INTRODUCTION
The recognition of negative effects arising in offspring of closely related parents (i.e. inbreeding depression) traces further back in history than any scientific observation of the phenomenon (Wright 1984). It is unknown exactly when inbreeding depression was acknowledged in society, but for several centuries it was a common belief among breeders of domestic plants and animals that close inbreeding often resulted in offspring with reduced viability (Knight 1799). Furthermore, in the majority of human cultures, marriage between closely related individuals has long been condemned, which partly has biological foundations (Rosman et al. 2009). Already in the Bible (Deuteronomy 27:22) it is stated that: ‘Cursed be anyone who lies with his sister, whether the daughter of his father or the daughter of his mother’.
Today, the theoretical framework behind inbreeding depression and heterosis (i.e. increased fitness in offspring of unrelated parents) is scientifically well-established
(Wright 1984, Charlesworth & Charlesworth 1987). Quantifying the effects of inbreeding has been of scientific interest since early studies in evolutionary biology, but approaches and attitudes have changed considerably over time as the scientific field has emerged. Furthermore, un-derstanding the causes and consequences of inbreeding depression has been of major importance to the fields of agriculture, human health, evolutionary biology, and con-servation biology (Charlesworth & Willis 2009). In 1986, shortly after the emergence of the conservation biology framework, the early conservation biologists Michael Gilpin and Michael Soulé developed the extinction vortex model to describe interactions between human impact, inbreed-ing, loss of genetic diversity, and demographic instability in small populations of wild mammals (Gilpin & Soulé 1986). Loss of genetic variation can lead to lower indi-vidual survival and reproduction, which reduces population size further, resulting in even more pronounced drift and inbreeding, which may ultimately drive a population to REVIEW
Inbreeding in natural mammal populations: historical
perspectives and future challenges
Malin HASSELGREN* Department of Zoology, Stockholm University, 106 91 Stockholm, Sweden. Email: [email protected]
Karin NORÉN Department of Zoology, Stockholm University, 106 91 Stockholm, Sweden. Email: karin. [email protected]
Keywords
conservation genetics, genomics, global, heterozygosity-fitness, inbreeding coefficient, mammals, pedigree
*Correspondence author. Submitted: 2 May 2019
Returned for revision: 28 June 2019 Revision accepted: 1 July 2019 Editor: DR
doi: 10.1111/mam.12169
ABSTRACT
1. The awareness of inbreeding as a potentially harmful process dates several centuries back in time, and occurred early in various religions, cultures, and societies. However, it was not until the 18th Century that the phe-nomenon was first investigated systematically through breeding experiments in domestic animals and plants. Investigations were followed by the es-tablishment of the theoretical framework in the 19th Century by Darwin, Mendel and other pioneering evolutionary biologists.
2. Throughout the development of this field, from breeding experiments and discoveries of the mechanism of inheritance, via heterozygosity-fitness cor-relations, construction of pedigrees for natural populations, emergence of the conservation genetics field, to present-day whole genome sequencing of extinct species, mammals have played a crucial role as model organisms and flagship species.
3. In this review, we present a chronological overview of the theoretical de-velopment and empirical data on inbreeding in mammals, from the 18th Century to the present day. Furthermore, in relation to the current ana-lytical capacity, we identify gaps in the knowledge and future challenges in the study of inbreeding and inbreeding depression in mammals.
extinction (Gilpin & Soulé 1986, Caughley 1994, Waser & Williams 2001).
During the emergence of the theoretical framework as well as the assemblage of empirical data, mammals have played a key role as model systems. Though most pioneer-ing breedpioneer-ing experiments were conducted on plants, interest was soon transferred to domesticated mammals (Wright 1921, Bartley 1992) and later on, to free-ranging or wild mammals (Crampe 1883, Wallace 1889, Ritzema-Bos 1894, Davenport 1900). Furthermore, there has been a substantial interest, and taxonomic bias, towards mammals in the field of conservation biology (Clark & May 2002), where inbreed-ing and inbreedinbreed-ing depression constitute central components of the background theories. The contribution of mammals to this topic is illustrated by a Google Scholar search on ‘inbreeding depression’ and ‘conservation genetics’, in which mammals are identified as a study system in 28% and 31% of the first 100 results, sorted on relevance.
Aims
In this article, we summarise the theoretical development in relation to empirical research in order to highlight the contribution of mammals as model systems in the ad-vancement of the research field of inbreeding and inbreed-ing depression. We present a chronological overview of the subject, going from the 18th Century to the present. Furthermore, by combining the theoretical framework with present methodological development, we identify gaps in the knowledge and future challenges in the study of in-breeding and inin-breeding depression in mammals.
METHODS
We reviewed citations of seminal, theory-building papers using Web of Science and Google Scholar searches using the search terms: ‘inbreeding’, ‘inbreeding depression’, ‘in-breeding depression mammals’, and ‘conservation genetics’, and sorted them by relevance. In addition, we selected four scientific papers as starting points: Frankham (1995), Keller and Waller (2002), Kardos et al. (2016) and Allendorf (2017). Based on this, representative empirical papers were selected from titles and abstracts. We augmented our data set with specific searches of literature from the defined time periods below by specifying the time period in Google Scholar.
CHRONOLOGICAL OVERVIEW
1760–1860: the first hybridisation
experiments
In the middle of the 18th Century, the first experiments on hybridisation and outbreeding depression (i.e. lowered
fitness in the progeny of widely outbred individuals) were conducted, and laid the foundation for future discovery of inbreeding depression and heterosis. Joseph Gottlieb Kölreuter, a German botanist, was the first scientist to hybridise plants for scientific purposes and found that hybrids often were sterile (Kölreuter 1761). However, Kölreuter did not contemplate the fitness consequences of sterility or hybridisation in general, and even though the study was incomplete, it allowed other scientists to continue research within this framework (Knight 1799, Morgan 1932, Zirkle 1951). Although these studies paved the way for future scientific discovery of inbreeding de-pression, almost another century would pass before the findings of Kölreuter and Knight were placed in an evo-lutionary and genetic context.
1860–1900: Darwinian selection
Darwin (1876) was the first scientist to investigate the effects of inbreeding and outbreeding in a systematic man-ner. He documented that many plant species had mecha-nisms for avoiding self-fertilisation and argued that such adaptations could derive an evolutionary advantage. Through his experiments, Darwin found that some highly self-fertile species could be inbred for several generations without notably suffering from inbreeding depression. Alfred Russel Wallace would continue to contemplate this matter in his book Darwinism (Wallace 1889). Wallace exemplified this using a population of rabbits Oryctolagus cuniculus on the island Porto, where a female rabbit and her offspring were released in 1445 and started to multiply rapidly (Lodge 1993). Despite the whole population being derived from only two founders, Wallace stated that the animals were ‘in perfect health and vigour’ and reasoned that rigid selection eliminated weak individuals and claimed that with such selection, harmful effects of inbreeding could be prevented for a long time (Wallace 1889). This phenomenon would in later years come to be termed ‘purging’ (Hedrick 1994).
inbreeding among humans would often not result in any negative fitness consequences because of cultural factors: ‘The widely different habits of life of men and women in civilised nations, especially amongst the upper classes, would tend to counterbalance any evil from marriages between healthy and somewhat closely related individuals’ (Darwin 1876). A reason why Darwin was reluctant in acknowledging inbreeding depression in humans was per-haps that he married his own cousin Emma Wedgewood; together they produced 10 children. In fact, a recent study revealed inbreeding depression on reproductive traits for males in the Darwinian family tree (Álvarez et al. 2015).
Mendelian inheritance
During the same time period, but independently of Darwin, the Catholic monk and botanist Gregor Mendel studied inheritance and hybridisation in pea plants Pisum sp. Through extensive and well-planned experiments, where he crossed a vast variety of peas and conducted statistical analyses, Mendel was the first to discover dominant and recessive traits and in what ratios these were transferred over generations, today known as Mendel’s law. He also demonstrated that the frequency of heterozygosity was reduced by 50% in each generation as a result of self-fertilisation (Mendel 1865). Apart from discovering the concept of heritable characteristics, later termed genes, Mendel also recorded heterosis in the crossings of differ-ent pea varieties and reported that the crossings had ‘… greater luxuriance that appears in all parts of the plants…’ (Mendel 1865). Mendel's research on inheritance was, however, rejected for many years.
Scientific studies on inbreeding depression in mammals were, at this time, still scarce, but important pre-Mendelian experiments on inbreeding in animals were those of Crampe (1883) and Ritzema-Bos (1894) with rats Rattus sp., and Davenport (1900) with mice Mus sp. They all recorded deterioration in fertility and the appearance of morpho-logical abnormalities after a few generations of inbreeding but did not relate this to the mechanism of Mendelian heredity. However, the rediscovery of the Mendelian law of inheritance in 1900 came to provide the crucial genetic basis for interpreting the effects of inbreeding vs. outcross-ing in the future research (Gliboff 1999).
1900–1920: the rediscovery of Mendel's law
In 1900–1901, Mendel’s work was re-discovered after four researchers repeated his experiments independently of each other, all claiming to be unaware of Mendel's previous discoveries (Correns 1900, De Vries 1900, Tschermak 1900, Spillman 1902). It has been questioned how unaware they actually were, and it seems that at least De Vries and
Tschermak learned from Mendel’s work before they pub-lished their findings (Sturtevant 1965). This led to a general acceptance of Mendel’s law, and the zoological interest for genetics started to increase. The re-discovery of Mendel’s work aided the zoologists Theodor Boveri and Walter Sutton to simultaneously study chromosomes and inherit-ance; they discovered that maternal and paternal chro-mosomes exist in homologous pairs and reasoned that: ‘chromosomes may constitute the physical basis of the Mendelian law of heredity’. The theory which identified chromosomes as the carriers of genetic material was termed the Boveri-Sutton chromosome theory (Gilbert 1978).
The dominance/overdominance hypothesis
Pearl 1916), such as Jersey cattle Bos taurus and domestic fowl (Gallus gallus, but a quantitative measure of in-breeding was still lacking.
1920–1940: the inbreeding coefficient
The geneticist Sewall Wright was the first scientist to define a coefficient expressing the degree of inbreeding (Wright 1921). Wright examined pedigrees of populations of short-horn cattle B. taurus and carried out pedigree-based ex-periments on guinea pigs Cavia porcellus. Based on this, his aim in deriving the inbreeding coefficient (f) was to facilitate the avoidance of close inbreeding in domestic and laboratory animals (Wright 1922). Wright was mainly considering quantitative traits (i.e. cumulative actions of many genes on a phenotype), and therefore the inbreeding coefficient was originally calculated as the correlation of additive genetic values between two uniting gametes (Wright 1921). Using Wright’s definition, the inbreeding coefficient naturally takes a negative value if two individuals are less related than random. Today, however, the inbreeding coef-ficient is generally defined as the probability of identity by descent of two alleles at a given locus, based on Mendelian ratios (i.e. the probability that a locus carries two gene copies that both originate from a single copy in a common ancestor, Malécot 1948). Wright’s formula-tion of the inbreeding coefficient greatly facilitated the quantification and subsequent understanding of inbreeding depression using pedigree records. Although the formula has been modified since, the invention influenced all future discussion on inbreeding depression (Hill 1996).
Several studies on inbreeding through detailed pedigrees in guinea pigs, rats, mice, and livestock were conducted during the 1930s (Livesay 1930, Marshak 1936, Grüneberg 1939, Wright 1984), and the dominance and overdomi-nance hypotheses were universally accepted as underlying mechanisms (Haldane 1937, Wright 1984). The scientific field experienced another major progress event in 1930, when statistician and biologist Ronald Fisher published the book The Genetical Theory of Natural Selection (Fisher 1930). This was the first work to combine Darwinian natural selection with Mendelian genetics, and the pub-lication was a key contributor to the development of evolutionary framework connected to inbreeding depression.
1940–1960:
F
-statistics
The formulation of the inbreeding coefficient and the major progress in evolutionary and genetic fields was followed by the recognition that the spread of genes is spatially restricted, which was considered to be a likely cause of inbreeding in many populations. Wright (1943)
suggested that evolution is influenced by an interplay between population structure, inbreeding and random genetic drift. He was the first to recognise that genetic drift reduces the total amount of genetic variation, and that evolution is not exclusively driven by selection but also by stochastic processes. Wright developed methods for describing genetic structure within and among popu-lations, termed F-statistics (Wright 1943, 1965); a concept that has been used widely to estimate inbreeding at both individual and population levels (Wright 1965, Nei 1977, Weir & Clark Cockerham 1984). The emergence of F statistics accelerated attempts to measure the effect of inbreeding on life-history traits and behaviour in both captive (Smith 1986) and wild (Selander 1970) mammal populations.
1960–1980: allozymes as molecular markers
1980–2000: DNA as molecular marker
Although the use of allozymes led to an explosion in empirical studies of inbreeding in mammals (Wayne et al. 1986, O’Brien & Evermann 1988, Brewer et al. 1990), the method has some major drawbacks, not least when study-ing inbreedstudy-ing. Allozymes measure genetic variation as protein variation, and heterozygosity may thus be under-estimated due to the redundancy of the genetic code. Furthermore, protein-coding genes are usually under strong selection pressure and the variation between individuals is generally low (Mitton 1994). Also, fresh and relatively large quantities of tissue are needed, which often requires destructive sampling, which is particularly problematic in the endangered populations that are often the scope of interest regarding inbreeding. Using DNA sequences, on the other hand, allows direct measures of genetic varia-tion, and mutations on sequence level can be detected. In 1983, the polymerase chain reaction was invented, which enabled amplification of millions of copies of DNA se-quences in many individuals through a relatively straight-forward method. This invention was the most important contributor during the shift from allozymes towards DNA as molecular markers (Schlötterer 2004). The revolution allowed researchers to address the relationship between DNA and inbreeding by estimating relatedness and genetic variation, e.g. in the grey wolf C. lupus (Ellegren 1999) and the naked mole rat Heterocephalus glaber (Faulkes et al. 1997).
Microsatellites and heterozygosity-fitness
correlations
In the 1990s, the analysis of microsatellites (sections of repetitive DNA that are typically neutral and highly vari-able) was developed, which facilitated more accurate heterozygosity-fitness measures (Allendorf 2017). Microsatellites were used as a proxy to estimate inbreeding levels and potential fitness effects in natural mammal populations. Two different hypotheses were proposed to account for correlations between heterozygosity and fitness in non-coding regions: either microsatellite heterozygosity reflects genome-wide heterozygosity (i.e. identity disequi-librium; Szulkin et al. 2010), or microsatellite loci are non-randomly physically associated with functional genes (i.e. linkage disequilibrium; Slatkin 2008).
At the end of the 20th and early 21th Century, studies on mammal species such as red deer Cervus elaphus (Slate et al. 2000), Soay sheep Ovis aries (Coltman et al. 1999), Arabian oryx Oryx leucoryx (Marshall & Spalton 2000), and California sea lions Zalophus californianus (Acevedo-Whitehouse et al. 2003) reported significant correlations between multi-locus heterozygosity and a variety of fitness
traits (Table 1). However, many of the studies using this approach found the correlations to be weak (Coltman & Slate 2003, Slate et al. 2004, Balloux et al. 2004, Pemberton 2004). The strength of the correlation depends on the variance of inbreeding coefficients as well as on the number of genotyped loci, but even when ~100 loci were used, heterozygosity-inbreeding correlations were still weak (Slate et al. 2004, Pemberton 2004). Therefore, genetically veri-fied pedigrees are regarded as a more informative way to measure inbreeding levels (Pemberton 2004, Slate et al. 2004, Pemberton 2008). Constructing a pedigree of a wild population along with collection of relevant fitness traits, however, comes with major difficulties, and heterozygosity-fitness measures are therefore still widely used to estimate inbreeding depression (Da Silva et al. 2009, Ruiz-López et al. 2012, Annavi et al. 2014).
Microsatellites and pedigrees of natural
populations
Table 1. Studies of natural mammal populations in which a significant effect of inbreeding on fitness was found
Species Method n Pop size Trait Effect size
Lethal
equivalents Ref
Red deer HFC 209 1175–1724 Life time breeding success
– – Slate et al. (2000) Red deer PED 1848 Not specified Birth weight, juvenile
survival
77% lower survival for juveniles with F = 0.25 compared to non-inbred ones
4.35 lethal equivalents for birth weight
Walling et al. (2011)
Red deer Genomic F 2622 Not specified Breeding success 72% lower breeding success for females vs. 95% for males, with
F = 0.125
– Huisman et al. (2016)
Soay sheep HFC 980 600–2000 Susception to parasites Two times higher parasite burden for individuals in the bottom of heterozy-gosity quartile compared to the top during high density years
– Coltman et al. (1999)
Soay sheep PED, genomic F 6336 Not specified Hindleg length, weight, survival, breeding success
– – Bérénos et al. (2016)
Arabian oryx HFC 57 50–225 Juvenile survival – – Marshall and Spalton (2000) California sea
lion
Internal relatedness factor
371 Not specified Disease susceptibility – – Acevedo-Whitehouse et al. (2003) Roe deer
Capreolus capreolus
HFC 222 200–250 Juvenile survival – – Da Silva et al. (2009)
Iberian lynx HFC 20 70–127 Semen quality – – Ruiz-López et al. (2012) European
badger
Meles meles
HFC, PED 975 c.220 Juvenile survival – – Annavi et al. (2014)
Grey wolf PED 48 2–100 Juvenile survival Reduction of 1.15 pups per litter surviving until its first winter for each increase of 0.1 in F
6.04 lethal equivalents for juvenile survival
Liberg et al. (2005)
Grey wolf PED 1518 4–340 Pairing and breeding success
1.84 times higher pairing success 2.5 times higher breeding success in immigrant offspring
compared to inbred ones
– Åkesson et al. (2016)
Florida panther
PED 591 20–95 Juvenile survival, adult survival
2.13 times higher juvenile survival in immigrant F1
offspring compared to purebred kittens
– Johnson et al. (2010)
Florida panther
Hybrid identification
172 30–87 Juvenile survival, adult survival
Three times higher juvenile survival in hybrids compared to non-hybrids
– Pimm et al. (2006)
Species Method n Pop size Trait Effect size
Lethal
equivalents Ref Meerkat PED 963 Not specified Pup mass at
emergence, hind foot length, pup growth, juvenile survival
Comparison between individuals with
F = 0.078 and non inbred ones: 10.9% reduction in pup mass, 3.7% reduction in hind foot lenght, 9.3% reduction in pup growth, 2.46 times more likely to die as juveniles
– Nielsen et al. (2012)
Pronghorn PED 294 86–100 Juvenile survival, birth mass, foot length, condition
– 24.17–28.72 lethal equivalents for juvenile survival
Dunn et al. (2011)
Red wolf PED 764 4–100 Adult body size – 0 lethal equivalents for juvenile survival
Brzeski et al. (2014)
Arctic fox PED 205 4–50 Juvenile survival, reproduction, longevity
– 12.11 lethal equivalents for juvenile survival, 13.77 lethal equivalents for reproduction
Norén et al. (2016)
Arctic fox PED 543 4–100 Juvenile survival, breeding success
1.9 times higher juvenile survival
1.3 times higher breeding success in immigrant F1
offspring compared to inbred individuals
– Hasselgren et al. (2018)
Bighorn sheep PED 165 42 on average
Reproductive success, survival, dominance rank
2.6 times higher reproductive success in outbred
males, 2.2-fold increase for females
– Hogg et al. (2006)
Mountain pygmy possum Burramys parvus
Hybrid identification
131 60–130 Litter size, tail size, longevity
Two times higher fitness advantage for hybrids compared to native individuals, based on hybrid genotype frequency
– Weeks et al. (2017)
Black footed ferret
Mustela nigripes
PED 262 7–300 Sperm motility – – Santymire et al. (2018)
Harbour seal HFC basedon SNP's
60 Not specified Susception to parasites – – Hoffman et al. (2014)
Genomic F, genomic inbreeding coefficient; HFC, heterozygosity-fitness correlation; n, sample size; PED, pedigree.
pronghorn (Dunn et al. 2011), the red deer (Walling et al. 2011), the red wolf (Brzeski et al. 2014), and the arctic fox Vulpes lagopus Norén et al. 2016, Hasselgren et al. 2018; Table 1). Other important study systems for under-standing inbreeding in natural populations are isolated, island-living populations such as the Soay sheep on St Kilda (Coltman et al. 1999) and the grey wolf on Isle Royale, Michigan, USA (Wayne et al. 1991), where in-depth individual monitoring can be accomplished.
Conservation genetics
During the 1970s, an interest in studying inbreeding de-pression in captive zoo animal populations emerged. By analysing studbook data, inbreeding depression was docu-mented in many different captive mammal populations (Bouman 1977, Ralls et al. 1980, Roberts 1982, Templeton & Read 1984, Laikre & Ryman 1991), which made biolo-gists recognise that inbreeding depression could be a serious threat to small and endangered populations. During the 1980s, the field of conservation genetics began to accentu-ate this (Frankel 1974, Frankel & Soulé 1981, Simberloff 1988, Spellerberg 1993). Processes such as demographic stochasticity, genetic drift, and inbreeding depression that threaten small populations disproportionately were identi-fied and termed the small population paradigm (Caughley 1994). A central part of the small population paradigm is the extinction vortex model, predicting that the loss of genetic variation in inbred and small populations might ultimately drive them to extinction (Gilpin & Soulé 1986). The extinction vortex and the small population paradigm have been addressed in threatened species such as the Iberian lynx Lynx pardinus (Palomares et al. 2012), the Florida panther (Fergus 1991) and the American mink Martes americana (Lacy & Clark 1993). Though it is dif-ficult to demonstrate under natural conditions, small mam-mal populations usually exhibit genetic threats associated with a small population size and high extinction risk.
At the same time, however, a debate questioning the importance of genetic variation in the wild appeared in the scientific community (Clegg 1983, Thornhill 1993, Hedrick 1994, Frankham 1995, Lacy 1997). The main argument against inbreeding depression under natural conditions was the lack of supportive, empirical data (Caro & Laurenson 1994). This led to the claims that inbreeding depression was rare and of little importance in nature, due to purging (Lande 1988) and inbreeding avoidance (Pusey & Wolf 1996, but see Ralls et al. 1988).
2000–2020
The connection between genetics, conservation and extinc-tion of wild populaextinc-tions remained a controversial quesextinc-tion
for several years. By the beginning of the 21st Century, genetic analyses were easily accessible, and a compelling amount of evidence was gathered, demonstrating that in-breeding depression affects not only captive, but also natural populations (reviewed by Keller & Waller 2002, O’Grady et al. 2006). Today, members of the scientific community generally agree that inbreeding depression can be more severe under harsh environmental conditions (Pemberton et al. 2017), and, although it is theoretically possible for purging to mitigate inbreeding depression, purging does not seem to be effective for most small populations. For instance, if genetic drift is stronger than selection, which is often the case in small populations, there is a high risk that deleterious alleles become fixed before they are purged. Furthermore, purging will not be efficient if inbreeding depression occurs due to many slightly deleterious alleles, rather than due to a few lethal ones (Wang 2000, Boakes et al. 2007). For instance, Xue et al. (2015) found fewer loss-of-function mutations in populations of eastern gorillas Gorilla beringei, a species with a long-term small effective population size (Ne), than in western gorillas Gorilla go-rilla, a species with a larger effective population size. Since loss-of-function mutations are generally highly deleterious (Glémin 2003), they are also likely to be purged more efficiently from small and inbred populations. On the other hand, eastern gorillas had higher accumulation of missense mutations, which are often only slightly deleterious (Glémin 2003), and therefore more difficult to purge from small populations. The support for avoidance strategies prevent-ing inbreedprevent-ing depression is limited, but genetic studies on canids have shown that discrimination of first-order relatives belonging to the same natal group can slow down the development of population inbreeding levels (Geffen et al. 2011, Godoy et al. 2018).
The genomics era
Although combining genetically verified pedigrees with life-history data is a widely used method to quantify in-breeding depression, the approach has a number of draw-backs. First, and perhaps most importantly, pedigrees are often incomplete, with uncertain relatedness and limited depth (Wang 2016), and they often fail to record historical inbreeding and relatedness among founders, causing in-breeding levels to be underestimated (Hedrick & Garcia-Dorado 2016). Furthermore, since the pedigree inbreeding coefficient is the probability of identity by descent and not a direct measurement, variation due to Mendelian segregation, recombination, and genetic linkage among individuals with the same relatedness is not captured.
and functional regions of the genome (Morin et al. 2004). This innovation paved the way for the rapid and revo-lutionary development of the field of population genomics, which came to offer a unique opportunity to perform detailed studies of inbreeding depression without the re-quirement to construct pedigrees over several generations. The availability of large-scale genomic data has dramatically increased our opportunities to explore the genetic, ecologi-cal and evolutionary basis of inbreeding depression in natural populations (Kardos et al. 2016).
Pedigree-based approaches are still a cornerstone for studies on inbreeding depression, but this is currently changing, and the number of studies using genomics to explore inbreeding depression are rapidly increasing, al-though most of them are still focussed either on humans or on captive mammal populations (Wang 2016). Studies of inbreeding depression using genomic data in natural mammal populations are still rare. During recent years, however, studies have compared how well pedigree-based inbreeding coefficients and marker-based ones reflect in-breeding depression in the wild. Empirical studies on harbour seals Phoca vitulina (Hoffman et al. 2014), soay sheep (Bérénos et al. 2016), and red deer (Huisman et al. 2016) found compelling evidence that inbreeding depres-sion is more easily detected using marker-based estimates than using pedigrees, both in terms of effect size and in terms of number of traits affected. These studies used 13 000–37 000 single nucleotide polymorphisms to estimate the proportion of the genome that was identical by de-scent. A number of simulation-based studies have also all found that the inbreeding coefficient is more precisely measured with large number of loci than with pedigrees (Keller et al. 2011, Kardos et al. 2015, Wang 2016).
An even more informative approach to the study of the extent of inbreeding is to scan genomes for runs of homozygosity (ROH), and thereby measure the proportion of the genome that is identical by descent (Allendorf 2017). Long stretches of ROHs indicate recent events of inbreed-ing, whereas shorter ROHs indicate historical inbreedinbreed-ing, where ROHs have been broken up by recombination and mutations have arisen in one or both parental lineages (Browning & Browning 2015). Detecting ROHs generally requires mapping of complete or almost complete genomes. Whole genome sequencing is still expensive, although techniques are very rapidly developing and also becoming cheaper and more easily accessible. In a recent study, Kardos et al. (2018) scanned 97 complete wolf genomes for ROH and found many individuals with ROHs stretch-ing over entire chromosomes; they concluded that the majority of the inbreeding originated from common an-cestors less than ten generations ago.
Studies on extinct species have suggested that inbreeding depression played an important role in their extinction.
Palkopoulou et al. (2015) performed whole-genome sequenc-ing on two individuals of woolly mammoth Mammuthus primigenius, one ca. 45 000-year-old specimen from the mainland of north-eastern Siberia, and one ca. 4300-year-old specimen from Wrangel Island, Russia. The aim was to find out whether inbreeding depression could have contributed to the final extinction of the population on Wrangel Island, the last remaining mammoth population in the world. Their results revealed that the genome of the Wrangel Island mammoth consisted of 23% ROH compared to less than 1% ROH in the mainland mammoth’s genome. They con-cluded that the Wrangel Island population experienced a pronounced reduction in genetic diversity and an increase in inbreeding shortly before its extinction. Díez-del-Molino et al. (2018) point out the importance of using multi-temporal samples when evaluating genetic erosion of species that have experienced demographic declines.
THE FUTURE: OPPORTUNITIES AND
CHALLENGES
Although the study of inbreeding has been of great interest to biologists since the time of Darwin, many questions remain unanswered, and several new questions have emerged with the development of evolutionary biology, conservation biology, population genetics, and genomics.
Even today, little is known about the severity, cause, and consequences of inbreeding depression in natural mammal populations. Whether inbreeding depression is caused by a few alleles of large effect or by many alleles of small effect, and whether or not purging is usually efficient in wild populations are questions that have been widely debated for several decades and still remain unre-solved (Kardos et al. 2016). One future challenge will be to separate the dominance hypothesis from the overdomi-nance hypothesis, and assess their contribution to inbreed-ing depression. Genomic approaches, however, provide evidence for heterozygous advantage being due to pseudo-overdominance, i.e. linkage with advantageous alleles, rather than the heterozygous advantage (Charlesworth & Willis 2009). In a recent study, Robinson et al. (2019) performed whole-genome sequencing on wolves from Isle Royale, and compared the sequences with wolf genomes from various other populations. In that case, the cause of inbreeding depression was likely to be an increase in the homozygosity of strongly deleterious alleles.
containing recessive lethal alleles are likely to be more heterozygous than other sites (Allendorf 2017, Kardos et al. 2018). For wild mammal populations, this has so far only been accomplished in the Scandinavian wolf population, where ten regions were identified as containing potential candidate loci (Kardos et al. 2018). One caveat is that there is currently no consensus for how long a ROH should be, or how few heterozygous loci can exist for a segment to be classed as identity by descent. Only the longest ROHs are usually analysed; shorter ones are ex-cluded since it is more difficult to distinguish whether they are due to ancient bottlenecks, due to background relatedness, or if they simple arose by chance (Kardos et al. 2016). This issue could be resolved by using histori-cal samples as a baseline (Díez-del-Molino et al. 2018). Furthermore, the ROH method does not identify any slightly deleterious mutations that might become fixed in an inbred population and are likely to be significant con-tributors to inbreeding depression. A way to address this is to scan the genome for non-synonymous mutations, generally assumed to be weakly deleterious (Ohta 1992), which could be used to identify how many alleles are involved in inbreeding depression and how efficient the process of purging is (Díez-del-Molino et al. 2018). Using the two mammoth genomes mentioned earlier, Rogers and Slatkin (2017) compared the number of non-synonymous mutations by identifying deletions, retrogenes, and non-functioning point mutations, and found a higher accu-mulation of deleterious mutations in the individual from Wrangel Island, probably due to its long-term isolation and the small effective population size on Wrangel Island.
Unravelling the specific functions of genes that are con-nected to inbreeding depression is possible by using the gene set enrichment analysis on candidate loci (Huang et al. 2009). This method relies on pre-established libraries in which the functions of gene groups in model species are
identified. Rogers and Slatkin (2017) used this approach to identify the functions of the affected gene sequences found in the mammoth genomes. They found a loss of olfactory receptors and urinary proteins in the Wrangel Island mammoth, as well as a loss of functional mutations at a locus associated with coat quality. This approach could be used to study threatened and inbred modern-day spe-cies. Furthermore, as our understanding of the underlying genomic mechanism increases, the role of epigenetic factors highlighted as important players influencing inbreeding depression can be addressed in detail (Vergeer et al. 2012).
Another method is to identify associations between fitness characters and ROHs across the genomic landscape. This approach is called genome-wide association mapping, and has so far not been carried out in any natural population, but has been applied successfully in studies on both humans and livestock (Pickrell 2014, Pryce et al. 2014, Howard et al. 2015). Furthermore, since deleterious alleles contributing to inbreeding depression are initially expected to be rare, ge-nome-wide association has low power to detect deleterious alleles, but as a population is exposed to genetic drift and as deleterious alleles of strong effect increase in frequency, this power increases with time (Kardos et al. 2016).
inbreeding depression and population demography would constitute valuable progress in conservation genetics. Moreover, developing methods to resolve more practical issues, such as choosing the most suitable individuals for translocation or captive breeding programs, would be a large and important step for the study of inbreeding de-pression in natural mammal populations.
CONCLUSIONS
Unravelling the effects of inbreeding has been of broad scientific interest for several centuries, and approaches used to study inbreeding have experienced a revolutionising de-velopment over the years, from experimental crossings of plants to whole-genome sequencing of extinct megafauna (Fig. 1). Mammals are usually considered to have limited experimental potential due to their slow life histories and high complexity, and are therefore not usually the first choice as model systems for studies of evolutionary pro-cesses. As demonstrated in this review, however, mammals have contributed to our fundamental understanding of inbreeding and inbreeding depression, and have been used to raise awareness of how genetic factors accelerate extinc-tion risk in small populaextinc-tions. In particular, mammals have been important systems for investigating inbreeding depres-sion in natural populations (Wayne et al. 1991, Coltman et al. 1999, Liberg et al. 2005, Johnson et al. 2010, Dunn et al. 2011, Walling et al. 2011, Nielsen et al. 2012, Brzeski et al. 2014, Åkesson et al. 2016, Norén et al. 2016, Hasselgren et al. 2018). Another benefit of using mammals is the strong interest in breeding domesticated mammal lineages, which has allowed parallel studies both in captivity and in the wild to augment our understanding of how inbreeding operates. At present, inbreeding has been studied in depth in a limited number of species, but considering the rapid loss of biodiversity through e.g. climate change accelerating habitat loss, inbreeding depression is likely to become a more severe problem in other species in the future (Pertoldi et al. 2007). Inbreeding depression thus remains a crucial area of study, as habitats worldwide are becoming increas-ingly fragmented and degraded (Kardos et al. 2016). Extrapolating the available knowledge to other populations to predict the future effects of inbreeding depression on population demography would constitute a major contribu-tion to adaptive management strategies of populacontribu-tions or species at risk in future generations.
ACKNOWLEDGEMENTS
We are grateful to Love Dalén for useful comments on earlier drafts, to Rasmus Erlandsson for drawing the figure, and to two anonymous reviewers for improving the
manuscript. The study was funded by the Swedish Research Council FORMAS (2015–1526).
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