Opinion
Inbreeding avoidance, tolerance,
or preference in animals?
Marta Szulkin1,2*, Katie V. Stopher3, Josephine M. Pemberton3, and Jane M. Reid4*
1
Centre d’Ecologie Fonctionnelle et Evolutive, UMR 5175 Campus CNRS, 1919 Route de Mende, 34293 Montpellier cedex 5, France
2
Edward Grey Institute, Zoology Department, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK
3
Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh,
EH9 3JT, UK
4
Institute of Biological & Environmental Sciences, School of Biological Sciences, University of Aberdeen, Zoology Building,
Tillydrone Avenue, Aberdeen AB24 2TZ, UK
Animal ecologists commonly assume that the reduced
fitness that often afflicts inbred offspring will inevitably
cause selection for inbreeding avoidance. Although ear-
ly empirical studies often reported inbreeding avoid-
ance, recent studies suggest that animals sometimes
show no avoidance or even prefer to mate with relatives.
However, current theory is insufficient to predict wheth-
er animals should avoid, tolerate, or prefer inbreeding
and hence to understand overall inbreeding strategy.
Furthermore, quantifying inbreeding strategy is chal-
lenging, requiring relatedness among unbiased sets of
actual and potential mates to be accurately estimated.
Here, we highlight key limitations of current theory and
empirical tests, and summarise the advances required
to predict, quantify, and understand animal inbreeding
strategies.
Inbreeding depression and inbreeding avoidance
Inbreeding depression (the reduced fitness of inbred indi-
viduals resulting from reproduction among relatives) is
widespread in wild, domesticated, and experimental popu-
lations [1,2]. Animal ecologists commonly assume that
inbreeding depression will drive the evolution of reproduc-
tive strategies that result in inbreeding avoidance, includ-
ing sex-biased dispersal, specific choice of unrelated mates,
and associated mechanisms of kin recognition and sperm
selection [3–5]. The assumption that inbreeding avoidance
must be adaptive because inbreeding reduces offspring
fitness pervades numerous hypotheses explaining repro-
ductive strategy and the design and interpretation of
empirical tests [4,6–9]. By contrast, long-standing theory
shows that the basic assumption that inbreeding depres-
sion will inevitably cause selection for inbreeding avoid-
ance is simplistic because it ignores the potential benefits
of inbreeding and costs of inbreeding avoidance [10–13].
Against this conflicting backdrop, there has been a recent
surge of interest in testing for departures from the distri-
bution of inbreeding expected given random mating in
animals, and empirical studies report diverse patterns
(e.g., [6,11,14–18]) (Figure 1). However, predicting and
Corresponding author: Szulkin, M. (marta.szulkin@zoo.ox.ac.uk)
Keywords: inbreeding avoidance; inbreeding preference; inbreeding strategy;
inclusive fitness; mate choice; relatedness.
*
These authors contributed equally to this work.
0169-5347/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tree.2012.10.016 Trends in Ecology and Evolution, April 2013, Vol. 28, No. 4
testing whether animals should or do avoid, tolerate, or
prefer relatives as mates is challenging. Few, if any, em-
pirical tests have an explicit theoretical basis or fulfil all
key requirements to quantify and interpret departures
from random inbreeding. Consequently, general conclu-
sions regarding inbreeding strategies (see Glossary) can-
not yet be drawn.
In many populations, dispersal reduces the probability
that inbreeding will occur and thereby causes inbreeding
avoidance without the need for mating bias against rela-
tives during post-dispersal life stages [19–21]. This out-
come arises irrespective of whether dispersal evolved due
to selection for inbreeding avoidance or due to other forces
such as local resource availability or kin competition
[13,22]. However, when dispersal is limited, inbreeding
strategy, consisting of inbreeding avoidance, tolerance,
or preference, could be expressed through mate choice
among philopatric relatives [3,11,23]. Here, we highlight
limitations of existing theory and data that impede the
clear prediction and analysis of inbreeding strategies in
philopatric animals, and outline theoretical and empirical
Glossary
Coefficient of inbreeding f: the probability that two alleles at any locus in an
individual are identical by descent (i.e., that they are copies of the same alleles
in a common ancestor). Inbreeding, or mating between relatives, produces
offspring with f > 0.
Coefficient of kinship k: the probability that homologous alleles sampled from
two individuals are identical by descent. Usually equal to half the coefficient of
relatedness between the two individuals, and to the coefficient of inbreeding of
offspring that would be produced if the two individuals mated.
Coefficient of relatedness r: the proportion of all alleles that two individuals
share that are identical by descent due to recent common ancestry (Box 2).
Identity by descent: alleles that are identical because they are copies of the
same ancestral allele.
Identity by state: identical alleles that are not necessarily copies of the same
ancestral allele.
Inbreeding strategy: the overall system of inbreeding, comprising inbreeding
avoidance (avoidance of mating with relatives, resulting in negative deviation
from relatedness expected under random mating), inbreeding tolerance
(absence of inbreeding preference or avoidance, resulting in inbreeding
occurring to the degree that would be expected given random mating among
population members), or inbreeding preference (preference for mating with
relatives, resulting in positive deviation from relatedness expected under
random mating).
Inclusive fitness: the relative number of gene copies an individual leaves in the
next generation through its own direct reproduction and through reproduction
of relatives.
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 Opinion
(a) (b)
(e) (f)
Figure 1. Empirical studies of inbreeding strategy have reported inbreeding avoidance in (a) red-winged fairy wrens Malurus elegans [6]; (b) bank voles Myodes glareolus
[15]; (c) zebra finches Taeniopygia guttata [14]; (d) grey wolves Canis lupus [30]; inbreeding tolerance in (e) New Zealand robins Petroica australis [11]; (f) bighorn sheep
Ovis canadensis [16]; (g) great tits Parus major [17] (but see [66,67]); and inbreeding preference in (h) cichlid fish Pelvicachromis taeniatus [18]. Photographs reproduced,
with permission, from L. Brouwer (a), M.D. Thom (b), J. Rutkowska (c), S. Kabessa (d), I. Jamieson (e), A.M. Martin (f), T. Veen (g), and H. Kullmann (h).
advances that are required to understand the occurrence,
ecology, and evolution of such strategies.
Theory of inbreeding strategy
Inbreeding and inclusive fitness
The widespread presumption that inbreeding depression
will inevitably cause net selection for inbreeding avoidance
ignores the inclusive fitness benefit of inbreeding that exists
because parents are more closely related to inbred offspring
than to outbred offspring. This increased relatedness arises
because inbred offspring, by definition, inherit alleles from
one parent that are identical by descent to those carried by,
and potentially inherited from, the other parent. Inbreeding
thereby increases the expected proportion of alleles that any
one parent shares with its offspring, including alleles
favouring inbreeding [10,24,25]. This inclusive fitness ben-
efit underpins extensive theory predicting the evolution of
self-fertilisation in plants [10,25–27]. That this same benefit
could also cause the evolution of biparental inbreeding was
suggested three decades ago (Box 1) [28,29], but remained
widely ignored by animal ecologists. The assumption that
inbreeding depression inevitably causes selection for in-
breeding avoidance continued to pervade hypotheses
explaining dispersal, mate choice, and polyandry
[3,5,6,11,30–32]. Recent theory has re-emphasised that bi-
parental inbreeding could be adaptive, even given inbreed-
ing depression, with the inclusive fitness benefit phrased as
‘helping relatives to breed’ and hence kin selection
[12,13,33]. However, existing theory does not predict what
sex-specific inbreeding strategies should evolve in popula-
tions of potentially inbreeding animals.
Limitations of current theory
Early models of biparental inbreeding demonstrated the
fundamental conceptual point that there will only be se-
lection for inbreeding avoidance if the costs of inbreeding
exceed the inclusive fitness benefit (Box 1) [28,29,34,35].
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(c) (d)
(g) (h)
TRENDS in Ecology & Evolution
They also highlighted that selection depends on the fitness
costs of avoiding inbreeding, for example through dispersal
or searching for unrelated mates, and on the degree to
which mating with a relative impedes mating with other
non-relatives (Box 1) [34]. Recent extensions consider si-
multaneous versus sequential mate choice and relative
investment in parental care, and show that the magnitude
of inbreeding depression under which inbreeding could be
adaptive is even greater than previously predicted [12].
Links between costs of inbreeding avoidance, life history
and ecology have also been further emphasised, but such
discussions have not always incorporated the inclusive
fitness benefit of inbreeding [11,23].
However, key limitations of existing theory inhibit
prediction of inbreeding strategies. First, basic models
predict sexual conflict over inbreeding; females should
tolerate less inbreeding than males under certain cir-
cumstances (Box 1) [28,34]. However, these ‘battle-
ground’ models do not consider the resolution of
sexual conflict or predict what sex-specific inbreeding
strategies might consequently evolve [35]. One notable
model showed that female mate choice for inbreeding
avoidance could drive male-biased dispersal, but attrib-
uted mating decisions entirely to singly mated females
and did not consider selection on inbreeding in males
[13]. A recent analysis of optimal inbreeding also consid-
ered female fitness only [33].
Second, existing models consider inbreeding between
two relatives within an otherwise outbred population
[12,28,34], or consider mean population-level relatedness
and inbreeding [13,33]. They do not explicitly consider
populations comprising individuals of varying relatedness,
in which different degrees of biparental inbreeding are
likely and selection on inbreeding strategy could be strong.
This restriction is important because selection on inbreed-
ing avoidance might depend on the probability that a
potential mate that a focal individual rejects to avoid
Opinion
Box 1. Predicting conditions favouring sex-specific inbreeding avoidance or preference
A basic model predicting conditions under which females and males
should prefer or avoid inbreeding balances the inclusive fitness
benefit of inbreeding (which arises because parents are more closely
related to inbred offspring) against fitness costs of avoiding inbreed-
ing, reduced fitness in inbred offspring (inbreeding depression), and
any reduction in further matings due to inbreeding [34]. Assuming an
otherwise outbred population, females and males should mate with a
relative if (Equations [I] and [II]):
d < 1 þ ½DNf ðcf  1Þ þ rDNm ðcm  1Þ=ð1 þ rÞ; [I]
d < 1 þ ½DNm ðcm  1Þ þ rDNf ðcf  1Þ=ð1 þ rÞ; [II]
respectively, where d is the magnitude of inbreeding depression (the
reduction in reproductive success through inbreeding relative to
outbreeding), DNf and DNm are the degrees to which females and
males forfeit effective outbred matings by inbreeding, respectively, cf
and cm are the costs of avoiding inbreeding for females and males,
respectively, and r is the relatedness between prospective mates.
If there is zero cost of inbreeding avoidance (cf = cm = 0) and both
sexes forfeit all other reproductive opportunities by inbreeding (DNf =
DNm = 1, i.e., strict monogamy), both expressions reduce to d < 0 and
both sexes should avoid inbreeding unless inbreeding depression is
negative (i.e., reproductive success is greater through inbreeding than
outbreeding).
However, if mating with a related female does not restrict the
mating opportunity of a male (DNm = 0, i.e., polygyny), then d < r/(1+r)
and d <1/(1+r) for females and males, respectively (Figure I). Conse-
quently, fitness can be maximised by inbreeding rather than by
outbreeding, even with inbreeding depression (d > 0). Furthermore,
the value of d at which fitness is maximised by outbreeding rather than
by inbreeding differs between males and females. Given the option to
mate with a brother (r = ½) or outbreed, female fitness is maximised
by inbreeding if d < 1/3, because the inclusive fitness benefit out-
inbreeding will subsequently mate with a different relative
and hence inbreed anyway.
Third, existing models of biparental inbreeding in ani-
mals do not consider coevolution of inbreeding and inbreed-
ing depression, or their coevolution with other
reproductive strategies, such as polyandry, that could
potentially facilitate inbreeding preference or avoidance
[31]. Predictions of plant selfing theory change substan-
tially when analogous constraints are relaxed, for example
by allowing purging of inbreeding load, reduced outcross-
ing given selfing (‘pollen discounting’), or ecological varia-
tion in cross-fertilisation failure or inbreeding depression
[10,26,27].
Given that sexual conflict over selfing is not expected,
existing selfing theory cannot be directly applied to predict
the evolution of biparental inbreeding strategies (Box 1).
New theory is therefore required. Existing analytical mod-
els should be extended to quantify the effects of variation in
relatedness on sex-specific selection on inbreeding strate-
gy. Models predicting the evolutionary resolution of sexual
conflict should then be formulated. The need to track sex-
specific selection and feedbacks between inbreeding strat-
egy and relatedness, and complex genetic architecture
underpinning the coevolution of inbreeding strategy and
inbreeding depression, means that analytical solutions
might be restrictive. Individual-based models will then
prove fruitful, especially given the need to consider small
populations where variation in relatedness and inbreeding
risk prevail (e.g., [36]).
Trends in Ecology and Evolution April 2013, Vol. 28, No. 4
weighs inbreeding depression. By contrast, male fitness is maximised
by inbreeding with a sister if d < 2/3. This basic model therefore
predicts sexual conflict over inbreeding; females should avoid
inbreeding given smaller d than males [34,35] (Figure I).
This basic ‘battleground’ model demonstrates that inbreeding
should not necessarily be avoided even if d > 0, and identifies key
parameters on which sex-specific thresholds for inbreeding avoid-
ance depend. However, it does not predict equilibrium inbreeding
strategies that might evolve in animals with biparental inbreeding.
Existing models of selfing are not directly applicable because they
consider the special case of r = 1, where there is no sexual conflict
(Figure I).
1.0
Magnitude of inbreeding depression
0.9
0.8
0.7
0.6
(δ)
0.5
0.4
0.3
0.2
0.1
0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Relatedness to potenal mate (r)
TRENDS in Ecology & Evolution
Figure I. Under polygyny, inbreeding is favoured in both sexes in the grey zone of
the parameter space, males only (black zone) or neither sex (white zone). There is
therefore sexual conflict over inbreeding in the black zone. Adapted, with
permission, from [34].
Quantifying inbreeding strategy: approaches and
challenges
Both observational and experimental studies will be re-
quired to quantify whether animals avoid, tolerate, or
prefer inbreeding across a range of biologically relevant
conditions and, hence, to test evolutionary predictions.
Such studies should comprise five key components: (i) an
appropriate system; identification of (ii) actual and (iii)
potential mates; (iv) accurate measurement of relatedness;
and (v) appropriate statistical analyses and interpretation.
These requirements might seem obvious, but arguably no
empirical studies have fulfilled them all, especially in wild
populations. We highlight key approaches and pitfalls, and
thereby outline a framework for future empirical test.
Choosing an appropriate study system
There is limited value in testing for inbreeding preference
or avoidance in systems where there is little variation in
relatedness between potential mates and, therefore, little
opportunity to express inbreeding strategy. This applies to
populations whose sizes, ecologies, or life histories mean
that relatives rarely interact [17,37,38], and to highly
inbred populations comprising uniformly close relatives
[8]. Rather, the most informative and powerful tests will
concern populations where there is sufficient variation in
relatedness to create opportunity for inbreeding preference
and avoidance, and for tolerance to occur as an evolved
rather than an inevitable constrained outcome. Studies
would usefully focus on populations that have experienced
207
Opinion
such conditions for numerous generations, and hence ex-
perienced long-term selection on inbreeding strategy.
Small populations where there is currently opportunity
for inbreeding, but which recently derived from large
panmictic populations where inbreeding was previously
rare, might not support useful hypothesis tests regarding
predicted inbreeding strategy [11,23,37].
Identifying actual mates
Directly quantifying inbreeding strategy requires the ac-
tual mate of all (or an unbiased sample of) breeding
individuals, or the parents of an unbiased sample of result-
ing offspring, to be identified. Mate identities are known
when specific monogamous pairings are experimentally
created [18], but often unknown in observational studies
and systems with multiple mating. Whereas mothers are
often identifiable from parental behaviour, paternity fre-
quently remains unassigned, uncertain, or misassigned.
Accurate molecular genetic paternity assignment is ham-
pered by incomplete or biased sampling of offspring and
potential parents, and insufficient or inaccurate genotyp-
ing [39,40]. Furthermore, and critically given the aim of
quantifying inbreeding strategy, the ability to identify
sires can vary with relatedness. The presence of relatives
within a population means that multiple individuals share
numerous alleles, resulting in error-prone parentage as-
signment [41]. Immigrant or transient males might be less
likely to be sampled and also less closely related to resident
females [42]. These biases are likely to arise in exactly the
populations with high variance in relatedness where adap-
tive inbreeding strategies might be expected. Moreover,
early-life mortality due to inbreeding depression can bias
sampling against inbred offspring, meaning that inbred
matings go undetected [4]. Mate and parentage assign-
ment for the purpose of quantifying inbreeding strategy
therefore requires particularly comprehensive or carefully
structured sampling of offspring and parents, and infor-
mative molecular markers, to ensure that identified mates
are unbiased with respect to relatedness.
Identifying potential mates
A further major requirement is to provide or identify
appropriate sets of potential mates for focal individuals.
Specific sets of potential mates can be experimentally
provided, but should be carefully chosen to ensure biologi-
cal relevance. Combinations of non-relatives and familiar
or unfamiliar full- or half-sibs are often provided, probably
because such individuals can be readily produced through
single generations of controlled breeding [18,37,43]. How-
ever, dichotomous choice experiments involving sibs and
non-relatives will be hard to interpret if sib encounters are
rare in nature, and cannot reveal nonlinear inbreeding
preference or avoidance. Experiments that provide a great-
er diversity of relatives as potential mates might therefore
be more insightful, but will require more complex breeding
designs to create appropriate individuals of standardised
age and reproductive history.
A major challenge facing observational studies is to
identify all the potential mates of an individual, or an
unbiased sample of those mates with respect to related-
ness, and hence define appropriate null models against
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Trends in Ecology and Evolution April 2013, Vol. 28, No. 4
which the relatedness of an individual to its actual mate
can be tested. Identifying potential mates is harder than
identifying actual mates because, by definition, rejected
mates cannot be identified by observing pairings or par-
entage. Moreover, it is often simplistic to assume that all
opposite-sex adults are available as potential mates [44].
Identifying potential mates therefore requires detailed,
individual-based knowledge of population ecology and life
history, ideally including the location of each individual
[6,17,45], timing of settlement [46], age [16,47], and cur-
rent or previous reproductive status [17,23]. Truly unbi-
ased sampling with respect to such variables, which might
co-vary with relatedness, is hard. Field studies are almost
inevitably spatially restricted, meaning that distant or
transient potential mates are missed. If relatedness varies
spatially, which is likely when dispersal is restricted and
inbreeding occurs, unidentified potential mates might be
relatively unrelated to focal individuals [45]. Individuals
that fail to mate and breed, or whose breeding attempt fails
early, might also go unobserved and hence be excluded as
potential mates [48]. This is problematic if such individuals
are non-random with respect to relatedness, for example
due to kin-selected cooperative breeding or reproductive
suppression, or if breeding attempts involving relatives are
more or less successful. Naive sampling can also confound
sets of more or less related individuals that are not repro-
ductively active simultaneously and consequently bias
inferred sets of potential mates [49]. Unbiased tests of
inbreeding strategy might therefore require populations
where all or almost all adults can be identified with high
confidence, and where detailed individual-based data can
be used to define appropriate null models [6,11,23,50].
Given that such models will rarely be demonstrably unbi-
ased, the most robust approach might be to formulate sets
of increasingly constrained models and verify whether
conclusions regarding inbreeding strategy are consistent
[16,17,23,30,47,51]. Meeting these conditions might re-
quire studies that are explicitly designed to identify unbi-
ased sets of potential mates rather than simply relying on
existing data. When potential mates cannot be identified,
an alternative approach is to compare relatedness between
an individual and its first and subsequent mates, or social
and extra-pair mates [6,52,53]. This approach has the
advantage that both mates can be identified, but the dis-
advantages that it measures conditional rather than abso-
lute inbreeding strategy and excludes singly mated
individuals.
Estimating relatedness
Quantifying inbreeding strategy requires the relatedness
between an individual and its actual and potential mates,
or the inbreeding coefficients of actual and potential off-
spring, to be estimated. Expected coefficients of related-
ness between any two individuals, and corresponding
offspring inbreeding coefficients, can be calculated from
pedigree data irrespective of whether the individuals mat-
ed or reproduced (Box 2) [16,17,23]. However, reconstruct-
ing pedigrees of sufficient depth, completeness, and
accuracy to quantify inbreeding strategy is challenging,
especially for wild populations. A single pedigree must link
not only focal individuals and their actual mates, but also
Opinion
Box 2. Approaches to estimating relatedness
The broad concept of ‘relatedness’ can be refined and measured in
numerous ways, with different assumptions and utilities [58,68,69].
Understanding inbreeding strategy requires estimation of the
coefficient of relatedness (r) defined as the proportion of alleles
that individuals share that are identical by descent due to recent
common ancestry and, hence, relative to a defined baseline
population [58].
Expected coefficients of relatedness (r) and kinship (k) between
pairs of individuals, and coefficients of inbreeding ( f) of actual or
hypothetical offspring, can be calculated from pedigree data
assuming Mendelian inheritance, where individual alleles have
parent–offspring transmission probabilities of 0.5. Expected r
between any two individuals x and y is (0.5)n, where n is the
number of pedigree links connecting x and y. When x and y have
multiple common ancestors, coefficients stemming from multiple
paths are summed. Expected k and f equal r/2 for any pair or
offspring, respectively. These calculations can incorporate multiple
common ancestors and hence inbreeding, but values are relative to
defined pedigree founders that are typically assumed to be outbred
and unrelated. Distant common ancestors contribute little variation
to r and k among contemporary individuals, or f of potential
offspring, because n is large. Consequently, pedigrees covering c. 5–
10 generations might be sufficient to detect relatively subtle
inbreeding strategy.
Realised relatedness can be estimated directly from co-dominant
marker genotypes (e.g., microsatellites or SNPs) because more
closely related individuals are expected to share more alleles. The
simplest estimator is ‘allele sharing’: the number of alleles shared
by two individuals divided by the total number of loci considered.
However, individuals can have identical alleles at a given locus
(identity by state) because of their pedigree relation (identity by
descent) or recurrent mutation.
Identity by state is common if an allele occurs at high frequency in
the focal population. Therefore, more advanced relatedness esti-
mators incorporate information on population allele frequencies
(estimated from sampled individuals) [70]. These comprise ‘like-
lihood’ estimators [71] and the widely used ‘method-of-moments’
estimators. The latter comprise ‘correlation’ estimators that provide
a single relatedness estimate for each pair of individuals [72], and
‘regression’ estimators that compare the genotype of one individual
to another and can thus generate different estimates depending on
which individual is compared to which [73,74]. These various
relatedness estimators have different precision and bias, which
depend on characteristics of the study system. Extensive compar-
isons, reviews, and critiques of these methods are available
[57,70,75].
all (or an unbiased sample of) their potential mates, and
encompass sufficient generations to quantify subtle varia-
tion in relatedness (Box 2). These specifications are more
demanding than those required to estimate inbreeding
depression: given that individuals with unknown ancestors
are still the actual or potential mates of other individuals,
they cannot simply be excluded from analyses (e.g.,
[42,54]).
Pedigree reconstruction involves assembling numerous
parentage assignments. Assignment error can therefore
introduce error and bias into pedigree relatedness esti-
mates throughout descendent generations. When an indi-
vidual has no assigned sire (or dam), relatedness is
typically calculated assuming that the unknown parent
is unrelated to all other pedigree members [42,54]. If
parentage assignment failed precisely because there are
multiple closely related potential parents [41], estimates of
relatedness among descendants will probably be down-
wardly biased. Similarly, if multiple offspring were sired
Trends in Ecology and Evolution April 2013, Vol. 28, No. 4
by the same unsampled or unidentified male, the standard
assumption that each offspring had a different unrelated
sire will also cause underestimation of relatedness. Ana-
lysts should minimise these biases by reconstructing sib-
ships [55], implementing ‘gene-dropping’ analyses [56],
assuming population mean rather than zero relatedness
among unidentified parents [56], or by applying integrated
analyses that allow parentage and effects of relatedness on
parentage to be jointly estimated [39]. The increasing
availability of abundant and accurate SNP markers will
allow improved parentage inference. However, even so-
phisticated genomic analysis is unlikely to provide a quick
or infallible substitute for meticulous collection of the
comprehensive multigenerational behavioural, life-histo-
ry, and genotypic data that allow complete, accurate pedi-
gree reconstruction [48]. Given that such data are also
needed to define appropriate sets of potential mates, stud-
ies that fail to collect them might struggle to elucidate
inbreeding strategy.
The difficulty of pedigree reconstruction has prompted
attempts to estimate pairwise relatedness directly from
genotype data (Box 2). This approach appears beguilingly
easier; it requires similar genotypic data but eliminates
the parentage assignment step. Several methods for esti-
mating relatedness from marker data have been proposed
(Box 2) [57]. Such methods assume that identity by state is
correlated with identity by descent, and that available
markers accurately measure genome-wide identity by
state. In fact, microsatellites frequently mutate to new
alleles that are indistinguishable from existing alleles,
impeding inference of identity by descent from identity
by state, and small sets of markers do not always reliably
capture genome-wide identity by state [58]. Although
marker-based estimators will capture coarse variation in
relatedness in populations where substantial variation
exists, they are unlikely to detect subtle variation in
relatedness that is of interest in the context of inbreeding
strategy [57]. The approach of estimating parental relat-
edness from marker-based offspring heterozygosity (e.g.,
[59]) is even less precise than estimating pairwise related-
ness from the genotypes of both parents because it uses
half the information. Indeed, the marker heterozygosity of
an individual often only weakly predicts its coefficient of
inbreeding [60,61]. Finally, estimates of parental related-
ness can be biased if the same markers are used to assign
paternity and estimate relatedness. Such spurious asso-
ciations can be avoided by using independent markers for
the two tasks [62,63].
Although the most informative approach to estimating
relatedness in non-model organisms is currently to use
complete pedigree data, high-density genotyping or se-
quencing might in future provide more precise estimation.
Pedigree analysis provides unbiased estimates of expected
pairwise relatedness but, except for parent–offspring rela-
tionships, realised relatedness varies due to recombination
and sampling variance [64]. High-density genotyping
allows estimation of realised rather than expected related-
ness. This approach might be particularly valuable in
evaluating inbreeding strategies in species with long gen-
eration times, where sufficient pedigree data cannot real-
istically be collected.
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Opinion
Statistical analyses and interpretation
Once actual and potential mates are identified and related-
ness estimated, the final hurdle is appropriate statistical
analysis. The most obvious and widely implemented analy-
sis is simply to quantify the mean difference in relatedness
between the actual versus potential mates of an individual
[17,23]. However, several extensions are desirable. It might
prove insightful to quantify other metrics of relatedness
distributions, such as the variance. If some individuals avoid
inbreeding whereas others preferentially inbreed, mean
observed relatedness could match null expectation but with
increased variance. Furthermore, unexpected patterns of
specific types of inbreeding could occur even when mean
relatedness does not differ from expectation [16,17,23].
Frequencies of specific categories of inbreeding, including
between familiar and unfamiliar relatives, should therefore
be quantified. This could facilitate detection of inbreeding
preference mediated by differential imprinting on familiar
versus unfamiliar relatives [17,23,65]. Moreover, because
overall inbreeding strategy often stems from multiple mate
choice events occurring at different stages throughout an
individual’s life history (e.g., [17,21,66,67]), analyses should
ideally consider these stages separately as well as combined
across its lifetime. Inbreeding strategies manifested at dif-
ferent life stages should therefore be quantified indepen-
dently. Finally, observed inbreeding strategy should be
interpreted in the context of current and historical popula-
tion ecology because behaviours that previously generated
inbreeding avoidance or preference might be less effective in
contemporary ecological conditions [30].
When neither inbreeding avoidance nor preference is
detected, appropriate power analysis should be provided to
quantify the ability of a study to detect departure from
random inbreeding. Such power analysis is required to
support any inference of inbreeding tolerance and, depend-
ing on the null hypothesis, might require simulations that
are tailored to specific data sets [8,16,23]. Potential bias
and uncertainty stemming from error and uncertainty in
mate identities and associated relatedness estimates
should also be explicitly considered. Sampling variance
associated with marker-based relatedness estimates might
mean that many studies have little power to detect in-
breeding strategy. Integrated analyses that simultaneous-
ly estimate uncertainty in paternity and/or relatedness,
and in resulting relationships between mating or parent-
age and relatedness might provide the most powerful and
robust statistical approach. This will require existing an-
alytical frameworks to be extended [39].
Concluding remarks
Inbreeding shapes genotypic variation and has profound
implications for mating system evolution [1,10,27,28,42].
Consequently, understanding inbreeding strategy is cen-
tral to evolutionary ecology, but substantial advances in
theory and empirical tests are still required. Explicit the-
oretical predictions pertaining to inbreeding strategy in
nonselfing animals are scant. Models that predict the
evolution of inbreeding strategy under sexual conflict,
including feedbacks with population relatedness structure
and inbreeding depression, need to be formulated. Existing
empirical studies do not fulfil all key requirements for
210
Trends in Ecology and Evolution April 2013, Vol. 28, No. 4
rigorous and informative test and are vulnerable to bias,
meaning that existing conclusions regarding inbreeding
strategy should be interpreted cautiously. However, oppor-
tunities to overcome these challenges are available in the
form of individual-based field studies, carefully designed
experiments, and molecular genetic tools that, in combina-
tion, should allow relatedness between individuals and
actual or potential mates to be adequately estimated. This
objective requires detailed knowledge of the biological
system, comprehensive or carefully designed data collec-
tion, and sophisticated molecular genetic and statistical
analysis; empiricists should no longer be content to accept
current standards with their inherent and typically
unquantified biases and uncertainty. Meanwhile, the ulti-
mate aim of discerning general principles will be facilitated
if authors objectively document the specific strengths and
limitations of their data, analysis, and inference.
Acknowledgements
We thank Magdalen College, Oxford and an IEF Marie Curie Fellowship
(M.S.), NERC (K.V.S.), the Royal Society (J.M.R.), and the ERC (J.M.P.)
for funding and Deborah Charlesworth, Lukas Keller, Ashleigh Griffin,
and two anonymous referees for helpful comments.
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