5

Genetic Diversity in Free-Ranging Horse and Burro Populations

This chapter reviews the relationship between genetic diversity and the long-term health of free-ranging horse and burro herds. It does that by reviewing genetic studies conducted on 102 horse Herd Management Areas (HMAs) and 12 burro HMAs under the jurisdiction of the Bureau of Land Management (BLM) and comparing the results with those of studies of other species and herds for evidence of an optimal level of genetic diversity that might be used as a management target. It also examines the idea that BLM’s free-ranging horse and burro herds can be considered a metapopulation, or a “population of populations that are spatially discrete but connected through natural or assisted immigration” (Levins, 1969). Metapopulation theory can be used to suggest directions for management activities that might be undertaken to attain and maintain the level of genetic diversity that is needed for continued survival and reproduction and for adapting to changing environmental conditions.

THE CONCEPT AND COMPONENTS OF GENETIC DIVERSITY

Genetic studies provide essential data for the management of populations, including estimates of the levels and distribution of genetic diversity, assessments of ancestry, and the detection of genetically distinct populations. At the population level, genetic diversity can be measured as the mean number of variants of a gene (alleles) or as the proportion of individuals that have different variants of a gene (heterozygosity). Theoretical and empirical studies have demonstrated substantial fitness costs associated with the loss of genetic diversity in both free-ranging and captive populations (Lacy, 1997; Saccheri et al., 1998; Crnokrak and Roff, 1999; Slate et al., 2000; Brook et al., 2002; Keller and Waller, 2002; Spielman et al., 2004). In small populations or populations that suffer size bottlenecks,1 allelic diversity is lost relatively quickly through random genetic drift, but heterozygosity is less affected. In small populations that are isolated, inbreeding is inevitable and occurs within only a few

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1 A population bottleneck is a large reduction in population size over one or more generations.



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5 Genetic Diversity in Free-Ranging Horse and Burro Populations T his chapter reviews the relationship between genetic diversity and the long-term health of free-ranging horse and burro herds. It does that by reviewing genetic ­studies conducted on 102 horse Herd Management Areas (HMAs) and 12 burro HMAs ­ nder u the jurisdiction of the Bureau of Land Management (BLM) and comparing the results with those of studies of other species and herds for evidence of an optimal level of genetic d ­ iversity that might be used as a management target. It also examines the idea that BLM’s free-ranging horse and burro herds can be considered a metapopulation, or a “popula- tion of populations that are spatially discrete but connected through natural or assisted immigration” (Levins, 1969). Metapopulation theory can be used to suggest directions for management activities that might be undertaken to attain and maintain the level of genetic diversity that is needed for continued survival and reproduction and for adapting to chang- ing environmental conditions. THE CONCEPT AND COMPONENTS OF GENETIC DIVERSITY Genetic studies provide essential data for the management of populations, including estimates of the levels and distribution of genetic diversity, assessments of ancestry, and the detection of genetically distinct populations. At the population level, genetic diversity can be measured as the mean number of variants of a gene (alleles) or as the proportion of indi­ viduals that have different variants of a gene (heterozygosity). Theoretical and empirical studies have demonstrated substantial fitness costs associated with the loss of genetic diver- sity in both free-ranging and captive populations (Lacy, 1997; Saccheri et al., 1998; ­Crnokrak and Roff, 1999; Slate et al., 2000; Brook et al., 2002; Keller and Waller, 2002; ­ pielman et al., S 2004). In small populations or populations that suffer size bottlenecks,1 allelic diversity is lost relatively quickly through random genetic drift, but heterozygosity is less affected. In small populations that are isolated, inbreeding is inevitable and occurs within only a few 1  population A bottleneck is a large reduction in population size over one or more generations. 143

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144 USING SCIENCE TO IMPROVE THE BLM WILD HORSE AND BURRO PROGRAM generations. Whereas inbreeding does not change allele frequencies, it results in a change in the proportion of individuals that carry two alleles at a locus that are identical by descent and decreases heterozygosity. Thus, it is important to measure and monitor allelic diversity, observed and expected heterozygosity (Ho and He), and ­ oefficients of inbreeding (Fis) in c managed populations. Genetic diversity in a population results from a number of evolutionary forces: muta- tion, natural selection, gene flow, and genetic drift. Although mutation is the ultimate source of all genetic variation, mutation rates of most genes are low and cannot replenish diversity quickly once it is lost (Lande, 1995). The effects of natural selection depend on whether it is directional, stabilizing, or balancing selection.2 Regardless of the kind of natu- ral selection exerted on a population, when a population is small, only strong selection will affect the level of diversity (Frankham et al., 2010). In contrast, the recruitment of even a small number of unrelated breeding individuals into a population (gene flow) can increase genetic diversity or prevent its loss. Genetic drift—random change in allele frequencies between generations—is a strong force in small populations and can result in rapid loss of genetic diversity (Frankham et al., 2010). A related issue is the detection of populations that are genetically distinct because of low gene flow and are thus functioning independently (Moritz, 1994). In such isolated populations, genetically based adaptations to local environmental conditions may arise. If management actions involve translocations (movement) of individuals among popula- tions, genetic data will help to guide the choices of donor and recipient populations. RESEARCH ON GENETIC DIVERSITY IN FREE-RANGING POPULATIONS SINCE 1980 In the late 1970s, when the National Research Council Committee on Wild and Free- Roaming Horses and Burros reviewed the state of the science, nothing was known about the genetics of free-ranging equids. The committee’s 1980 report found that “no informa- tion exists about these populations concerning . . . the amount of genetic variation within populations, the amount of genetic differentiation between populations, and the pattern of genetic relatedness (‘phylogeny’) of the wild populations and the domestic breeds” (NRC, 1980, p. 93). Furthermore, no information on the amount of genetic variation within or between breeds of domestic equids existed (NRC, 1980). Therefore, that committee recom- mended that genetic studies be conducted to assess the genetic health of the herds. The lack of information regarding the ancestry and lineages of free-ranging equids was also identified as a concern. As a result, BLM awarded a grant to the University of California, Davis (UC Davis) for a study of free-ranging horse genetics. From December 1985 to October 1986, research- ers collected 975 blood samples from five horse populations under BLM management in Oregon and Nevada. A total of 19 genetic loci known to be polymorphic3 in domestic horses were screened (seven red-cell antigens and 12 isoenzyme and serum proteins) and used to estimate levels of genetic diversity and differentiation among herds and to investigate herd 2  Natural selection can take three forms in a population. In directional selection, the frequency of an allele increases because of its greater fitness (its ability to help the individual survive and reproduce). Stabilizing selec- tion decreases the frequency of alleles that have lower fitness, that is, alleles that hinder an individual’s chances to reproduce. Directional and stabilizing selection can continue until a beneficial allele is fixed in the population or the detrimental allele is eliminated. In balancing selection, more than one variant of a gene is maintained in the population, and individuals carrying more than one variant of a gene may have a genetic advantage in their environment. 3  Containing more than one allele.

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GENETIC DIVERSITY IN FREE-RANGING HORSE AND BURRO POPULATIONS 145 ancestry. The results, which were reviewed by the Committee on Wild Horse and Burro Research and published in the National Research Council’s report Wild Horse Populations: Field Studies in Genetics and Fertility (NRC, 1991), indicated that free-ranging herds did not differ from domestic herds with respect to levels of genetic diversity (heterozygosity and allelic diversity) and that differentiation among herds was less than that among breeds of domestic horses. With regard to herd ancestry, the results were consistent with the hypoth- esis that herds originated from escaped or released domestic horses. Studies by E. Gus Cothran at the University of Kentucky and Texas A&M University have been conducted since 2000 to monitor genetic diversity in individual free-ranging horse herds and assess their genetic similarity to domestic horse lineages. The earliest of these studies used the same types of genetic loci used by the UC Davis researchers (17 isozyme and serum proteins), but more recent studies have used 12 highly polymorphic micro­ atellite s DNA loci4 (Goldstein and Pollock, 1997). The more recent studies have made substantial progress in comparing existing populations with exemplars of New World and Old World domestic breeds and have yielded valuable information about herd ancestry and lineages. Furthermore, although the 1980 National Research Council report identified a lack of infor- mation on the genetic variation of both free-ranging horse and burro herds, the UC Davis study did not include samples from burros. Cothran has studied 12 burro herds with nine microsatellite loci. This chapter reviews the results of Cothran’s studies, comparing them with published results on genetic diversity of free-ranging donkey populations in Spain and Sicily (Aranguren-Mendez et al., 2001, 2002; Guastella et al., 2007; Bordonaro et al., 2012). THE RELEVANCE OF GENETIC DIVERSITY TO LONG-TERM POPULATION HEALTH The probability of natural gene flow in free-ranging horses and burros varies among herds. In some herds, management actions have included removals that had unknown effects on the levels and distribution of genetic diversity. Isolation and small population size, in combination with the effects of genetic drift, may reduce genetic diversity to the point where herds suffer from the reduced fitness often associated with inbreeding. That would compromise the ability of herds to persist under changing environmental conditions. Inbreeding Inbreeding depression, defined as a reduction in fitness due to the loss of diversity and the expression of deleterious genes that can accompany inbreeding, can be difficult to detect, especially in wild populations, and the relationship between inbreeding depression and extinction risk is not clear (Lacy, 1997). Crnokrak and Roff (1999) reviewed the litera- ture on wild populations known to be inbreeding to determine what levels of inbreeding depression were occurring and whether they had important fitness effects. They found that most estimates of inbreeding depression (169 estimates in 35 species and 137 traits) were high enough to be biologically important, and most of the traits that they surveyed were directly related to fitness; this allowed them to conclude that inbreeding depression is detectable 54 percent of the time in species known to be inbred. Keller and Waller (2002) established that inbreeding depression occurs in the wild, is measurable, and can influence population viability. They cited literature on agricultural systems that demonstrated that the cost of a 10-percent increase in inbreeding leads to a 5- to 10-percent loss of fitness. 4  Microsatellite loci contain tandem repeats of one to six base pairs and are commonly used as molecular markers to detect genetic variation and relatedness among individuals in a population.

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146 USING SCIENCE TO IMPROVE THE BLM WILD HORSE AND BURRO PROGRAM In fact, Lacy (1997) could find no evidence that any mammalian species is unaffected by inbreeding. In addition to diseases related to genetic mutations, a species may demonstrate con- ditions or abnormalities and reduced fitness due to inbreeding. Some of the evidence on inbreeding depression or correlations between low genetic diversity and fitness traits in ungulates is reviewed below. There is evidence in horses that inbreeding avoidance occurs in the harem band as fathers and stepfathers avoid copulating with related young mares (Berger, 1986; Berger and Cunningham, 1987). However, that does not preclude inbreeding at the population level inasmuch as both sons and daughters disperse from the natal group and may associate later in life as adults. Reproductive Physiology, Reproductive Success, and Offspring Survival Inbreeding results from reproduction by two related parents. If the ancestries of the p ­ arents are known with a high degree of certainty, a pedigree can be constructed, and the coefficient of relatedness (the inbreeding coefficient, F) of the offspring can be calcu- lated on the basis of the relatedness of the parents. In free-ranging populations, however, relatedness among breeding individuals is rarely known but can be estimated by using biparentally inherited DNA markers such as microsatellite loci (Eggert et al., 2010) or s ­ ingle-nucleotide polymorphisms (SNPs; Li et al., 2011). The use of genetic markers to estimate pairwise relatedness between individuals can be problematic primarily because of incomplete sampling, the overall low variance in relatedness among individuals in natural populations, and the need for large numbers of markers to produce precise esti- mates (­ sillery et al., 2006; Pemberton, 2008; Li et al., 2011). Genetic estimates of inbreed- C ing coefficients at the population level can also be problematic; they have been found to be strongly affected by the size, history, and genetic diversity of the founders (Ruiz-Lopez et al., 2009). Thus, although there are potential problems with both pedigree-based and molecular genetics-based estimates of inbreeding, both can provide information about inbreeding that is useful for population management. Data on inbred ungulates suggest a negative relationship between inbreeding and reproductive health. In Cuvier’s gazelle, Gomendio et al. (2000) found an inverse relation- ship between inbreeding levels and ejaculate quality. The Texas state bison herd, which was founded by only five individuals in the 1880s, has statistically significantly lower genetic diversity than herds in Yellowstone and Theodore Roosevelt National Parks. Halbert et al. (2004) found semen abnormalities in four of eight tested bulls from the Texas state herd. In Przewalski’s horse mares, Collins et al. (2012) found a significant association between mean urinary estrogen over an ovulatory cycle and mean kinship, a measure used to quantify relatedness between individuals in a population (Lacy et al., 1995). Mares that had higher mean kinship had lower estrogen concentrations. In a study of sequential ejaculates from Shetland pony stallions, van Eldik et al. (2006) found that higher inbreeding coefficients based on pedigree data correlated with lower sperm quality in the form of lower percentages of progressively motile and morphologi- cally normal sperm. Those effects were apparent even at relatively low inbreeding levels (F = 0.02) and worsened with increasing inbreeding. In contrast, Aurich et al. (2003) studied single ejaculates from Noriker draught horse stallions and found no correlations between semen quality and heterozygosity at microsatellite loci. Luis et al. (2007) analyzed the genetic structure of the Sorraia horse breed, which has populations in Germany and Portugal and is characterized by relatively high levels of inbreeding (F = 0.363). They found low genetic diversity compared with other breeds and

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GENETIC DIVERSITY IN FREE-RANGING HORSE AND BURRO POPULATIONS 147 stated that further analysis showed that inbreeding levels correlated negatively with adult fertility and juvenile survival (C. Luis, Universidade de Lisboa, Portugal, unpublished results). In addition to abnormalities in semen quality, the Texas state bison herd was characterized by lower natality and higher calf mortality than other captive bison herds (Halbert et al., 2004). In a study of red deer on the Isle of Rhum, Scotland, Slate et al. (2000) found that lifetime breeding success in both females and males (as measured by the number of calves produced) correlated positively with heterozygosity at nine microsatellite loci. Finally, in a study of 12 species of ungulates maintained in zoos, Ballou and Ralls (1982) demonstrated that infant mortality was higher in inbred than in noninbred offspring in 11 of 12 species of ungulates and that inbreeding was the only possible explanation for the observed differences. Disease Sasidharan et al. (2011) found that populations of mountain zebra affected by sarcoid tumors, which are known to have a partially genetic basis, had lower genetic polymor- phism, lower expected heterozygosity, and lower gene diversity and higher values of inter- nal relatedness and homozygosity than populations that were not affected by these tumors. Although the trends were clear, the differences were not statistically significant. Ragland et al. (1966) described an outbreak of sarcoids in horses in which affected animals were related and originated from a highly inbred family line. Congenital Defects Zachos et al. (2007) conducted a genetic analysis of a herd of about 50 red deer known to have descended from no more than eight individuals. The genetic diversity shown by data that the authors provided did not appear to be significantly lower than that in other red deer populations, but in this population a number of cases of brachygnathy,5 which is believed to be associated with inbreeding in deer (Renecker and Blake, 1992), have been observed. In horses, the condition known as club foot is defined as “a flexural deformity of the coffin joint resulting in a raised heel; not to be confused with the club foot deformity of humans” (Siegal, 1996). Although the condition is suspected to have a genetic basis, to the committee’s knowledge this has not been confirmed. Club foot has been reported in free- ranging horse herds, but it is not a life-threatening or “limited-use” condition. Clinical Issues Related to Genetics in Horses Aside from concerns about the deleterious effects of inbreeding, there are concerns related to the genetics and health of horses. Similar concerns may exist for burros, but the committee could find no publications about clinical issues related to genetics. According to Brosnahan et al. (2010) and Finno et al. (2009), 10 or 11 conditions in horses are known to be caused by genetic mutations. All are single-gene, autosomal muta- tions inherited in a Mendelian fashion (Brosnahan et al., 2010). Although all are consid- ered rare, they have had important effects on major breeds. Some of the conditions are lethal, but others are not, so the mutations can spread in herds, especially when inbreeding occurs. Commercial testing is available for all except the mutation involved in lavender foal 5  Brachygnathy, also known as parrot mouth, is the underdevelopment of the lower jaw.

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148 USING SCIENCE TO IMPROVE THE BLM WILD HORSE AND BURRO PROGRAM syndrome. Very few of the conditions present clinical signs that would be unambiguous and discernible during a gather of horses that includes large numbers of unknown animals that are grouped for relatively short periods (e.g., days) and are not under constant, indi- vidual observation. However, because many of the conditions can be diagnosed via genetic screening of blood or hair samples, surveillance of the genetic mutations underlying them is possible in HMAs. Screening of samples from gathered horses could be used to generate frequencies of the alleles involved in these disorders, and the frequencies could be moni- tored during later gathers in order to determine whether a particular HMA has a higher occurrence of a given mutation that might affect the fitness of the herd. The conditions that seem to be immediately discernible on observation are discussed below on the basis of clinical data provided by Brosnahan et al. (2010) and Finno et al. (2009). Junctional epidermolysis bullosa is a trait known to affect Belgians, other draft breeds, and American Saddlebred horses. The condition is most often observed in foals, which dem- onstrate irregular, reddened erosions and ulcerations on the skin and mouth over pressure points. Ocular and dental abnormalities co-occur in some cases. Another notable manifesta- tion of the condition is complete sloughing of the hooves in foals, which is terminal. Overo lethal white foal syndrome or ileocolonic aganglionosis presents in the form of an all white or nearly all white hair coat in foals and an underlying intestinal obstruction. Affected breeds include American Paint Horse, Quarter Horse, and rarely Thoroughbreds. Diagnosis of the condition is difficult because of the wide variation in phenotype in these breeds and associated ambiguous language related to color patterns. The condition is terminal. Grey horse melanoma is found in many breeds and is manifested as a gray coat in conjunc- tion with dermal melanomas. The melanomas themselves are not typically life-­ hreatening, t but they may metastasize to other organs. Arabian horses are the primary breed affected by lavender foal syndrome or coat color dilution lethal. Affected animals’ coats appear silver, pink, or lavender. Other clinical signs include seizures, dorsiflexion of the head and neck, hyperaesthesia, and recumbency. Pro- gressive neurological dysfunction is also observed. The condition is terminal. Testing is not available commercially but was in development at the time of the committee’s study. Hereditary equine regional dermal asthenia is known to affect Quarter Horses and horses from a Quarter Horse lineage. Signs include seromas, hematomas, open wounds, scars, and sloughing of the skin. In addition, the skin is loose and is easily separated from the underly- ing fascia. In areas of hair regrowth, white hairs are typical of this condition. Skin lesions can be treated, but euthanasia is the typical outcome. Testing is available. For most of those clinical conditions, an aberration in coat color pattern is the most discernible and unambiguous cue. Although limb deformities or abnormal gait patterns are clinical signs in some conditions, they may be due to nongenetic factors. Regardless of the underlying causes, phenotypic data have not been recorded and integrated into the genetic management of free-ranging herds. Recording the occurrence of phenotypic data associated with diseases and clinical issues along with information on the age and sex of the affected animals would allow BLM to monitor the distribution and prevalence of a number of genetic conditions that have direct effects on herd health. Genetics and Population Viability The maintenance of genetic diversity in a population is a function of the genetic effec- tive population size (Ne; Wright, 1931, 1938), which is defined as the size of an idealized

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GENETIC DIVERSITY IN FREE-RANGING HORSE AND BURRO POPULATIONS 149 population that would experience the same magnitude of random genetic drift as the popula- tion of interest (Conner and Hartl, 2004) and can be estimated with genetic or demographic data. Populations that have experienced fluctuating sizes between generations, unequal sex ratios, or high variance in reproductive success are likely to have effective population sizes that are lower than the number of animals present; Frankham’s (1995) review of effective population size estimates in wildlife concluded that they are usually at least an order of magnitude lower. It was originally thought that an effective population size of at least 50 was necessary to avoid short-term inbreeding depression, but empirical work suggests that if mainte- nance of fitness is important, effective population sizes much larger than 50 are necessary. Theoretical studies suggest that the figure could be closer to 5,000 for several reasons. First, new genetic variation from mutations is added to a population more slowly than originally thought (Lande, 1995). Mutations with large effects tend to be detrimental and are removed from the population by natural selection, so the overall mutation rate does not accurately predict the infusion of new genetic variation. Second, the effects of inbreeding depression are likely to be more severe in stressful environments (Jiménez et al., 1994; Pray et al., 1994). Finally, slightly deleterious mutations may accumulate in smaller populations and lead to a decline in fitness (Lynch and Gabriel, 1990; Charlesworth et al., 1993; Lande, 1994). A related concern is whether there is a general rule that would help managers to decide how large a population needs to be to remain genetically and demographically viable in the long term (Flather et al., 2011a,b). Flather et al. (2011b) argued that a general rule of thumb is not scientifically defensible given the variation among species, their evolutionary history, the habitats that they occupy, and the threats to their survival. However, they agreed with previous suggestions that multiple populations totaling thousands, rather than hundreds, of individuals will probably be necessary for long-term viability of species. At the time of the committee’s study, the total population of horses on BLM land exceeded 31,000. When that population is considered as a whole, concerns regarding minimum viable population (MVP) size are not important. However, this population exists in many smaller, fragmented units. Only a small fraction of the HMAs or HMA complexes contain more than 1,000 horses, so no single HMA or complex could be considered to have an MVP size for the long term, although the analyses cited above suggest that horse populations on HMAs or HMA complexes that are larger than 1,000 do have a greater than 50-percent probability of survival for 100 years. In addition, it does not appear to be realistic to attempt to manage each HMA or HMA complex with a goal of a minimum of 5,000 animals. Therefore, man- agement of the HMAs as a metapopulation, in the form of natural and assisted movement of animals between HMAs, will be necessary for long-term persistence of the horses at the HMA or HMA-complex level. Movement of animals will need to be guided by a number of genetic, demographic, behavioral, and logistical factors, discussed later in this chapter. In contrast with horses, the total population of free-ranging burros is estimated at only about 5,000 and is therefore at what scientists would consider an MVP size. These animals exist in fragmented units, each of which has a population size well below the MVP size; as in the case of horses, it is unrealistic to consider increasing the population in each unit to 5,000. Genetic monitoring and movement of burros between HMAs is therefore more necessary than it is for horses to maintain the overall population for the long term. The same factors that would inform movements of horses would apply to movements of burros.

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150 USING SCIENCE TO IMPROVE THE BLM WILD HORSE AND BURRO PROGRAM IS THERE AN OPTIMAL LEVEL OF GENETIC DIVERSITY IN A MANAGED HERD OR POPULATION? In a survey of genetic diversity levels in mammals, Garner et al. (2005) found an aver- age heterozygosity value of 0.677 ± 0.010 in healthy populations. For 16 species, they com- pared healthy populations with ones that had experienced a demographic challenge and found a strong association between demographic threats and the loss of heterozygosity (healthy mean, 0.715 ± 0.240; demographically challenged mean, 0.525 ± 0.040). They also found evidence of differences in genetic diversity among families within orders of mam- mals. Genetic diversity in free-ranging horses and burros in HMAs should be compared with genetic diversity detected in other free-ranging and domestic herds to determine the health of a herd or population, depending on the management goal. Genetic Diversity in Free-Ranging Horses Table 5-1 compares estimates of genetic diversity of free-ranging populations (Sable Island, eastern Canada; Colonial Spanish horses known as the Marsh Tacky, found in South Carolina, the Florida Cracker, and populations on Shackleford Banks, Corolla, and Ocracoke Islands, North Carolina; southern European native horse breeds; and Assateague Island, Maryland), domestic breeds, and the endangered Sorraia horse breed (Portugal and Germany), which was founded in 1937 with three stallions and seven mares. The studies have shown that the mean observed heterozygosity was below that observed in healthy mammal populations for the Sorraia and Colonial Spanish horse populations and for some domestic breeds. Observed heterozygosity was on a par with that in healthy mammal populations of free-ranging horses on Sable Island and Assateague Island, breeds from Canada and Spain, some domestic breeds, and some southern European native breeds. TABLE 5-1  Estimates of Genetic Diversity of Free-Ranging and Domestic Horses Observed Population Allelic Diversity Heterozygosity Fis Reference Sable Island 5.60 ± 1.35 SD 0.647 ± 0.035 SD 0.070 Lucas et al., 2009 Sorraia 3.32 ± 0.95 SD 0.450 ± 0.212 SD –0.061 to 0.018 Luis et al., 2007 Domestic breeds from 5.50 ± 0.42 SE to 0.66 ± 0.02 SE to –0.046 to 0.083 Plante et al., 2007 Canada and Spain 8.25 ± 0.57 SE 0.79 ± 0.04 SE Southern European 5.75 ± 1.54 SD to 0.687 ± 0.170 SD to Not estimated Solis et al., 2005 native horse breeds 8.08 ± 1.93 SD 0.772 ± 0.099 SD Domestic breeds (10 3.6 ± 0.3 SE to 0.494 ± 0.057 SE to Not estimated Vilà et al., 2001 breeds, 191 individuals) 4.5 ± 0.4 SE 0.626 ± 0.058 SE Colonial Spanish horse 4.00 ± 1.27 SD to 0.54 ± 0.18 SD to –0.069 to 0.058 Conant et al., 2012 populations (five) 7.73 ± 2.05 SD 0.74 ± 0.10 SD Assateague Island 7.4 ± 1.8 SD 0.794 ± 0.102 SD Not estimated Eggert et al., 2010 NOTE: SD = standard deviation; SE = standard error.

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GENETIC DIVERSITY IN FREE-RANGING HORSE AND BURRO POPULATIONS 151 Genetic Diversity in Horses Managed by Bureau of Land Management Genetic studies have been conducted by E. Gus Cothran for many of the HMAs (Table 5-2). The results of the studies have shown that genetic diversity varies among HMAs. Allelic diversity values range from 2.58 (Liggett Table, OR) to 8.00 (Warm Springs, OR, and Paisley Desert, OR), observed heterozygosity values range from 0.497 (Cibola- Trigo, AZ) to 0.815 (Hog Creek, OR), and inbreeding coefficient values range from –0.230 (Nut Mountain, CA) to 0.133 (Lahanton Reservoir, NV). The lowest allelic diversity and heterozygosity found in the HMAs are consistent with those in the endangered Sorraia breeds and the Colonial Spanish horse populations, all of which are small, isolated herds. The management goal, as stated in the BLM’s Wild Horses and Burros Management Hand- book (BLM, 2010), is to keep the observed heterozygosity (Ho) of all herds no lower than one standard deviation below the mean in the BLM herds. In the 2012 Cothran reports, the free- ranging feral horse mean Ho was listed at 0.716 with a standard deviation of 0.056, and the value below which a herd was considered at critical risk was listed in the BLM handbook as 0.66 for DNA estimates and 0.31 for blood group estimates. By those standards, herds in eight HMAs listed in Table 5-2 are at risk because of low heterozygosity. If the same criterion is applied to allelic diversity (mean number of alleles [MNA]), the goal would be 4.97 alleles/locus (mean, 6.06; standard deviation, 1.09), and an additional HMA would fall below the acceptable level. An examination of Table 5-2 reveals that herds in 34 HMAs have observed heterozygosity or allelic diversity values between the mean and the value at which a herd is considered at critical risk and should be managed and monitored routinely to detect decreases in diversity or improvements as the result of management actions. One HMA—Liggett Table, OR—has low heterozygosity, extremely low allelic diversity, and a small appropriate management level (AML). Its low inbreeding coefficient is surprising, in that it is inconsistent with expectations under those conditions. The Cothran report for this HMA notes that one horse was destroyed because of an unspecified congenital defect. Each of the Cothran reports includes information on the percentage of variants (micro­ satellite alleles) that have frequencies below 0.05 because these rare variants are the ones most likely to be lost if population size declines or not all individuals reproduce equally. It is important to note that although some microsatellite loci have been implicated in human disease (Wooster et al., 1994), the dinucleotide (2-bp repeat motif) microsatellite loci used in the Cothran studies are found in regions of DNA that are unlikely to directly affect the fitness of individuals. In those studies, microsatellite loci were used as proxies to test for overall levels of genetic diversity and to assess levels of inbreeding. Managing for the pres- ervation of microsatellite alleles that are rare in an HMA would not be expected to increase fitness, and this approach is not recommended in any of the Cothran reports. Evidence of Strong Associations with Spanish Bloodlines Phenotypic similarities and historical records have suggested that several HMAs have high concentrations of old Spanish blood and thus may be assigned high priority for con- servation. Cothran’s studies have addressed that, using both blood group polymorphisms, which reveal alleles that have strong associations with Spanish bloodlines, and micro­ satellite loci. Because the blood group polymorphisms provide clear evidence and the micro­satellite loci do not, the results that he presented to the committee were based only on blood group data. He found evidence of Spanish blood in the Cerbat Mountains, AZ; Pryor Mountains, MT; and Sulphur, UT, HMAs. The Cerbat Mountains herd is largely isolated, but the reports show that the Pryor Mountain and Sulphur herds both have Spanish blood

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TABLE 5-2  Genetic Diversity in Free-Ranging Horses in Herd Management Areas Determined by Using 12 Microsatellite Loci 152 Year Herd Management Area State  N Sampled AML Ho He Fis MNA Cothran Report Date—Comments Cerbat Mountains AZ 90 Evidence of Spanish blood Cibola-Trigo AZ 24 2005 150 0.497 0.490 –0.014 4.17 04/15/08 Arizona mean value 0.497 0.490 –0.014 4.17 Bitner CA 21 2012 25 0.734 0.713 –0.029 5.92 05/15/12 Buckhorn CA 31 2010 85 0.806 0.749 –0.076 6.42 10/20/10 Carter Reservoir CA 60 2009 35 0.689 0.688 –0.001 6.33 10/04/10 Centennial CA 69 2001 168 0.665 0.709 0.062 6.92 11/19/01 Chicago Valley CA 12 Coppersmith CA 53 2009 75 0.708 0.703 –0.007 6.33 10/05/10 Fort Sage CA 29 Fox Hog CA 115 2012 220 0.717 0.730 0.018 7.42 05/18/12 High Rock Canyon CA 35 2012 120 0.774 0.773 –0.001 7.75 05/17/02 Massacre Lakes CA 35 New Ravendale CA 25 Nut Mountain CA 47 2011 55 0.770 0.752 –0.230 7.33 05/14/12 Piper Mountain CA 17 Red Rock Lakes CA 25 Round Mountain CA 10 Twin Peaks - Gilman CA 13 2011 758 0.724 0.764 0.052 6.67 04/28/11 Twin Peaks - S Observ CA 52 2011 758 0.710 0.754 0.058 7.67 04/28/11 Twin Peaks - Skedaddle/Dry Valley CA 28 2011 758 0.673 0.744 0.096 6.92 04/28/11 Wall Canyon CA 14 2012 25 0.708 0.709 0.001 6.00 05/23/12 California mean value 0.723 0.732 0.005 6.81

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Little Book Cliffs CO 29 2002 150 0.745 0.721 –0.034 6.25 05/28/03 Piceance-East Douglas Creek CO 32 2006 235 0.635 0.640 0.007 4.67 06/01/10 Sand Wash Basin CO 50 2001 362 0.730 0.723 –0.009 6.50 04/16/02 Spring Creek Basin CO 15 2007 65 0.689 0.702 0.018 5.08 06/21/10 Colorado mean value 0.700 0.696 –0.005 5.63 Black Mountain ID 25 2010 60 0.720 0.704 –0.023 6.42 03/03/11 Challis ID 46 2002 253 0.743 0.735 –0.010 6.42 09/15/03 Four Mile ID 25 2003 60 0.740 0.691 –0.071 5.75 06/14/04 Hard Trigger ID 30 2010 130 0.764 0.733 –0.042 6.50 03/01/11 Sand Basin ID 25 2003 64 0.782 0.726 –0.077 6.33 06/14/04 Saylor Creek ID 50 2010 50 0.767 0.748 –0.025 6.42 12/17/10 Idaho mean value 0.753 0.723 –0.041 6.31 Pryor Mountains MT 103 2009 120 0.757 0.762 0.007 6.58 09/02/10 Evidence of Spanish blood Montana mean value 0.757 0.762 0.007 6.58 Antelope NV 324 Antelope Valley NV 28 2011 259 0.765 0.756 –0.012 6.75 06/13/11 Augusta Mountains NV 29 2011 308 0.770 0.756 –0.018 6.58 04/26/11 Bald Mountain NV 97 2009 215 0.759 0.775 0.021 7.58 08/13/10 Black Rock East NV 31 2012 93 0.710 0.753 0.057 7.00 05/30/12 Black Rock West NV 19 2012 93 0.675 0.654 –0.032 5.67 05/30/12 Monitor closely and consider introducing two to four new mares Buffalo Hills NV 51 2009 314 0.724 0.729 0.008 7.00 08/05/10 Calico Mountains NV 40 2012 333 0.748 0.723 –0.035 6.92 05/24/12 Callaghan Austin Allot NV 40 2009 237 0.742 0.780 0.049 7.25 08/12/10 153 continued

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164 USING SCIENCE TO IMPROVE THE BLM WILD HORSE AND BURRO PROGRAM numbers of females in the population will increase variance in family size in that many more females than normal will fail to produce offspring. Because Ne is inversely propor- tional to variance in family size, any increase in the number of nonreproducing but surviv- ing females will decrease effective population size. Second, any movement of the sex ratio of breeders from 1:1 will also decrease effective population size. That effect can be subtle in polygynous species, such as horses and burros, inasmuch as the number of breeding males is usually less than the number of breeding females. Thus, although reducing the number of breeding females through female contraception may move the ratio closer to 1:1, the reductions in total numbers of breeders and increases in the variance in family size may still lead to an overall reduction in Ne. Alternatively, if population size is reduced by decreasing the number of males, it might not reduce Ne depending on the pool of bachelor males available to become harem stal- lions. If the pool is large, it will leave the number of breeders and variance in family size unaffected. It is important to consider those effects in the planning phase of management actions. A modeling approach (see Chapter 6) will allow managers to consider the effects of p ­ opulation-size reduction by using fertility-control methods and other important factors. Individual-Based Genetic Management Maximum retention of genetic diversity in each HMA (or HMA complex) and in the population as a whole could be achieved if horses and burros were managed as indi­ viduals. That entails knowing all individuals in the population unit, their relationships, and their reproductive performance over time. The detailed population monitoring and record- keeping required to accomplish this has been possible in some barrier-island horse popula- tions, including Assateague Island (Eggert et al., 2010) and Shackleford Banks. This level of management would entail an important departure from a truly wild population that is subject to natural selection, a distinction that would need to be made clear to all interested parties. It would also differentiate the management of free-ranging horses and burros from that of other species in the landscape, with the exception of cattle. The committee believes individual-based genetic management might be possible in some HMAs in which habitat conditions and local or BLM knowledge of individual animals make it possible to track individuals (for example, Pryor Mountains). In addition to monitoring the genetics (via pedigree) and demographics of the popu- lation, individual-based genetic management would require actively controlling repro- duction of individual animals so that they contribute to the gene pool equally and rare alleles or genotypes are not lost. The barrier islands of Assateague and Shackleford Banks provide some models of attempts to maximize genetic diversity in free-ranging animals through targeted contraception. Nuñez (2009) summarized the evolution of the contracep- tive management programs on those islands. Software tools for keeping track of animal pedigrees, analyzing genetic relationships, and monitoring population demography were developed for captive populations of animals in zoos; their application to the Assateague Island population was described by Ballou et al. (2008) and Eggert et al. (2010). In HMAs in which known individual animals can be reliably contracepted, either temporarily or permanently, this type of genetic management is possible. In HMAs in which following individual animals and managing their individual reproductive performance is not fea- sible, a less labor-intensive approach to genetic management is possible with the use of translocations.

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GENETIC DIVERSITY IN FREE-RANGING HORSE AND BURRO POPULATIONS 165 Translocation for Genetic Restoration HMAs exist in a mosaic of ecological habitats, anthropogenic effects, political jurisdic- tions, free-ranging horse and burro protection status, and property-ownership arrays. The likelihood of natural migration between HMAs is affected by many factors. Two of the most important are the distance over which dispersing animals must travel to reach other HMAs and the quality of the intervening habitat. Hampson et al. (2010) used GPS collars to study travel distances in two populations of free-ranging horses (12 horses) in Australia over 6-7 days at two sites that differed in vegetation and water abundance. There were no differences in daily travel distances between the two sites, but there was a wide range: 8.1-28.3 km. The mean daily travel distance was 15.9 km (18.2 km for males, 14.8 km for females). Some animals in the study were observed walking for 12 hours to reach water. Hampson et al. (2010) cited data from previous studies that showed travel distances of 17.9 km/day by free-ranging horses in Australia, 8.3 km/day by wild asses and 3.5 km/day by Przewalski’s horses in Mongolia, and 15 km per 12 hours by female zebras. Given the distances between many pairs of HMAs, movement of horses and burros for ­ genetic or demographic reasons would probably need to be facilitated by BLM. The practice of moving individual animals between populations for genetic restoration, or transloca- tion, is justified scientifically. Perhaps the most famous case is that of the Florida panther (Puma concolor coryi). Details on the background of that population, the issues and decision- making processes involved in the genetic restoration, and the outcomes may be found in Hedrick (2001), Pimm et al. (2006), Johnson et al. (2010), and Benson et al. (2011). Briefly, this subspecies of puma was reduced to a population of about 25 in the early to mid-1990s and demonstrated lower genetic diversity than other North American puma populations (Culver et al., 2000) and a number of traits that suggested that the influences of inbreed- ing and genetic drift had completely or nearly fixed genes for potentially deleterious traits that were previously rare. Introduction of female Texas panthers (Puma concolor stanleyana) into the population in 1995 resulted in the production of offspring (Land and Lacy, 2000) that lacked several of the deleterious traits (Shindle et al., 2000; Hedrick, 2001; Johnson et al., 2010). Offspring, particularly females, had survival rates almost twice those previously observed in the population (Pimm et al., 2006; Benson et al., 2011), and increases in survival rates were correlated with increased heterozygosity (Benson et al., 2011). Despite these successes in population growth and apparent health, Johnson et al. (2010) pointed out that the future of the Florida panther will require continuing intensive management, including regular infusions of new genetic material, in the face of anthropogenic threats, habitat loss, infectious diseases, and continued inbreeding. Other case studies of translocation providing genetic and demographic benefits include African lions (Trinkel et al., 2008), adders (Madsen et al., 1996, 1999, 2004), and prairie chickens (Westemeier et al., 1998). Studies by Vilà et al. (2003), Seddon et al. (2005), and Adams et al. (2011) described the favorable effect of a single natural immigrant into a wolf population. The case studies are supported by laboratory studies that have demonstrated genetic benefits, fitness benefits, or both of the infusion of new genetic material into small, inbred populations (e.g., Spielman and Frankham, 1992; Ebert et al., 2002; Saccheri and Brakefield, 2002). Selection of Animals for Translocation As early as the 1930s, it was established that inbreeding depression in small, isolated populations could lead to loss of fitness and increased risk of extinction (Wright, 1931).

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166 USING SCIENCE TO IMPROVE THE BLM WILD HORSE AND BURRO PROGRAM Wright’s analyses led him to conclude that even small amounts of gene flow between isolated small populations could offset the adverse effects of genetic drift and inbreeding. That conclusion gave rise to a large body of work aimed at determining exactly how much gene flow, in the form of immigrants per generation, was necessary to offset the adverse effects of genetic deterioration. A rule of thumb of one immigrant per generation emerged (Kimura and Ohta, 1971; Lewontin, 1974; Spieth, 1974) and has been widely adopted in conservation practice. More recently, that rule of thumb has been challenged on the basis of the simplistic assumptions that were used in deriving it (e.g., Mills and Allendorf, 1996; Vucetich and Waite, 2000). At the time this report was prepared, it seemed likely that in real- world applications, one immigrant per generation would be an absolute minimum. Mills and Allendorf (1996) outlined a number of scenarios in which the number of immigrants per generations should probably exceed one, including scenarios in which at least one of the following is the case: • Inbreeding depression is believed to be occurring already. • Immigrants are closely related to each other or to the receiving population. • Effective population size is much lower than the number of animals present. • Social, behavioral, ecological, or logistical factors prevent single animals from immigrating successfully. • Immigrants are at a disadvantage in probability of survival and reproduction. • The receiving population has been isolated for many generations. • Extinction risk due to demographic or environmental variation is deemed to be very high unless there is aggressive supplementation. The authors concluded that up to 10 immigrants per generation might be necessary to effect genetic restoration in those situations. Vucetich and Waite (2000) extended the analyses by modeling variation in population fluctuation and suggested that more than 20 immigrants per generation may be necessary if high population fluctuation leads to drastically reduced effective population size. In addition to the number of animals to translocate, the interval for doing so must be determined. There are important practical and logistical considerations involved, but the translocation of animals for genetic restoration is usually thought of as being conducted on a per-generation basis. Therefore, one starting point is to determine the generation time of free-ranging horses. Eggert et al. (2010) constructed a pedigree for the Assateague Island horse population on the basis of molecular analyses and herd records and derived an estimate of 10 years. Goodloe et al. (1991) also derived an estimate of 10 years for horses on Cumberland Island, Georgia. Similarly, historical pedigree data on zoo populations of wild equids in North America all have generation time estimates of about 10 years (range 9.6 years in Somali wild ass to 10.4 years in Hartmann’s zebra6). Thus, it would be valid to consider 10 years as an appropriate interval for translocating animals between popula- tions for genetic restoration. On the basis of the literature, it appears that translocation of 10 animals between populations every 10 years would be appropriate. BLM is already experienced in the capture and transport of animals for population management, and the protocols for translocation would be similar to those currently used for gathers; only the destination of the removed animals would differ. Although the movement of animals among HMAs has the potential to facilitate the spread of pathogens (Champagnon et al., 2012), the probability of that could be minimized through observation 6  Association of Zoos and Aquariums website, www.aza.org. Accessed April 16, 2012.

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GENETIC DIVERSITY IN FREE-RANGING HORSE AND BURRO POPULATIONS 167 and advance testing of source and target herds. Below is an outline of some factors to con- sider in selecting animals for translocation for genetic management. Genetic Factors. Because the goal of translocation is to supplement the genetic diversity in a herd and reduce the probability of inbreeding, it is advisable to select animals that are unrelated to the target herd. In most cases, pedigree information on free-ranging horse and burro populations will not be available, so absolute genetic relationships among individual animals will be unknown. The use of genetic information, however, will make it possible to choose individual animals that have moderate levels of differentiation from the target population. The term outbreeding depression is used to describe a decrease in fitness due to hybrid- ization between individuals from populations that have differentially adapted genomes (Frankham et al., 2011). Frankham et al. (2011) used empirical data and modeling to develop a decision tree for predicting the probability of outbreeding depression. Their tree proved robust when crosses that had known outcomes were used, and it suggested that outbreed- ing depression is likely when the populations being crossed are of different species, exhibit fixed chromosomal variants, have not exchanged genes in 500 years, or inhabit differ- ent environments. None of those risk factors seems to apply to free-ranging horses and b ­ urros in HMAs. Environments may differ between HMAs, but Frankham et al. (2011) suggested that environmental differences need to be substantial enough to select for differ- ent traits among populations. They recommended paying particular attention to the needs and resources to which a species is most sensitive and to the range of variation in impor- tant features of the environments under consideration. The adaptability of the horse and its associated ability to live in various environments appears to lessen the concern about environmental differences between possible translocation sites. By using the genetic data generated for the evaluation of level of genetic diversity, it is possible to estimate the level of differentiation among HMAs. The fixation index (Fst) is a measure of genetic distance, or population differentiation, that is based on genetic poly- morphisms (Wright, 1931). Polymorphic microsatellite loci constitute a powerful tool for predicting which populations are so similar (low Fst value) that translocating animals will probably not be successful in supplementing genetic diversity and which are so different (high Fst value) that genetic compatibility between individuals may not be optimal and may reduce the probability of successful translocation. Matrices of pair-wise Fst values for horses and burros based on genetic data from BLM herds are in Appendix F and could be used to identify the mixtures that might be most successful because they exhibit moderate Fst values. New genetic variation needed by an HMA does not necessarily need to come from another HMA. Mares in long-term holding facilities could also be used as sources of genetic diversity if necessary, assuming that they present no novel disease risk for free-ranging horses and burros. The genetic tools described above can be used to identify free-ranging horses and burros on other public lands, in private sanctuaries, and in long-term holding facilities that could be used to infuse new genetic variation into an HMA. Behavior and Social Factors. Given the harem social structure of free-ranging horses and the fact that this structure means that more sexually mature females than sexually mature males are breeding at any one time, it appears that the most rapid way to infuse new genetic material into an HMA via translocation would be to move young, sexually mature mares between HMAs. Young mares new to an HMA are likely to be courted by bachelor males and to be open to forming consortships with them. Older mares would also probably be bred relatively quickly, but they may be more selective in forming consortships with

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168 USING SCIENCE TO IMPROVE THE BLM WILD HORSE AND BURRO PROGRAM bachelor males. Ideally, translocated mares would already be familiar with one another, if possible originating from the same harem. Kaseda et al. (1995) found that mares that had long-term bonds to harem stallions had higher reproductive success than mares that wan- dered between bands regularly or that had shorter bonds to stallions. Linklater et al. (1999) also found that single mares that were dispersing between bands had lower fecundity, reproductive success, and body condition; had higher parasite levels; and received more aggression from bachelor males than mares in established harems. Moving established groups of females may buffer some of those adverse effects, but it is possible that trans­ locating bonded females without a harem stallion will lead to dissolution of bonds between mares (Rubenstein, 1994). A second option and one that might further lessen adverse effects is to move intact harems if the harem members and associated stallions can be reliably identified during gathers. That would immediately add new genetic material to the site, but there would be a longer delay in getting that material into the gene pool because foals born into the harem would have to grow up, disperse, and interbreed with members of the resident population. A third option would be to move bachelor males. This option carries the most risk with respect to getting new genes into the resident population. Stallions that have harems may be quite successful in spreading their genes rapidly via breeding with multiple mares, but obtaining a harem is not easy, and bachelor males may not survive to realize breeding opportunities. Immediate and long-term infusion of new genetic material may be most likely if intact harems or groups of young mares (immediate) are translocated with a number of males (long term). Burros are characterized by a less cohesive social structure in which the only long- term relationships are between females and their dependent offspring. Thus, there would be fewer challenges in integrating new females into a burro population, so females would be the first choice for translocation of animals for genetic restoration of burro populations. Males would also be viable candidates for genetic restoration, but introduced males would have to compete with resident males for access to breeding females. Fertility Control and Implications for Translocation Introductions of males or females are likely to have different consequences in that add- ing new females will increase numbers exponentially over time. If population regulation involves female contraception, adding new fertile females could be counterproductive, so adding novel males may be the best way to increase genetic diversity without increasing population size. However, to ensure that the new males become breeders, either a large number would need to be translocated or some of the resident males would need to have their fertility reduced. Alternatively, if curtailing male fertility is the preferred means of population regulation, either a smaller number of novel males can be added to replace a disproportionate number of resident males that are made sterile, or novel females can be added inasmuch as whenever one of the few remaining resident males breeds, he will sire offspring that have genes from the novel females.   Which type of translocation is best to use will depend on a variety of factors, many of which can be tested with a modeling approach in the planning phase (see Chapter 6). Population size, fertility-control methods, and the effects of translocation on Ne will need to be considered. Although translocating males may require fewer total introductions when population size is being regulated because male additions increase the population growth arithmetically rather than exponentially, novel males may find it difficult to obtain harems

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GENETIC DIVERSITY IN FREE-RANGING HORSE AND BURRO POPULATIONS 169 (horses) or territories (burros), which are prerequisites for siring many offspring. Many tradeoffs will require sensitivity to context in designing effective translocation strategies that enhance genetic diversity without upsetting existing population regulatory strategies. CONCLUSIONS Genetic diversity is an important component of the health of free-ranging horses and burros on HMAs, in that it provides the raw material needed to respond to environmental changes. Maintenance of genetic diversity is a function of the effective population size (Ne), which is probably at least an order of magnitude lower than the number of animals present. Factors that reduce Ne include unequal sex ratios, variance in family sizes, and high vari- ance in population sizes between generations. In small, isolated herds, inbreeding is inevi- table and will occur within only a few generations. It is important to measure and monitor allelic diversity, observed and expected heterozygosity (Ho and He), and coefficients of inbreeding (Fis) in HMAs to detect the loss of diversity before the reduction in fitness that has been observed in many inbred populations becomes a problem. In recognition of the importance of monitoring genetic diversity, and as recommended in previous National Research Council reports, BLM has collaborated with outside scien- tists since 1985 to monitor herd-specific diversity on the basis first of isozyme and serum proteins and later of nuclear microsatellite loci. The committee recommends that BLM continue to monitor genetic diversity as part of the routine management of both horse and burro HMAs. The BLM Wild Horses and Burros Management Handbook does not clearly state which HMAs should be monitored and how often studies should be repeated. The commit- tee recommends routine monitoring at all gathers and collection and analysis of a sufficient number of samples to detect losses of diversity. Genetic concerns involve both the potential for the reduced fitness associated with inbreeding and the effects of mutations that can cause phenotypic conditions that affect the fitness of a herd. The Cothran studies are excellent tools for BLM to use in managing herds to reduce the incidence of inbreeding, but they do not provide information about the effects of specific genes known to cause genetically based conditions. To the committee’s knowledge, no tests have been conducted to detect the presence of genetic mutations asso- ciated with those types of conditions. The committee recommends that BLM document the incidence of coat color or other morphological anomalies that may indicate the presence of deleterious mutations during all gathers. For herds in which phenotypic data suggest the presence of genetically based disorders, the committee recommends testing and consulta- tion with geneticists and equine veterinarians to devise appropriate management actions. Monitoring of genetic diversity in burro HMAs has been conducted in only one herd since 2005. Genetic diversity in burro herds is lower than that in Spanish and Sicilian breeds, including endangered breeds, and many of the AML numbers are low to very low. The committee recommends that BLM resume the genetic monitoring of burro HMAs. Although the available literature does not report clinical issues in burros, the committee recommends that BLM routinely monitor and record the incidence of any morphological anomalies that may indicate the deleterious effects of inbreeding. The committee recognizes that genetic management of some HMAs is complicated by other considerations. For herds that have strong associations with Spanish bloodlines— such as those of the Cerbat Mountain, AZ; Pryor Mountains, MT; and Sulphur, UT—or herds that contain unique morphological traits—such as the Kiger, OR, herd—BLM will need to balance concerns about maintaining breed ancestry with the need to maintain optimal genetic diversity. Herds that remain isolated over the long term will inevitably

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170 USING SCIENCE TO IMPROVE THE BLM WILD HORSE AND BURRO PROGRAM lose genetic diversity inasmuch as maintaining or slightly increasing herd sizes will not offset the effects of genetic drift. The public is interested in these herds, and it is particu- larly important that BLM seek opportunities to discuss the complexity of the situation with interested parties. It is true that the existence of a few genetic markers may indicate Spanish origin, but the remainder of the genome may not; rather, it may reflect horses that are well adapted to local conditions. If the latter is the case, isolation of the herd to maintain purity may be mistaken and may lead to unnecessary loss of genetic diversity. The committee recommends that BLM examine in more depth the genetic constitution of these herds and share the findings with the public so that informed decisions about the sustainability of the populations can be made (see Chapter 8). The committee recommends that BLM consider some groups of HMAs to constitute a single population and manage them by using natural or assisted migration (trans­ ocation) l whenever necessary to maintain or supplement genetic diversity. Although there is no magic number above which a population can be considered forever viable, studies sug- gest that thousands of animals will be needed for long-term viability and maintenance of genetic diversity. Very few of the HMAs are large enough to be buffered against the effects of genetic drift, and herd sizes must be maintained at prescribed AMLs, so manag- ing the HMAs as a metapopulation will reduce the rate of reduction of genetic diversity in the long term. Finally, the committee recommends that BLM stay abreast of advances in population genetics and genomics. New laboratory and data-analysis tools promise to reduce costs while providing more powerful methods for monitoring genetic diversity and resolving breed relationships. The 12 nuclear microsatellite loci that are currently used for estimating genetic diversity and genetic differentiation among herds were chosen largely from those approved by the International Society of Animal Genetics for their informativeness in equine genotyping. Thus, they are useful tools for estimating overall genetic diversity and population divergence. However, the small number of loci and the uncertainty about their evolution limit their power to resolve relationships among closely related lineages, such as equid breeds. Recently, the Illumina 50K SNP Beadchip, an equine SNP ­ enotyping array g with over 50,000 polymorphic loci, was developed and found to be informative in several equid species (McCue et al., 2012). The Equine Genetic Diversity Consortium successfully used that array to assess the effects of inbreeding and natural selection in 36 breeds from around the world, to infer relationships among breeds, and to detect signals of ancestral admixture (Petersen et al., 2012a). Genomic tools are also being used to detect the genetic underpinnings of traits that are under positive natural selection (Petersen et al., 2012b) and mutations that are responsible for genetically based diseases, such as lavender foal syndrome (Brooks et al., 2010). Genomic analysis can provide much finer resolution of questions about breed associations and will soon be the method of choice for population- level analysis. REFERENCES Adams, J.R., L.M. Vucetich, P.W. Hedrick, R.O. Peterson, and J.A. Vucetich. 2011. Genomic sweep and potential genetic rescue during limiting environmental conditions in an isolated wolf population. Proceedings of the Royal Society of London Series B 278:3336-3344. Aranguren-Mendez, J., J. Jordana, and M. Gomez. 2001. Genetic diversity in Spanish donkey breeds using micro- satellite DNA markers. Genetics Selection Evolution 33:433-442. Aranguren-Mendez, J., M. Gomez, and J. Jordana. 2002. Hierarchical analysis of genetic structure in Spanish donkey breeds using microsatellite markers. Heredity 89:207-211.

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