. oral pres.; 2nd European Congress of Conservation Biology, Prague, Czech Rep.;

Gedeon, C., Hoffmann, I., Váczi, O., and Suchentrunk, F. 2009. The effect of population density and isolation on the mating system of Spermophilus citellus. poster; International Ethological Conference, Rennes, France;

Gedeon, Cs., Markó, G., Németh, I., and Altbäcker, V. 2008.Nest material selection affects insulation quality in the EGS. oral pres.; 2nd EGSM, Svaty Jan pod Skalou, Czech Rep.;

Gedeon, C., Hoffmann, I., and Suchentrunk, F. 2008. Population genetics of the European ground squirrel (Spermophilus citellus) from central and southeastern Europe. poster, 82nd DGS, BOKU, Vienna, Austria;

Říčanová, Š., Gedeon, C., Bryja, J., Cosson, J.-F., Choleva, L., Ambros, M. 2008. Microsatellite and MHC genetic diversity in the EGS in central Europe. oral pres.; University Conference, Ceske Budejovice, Czech Rep.;

134 Váczi, O., Gedeon, Cs., and Altbäcker, V. 2007. Theory & practice of the Spermophilus c. reintroductions. oral pres.; Conservation efforts for the benefit of Vertebrates in Hungary, Nimfea Assoc., Túrkeve, Hungary;

Szenczi P., Bánszegi O., Dúcs A, Gedeon, Cs., Markó, G., Németh, I., Altbäcker, V. 2007. Position of nests and mound morphology in relation to different soils in the mound-building mouse (Mus spicilegus). oral pres.; Xth Congress of the Hungarian Ethological Society, Göd, Hungary;

Szenczi P., Bánszegi O., Dúcs A, Gedeon, Cs., Markó, G., Németh, I., Altbäcker, V. 2007. The mound-building mouse as a “family entrepreneur”: reduced reproduction and aggression as the basics for cooperative survival. oral pres.; Xth Congress of the Hungarian Ethological Society, Göd, Hungary;

Gedeon, Cs., Attila, S. 2006. Conservation of the Spermophilus citellus. workshop moderator; 1st European Ground Squirrel Meeting, Felsőtárkány, Hungary;

Gedeon, Cs., Drickamer, L.C., Meador, A. S. 2005. Behavioural aspects of Gunnison’s prairie dog (Cynomys gunnisoni) mounds. oral pres.; XXIXth International Ethological Conference, Budapest, Hungary;

Publications not related to the thesis

Szenczi P., Bánszegi O., Dúcs A, Gedeon, Cs., Markó, G., Németh, I., Altbäcker, V. 2011. Communal mounds enhance successful overwintering in Mus spicilegus. Manuscript is accepted to Journal of Mammalogy.

Hulová, Š., Bryja, J., Cosson, J. F., Gedeon, Cs., Choleva, L., Ambros, M., and Sedláček, F. 2011. Depleted genetic variation of the European ground squirrel on both microsatellites and major histocompatibility complex gene as a result of strong population fragmentation in Central Europe. Accepted (online published) to Conservation Genetics.

135 Banczerowski J. Fekete M., Gedeon Cs., Hegedűs K., Horváth Cs., Jolánkai M., Idei M. 2009 (eds.). A Magyar Tudományos Akadémia kutaóhelyeinek 2008.évi tudományos eredményei II., III. Kötetek. [Scientific results of the research Institutes of the Hungarian Academy of Sciences, 2nd and 3rd Volumes] Akaprint Press, Budapest, pp. 276 és pp. 338.

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