Assessing inbreeding and loss of genetic variation in canids, domestic dog (Canis familiaris) and wolf (Canis lupus), using pedigree data

Transcription

1 Assessing inbreeding and loss of genetic variation in canids, domestic dog (Canis familiaris) and wolf (Canis lupus), using pedigree data Mija Jansson DEPARTMENT OF ZOOLOGY DIVISION OF POPULATION GENETICS STOCKHOLM UNIVERSITY 2014

2 Cover illustration: Mija Jansson Mija Jansson, Stockholm 2014 ISBN Printed in Sweden by US-AB, Stockholm 2014 Distributor: Department of Zoology, Stockholm University

3 Om saker och ting rullar på för enkelt är du bergis mitt i en utförsbacke. Claes Wiberg

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5 Abstract Genetic variation is necessary to maintain the ability of wild and domestic populations to genetically adapt to changed selective pressures. When relationships among individuals are known, conservation genetic management can be based on statistical pedigree analysis. Such approaches have traditionally focused on wild animal conservation breeding in captivity. In this thesis, I apply pedigree-based techniques to domestic and wild animal populations, focusing on two canids the domestic dog and the wild wolf. Main objectives include to 1) develop a means for making any pedigree fit the input requirements of the software Population Management x (PMx) and to use this program to 2) investigate rate of inbreeding and loss of genetic variation in dog breeds, including possible correlations between recent inbreeding and health problems, 3) estimate effects on inbreeding of the 2010 hunt of the endangered Swedish wolf population, and to 4) evaluate the potential to genetically support this wolf population through cross-fostering releases of zoo bred pups from a conservation breeding program. Results include successfully developing the converter program mped (Paper I) and applying both mped and PMx to dog and wolf pedigrees. I found extensive loss of genetic variation and moderate rates of recent inbreeding in 26 dog breeds, but no major difference in these parameters between breeds classified as healthy vs. unhealthy (Paper II). I found average inbreeding coefficients to more than double (from F=0.03 to 0.07) and founder genetic variation to decrease by c. 30 percent over the past few decades in traditional Swedish dog breeds identified as being of conservation concern (Paper IV). Hunting will make it less likely to reach genetically based Favourable Conservation Status criteria for the Swedish wild wolf population (Paper III), but release of zoo bred wolves through cross-fostering may potentially almost double founder genetic variation of this population (Paper V).

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7 This thesis is based on the following papers, which in the text will be referred to by their corresponding Roman numerals. I: Jansson, M., Ståhl, I., Laikre, L. (2013) mped: a computer program for converting a text file into a pedigree file used by the PMx-software for conservation genetic analysis. Conservation Genetics Resources, 5: II: III: IV: Jansson, M., Laikre, L. (2013) Recent breeding history of dog breeds in Sweden: modest rates of inbreeding, extensive loss of genetic diversity, and lack of correlation between inbreeding and health. Journal of Animal Breeding and Genetics, Article first published online: 2 DEC 2013; DOI: /jbg Laikre, L., Jansson, M., Allendorf, F. W., Jakobsson, S., Ryman, N. (2013) Hunting threatens achieving Favourable Conservation Status for an isolated, highly inbred wolf population. Conservation Biology, 27: Jansson, M., Laikre, L. Monitoring rate of inbreeding and loss of genetic variation in traditional Swedish dog breeds of conservation concern using pedigree data. (Manuscript) V: Jansson, M., Amundin, M., Laikre, L. Supportive release from a zoo population by cross-fostering can significantly increase genetic variation in the highly inbred wild Swedish wolf population. (Manuscript) Paper III is reprinted with kind permission of the original publisher, Conservation Biology, which owns the copyrights.

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9 Assessing Inbreeding and Loss of Genetic Variation in Canids, Domestic Dog (Canis familiaris) and Wolf (Canis lupus), Using Pedigree Data Mija Jansson Division of Population Genetics, Department of Zoology, Stockholm University, SE , Stockholm, Sweden INTRODUCTION Conserving Genetic Diversity The Founders of a Population Assessing Inbreeding Levels NATIONAL AND INTERNATIONAL CONSERVATION POLICY OBJECTIVES STUDY SPECIES The Domestic Dog The Wolf INBREEDING DEPRESSION Inbreeding Depression in the Domestic Dog Inbreeding Depression in the Wild Wolf Inbreeding Depression in the Zoo Wolf MATERIALS AND METHODS Paper I: The mped Converter Program Paper II: Recent Inbreeding and Health in Dogs Paper III: Genetic Effects of Wolf Hunting Paper IV: Swedish Native Dog Breeds Paper V: Supportive Release from a Zoo Population RESULTS mped (Paper I) Inbreeding and Health in Dogs (Paper II) Genetic Effects of Hunting the Wild Swedish Wolf Population (Paper III) Inbreeding and Genetic Variation in Dog Breeds of Conservation Concern (Paper IV) Supportive Release from a Zoo Population (Paper V) DISCUSSION AND CONCLUSIONS ACKNOWLEDGEMENTS

10 REFERENCES APPENDIX 1 CONSERVATION GENETIC CONCEPTS IN PEDIGREE ANALYZIS APPENDIX 2 INBREEDING DISTRIBUTION, SCHILLER HOUND APPENDIX 3 INBREEDING DISTRIBUTION, BULLDOG APPENDIX 4 MEAN KINSHIP DISTRIBUTION, SCHILLER HOUND APPENDIX 5 MEAN KINSHIP DISTRIBUTION, BULLDOG APPENDIX 6 AUTOSOMAL RECESSIVE DISEASE IN DOMESTIC DOGS APPENDIX 7 CHAMPIONSHIPS

11 INTRODUCTION Conserving Genetic Diversity Many old, domestic breeds, which are not used in large scale commercial production, typically have a small population size, and many are considered threatened (Lannek 2007). Domesticated animals are under strong selective breeding that result in a loss of genetic variation (Johansson and Rendel, 1968), and during recent years conservation genetic focus has increased in domestic animal populations (Teinberg et al. 1995, Stephens and Splan 2013, Joly et al. 2012, Leroy et al. 2011). This attention includes both scientific efforts and international and national policy work, including the Global Plan of Action for Animal Genetic Resources adopted in Interlaken, Switzerland (FAO, 2007). Traditionally, pedigree analysis for conservation management has focused on zoo populations of threatened wild animals (Frankhamn et al 2004); available software has been developed in that context (Ballou et al. 1995). The necessity of conserving genetic variation in wild animals has been recognized and studied for quite some time (e.g. Chesser et al. 1982). As conservation genetic management extends to include domestic populations, and as pedigree data becomes increasingly available also for wild populations, there is an increasing need for methods, including software, developed for zoo populations (Koch et al 2008, Naish and Hard 2008) to be applied to populations outside the zoo community. In statistical pedigree analysis, exact genotypic probabilities are often too complex to compute even for modern day personal computers. Computer simulations thus become a valuable tool (Ballou et al. 1995). For example, the Gene dropping (MacCluer et al. 1986) computer simulation application is frequently used to analyze loss of genetic variation over time in populations bred for conservation purposes in zoos. Gene dropping simulates how alleles from individual founders are spread to descendants in the pedigree, and may be used to address a series of questions relating to allele diversity retention and genotypic similarity among various groups of individuals in the pedigree (Lacy 1989; Geyer et al. 1989). Examples of the use of gene dropping include (MacCluer et al. 1986); estimating inbreeding coefficients and the amount of existing genetic variation in the population, as well as predicting the risk of future loss of genetic variation (for conservation genetic concepts in pedigree analysis, see further in Appendix 1). The Founders of a Population The population founders limit the genetic potential of a population: the amount of genetic variation in a population cannot exceed that contributed by the founding individuals. In dog breeds, because of the closed gene pools, founder effects are responsible for several breed related diseases (Ubbink et al. 1998). Today s modern breeds have closed gene pools, and 99 % of 414 dogs from 85 breeds are correctly assigned to their breed in a cluster analysis, meaning 11

12 that the relationships within a breed are not really questionable. This results in a reduced population size and an overall increase in genetic drift among domestic dogs (Wayne & Ostrander 2007). The increase in genetic drift results in a loss of genetic diversity within breeds and greater divergence among them. In some breeds, genetic variation has been further reduced by catastrophic events such as World War II (Wayne & Ostrander 2007), which reduced the number of pure bred dogs during that time. The variation (or lack of variation) in dog traits also seems to be driven by artificial selection (Vilà et al. 1999). It is possible that the intense inbreeding during the founding of the breeds made the deleterious recessive alleles widely spread already within the breeds (Vilà et al. 1999). For many wild species, it is natural to identify the subgroups within a geographical region, but this is less natural for purebred dog populations, mainly due to the effective use of popular sires (Calboli et al. 2008). The wild wolves of Scandinavia, like many other threatened wolfpopulations in the world, suffer from geographic isolation and fragmentation (Liberg and Sand 2009) which leads to no or minimal immigration and that the population could never extend its original potential. Assessing Inbreeding Levels Indications of inbreeding depression has been shown among both domestic dogs and the wild wolf-population of Scandinavia (e.g. Olafsdottir and Kristjansson 2008; Liberg et al. 2005; Räikkönen et al. 2006). Many pedigree-dogs have high coefficients of inbreeding (Calboli et al. 2008), and the wild wolves of Scandinavia are, on average, more related than siblings (Paper III). Given a situation with no immigration (and a finite population size), inbreeding levels increase over time. Pedigree analysis is useful to estimate loss of genetic variation due to inbreeding increases (Calboli et al. 2008). Even though homozygosity in smaller numbers of loci and inbreeding coefficients or in extreme inbreeding situations (such as selfing) are sometimes reported to be significantly associated, heterozygosity measured by molecular markers and inbreeding coefficient are generally uncorrelated (Bolloux et al. 2004). NATIONAL AND INTERNATIONAL CONSERVATION POLICY Both wild and domestic animals are explicitly mentioned in the United Nations Convention on Biological Diversity (CBD; and the National Swedish Environmental Objectives. Other policies handle either wild (The Habitats Directive, 92/43/EEC, available at: or domestic populations (Global Plan of Action for Animal Genetic Resources, available at: 12

13 The CBD aims at conserving biological diversity, sustaining the use of the components of biological diversity and fair and equitable sharing of the benefits arising from the utilization of genetic resources. The importance of conserving genetic variability of domestic populations of animals and plants is becoming increasingly recognized. The CBD, as well as the National Swedish Environmental Objectives ( explicitly state that domesticated animals, and the genetic resources they represent, are part of the biological diversity that should be conserved, monitored, and sustainably used. Domesticated animals are mentioned as an indicator for assessing biologic diversity trends ( and Target 13 of the new Strategic Plan for explicitly focuses on genetic variation of domestic populations ( The Habitats Directive (92/43/EEC) is the central biodiversity legislation within the European Union, which forces all member countries to promote a Favourable Conservation Status (FCS) of certain listed habitats and species, including the wolf (except for a Spanish and a Greek population). FCS of a species is defined in Article 1i of the Habitats Directive as: conservation status of a species means the sum of the influences acting on the species concerned that may affect the long-term distribution and abundance of its populations within the territory referred to in Article 2; The conservation status will be taken as favourable when: population dynamics data on the species concerned indicate that it is maintaining itself on a long-term basis as a viable component of its natural habitats, and the natural range of the species is neither being reduced nor is likely to be reduced for the foreseeable future, and there is, and will probably continue to be, a sufficiently large habitat to maintain its populations on a long-term basis The Interlaken Declaration on Animal Genetic Resources for food and agriculture has been signed by 109 countries, including Sweden. The declaration recognizes that there are significant gaps and weaknesses in national and international capacities to inventory, monitor, characterize, sustainably use, develop and conserve domestic animal genetic resources, and this needs to be addressed urgently. It also calls for mobilization of substantial financial resources and long-term support for national and international animal genetic resources programs. Wolf conservation is a controversial issue in Sweden, heavily and constantly debated. In this debate, unfortunately, scientific results are often used to legitimize different, and often conflicting, positions (Möller-Hansen et al. 2011). 13

14 OBJECTIVES The aim of my work is to generate knowledge that will contribute to a sound conservation genetic management of the domestic dog and the wild wolf, for both species pedigrees exists. For this thesis, I used pedigree data to address questions relating to rates of inbreeding and loss of genetic variation (measured in terms of founder alleles; Lacy 1989). The major objectives were: To develop a converter that can transform a studbook from a text file (.txt) to an input file for the pedigree analysis software PMx (.ped; Paper I) which can be analyzed in the free software Population Management x (Papers II, III, IV, V). To examine levels and rates of inbreeding and degree of retention of genetic variation in dog and wolf pedigrees with the specific objective of addressing the following questions: Is there a difference in inbreeding levels and retention of genetic variability between healthy versus unhealthy dog breeds that may imply that recent genetic management affects their health status? (Paper II.) Were levels of inbreeding, kinship and retention of founder genetic variation in the Scandinavian wild wolf population affected by the hunt in 2010? (Paper III.) What are the rates of inbreeding and loss of genetic variation measured in terms of founder alleles in 12 dog breeds originating in Sweden, including the 10 breeds identified as of conservation concern? (Paper IV.) What is the potential of a zoo population of wolves, bred for conservation purposes, to provide genetic support for the genetically weak wild Scandinavian wolf population? (Paper V.) 14

15 STUDY SPECIES The model organisms used in this thesis are the domestic dog (Canis familiaris) and the wolf (Canis lupus; Figure 1). I provide a brief description of these species relating to this thesis. Figure 1. Study species of this thesis, wolf left (Canis lupus; photo by Lovisa Häggström) and domestic dog right (Canis familiaris) as exemplified by a Norwegian elkhound, grey (photo by Sannse and obtained from en.wikipedia.org). The Domestic Dog Although domesticated animal breeds account for only a small number of species, they have had profound effects on the evolution of human societies and on the course of human history (Ruane 2000). The dog is considered the first domestic animal. Its wild ancestor, the wolf, was probably domesticated by mobile hunters/gatherers rather than by settled farmers (Savolainen 2007). The domestication of the dog occurred around 14,000-15,000 years ago (Sundqvist et al. 2006, Savolainen et al. 2002). Up until 200 years ago, dogs were primarily selected for breeding based on practical use for hunters and herders, but for a long time dogs have also been used for other practical purposes such as pulling sledges, guarding property, and as lapdogs to provide warmth (Beilharz 2007). In the last centuries, morphology has become the primary focus of selection. Sundqvist et al. (2006) suggest that most modern dog breeds have a recent origin, probably less than 200 years old. They also show that there was an unequal contribution of sexes in the origin of modern dog breeds with fewer males than females contributing genetically. 15

16 According to phylogenetic studies, dog breeds are organized into a distinct evolutionary hierarchy with the following primary groups (Wayne and Ostrander 2007; Vilà and Leonard 2007, Table 1, materials and methods, below): Herding Mastiff (including e.g. some terriers) Modern European (from the 1800th) Mountain (including e.g. German shepherd and some spaniels) There are associations of dog haplotypes with wolf lineages which indicate admixture between wolves and dogs which could have been an important source of genetic variation for domestic dogs (Vilà et al. 2003). Some North Scandinavian/Finnish dog breeds were recently proven to have a different origin than the rest of domestic dog breeds. These breeds are the results of wolf and dog crossings a few hundred to a few thousand years ago, rather than from one single domestic event (Klütsch et al. 2009). The breeds are Finnish spitz, Norwegian elkhound (grey), Norwegian elkhound (black), and Finnish lapphund. With respect to domestic animal populations, the Swedish Board of Agriculture has identified a number of traditional Swedish breeds of particular conservation concern, including ten dog breeds (Lannek 2007). In Paper V, we describe the conservation genetic situation of the dog breeds that are Sweden s conservation responsibility. The Wolf Recent studies have shown that large carnivores have even greater effects on eco-system than previously assumed and the grey wolf has been shown to affect not just the fauna but the flora as well (Ripple et al. 2014) the wolf is an important species. The wolf (Canis lupus L.) is a native species of Sweden since the last ice age, but it is currently classified as Endangered (Swedish Species Information Centre; It was hunted to extinction during the first part of the 20 th century. In the mid-1960s the species became protected when only occasional individuals were observed, but unfortunately this did not prevent their extinction in the first part of the 20 th century. In the winter of , a breeding pair was established in the border region between the Province of Värmland and Norwegian Hedmark and provided the foundation for the return of the species to the Swedish fauna (Liberg and Sand 2009). These two individuals, together with a male wolf which immigrated from Finland/Russia around 1990, constitute the primary genetic basis for the current population more than 300 wolves inhabiting the central part of the Scandinavian Peninsula. For more than 18 years, none of at least ten wolves that immigrated via Finland to Northern Scandinavia were able to reach the population in Mid- Scandinavia and contribute genes to the population (Liberg and Sand 2009). New genes were added in 2008 when two unrelated immigrating male wolves reproduced with females from the Scandinavian population (Åkesson and Bensch 2010). 16

17 Figure 2. The location of the breeding sites for the first five founders of the Swedish wolf population. Nyskoga refers to a mating couple from Nyskoga, all other founders were males. Every immigrant is important to the isolated Scandinavian wolf population. Since 2008, only seven more wolves, apart from the original founders, have migrated into Scandinavia (Ann Dahlerus, Svenska rovdjursföreningen and Mikael Åkesson, Grimsö Wildlife Research Station, personal communication, January 2014.). Five of them have had contact with the Scandinavian wolf population. One of these wolves, a female that was first observed in the northern part of Sweden in the Province of Norrbotten and later in the Province of Jämtland in 2010 ( Kilbergstiken later Junselevargen ), caused damage to reindeer and was subsequently moved south three times only to travel north again. Her first partner was illegally shot following their first relocation. In 2012, the female settled near Junsele with a new male, but he was shoot by authorities after killing reindeer. Authorities stopped the hunt on the female after public complaints and due to EU regulations. The female has currently bonded with a male from Siljansringen. In 2013, a couple from Finland immigrated and were moved from northern Sweden to Tiveden where they settled down and had at least three, probably five, pups. Thus, there are currently seven and one potential founder of the existing Swedish wolf population. This is a low number from a conservation genetics perspective, because the amount of genetic variation in a population cannot exceed that contributed by the founding individuals. The precarious situation of the Swedish wolves has been pointed out for decades (e.g. Laikre 1999, Liberg et al 2005, Hagenblad et al 2009). Rapid population growth and gene flow from neighboring populations are critical for achieving long-term population viability. Nevertheless, population levels have been kept low and in 2009, the Swedish Parliament temporarily decided 17

18 that the population should be kept below 210 individuals (Swedish Government 2008/09:210). In order to achieve this, 28 wolves were culled in 2010 and 19 in In addition, close to 30 animals were shot in these two years for other reasons (e.g., causing damage to livestock), 16 were killed in traffic and two drowned because of sedation after being tagged with radio transmitters. In 2014, the Swedish Government decided on a new licensed killing of 30 wolves in the provinces of Dalarna, Värmland and Örebro even though it is against both Swedish and EU legalization and after appeals to the administrative court, the Government had to call off the hunt, at least temporarily. For the past decades, the issue of viable carnivorous population has been discussed in Sweden. A population's viability is affected by both external factors, such as climate, food sources and diseases, and, for populations less than thousands of animals, the population size in itself is considered a risk as inbreeding leads to less evolutionary potential and inbreeding depression (Ebenhard and Höggren, 1999). INBREEDING DEPRESSION Conservation is particularly complicated in small populations (Franklin1980) because they inevitably suffer from inbreeding and loss of genetic variation, which may cause inbreeding depression and loss of evolutionary potential (i.e. the ability to adapt to changes in the environment; Gyllensten and Ryman 1985). Virtually all the genetic effects which arise in small populations result from the random sampling of alleles from parents to offspring. This process is known as genetic drift. In a small population, gene frequencies change rapidly from one generation to another because of this random process. A small population size may have genetic effects in both a long-time and short- time perspective. Inbreeding depression may occur over a short period. More long-term, sampling effects causing fluctuations in gene frequencies have important consequences for the future evolution of the species (Crow and Kimura 1970). Long-term species survival is dependent on retaining enough genetic diversity both within and between populations to accommodate new selection pressure brought about by environmental change (Schonewald-Cox et al. 1983). But there are examples of wild populations with a longterm small and effective population size with small genetic variations that are viable (Rodríguez et al. 2011). Inbreeding Depression in the Domestic Dog In the domestic dog, several breed-related diseases have been attributed to founder s effect (Ubbink et al. 1998). Such diseases constitute a huge problem in many dog populations and are probably threatening their survival (Ubbink et al. 1998). For example, a population of Dutch Labrador retrievers (with elbow dysplasia) showed estimates of relatedness to seven related ancestors when modeling the most likely pattern in passage of genetic risk for the disease over generations (Ubbink et al. 1998). In investigations of cone degeneration (achromatopsia; Yeh et 18

19 al. 2013) in Siberian husky and Alaskan sled dogs and miniature Australian shepherd, all affected alleles were shown to be identical by descent, strongly suggesting a founder effect. Since the miniature Australian shepherd is not known to be genetically related to the Alaskan Malamute, other breeds may potentially carry the same allele and be affected by cone degeneration (achromatopsia). English keeshounds have problems with canine epilepsy. By calculating inbreeding and identifying common ancestors (under the hypothesis that both parents of an epileptic pup were themselves carriers; Hall & Wallace 1996), it could be assumed that the predisposition to epilepsy in keeshounds is determined by a single autosomal recessive gene. In the Icelandic sheepdog, hip dysplasia was also found to be significantly related to inbreeding depression (Ólafsdóttir & Kristjánsson 2008). P G C Bedford, at the Royal Veterinary College in UK, states in an editorial comment in Animal Welfare 1999 that: Within the world of the pedigree dog, competition is extreme and breeding policy based on dedication to breed type has resulted in the appearance of some 300 inherited diseases among canine species worldwide. In appendix 6, I have compiled 145 diseases in the domestic dog with autosomal recessive inheritance. Inbreeding Depression in the Wild Wolf The genetic problems of the wild wolf population of Scandinavia include inbreeding depression (Liberg et al. 2005; Räikkönen et al. 2006). The number of surviving pups per litter during their first winter after birth are strongly correlated with inbreeding coefficients of pups (R 2 =0.39, p=0.001; Liberg et al. 2005). Several congenital malformations of the backbone have been shown (Räikkönen et al. 2006). Immigration of wolves from eastern populations is essential in order to counteract the possible manifestation of high frequencies of deleterious traits (Räikkönen et al. 2006). Inbreeding Depression in the Zoo Wolf Previous studies on captive wolves have shown negative effects of inbreeding (Laikre and Ryman 1991, Laikre et al. 1993). These effects are expressed as a reduction of juvenile weight, reproduction, longevity and a hereditary form of blindness. Presently, there are no documented negative effects of inbreeding in the captive population of wolfs (personal communication January 2014, Mats Amundin, official studbook keeper within the framework of the Swedish Association of Zoos and Aquaria). 19

20 MATERIALS AND METHODS Paper I: The mped Converter Program In Paper I, I present the mped program which we developed to convert studbook data into a format that can be used in the Population Management x software (PMx; Ballou et al. 2011; Lacy et al. 2011). PMx is a free software for analysis and conservation management of pedigreed zoo populations. It provides tools for optimal genetic management of populations for which preserving genetic diversity is a primary goal. The software can handle large data set outputs of several parameters relating to inbreeding and loss of genetic variation. PMx is most easily used as an accessory program to the SPARKS software for studbook management, but it can also be used as a stand-alone software for population management of the demographic and genetic data The data must first be prepared in a specific format (.ped, the PMx input file). It is quite timeconsuming, to the point where it is impossible, to create.ped files from else than SPARKS s studbooks, with many manual steps. Paper II: Recent Inbreeding and Health in Dogs Paper II focuses on the investigation of potential correlations between recent inbreeding and health in populations of dog breeds recorded in Sweden and those we obtain from the Swedish Kennel Club. In total, 332,784 individuals from 26 pedigrees (breeds) of domestic dog were analyzed (Table 2). I selected populations for analysis by first identifying healthy and unhealthy breeds based on information from insurance companies. Statistics reflecting the extent of veterinary care per dog breed were obtained from Sweden s four most prominent pet insurance companies for pets (Agria; Folksam; If; and Sveland; The companies Agria, Folksam and Sveland use six price categories (six being the highest costs for veterinary care per dog, and one the lowest). The If insurance company uses eight price categories (where eight represents the highest costs for veterinary care per dog, and one the lowest). I ranked dog breeds based on the classifications from the four companies, respectively, and defined unhealthy breeds as those classified as most expensive with respect to veterinary care by at least three of the four companies. The opposite was done to identify healthy breeds (breeds classified in the category of lowest veterinary care expenses by at least three of the four companies). I identified 15 unhealthy and 11 healthy breeds. These breeds are presented in Table 1, together with the classification of each breed with respect to type of dog made by the international kennel club FCI ( Parker et al. (2004), and Wayne and Ostrander (2007), respectively. The number of individuals per pedigree is shown in Table 2. 20

21 Table 1. Unhealthy/healthy breeds were chosen from data from insurance companies. The type of dog is based on the nomenclature from the international kennel club (FCI). The final classification was based on analysis in the publications of Parker et al. (P. et al. 2004) and Wayne and Ostrander (W. and O. 2007). We have used the categories of W. and O., namely: Ancient breeds (A), Herding (H), Mastiff (M), Modern European (E), Mountain (X). In Paper II, only a certain type of bull terrier - the miniature bull terrier - was studied. Type of breed Classification Class Breed (FCI Breeds nomenclature) P. et al. W. and O. Unhealthy bull terrier Bulltype terrier M M Unhealthy bulldog Molossioid breed M M Unhealthy bullmastiff Molossioid breed M M Unhealthy dogo Argentino Molossioid breed - - Unhealthy German boxer Molossioid breed M M Unhealthy great Dane Molossioid breed E H Unhealthy mastiff Molossioid breed M M Unhealthy Neapolitan mastiff Molossioid breed - - Unhealthy Rottweiler Molossioid breed M X Unhealthy shar pei Molossioid breed A A Unhealthy French bulldog Small Molossian type M M Unhealthy Bernese mountain dog Mountain Dog M X Unhealthy dobermann Pinscher E E Unhealthy deer hound Sighthound - - Unhealthy Irish wolfhound Sighthound H H Healthy coton de Tuléar Companion and Toy dog - - Healthy Hamilton hound Scenthound - - Healthy Schiller hound Scenthound - - Healthy Småland hound Scenthound - - Healthy Finnish lapphund Spitz type - - Healthy Finnish spitz Spitz type - - Healthy Norrbottenspitz Spitz type - - Healthy Norwegian buhund Spitz type - - Healthy Norwegian elkhound, black Spitz type - - Healthy Norwegian elkhound, grey Spitz type E E Healthy Siberian husky Spitz type A A 21

22 Bulltype terriers are terriers that originally were used for fighting (Bull terriers and Staffordshire terriers). Molossers are solidly built, generally quite large dog breeds mostly used for guarding people or livestock. Mountain dogs are molossoid breeds but more of a farm dog. Pinschers (and schnauzers) are continental dogs of the type that in Great Britain are called terriers but unlike the terriers the pinschers (and schnauzers) were farm dogs. Sighthounds hunt primarily by speed and sight. Companion and Toy dogs are small in body size and are primarily used as companions. Scenthounds hunt primarily by scent. Table 2. The number of individuals in the pedigree for each of the 26 dog breeds included in Paper II. The type of breed is based on Federation Cynologique Internationale (FCI) Breeds nomenclature. Status Full name No. in ped. Healthy coton de Tuléar 3563 Healthy Hamilton hound Healthy Schiller hound Healthy Småland hound 7118 Healthy Finnish lapphund 6465 Healthy Finnish spitz Healthy Norrbottenspitz 8176 Healthy Norwegian buhund 4123 Healthy Norwegian elkhound, black 994 Healthy Norwegian elkhound, grey Healthy Siberian husky Unhealthy Bull terrier 4870 Unhealthy Bulldog 5830 Unhealthy Bullmastiff 3749 Unhealthy dogo Argentino 1295 Unhealthy German boxer Unhealthy great Dane Unhealthy mastiff 1678 Unhealthy Neapolitan mastiff 1207 Unhealthy Rottweiler Unhealthy shar pei 2475 Unhealthy French bulldog 8933 Unhealthy Bernese mountain dog Unhealthy Dobermann pinscher Unhealthy deer hound 1555 Unhealthy Irish wolfhound

23 The health problems occurring in the breeds classified as unhealthy have been noted by the Swedish Kennel Club (SKC). Attention to these health issues caused by exterior exaggerations has been noted for instance in the publication Special Breed Specific Instructions (BSI) - regarding exaggerations in pedigree dogs for both the individual dog and the development of the breed as a whole. BSI is handed out to every judge at Swedish dog shows. The aim is to identify areas of risk and to prevent possible future problems. 10 of the 15 unhealthy breeds in this study are noted in the BSI, i.e.: Bull Terrier (and Miniature Bull Terrier), Bulldog, Bullmastiff, German Boxer, Great Dane, Mastiff, Neapolitan Mastiff, Shar Pei, French Bulldog, and Irish Wolfhound. The origins of the dog breed exteriors are explained by their original tasks, even though the interpretations of the breed standards do not always reflect their original task. The molossoid and bulltype breeds are typically heavy and solid. When these physical characteristics are exaggerated, the exterior becomes unhealthy. The pincher and sighthounds included in the study are very large in body size for their group of breeds. Typical molossoid breed diseases are shown in Table 3. Though molossoid breeds show common problems (they all have skin problems), it is unknown whether this caused by a common genetic background. To address the issue of possible temporal trends in average inbreeding and retention of founder genetic diversity, we analyzed levels of inbreeding and loss of founder genetic variation at three points in time including dogs alive at 31th of December, 1980, 1995, and 2010, respectively. The pedigrees of December 31, 2010, represent the full pedigree of each breed (Table 2). The number of individuals per full pedigree varied from 994 (Norwegian elkhound, black; Table 1) to (Norwegian elkhound, grey). See further Appendix

24 Table 3. Typical health problems of some of the breeds classified as unhealthy. Data were obtained from Breeds Specific Strategies available from the Swedish Kennel Club ( [in Swedish]). Breed type classification is based on Parker et al (P et al) and Wayne and Ostrander 2007 (W & O); Mastiff (M) and Mountain (X). Breed P et al W & O Health problems bull terrier M M immunological diseases (mainly skin diseases), patellaluxation, heart and kidney problems bulldog M M bullmastiff M M German boxer M M mastiff M M Rottweiler M X HD, ED, epilepsi, thyroid diseases French bulldog M M Bernese mountain dog M X weak contractions, HD 1, a large number of skin diseases, eye problems, problems with circulation and breathing, epilepsy cancer (lymphoma), skin diseases, eye problems, ligament injuries, HD, ED 2, aortic stenosis HD, osteoarthritis, PNP (kidney disease), heart problems, skin problems, epilepsy, Cryptorchidism, spondylosis, weak contractions ear and eye problems, obstetric complications, HD, ED, cruciate ligament injuries, gastric torsion/bloat stomach, skin problems respiratory problems, eye problems, ear infections/problems, allergies (including skin), patella dislocationtumors of the mouth/throat sarcoma lymphon, back pain, epilepsy, othematom, problems in the lower urinary tract HD, ED, PNP (kidney disease), umbilical hernia, tumors, pyometra, cruciate ligament injuries 1 Hip dysplasia 2 Elbow dysplasia 24

25 Paper III: Genetic Effects of Wolf Hunting Paper III aimed to investigate if levels of inbreeding, kinship and retention of founder genetic variation in the Scandinavian wild wolf population were affected by the hunt performed in 2010 and to assess whether the Swedish wolf hunts in 2010 and 2011 were in line with national and international policy agreements (including agreements within the European Union [EU] of which Sweden is a part).the study is based on a studbook of the Swedish wild wolf population that has been generated by the Skandulv Research Project ( The Swedish wolf population has been monitored closely since the establishment in the 1980s. Tracking data in combination with molecular genetic analysis of collected blood, hair, and other biological material including tissue from dead animals has resulted in an almost complete pedigree of the population that is maintained by the Skandulv Project (Liberg et al. 2005). We analyzed the genetic effects of the 2010 wolf hunt using the pedigree data from the Skandul Project as of November 2010 obtained from the Skandulv Project. We used the Population Management 2000 software ( to compute inbreeding coefficients, mean kinship, to analyze founder contribution and loss of founder genetic variation. Paper III also relates wolf hunting and the genetic situation of the Swedish wolf population to existing international and national conservation policies, particularly the EU Habitats Directive ( the UN Convention on Biological Diversity ( and the Swedish national environmental goals (Swedish Government Bill 2004/05:150, Environmental Quality Objectives - A Shared Responsibility, adopted by the Swedish Parliament in November 2005). Paper IV: Swedish Native Dog Breeds In Paper IV, we described the rates of inbreeding and loss of genetic variation, measured in terms of founder alleles in 12 dog breeds originating in Sweden, including the 10 breeds identified as being of conservation concern. The pedigrees of nine of the 12 breeds date back to the 1960s or before. Four of the breeds were recognized as pedigree breeds relatively late by the Swedish Kennel Club the Danish Swedish Farmdog (1987; Table 1), the Gotland hound (1990), the Swedish white elkhound (1993), and the Hällefors hound (2000). The pedigrees of these breeds date back to the 1970s for Hällefors hound and to the 1980s for Danish Swedish farmdog, Gotland hound, and Swedish white elkhound. To monitor possible temporal trends, we analyzed levels of inbreeding and loss of founder genetic variation (see below) at five points in time including dogs alive on December 31 in the years of 1980, 1990, 2000, 2006, 2012, respectively. To determine the number of live dogs at the three points in time, we had to make assumptions about the longevity of dogs and we only considered dogs with a Swedish registration number as being alive. Paper V: Supportive Release from a Zoo Population This paper describes the potential of a zoo population, bred for conservation purposes, to provide genetic support for the genetically weak wild Scandinavian wolf population. The pedigree of the wild Scandinavian wolf population is maintained by the Skandulv Project. We obtained studbook data as of January 2012, and this pedigree included a total of 740 individuals, 376 of which were 25

26 classified as being alive. This classification was based on the assumption that identified individuals that have not yet been documented as dead are alive unless they are unreasonably old and/or have not been seen for many years. The pedigree of the captive population is maintained by Mats Amundin, a co-author who maintains the official studbook keeper within the framework of the Swedish Association of Zoos and Aquaria. We used pedigree records per January 2012 comprising a 132 total of 1229 individuals out of which 145 were alive at that time. We used the Population Management x software to obtain the quantities assessed, and mped to convert the pedigree of the wild wolf population into PMx input format (see Paper 1 above). Input files to PMx for the captive population pedigree were generated through PopLink 2.3 (Faust et al. 2012). PopLink is a computer program designed for management and analysis of studbook databases. (Zoo studbooks include information on each individual in a population, including pedigrees, and dates of birth, death and transfers between institutions. The studbook traces the entire history of each individual in a population; these collective histories describe the population's genetic and demographic identity.) PopLink can help maintain, analyse and export data for a captive population which are relevant to their genetic and demographic management. PopLink imports a studbook from SPARKS, the current software used to manage studbook datasets in Zoos. (Faust et al ) RESULTS In this section, the results are presented by paper. mped (Paper I) To simplify the creation of new ped-files from databases which were not originally constructed for PMx, we developed a converter called mped (make ped file) in the C programming language. The converter we developed (Paper I), proved to be useful in transforming studbook data into a pedigree file (a.ped file) which can be used by Population Manager x. We had several problems with some dog pedigrees obtained, including too extensive data for PMx (Table 2), which typically have an upper limit of about 20,000 individuals, but also depending on the complexity of the pedigree. mped was constructed to reduce the file by deleting dead individuals which have no descendants in the living population. Also, data on date of birth are sometimes missing in the SKC studbooks, and mped can help estimate birthdates in such cases. Similarly, the SKC databases do not include dates of death, but mped can provide estimated dates. There are also other ways in which mped can modify pedigrees, and in addition to providing input files for PMx, mped can produce input data to the Vortex Population Viability Analyses Software simulation program ( mped was successfully used to provide pedigree files used for Papers II and III. 26

27 Inbreeding and Health in Dogs (Paper II) We found extensive loss of genetic variation and moderate rates of inbreeding in all the 26 breeds examined, but no strong indication of a difference in these parameters between healthy and unhealthy breeds (Table 4). Thus, we conclude that recent breeding history with respect to inbreeding levels and maintenance of founder alleles does not appear to be a main cause of poor health in some dog breeds. Table 4. Summary of results from Paper II regarding healthy and unhealthy breeds at the three different points of time. Mean values, for all breeds, of founder, founder genome equivalents (fge), mean kinship (MK) and inbreeding (F). Health status Year Founders fge Lacy1995 MK F Healthy Healthy Healthy Unhealthy Unhealthy Unhealthy Appendices 2-3 show an example of distribution of F for a healthy and an unhealthy breed and Appendices 4-5 show MK for the same breeds. MK typically varies less than F when the mean kinship is the theoretical F for the next generation. Figure 3 includes a figure for inbreeding level and one for genetic variation for all breeds during the years, grouped in unhealthy and healthy breeds. The inbreeding Figure (3a) illustrates the lack of a linear trend in inbreeding. 27

28 Figure 3. Left (a) - Mean inbreeding for breeds, classified as healthy (light) and unhealthy (dark). The classification of healthy or unhealthy was based on statistics on extent of veterinary care obtained from Sweden s four largest insurance companies for pets. Right (b) - The Loss of Genetic Variation measured as Founder Genome Equivalents (fge) per Founder for each breed, grouped as unhealthy breeds (dark) and healthy breeds (light). Genetic Effects of Hunting the Wild Swedish Wolf Population (Paper III) Prior to the 2010 hunt, the wild Swedish wolf population consisted of 209 individuals. Fourteen of these wolves were protected from hunting because they represented the territories of the two males that immigrated into Sweden in 2007/2008. This implies that 195 animals were subjected to hunting. The mean inbreeding coefficients among the 195 animals was F=0.29. Among the 28 wolves which were killed, the average inbreeding was F=0.26. This was significantly less than what would be expected if the 28 animals killed had been selected at random. Average F was at 0.27 after the 2010 hunt. If pedigree data had been used to identify the most inbred individuals, average F could have been reduced to However, reducing the average level of inbreeding 28

29 should not be the only objective of genetic management in a case like this; it is equally important to maintain as much as possible of the remaining allelic diversity from the five founders of the population. Maximizing the retention of their alleles includes reducing further loss of genetic variation and striving to spread the genes of the two most recent immigrant males to make their genetic contribution similar to that of the three original founders. During the 2010 hunt, offspring from the two immigrant males were protected. Thus, the genetic contribution from these founders was not reduced, but the proportion of lost variation measured as founder allele survival as calculated from the pedigree increased from 18% to 20% for the two original founders and from 4% to 5% for the male which immigrated in Inbreeding and Genetic Variation in Dog Breeds of Conservation Concern (Paper IV) Inbreeding is relatively extensive in these breeds with the average F across all breeds exceeding that of first cousin mating in both 2006 and 2012, and for 8 of the 12 breeds the average F of the living population exceeds at one or more points over time. Similarly, the ranges of F within breeds show conspicuously high inbreeding coefficients for at least individual dogs within all breeds. There is no correlation between average F and population size and there is no correlation between F and the number of founders at any of the points in time, indicating that, contrary to what would be expected; number of founders or population size does not explain inbreeding levels. MK is used in conservation breeding to choose breeding animals; by prioritizing low MK, individual inbreeding and loss of founder genetic variation is minimized. Average and range of MKs among the Swedish dog breeds show that such prioritization has typically not been carried out for these breeds. In many cases, average MK at one point in time is lower than average F for the next time point. For instance, average F for the Swedish lapphund is consistently larger than the average MK for the prior time point. In contrast, for the Swedish white elkhound, the Norrbotten spitz, the Danish Swedish farmdog, and the Gotland hound, the average F is below the MK of the preceding time step, indicating that dogs chosen for breeding have had lower MK than the average among living dogs. Comparing ranges of MK to ranges of F, however, indicates that for none of these dogs, breeding animals are consistently chosen to primarily reduce inbreeding and retain genetic variation. Genetic variation is measured in relation to population founders as the proportion of founder alleles that remain among living animals at separate points in time, and the results show that the loss of such variation is extensive. Similar results are observed when retention of founder alleles is quantified as founder genome equivalents. The result indicate that current gene pools of separate Swedish traditional dog breeds represents variation of less than 20 unrelated founders, and in many cases less than 10 unrelated founders. 29

30 Supportive Release from a Zoo Population (Paper V) We combined the pedigrees of the captive and wild populations to investigate the potential for genetic support from the zoo to the wild. The joint population has 15 founders and 21 of their alleles remain, fgelacy1995= 3.2 and fgelacy1989=4.8. Thus, founder genetic variation of the wild wolf population can be almost doubled using genetic support from the zoo. We computed target values for genetic contribution of separate founders taking their retention of genetic variation into account, and found that for maximum founder allele retention, the zoo founders should contribute just below 50 percent to a joint population. We investigated how much of the genetic variation of the zoo population would remain in five or ten hypothetical offspring from each of eight established captive breeding pairs existing at the time of our data collection. For separate PMx runs, we assumed that only hypothetical offspring (5 or 10) from a particular pair was alive. The amount of remaining founder genetic variation is consistent and not much affected by the number of offspring (5 or 10). The remaining number of founder alleles is around 3.5, representing 30 percent of current levels of variation. Further, if all eight pairs produced either 3 or 5 offspring, resulting in 24 or 40 hypothetical pups and assuming only these pups remain alive, the number of retained founder alleles is 9.9 or 10.3, fge Lacy1995 is 3.0 or 3.1, and fge Lacy1989 is 4.6 or 4.7, respectively. Thus, both 3 and 5 pups per pair retain a large proportion of the remaining genetic variation of the zoo population. Founder allele retention can be computed for a large number of hypothetical combinations of reproducing pairs. Our limited analysis indicates that a relatively large proportion of the founder allelic gene pool of the captive population can be maintained by a limited part of the population. DISCUSSION AND CONCLUSIONS Even though pedigree information can be used to monitor and control inbreeding in a population, molecular data can also be used. A study shows that the heterogeneity found when estimating the alleles identical by descent between pairs of dogs ranged from 0 to 0.60 within the same breed (Pertoldi, 2013). The 0.6 value is much higher than common inbreeding levels (and this is probably due to early inbreeding in the origin of breeds). Despite this, pedigree analysis is more cost-effective when investigating recent inbreeding in large dog populations. In this section, I will briefly go through the conclusions for the different papers followed by some discussion. Developing a converter that can transform a studbook from a text file (.txt), to a input file for the PMx pedigree analysis software (.ped; Paper I): Me and my colleagues were able to develop the mped converter program which allows for a broad range of pedigrees outside the zoo community 30