Update on Hybridization

Hybridization in Plants: Old Ideas, New Techniques[OPEN]

Benjamin E. Goulet, Federico Roda, and Robin Hopkins*
Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts
02138 (B.E.G., F.R., R.H.); and Arnold Arboretum of Harvard University, Boston, Massachusetts 02131 (R.H.)
ORCID ID: 0000-0002-6283-4145 (R.H.).

Hybridization has played an important role in the evolution of many lineages. With the growing availability of genomic tools
and advancements in genomic analyses, it is becoming increasingly clear that gene flow between divergent taxa can generate

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new phenotypic diversity, allow for adaptation to novel environments, and contribute to speciation. Hybridization can have
immediate phenotypic consequences through the expression of hybrid vigor. On longer evolutionary time scales, hybridization
can lead to local adaption through the introgression of novel alleles and transgressive segregation and, in some cases, result in
the formation of new hybrid species. Studying both the abundance and the evolutionary consequences of hybridization has deep
historical roots in plant biology. Many of the hypotheses concerning how and why hybridization contributes to biological
diversity currently being investigated were first proposed tens and even hundreds of years ago. In this Update, we discuss how
new advancements in genomic and genetic tools are revolutionizing our ability to document the occurrence of and investigate
the outcomes of hybridization in plants.

In natural populations, hybridization can act in begun in 1716, when Cotton Mather described corn/
opposition to divergence, introduce adaptive varia- maize (Zea mays) and squash (Cucurbita spp.) plants as
tion into a population, drive the evolution of stronger being of hybrid origin (Zirkle, 1934). Around the same
reproductive barriers, or generate new lineages. Hy- time Thomas Fairchild produced what was likely the
bridization is purposefully employed in the breeding first intentional wild plant hybrid between two Dian-
of domesticated plants to take advantage of transient thus species (Zirkle, 1934). Over the next 300 years,
hybrid vigor, move desirable variation among line- botanists including J.E. Smith (1804), Wilhelm Olbers
ages, and generate novel phenotypes. With the advent Focke (1881), and Leonard Cockayne (1923) made
of next-generation sequencing and the availability of notable efforts to catalog natural hybridization
genomic data sets has come a tide of interest in hy- (Anderson and Stebbins, 1954; Stebbins, 1959). Until
bridization and introgression. This includes the de- the advent of molecular data, hybrids had to be iden-
velopment of methods for detecting gene flow and a tified by phenotypic comparisons, a practice that
steadily growing set of empirical studies of natural was eventually formalized into the hybrid index
hybridization (for review, see Payseur and Rieseberg, (Anderson, 1949).
2016) as well as a shift toward thinking of phylogenies Joseph Gottlieb Kölreuter (1766) is credited with
as reticulate webs rather than strictly bifurcating trees the first rigorous investigations of the consequences
(Mallet et al., 2016). One reason for this trend is that of hybridization, showing, for instance, that early-
genomic data are particularly well suited to address generation hybrids tend to be phenotypically interme-
the problem of detecting gene flow. Another is the diate between parents but may be more luxuriant,
growing recognition that hybridization is widespread while later-generation hybrids more closely resemble
and may have significant evolutionary consequences, parental forms. Following Kölreuter (1766), many bot-
a long-held belief about plants that is increasingly anists have introduced or developed major hypotheses
extended to animals (Mallet, 2005; Arnold, 2006; regarding the consequences of hybridization, including
Abbott et al., 2013; Vallejo-Marín and Hiscock, 2016). work on heterosis (Jones, 1917; East, 1936), transgres-
The study of hybridization in plants has a rich his- sive segregation and adaptive introgression (Lotsy,
tory. Verne Grant (1981) noted that much of the his- 1916), and hybrid speciation (Winge, 1917; Müntzing,
torical work on hybridization in plants could be 1930). Finally, Edgar Anderson (1949) and G. Ledyard
partitioned into cataloging the frequency of hybridi- Stebbins (1950) both synthesized and developed many
zation and exploring the evolutionary consequences of of these ideas, making major botanical contributions to
hybridization. To this day, our research on hybridi- the modern synthesis.
zation still focuses on these two themes. In plants, Our goal is to draw connections between the con-
scientific identification of hybrids is thought to have ception and development of ideas in plant hybridiza-
tion and the recent and future work in these areas. This
Update is not meant to be an exhaustive review of the
* Address correspondence to rhopkins@fas.harvard.edu. literature; rather, we hope to present a handful of
[OPEN]
Articles can be viewed without a subscription. research areas that combine rich histories of botani-
www.plantphysiol.org/cgi/doi/10.1104/pp.16.01340 cal and evolutionary thought with exciting recent
Plant PhysiologyÒ, January 2017, Vol. 173, pp. 65–78, www.plantphysiol.org Ó 2017 American Society of Plant Biologists. All Rights Reserved. 65
Goulet et al.

combined with classical experiments (i.e. to determine

the strength of selection in the field or the molecular
function of a particular allele).

IDENTIFYING HYBRIDIZATION
One of the greatest achievements of genomics is re-
vealing the fundamental role of hybridization in shap-
ing the history of life on earth. In spite of some
disagreement regarding the definition of hybridization
(Box 1), it is clear that a significant proportion of plant
and animal taxa have experienced hybridization and

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introgression (Mallet, 2005). The concept of genetic in-
trogression, defined as the movement of genetic mate-
rial between parental types through the production of
and mating with hybrids (Grant, 1981), predates the
genomic era and was founded upon observations of
increased phenotypic variation in areas of contact be-
tween plant species (Du Rietz, 1930; Marsden-Jones,
1930). Introgression was formerly inferred by using
hybrid indices and pictorialized scatter diagrams,
which scored individuals from putative hybrid popu-
lations based on the similarity to phenotypes of pa-
rental forms (Anderson, 1949; Grant, 1981). These
indices are based on the idea that parental phenotypes
are recombined in hybrids and that the proportion and
distribution of these phenotypes will reflect the amount
and nature of introgression. However, Anderson (1948)
lamented that “Gene flow from one species to another
may go far beyond any point which could be detected
by ordinary morphological techniques. We shall not be
able to assess the real importance of introgression until
we can study genetically analyzed species in the field
and determine the actual spread of certain marker
genes.”
As predicted by Anderson (1948), analyses of se-
quence divergence, haplotype structure, and allele
frequency distributions in genomic data have funda-
mentally improved our ability to detect hybridization
and even identify introgressed loci (Rieseberg et al.,
1993; Payseur and Rieseberg, 2016).
The evolutionary history of a population is reflected
in the genetic variation of its genomes. Model-based
methods are widely used to infer global (genome-
average) and local (locus-specific) ancestry from pop-
ulation variation data (Gompert and Buerkle, 2013; Liu
et al., 2013). For example, the program STRUCTURE
uses a hierarchical Bayesian model to identify sub-
populations and estimate global ancestry for each
sampled individual based on allele frequency data
(Pritchard et al., 2000; Porras-Hurtado et al., 2013) and
has been extended to estimate locus-specific ancestry
advancements. In particular, we consider the ways in (Falush et al., 2003). Maximum likelihood-based pro-
which genomic data have changed how we think about grams, like ADMIXTURE (Alexander et al., 2009), allow
hybridization in plants and highlight areas that we for less computationally intensive estimates of genetic
believe are especially accessible to genomic study. We ancestry. Model-based methods that infer locus-specific
also recognize that, while genomic data provide pre- ancestry (Falush et al., 2003; Sankararaman et al., 2008;
viously inaccessible insight into the evolutionary his- Paşaniuc et al., 2009; Price et al., 2009) are particularly
tory of plant populations, they are most powerful when useful for detecting hybridization and introgression
66 Plant Physiol. Vol. 173, 2017
 Hybridization in Plants

Several phylogenomic analyses have been developed

to infer introgression in spite of ILS. The ABBA-BABA
test is currently the most widely used and is based on
counts of ancestral (A) and derived (B) alleles in sets of
four samples with known phylogenetic relationships
(i.e. three ingroups and an outgroup). Two allele pat-
terns, ABBA and BABA, are incongruent with the spe-
cies tree BBAA and can be used to infer introgression
(Green et al., 2010). Under ILS, the two patterns should
be equally frequent; therefore, a significant excess of one
pattern over the other (as evaluated with Patterson’s D
statistic) is indicative of introgression (Fig. 1B). These
analyses have been used to successfully detect ancient

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and recent introgression in spite of high levels of ILS
(Pease et al., 2016; Ru et al., 2016).
Another approach to infer reticulate evolutionary
histories is to model phylogenetic networks in which
introgression is represented by nodes connecting

without requiring a priori assignment of samples into

different populations and can be used on taxa without a
reference genome (Vähä and Primmer, 2006; Porras-
Hurtado et al., 2013). For instance, such analyses have
been used to identify crop-wild introgression in chicory
(Cichorium intybus) and maize (Kiær et al., 2009;
Hufford et al., 2013). However, many of these model-
based analyses may have difficulty distinguishing be-
tween different evolutionary histories, as they do not
account for incomplete lineage sorting (ILS) or estimate
the timing of introgression (Falush et al., 2016).
Independent mutations accumulate in the genomes
of reproductively isolated taxa; therefore, the amount
and pattern of genetic differences between species
reveal the relative time of divergence between them.
Phylogenetics-based analyses utilize this property of Figure 1. Differentiating between introgression and ILS. A, Individual
genetic variation to infer hybridization and introgression gene trees may be incongruent with the species tree (outlined in black)
based on gene tree discordance and relative divergence due to either ILS (purple) or introgression (orange). Genetic divergence,
patterns. Specifically, a sequence that is introgressed is as indicated by total branch length, between taxa 2 and 3 is predicted to
expected to show less divergence than is expected based be shorter under introgression than ILS. B, The ABBA-BABA test is used
on the phylogenetic relationship of two lineages. A to detect an excess of one pattern of discordance relative to the other in
phylogenetic analysis of such loci will be discordant with four taxon phylogenies (three ingroup taxa and an outgroup) by com-
paring counts of allele patterns at polymorphic sites that differ from the
the species tree (Fig. 1A). But introgression is not the only
species tree (outlined in black). If the star symbol represents mutation
phenomenon that can cause discrepancies between gene from ancestral A alleles to derived B alleles, then in this example, in-
trees. The persistence of ancestral polymorphism after congruent ABBA allele patterns are due to either introgression (orange)
the divergence of two species can produce phylogenetic or ILS (purple). BABA allele patterns are due to ILS alone. An equal
signals that differ from the species tree. This phenome- number of incongruent ABBA and BABA allele patterns are expected
non, known as ILS, produces a signal of incongruence under ILS alone; therefore, a significant excess of ABBA allele patterns is
that, in some ways, mimics introgression (Fig. 1A). consistent with a history of introgression.

Plant Physiol. Vol. 173, 2017 67

Goulet et al.

hybridizing species in a phylogenetic tree (Bapteste trajectory of lineages. Although Kölreuter (1766) ob-
et al., 2013; Hahn and Nakhleh, 2016; Mallet et al., served hybrid vigor, he more generally concluded that
2016). These methods have proven particularly useful interspecific hybrids are usually difficult to produce
for inferring the timing, magnitude, and direction of and are frequently sterile. Hybrids are often inviable,
gene flow (Than et al., 2008; Solís-Lemus and Ané, sterile, or exceedingly rare, such that genetic exchange
2016). between species is not possible. Hybridization without
Because recombination breaks apart haplotypes over gene flow has fewer evolutionary consequences and,
time, recent introgression is expected to generate long- therefore, is not addressed here. Instead, we focus pri-
shared haplotype blocks between hybridizing species, a marily on how hybridization with gene flow affects the
pattern that is not predicted under ILS. Therefore, the genetic and phenotypic composition of populations
distribution of haplotype block sizes can be used to infer immediately and over longer evolutionary time scales.
introgression (Pool and Nielsen, 2009; Gravel, 2012; Our discussion starts with phenomena in F1 hybrids
Mailund et al., 2012; Harris and Nielsen, 2013). These (heterosis), continues to population-level processes

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methods are less widely used because they require (transgressive segregation and adaptive introgression),
haplotype data from multiple individuals as well as a and concludes with hybrid speciation and reinforce-
null distribution of expected haplotype sizes, which is ment.
not attainable in many systems.
Although tests to detect hybridization do not require Heterosis
the identification of exchanged genes, similar analyses
have been adapted to detect the targets of introgression It has long been observed that crossing two plant
(Rosenzweig et al., 2016). For instance the f statistic, an species or genotypes can create a hybrid with faster
expansion of Patterson’s D, is used to search for genomic growth rate, more biomass at maturity, and/or greater
regions with increased proportions of shared derived reproductive output than its parents. This counterin-
variants, likely exchanged by recent gene flow (Green et al., tuitive phenomenon is called hybrid vigor or heterosis.
2010; Durand et al., 2011). Methods to detect long-shared Both Kölreuter (1766) and Darwin (1876) described the
haplotypes also have been used to identify genes involved phenomenon of heterosis in their experimental crosses
in adaptive introgression (Pardo-Diaz et al., 2012; Racimo of plants, but neither offered explanations to the un-
et al., 2015; Dannemann et al., 2016). Finally, because derlying mechanism causing the pattern (Mayr, 1986;
introgressed loci will share a more recent common ancestor Chen, 2013). Following Shull’s (1908, 1911) pioneering
than the most recent common ancestor of hybridizing taxa, experiments in maize, determining the genetic mecha-
they should have a lower genetic distance in hybridizing nism causing heterosis became one of the earliest
taxa than nonintrogressed loci (Fig. 1A). problems in the new field of genetics. How does a hy-
Genomic methods have dramatically improved our brid that has an allele from each parent perform so
ability to detect introgression and have expanded the much better than either of the parental sources of the
number of taxa amenable to a detailed study of hybrid- alleles?
ization. However, there are still limits to what we can Early research on heterosis yielded two competing
learn from genomic data. For instance, the timing, di- hypotheses that we are still investigating today: domi-
rection, and magnitude of gene flow define the biological nance (Jones, 1917) and overdominance (East, 1936).
implications of hybridization. Calculating these param- The dominance model posits that recessive deleterious
eters is challenging and has traditionally been conducted alleles accumulated at different loci in each parental
by modeling population divergence using theoretical taxon and that, in F1 hybrids, these deleterious alleles
frameworks such as the isolation with migration model are masked by beneficial alleles from the other parent.
(Nielsen and Wakeley, 2001; Hey and Nielsen, 2004). The overdominance hypothesis posits that, at loci con-
These methods are computationally demanding and tributing to heterosis, the heterozygous genotype is
make controversial evolutionary assumptions (Sousa superior to both homozygous genotypes. Recent ad-
and Hey, 2013; Payseur and Rieseberg, 2016). Models of vances in genetic and genomic methods have allowed
phylogenetic networks (Than et al., 2008; Solís-Lemus for more thorough characterization of the mechanisms
and Ané, 2016; Wen et al., 2016) and the five-taxa ex- causing heterosis and also have implicated epistatic
tension of the ABBA-BABA test (Eaton and Ree, 2013; interactions among alleles at multiple loci, epigenetic
Pease and Hahn, 2015) have made progress toward modifications to the genome, and the activity of small
evaluating the direction and magnitude of introgression, RNAs (Chen, 2013). Despite more than a century of
and future efforts should continue to develop such research, the genetic basis of heterosis remains an open
methods. question. Early work tended to assume a single, com-
mon cause of heterosis (Crow, 1948), but it has become
clear that multiple causal mechanisms contribute to
heterosis (Grant, 1975; Kaeppler, 2012).
EVOLUTIONARY CONSEQUENCES
Quantitative trait locus (QTL) mapping experiments
OF HYBRIDIZATION
have been used to identify and then characterize loci
Identifying a history of hybridization still leaves the contributing to heterotic phenotypes. Such studies are
question of how hybridization affects the evolutionary limited by the density and genomic coverage of genetic
68 Plant Physiol. Vol. 173, 2017
 Hybridization in Plants

markers, so the most convincing genomic characteri- Transgressive Segregation

zations of heterosis come from genetic model systems
including rice (Oryza sativa), maize, cotton (Gossypium Similar to heterosis, transgressive segregation occurs
hirsutum), and Arabidopsis (Arabidopsis thaliana). These when phenotypic trait values in hybrid populations fall
genomic studies paint heterosis as the cumulative result outside the range of parental variation. Transgressive
of many loci that have a mixture of dominant, over- segregation demonstrates how hybridization can pro-
dominant, and epistatic effects (Tang et al., 2010; Zhou duce novel phenotypes and thus enable adaptation to
et al., 2012; Shen et al., 2014; Shang et al., 2015). There is new ecological niches, found new lineages, and play a
one notable exception to this pattern, a single locus significant creative role in evolution. Transgressive
controlling heterosis for yield in tomato (Solanum lyco- segregation is distinct from heterosis because it mani-
persicum). Krieger et al. (2010) show that tomato plants fests predominantly in the F2 generation and later and
heterozygous for a wild-type and a nonfunctional allele may persist indefinitely once established (Rieseberg
at SINGLE FLOWER TRUSS have significantly greater et al., 1999). This difference suggests possible distinct

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yield than either homozygote genotype. Thus, heterosis genetic mechanisms for the two phenomena.
for yield is driven by overdominance at a single locus. Transgressive segregation is common in hybrid plant
Recent genetic and genomic studies also have populations. Rieseberg et al. (1999) found that 97%
revealed that interactions between divergent epigenetic (110 of 113) of studies reporting parental and hybrid
regulatory systems contribute to heterosis in F1 hybrids trait values include at least one transgressive trait.
(Groszmann et al., 2013; Greaves et al., 2015). In Ara- Stebbins (1950) cites early observations of transgressive
bidopsis, Wang et al. (2015) demonstrate that F1 hy- segregation by Lotsy (1916) and Hagedoorn and
brids show gene expression levels outside of the Hagedoorn-Vorstheuvel La Brand (1921) and notes the
parental range for defense, abiotic stress, and hormone potential of transgressive traits to allow adaptation to a
response pathways, due in part to epigenetic regula- new ecological niche; however, he does not offer hy-
tion. In many cases, these pathways are down- potheses regarding the genetic mechanism underlying
regulated, consistent with the idea that there are this phenomenon. In the 1970s, it was assumed that the
tradeoffs between growth and defense or abiotic stress genetic mechanism was understood (Grant, 1975), and
response. There is also emerging evidence from Ara- yet now we realize that, like heterosis, there are multi-
bidopsis and rice that small RNAs, including micro- ple possible causes of transgressive segregation re-
RNAs and small interfering RNAs, may be involved in quiring continued investigation.
heterosis, as F1 hybrids often show small RNA ex- While a number of hypotheses have been proposed
pression levels outside of the parental range (Ng et al., (Rieseberg et al., 1999), the best-supported genetic
2012). Compellingly, Shen et al. (2012) were able to mechanisms causing transgressive segregation are
eliminate heterosis in F1 hybrids of Arabidopsis by complementary gene action and epistasis (Rieseberg
treatment with a DNA demethylating agent and by et al., 1999; Dittrich-Reed and Fitzpatrick, 2013). The
introducing a mutation that compromises gene regu- complementary gene action model requires that both
lation by small RNAs. parents harbor additive alleles of opposing sign at dif-
While the fundamental task of explaining the genetic ferent loci affecting a multilocus trait (some + and some 2),
basis of heterosis has persisted for over 100 years, recent which then sort in favor of one direction in the segregat-
genomic studies have made progress toward its solu-
ing hybrids. For example, a late-generation hybrid may
tion. In most cases, multiple genetic mechanisms, in-
acquire + alleles for a trait from both parents across dif-
cluding dominance, overdominance, epistasis, and
ferent loci (Fig. 2). Grant (1975) called this an oppositional
epigenetics, act simultaneously in F1 hybrids to pro-
duce heterotic phenotypes. The implication of multiple multiple gene system and credits Nilsson-Ehle (1911)
mechanisms and many loci is consistent with the find- with one of the earliest explicit proposals of the phe-
ing that levels of heterosis for different traits are not nomenon in wheat (Triticum aestivum). The epistasis
strongly correlated, suggesting that the basis of heter- model predicts that nonadditive interactions between loci
osis is largely trait specific (Flint-Garcia et al., 2009). from different parents can cause extreme trait values in
Future studies should follow-up genome-wide surveys hybrids. Recent advancements in genomic analyses have
with molecular studies of individual loci to confirm that suggested additional mechanisms underlying transgres-
heterosis is the result of multiple genetic models acting sive segregation, including a role for small interfering
in concert and to further our mechanistic understand- RNAs (Shivaprasad et al., 2012).
ing of how these different genetic models cause heter- Many QTL studies of transgressive traits find sup-
otic trait values. Although genetic and genomic studies port for complementary gene action, epistasis, or both
have the potential to improve our understanding of (deVicente and Tanksley, 1993; Hagiwara et al., 2006;
heterosis, it is also important to continue to assess the Mao et al., 2011). For an example in Helianthus spp., see
relative importance of heterosis in natural plant sys- Box 2. Interestingly, oppositional QTLs are more com-
tems. Phenotypic assessments of hybrid vigor versus mon for morphological traits than physiological traits
hybrid sterility and inviability in natural and controlled (Rieseberg et al., 2003b), suggesting that physiological
environments are key to determining the contribution traits are less likely to be transgressive in hybrids. It is
of heterosis in plant evolution more generally. unclear why this would be the case, but it may reflect
Plant Physiol. Vol. 173, 2017 69
Goulet et al.

and small RNA activity also may be important con-

tributors. Future work should continue to investigate
the genetic and molecular basis of transgressive segre-
gation, particularly in wild populations. Additionally,
experiments in the field demonstrating that transgres-
sive trait values facilitate the adaptation of a hybrid
lineage to a new ecological niche will bolster the case for
a creative role of hybridization in evolution and speci-
Figure 2. Complementary gene action causes transgressive segrega- ation. Finally, more research is needed on the factors
tion. Complementary gene action occurs when additive alleles for a determining the likelihood of transgressive segrega-
multilocus trait act in opposition to one another in both parent lineages
tion. Are there patterns in dominance effects of loci
but sort in favor of one direction of effect in segregating hybrids. Indi-
vidual loci contributing to a trait are indicated along a chromosome
underlying transgressive segregation? What is the his-
with their additive contribution to the trait value. The total trait value for tory of selection on loci implicated in transgressive

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each genotype is indicated by the boxed number. One possible hybrid traits, and have these histories potentiated transgres-
genotype is depicted that has acquired all + alleles and, therefore, has a sive segregation in hybrids?
transgressive trait value.

Adaptive Introgression
weaker selection on morphological traits that is more
permissive to the accumulation of antagonistic alleles. The production of hybrid offspring generates the
Can we predict the plausibility of transgressive seg- potential for gene flow between parent populations.
regation between two populations? It has been If hybrids are fertile, they may backcross with either
hypothesized that genetic distance is positively corre- or both of the parents, resulting in introgression.
lated with the frequency of transgressive segregation Excessive gene flow can lead to genetic swamping
(Rieseberg et al., 1999). Stelkens and Seehausen (2009) and the extinction of rare taxa (Levin et al., 1996;
found significant evidence in favor of a positive corre- Todesco et al., 2016); however, introgression also may
lation in eudicots; however, this correlation disappears serve as an evolutionarily creative force by intro-
when monocots are included in the analysis. Also, ducing new, possibly adaptive, genetic variation
Rieseberg et al. (1999) found transgressive segregation into a population. The idea that introgression can
to be significantly more common in intraspecific crosses move adaptive variation between populations is
than in interspecific crosses. One possible explanation first credited to the botanist Johannes Lotsy (1916;
for the somewhat ambiguous support for this hypoth- Stebbins, 1959), but the field’s most influential early
esis is that the accumulation of fixed differences caus- thinker was Edgar Anderson, who coined the
ing transgressive segregation is masked by the term “introgressive hybridization” (Anderson and
simultaneous accumulation of detrimental genetic in- Hubricht, 1938). Anderson (1948) emphasized how
compatibilities (Dittrich-Reed and Fitzpatrick, 2013). open ecological niches that recombined aspects of
Additionally, assuming that complementary gene ac- parental habitats would favor recombinant hybrid
tion is the most common mechanism, the frequency of offspring that could draw from the genetic variation
transgressive segregation should depend not only on present in both parents, an idea he called “hybridi-
divergence but also on the history of selection on traits zation of the habitat.” Furthermore, Anderson and
(Rieseberg et al., 2003b). Drift or stabilizing selection Stebbins (1954) proposed that introgression, unlike
increases the likelihood of fixing antagonistic alleles for spontaneous mutation, could introduce large blocks
a polygenic trait, whereas directional selection will tend of novel variation into a population, potentially
to fix alleles with effects in the same direction. Obser- moving an adaptive trait along with its modifiers to
vations of transgressive segregation for agriculturally allow rapid differentiation into a new ecological niche.
important traits in domestic plants (Hagiwara et al., Although a number of putative examples of adaptive
2006; Mao et al., 2011) further complicate this hypoth- introgression were proposed in the genera Tradescantia,
esis, because crops tend to be under strong directional Melandrium, and Helianthus (Anderson, 1949; Stebbins,
selection yet demonstrate extensive transgressive seg- 1950), empirical study of adaptive introgression was
regation. Finally, hybrids between inbred plants are limited by the difficulty of identifying introgressed loci
much more likely to show transgressive trait values underlying adaptations.
than hybrids between outbred populations (Rieseberg In order to demonstrate adaptive introgression, it
et al., 1999). Thus, describing the likelihood of trans- must be shown that a variant in one population is de-
gressive segregation is complex and depends on di- rived from gene flow with a second population and that
vergence, history of selection, and breeding system. this variant is adaptive. Demonstrating the latter
Transgressive segregation appears to have multiple involves well-established techniques such as recipro-
underlying genetic causes, which partially overlap with cal transplant experiments and common garden ex-
those found for heterosis. Complementary gene action periments to measure selection on traits. However,
and epistasis have amassed the most empirical support, work on adaptive introgression was historically lim-
and recent work suggests that epigenetic regulation ited to studying adaptive phenotypes thought to be
70 Plant Physiol. Vol. 173, 2017
 Hybridization in Plants

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introgressed without the ability to determine their ge- demonstrated in artificial hybrids of rice (Yang et al.,
netic basis. Genomic analyses can now demonstrate 2011) and sunflower (Helianthus annuus; Snow et al.,
that alleles contributing to an adaptive phenotype in 2003). Meanwhile, Hufford et al. (2013) found evidence
hybrids are in fact introgressed. However, the likeli- for adaptive introgression from wild teosinte (Zea spp.)
hood of determining the genetic basis and evolutionary into maize crops using a combination of genomic
history of an adaptive trait will depend on its genetic analyses and growth chamber experiments. Adaptive
architecture: adaptive introgressed traits with simple introgression also has been studied extensively in nat-
genetic architectures will be easier to detect than traits ural hybrid zones or swarms (Grant, 1981). Early re-
controlled by many loci. searchers observed that hybrid swarms were more
Much attention has been paid to the exchange of common in areas subject to human disturbance, high-
potentially adaptive variation between crop plants and lighting the importance of habitat in the formation and
their wild relatives (Kwit et al., 2011; Ellstrand et al., persistence of hybrids (Wiegand, 1935; Anderson,
2013; Warschefsky et al., 2014). For example, adaptive 1949). Indeed, multiple models proposed to explain the
introgression of transgenes conferring herbivore resis- persistence of stable hybrid zones invoke extrinsic se-
tance from crop plants to wild relatives has been lection for hybrid genotypes (Buerkle et al., 2003). Thus,

Plant Physiol. Vol. 173, 2017 71

Goulet et al.

many have advocated the use of natural hybrid zones and recent advancements due to innovative genomic
for studying the process of adaptive introgression analyses.
(Levin, 1979; Rieseberg and Carney, 1998). For a brief The notion that costly hybridization could favor in-
description of research done on adaptive introgression creased isolation is attributed to Alfred R. Wallace
in the genus Helianthus system, see Box 2. (1889), and the process used to be referred to as the
The advancements in genomic sequencing have en- Wallace effect (Grant, 1966). During the modern syn-
abled easier identification of introgressed loci and the thesis, Dobzhansky (1940) first clearly articulated how
detection of genomic signatures of selection in natural greater assortative mating could be favored by selection
hybridizing populations. For example, genome scans to decrease hybridization and maintain coadapted
for selection in Populus trichocarpa identified a number gene complexes. Although much of the formative re-
of candidate loci under strong selection (Geraldes et al., search on reinforcement was done in animal systems
2014). Further genomic analyses to detect introgressed (Dobzhansky and Koller, 1938; Blair, 1955; Dobzhansky
loci between P. trichocarpa and Populus balsamifera et al., 1964; Littlejohn and Loftus-Hills, 1968), there is a

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found that at least one of the genomic regions with a long history of botanical research on reinforcement as
history of strong selection also was introgressed from well (Grant, 1966; Levin and Kerster, 1967; McNeilly
P. balsamifera (Suarez-Gonzalez et al., 2016). Phenotypic and Antonovics, 1968; Paterniani, 1969; Whalen, 1978).
data for the sequenced individuals showed that P. tri- Reinforcement begins with mating between closely
chocarpa individuals with a P. balsamifera haplotype at related taxa. This hybridization is costly due to low
the region of interest had higher chlorophyll and leaf hybrid viability or fertility. Costly hybridization gen-
nitrogen contents than those with native P. trichocarpa erates selection favoring new traits that increase
haplotypes (Suarez-Gonzalez et al., 2016). Together, assortative mating. These novel trait values are selected
these genomic analyses suggest adaptive introgression. in sympatric populations because they decrease
Until recently, researchers interested in adaptive in- hybridization, but they are not necessarily favored in
trogression have been limited to investigations of phe- allopatry, thus generating a pattern of character dis-
notype, without knowledge of the underlying genetic placement (Howard, 1993; Servedio and Noor, 2003).
variation. The advent of molecular genetic and genomic The feasibility of reinforcement is controversial, be-
tools has allowed for the identification of introgressed cause gene flow between hybridizing taxa can prevent
loci associated with adaptive traits. Applications of the evolution of reproductive isolation in sympatry
genomic data, such as the examples highlighted above, (Felsenstein, 1981; Butlin, 1987). Thus, hybridization is
can be used to test proposed cases of adaptive intro- both the source of reinforcing selection and a major
gression and identify new cases, enabling an empirical hindrance to the success of reinforcement (Kirkpatrick,
assessment of the idea of Anderson and Stebbins 2000). In order for new alleles conferring assortative
(1954) that introgression is an important mechanism of mating to evolve, they must remain genetically associ-
adaptive evolution. At this time, the most convincing ated with alleles causing reduced hybrid viability or
evidence that an introgressed trait is adaptive in wild fertility (for review, see Servedio, 2009). Extensive the-
populations will still come from common garden oretical research has illustrated that the feasibility
and reciprocal transplant experiments. However, ge- of reinforcement is determined by a balance between
nomic data complement these classic experiments with the evolutionary forces of selection, gene flow, and
more extensive characterization of introgressed genetic recombination (Liou and Price, 1994; Servedio and
variation and its phenotypic effects. Future studies Kirkpatrick, 1997; Kirkpatrick and Servedio, 1999;
should address questions such as the following. How Kirkpatrick, 2000).
many traits are typically affected in cases of adaptive In light of this controversy, much of the empirical
introgression, and how is the genetic architecture of research on reinforcement has focused on demonstrat-
those traits distributed across the genome? And what ing that the process occurs. Only recently has research
are the frequency and importance of genotype-by- expanded to explore how and why reinforcement can
environment interactions in modulating the effects of occur. What mutations cause reinforcement? How
introgressed alleles? strong is reinforcing selection? How much gene flow
occurs during the process of reinforcement? Advance-
ments in genetic and genomic tools have revolutionized
Reinforcement our ability to address these questions. We are only just
starting to understand the genetic basis of reinforce-
Hybridization between diverged lineages often is ment (Ortíz-Barrientos and Noor, 2005; Saether et al.,
not adaptive. Hybrids may be inviable, sterile, or 2007), and flower color variation in Phlox spp. is the
maladapted, and this cost to hybridization can generate only case of reinforcement for which the causal genes
selection to decrease mating between diverged line- have been identified (Hopkins and Rausher, 2011). Yet,
ages. The process of increased reproductive isolation comparing genome scans for divergence and selection
due to selection to decrease hybridization is called between allopatric and sympatric populations may
reinforcement. Reinforcement in plants has been suggest new candidate genes underlying reinforcement
reviewed elsewhere (Hopkins, 2013), but we note here (Smadja et al., 2015). Once causal genes have been
its historical importance in the study of hybridization identified, future population genomic analyses can be
72 Plant Physiol. Vol. 173, 2017
 Hybridization in Plants

used to infer when and how the mutations evolved directly accountable for establishing reproductive iso-
(Barbash et al., 2004; Presgraves and Stephan, 2007; lation (Schumer et al., 2014). While homoploid hybrid
Tang and Presgraves, 2009; Sweigart and Flagel, 2015). speciation often is invoked upon the demonstration of
Understanding the balance between selection and hybrid origin and a distinct ecological niche, there are
gene flow during reinforcement requires a complement very few examples that draw a compelling mechanistic
of genomic and experimental inquires. In Phlox spp., we link between hybridization and the establishment of a
are beginning to understand the strength of these evo- new, isolated species (Schumer et al., 2014; Yakimowski
lutionary forces. Phlox drummondii evolved divergent and Rieseberg, 2014). Furthermore, there is evidence
flower color in sympatry with the closely related Phlox that genetic divergence affects the relative likelihood of
cuspidata due to reinforcement. Common garden field homoploid (more likely between less-diverged species)
experiments demonstrate that flower-color divergence or polyploid (more likely between more-diverged spe-
decreases hybridization by as much as 50% (Hopkins cies) hybrid speciation (Chapman and Burke, 2007).
and Rausher, 2012), and a population genetic model This is consistent with the idea that homologous chro-

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estimates strong selection driving flower-color varia- mosomes will pair with greater fidelity as the genetic
tion in this system (Hopkins et al., 2014). Genomic divergence between progenitors of an allopolyploid
analyses also have begun to reveal the extent of gene increases, leading to higher fertility (Stebbins, 1947).
flow between hybridizing sympatric species that ex- Differences in initial intrinsic reproductive isolation
perienced reinforcement (Kulathinal et al., 2009). More from parental species help explain why these two
analyses investigating the direction, amount, and tim- modes of hybrid speciation differ so dramatically in
ing of gene flow between sympatric species that expe- frequency. The process of genome doubling often pro-
rienced reinforcement are necessary to more fully duces some degree of immediate reproductive isolation
understand how and why reinforcement occurs. from parental lineages by virtue of the inviability or
sterility of interploid progeny. Unlike allopolyploids,
homoploid hybrids do not achieve instant reproductive
Hybrid Speciation isolation from their parental species. However, homo-
ploid hybrids may evolve partial intrinsic reproductive
Linnaeus (1760) first suggested that new species isolation from both parents relatively quickly. Follow-
arose by hybridization in Disquisitio de sexu plantarum, ing the refining of species concepts and Dobzhansky’s
and in so doing, he rejected the notion of immutability. (1940) emphasis on reproductive barriers, Müntzing
Hybridization is widespread, but the generation of a (1930) proposed that homoploid hybrids could become
unique, isolated hybrid lineage is likely very rare. New partially reproductively isolated by sorting and fixing
hybrid lineages must establish reproductive isolation genetic incompatibilities with both parents. Grant
and a unique ecological niche in order to overcome (1958) later described this process as recombinational
genetic swamping and competition from parental spe- speciation. Early work on recombinational speciation
cies. A new hybrid lineage may be formed through al- focused on describing how chromosomal rearrange-
lopolyploidy or through homoploid hybrid speciation. ments sort in hybrids (Fig. 3A; Grant, 1981), and this
Allopolyploid lineages may be formed by the fusion framework has since been applied to the sorting of
of unreduced gametes, genome doubling following genic incompatibilities as well (Fig. 3B; Schumer et al.,
hybridization, or via a triploid bridge (Ramsey and 2015). Recombinational speciation has been empirically
Schemske, 1998). Homoploid hybrid speciation de- confirmed in multiple systems (Grant, 1981), including
scribes the formation of a new, reproductively isolated the well-studied Helianthus hybrid species (Box 2).
hybrid lineage without a change in ploidy. Hybrid speciation is more likely to be successful if
Allopolyploid hybrid speciation is the more common hybrids can escape competition with initially much
and feasible form of hybrid speciation (Soltis and Soltis, more numerous parental genotypes through ecological
2009). A recent review found that 11% of species across differentiation (Buerkle et al., 2000). As early as 1894,
47 plant genera were likely of allopolyploid origin Anton Kerner emphasized the importance of hybrids
(Barker et al., 2016). Meanwhile, only a handful of ex- colonizing open habitat, unoccupied by parent popu-
amples of homoploid hybrid speciation have been lations, for their persistence (Grant, 1981). Early sup-
identified in animals (Mavárez et al., 2006; Lukhtanov port for the necessity of open habitat came from
et al., 2015), fungi (Leducq et al., 2016), and plants observing a high frequency of allopolyploid species in
(Rieseberg et al., 2003a). In agreement with the paucity regions subject to glacial disturbance (Anderson, 1953).
of well-supported examples, simulations suggest that It has since been hypothesized that the rapid evolution
hybridization more likely results in stable hybrid zones of genome structure and gene expression following
or the extinction of a parental species than homoploid polyploidy can contribute to novel trait expression
hybrid speciation (Buerkle et al., 2003). The apparent and ecological differentiation (Soltis and Soltis, 2009;
difference in frequency also may reflect a bias in the Madlung, 2013). Synthetic allopolyploids have been
likelihood of detecting polyploid versus homoploid created to verify the feasibility of this hypothesis (Gaeta
hybrid species. Verifying a case of homoploid hybrid et al., 2007). It has been demonstrated that, in some
speciation requires both demonstrating the hybrid ori- cases, allopolyploids can survive in a broader range of
gin of the lineage and showing that the hybridization is environments than their progenitors. This can be due to
Plant Physiol. Vol. 173, 2017 73
Goulet et al.

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Figure 3. Recombinational speciation contributes to homoploid hybrid speciation. Recombinational speciation involves the sorting of intrinsic in-
compatibilities between parents in a hybrid lineage. If some incompatibilities are resolved in favor of one parent and others are resolved in favor of the
other, the hybrid lineage will have some degree of intrinsic incompatibility with each parent not exceeding the level of incompatibility between the
parents. Incompatible alleles are connected by a dashed red line and an X. A, Chromosomal rearrangements such as inversions present in parent taxa
may be differentially fixed in hybrid populations conferring partial incompatibility with both parent lineages. B, Similarly, recombinational speciation
may involve the differential fixation of genic incompatibilities.

greater gene regulatory flexibility as a result of homolog- few thoroughly explored examples of homoploid hy-
specific gene regulation (Dong and Adams, 2011; brid speciation in plants, further investigations of pu-
Combes et al., 2012) or alternative splicing (Zhou tative cases that focus on demonstrating a role for
et al., 2011) in response to environmental perturbation. hybridization in establishing reproductive isolation
However, there remains a need for more empirical will elucidate which details of hybrid speciation are
evidence demonstrating ecological differentiation facili- general patterns and which are exceptional. For in-
tated by allopolyploidy (Abbott et al., 2013; Madlung, stance, what is the relative importance of intrinsic in-
2013; Soltis et al., 2014). compatibilities versus ecological differentiation in
Homoploid hybrid species are thought to adapt to establishing isolation from parent species? What is the
unique habitats primarily through the transgressive level of incompatibility between parental species that
segregation of parental alleles. Stebbins (1950) high- create a hybrid species? And, is it common for a pair of
lighted the potential of transgressive segregation to species to harbor genetic variation that could produce
produce new hybrid traits allowing the colonization of transgressive hybrid phenotypes facilitating ecological
a novel, rather than intermediate, ecological niche. The divergence?
Helianthus hybrid species provide the most detailed
analysis of ecological differentiation contributing to
homoploid hybrid speciation (Box 2).
CONCLUSION
The most recent advance in the study of hybridiza-
tion is the use of genetic and genomic data to detect As plant evolutionary biologists, we should not be
signatures of hybridization in wild populations. Ge- surprised by the growing realization that hybridiza-
nomic tools are particularly useful for identifying hy- tion occurs across the tree of life. Botanists have been
brid species because hybrid species are predicted to studying the existence and evolutionary conse-
have transgressive phenotypes, thus making pheno- quences of hybridization since the birth of taxonomy,
typic intermediacy an inadequate criterion for identi- through the modern synthesis, and into the genomic
fying hybrid lineages, and homoploid hybrids are not era. We now have access to a torrent of genomic data
necessarily expected to maintain equal proportions of unimaginable by earlier researchers in this field,
both parental genomes. However, the application of which is revealing that many genomes are mosaics of
genomic data to the identification of homoploid hybrid fragments with different ancestry, some of which are
species is currently plagued by the difficulty of dis- more reticent to gene flow than others (Nosil et al.,
tinguishing between introgression that occurred after 2009; Mallet et al., 2016; but see Cruickshank and
speciation and hybridization that was directly involved Hahn, 2014). As we begin to use these data to address
in the speciation process (Schumer et al., 2013). With so outstanding questions in evolutionary biology, we
74 Plant Physiol. Vol. 173, 2017
 Hybridization in Plants

mechanisms underlying phenomena such as hetero-

sis and transgressive segregation; we can better
understand how new combinations of alleles from pa-
rental taxa interact with novel environments to al-
low the persistence of hybrid lineages; and we can
infer the history of gene flow and selection across
specific genomic regions simply by looking at pat-
terns of genetic variation. In addition to testing
old hypotheses about how hybridization generates
novel phenotypes and lineages, we are generating
new hypotheses based on phenomena, such as small
RNAs and epigenetics, that have been discovered
only recently. Many of the outstanding questions

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about hybridization (see Outstanding Questions) are
the same questions that plagued the founders of
genetics, but our new tools give promise of new
answers.

ACKNOWLEDGMENTS
We thank James Mallet and Heather Briggs for helpful comments, Loren
Rieseberg for assistance with Box 2, as well as Alice Cheung, Richard Amasino,
Cris Kuhlemeier, and Thomas Dresselhaus for the invitation to participate in
this special issue.
Received August 30, 2016; accepted November 24, 2016; published November
28, 2016.

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