Review

Tansley review
The origins of reproductive isolation in plants

Author for correspondence: Eric Baack1, Maria Clara Melo2, Loren H. Rieseberg3,4 and
Daniel Ortiz-Barrientos
Tel: +61 7 3365 1767 Daniel Ortiz-Barrientos2
Email: d.ortizbarrientos@uq.edu.au 1
Department of Biology, Luther College, Decorah, IA 52101, USA; 2School of Biological Sciences, The University of Queensland, St
Received: 9 January 2015 Lucia, QLD 4072, Australia; 3Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; 4Department
Accepted: 16 March 2015
of Biology, Indiana University, Bloomington, IN 47405, USA

Contents

Summary 968 V. The timing of reproductive barriers 979

I. Introduction 968 VI. Synthesis and future directions 980

II. Reproductive barriers in plants 970 Acknowledgements 981

III. Geographic patterns and the origin of reproductive isolation 971 References 981

IV. Genetic changes and evolutionary drivers behind reproductive 973

isolation

New Phytologist (2015) 207: 968–984
doi: 10.1111/nph.13424
Key words: genetic architecture, geography
of speciation, local adaptation, plants,
reproductive isolation, speciation.
Summary
Reproductive isolation in plants occurs through multiple barriers that restrict gene flow between
populations, but their origins remain uncertain. Work in the past decade has shown that
postpollination barriers, such as the failure to form hybrid seeds or sterility of hybrid offspring, are
often less strong than prepollination barriers. Evidence implicates multiple evolutionary forces in
the origins of reproductive barriers, including mutation, stochastic processes and natural
selection. Although adaptation to different environments is a common element of reproductive
isolation, genomic conflicts also play a role, including female meiotic drive. The genetic basis of
some reproductive barriers, particularly flower colour influencing pollinator behaviour, is well
understood in some species, but the genetic changes underlying many other barriers, especially
pollen–stylar interactions, are largely unknown. Postpollination barriers appear to accumulate at
a faster rate in annuals compared with perennials, due in part to chromosomal rearrangements.
Chromosomal changes can be important isolating barriers in themselves but may also reduce the
recombination of genes contributing to isolation. Important questions for the next decade
include identifying the evolutionary forces responsible for chromosomal rearrangements,
determining how often prezygotic barriers arise due to selection against hybrids, and establishing
the relative importance of genomic conflicts in speciation.

morphological gaps, trait evolution within populations does not

I. Introduction
The plant kingdom is unmistakably discontinuous. From the
diverse forms in the Hawaiian silversword alliance (Baldwin &
Sanderson, 1998) to the many eucalypts of Australia (Steane et al.,
2011), gaps in morphology often separate groups of organisms that
we call plant species. Although natural selection can create such
always cause populations to evolve independently from one another
due to gene flow (Mayr, 1942). Evolutionary independence of co-
occurring populations requires the evolution of reproductive
barriers, which in turn can facilitate the accumulation of morpho-
logical differences. Thus, reproductive isolation, whether partial or
complete, is an essential cause of plant biodiversity.

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Ideas about reproductive isolation are old, and were not always
evolutionary. For a time, Buffon (1749) considered that sexual
reproduction within a group, but not between groups, delineated
unchanging species (Gayon, 1996). It was Darwin (1859) who first
saw clearly the changing nature of species, and suggested that
speciation was a continuous evolutionary process where forces
acting during the early stages of the process (e.g. the origin of
varieties) eventually resulted in the formation of distinct groups of
organisms that lack intermediates, or species, adapted to specific
niches (Darwin, 1859; Mallet, 2008). These morphological gaps
between sympatric taxa suggested to Darwin that reproductive
barriers had formed as a byproduct of evolution by natural
selection, but reproductive isolation was peripheral to his central
arguments on speciation. In the decades following Darwin,
biologists examined reproductive isolation in the context of
speciation (e.g. Poulton, 1908; Bateson, 1909; Turesson, 1922),
with Dobzhansky (1937) and Mayr (1942) fully bringing speci-
ation into the modern evolutionary synthesis. Unlike Darwin, they
saw speciation as largely synonymous with the accumulation of
reproductive barriers, ultimately leading to the formation of
irreversible biological species (Coyne & Orr, 2004).
When Stebbins (1950) extended the modern synthesis to plants,
he adopted the biological species concept and largely agreed with
Dobzhansky and Mayr on the relevance of reproductive barriers.
Indeed, his list of barriers (p. 196) will seem familiar to
contemporary students of speciation (Coyne & Orr, 2004;
Rieseberg & Willis, 2007; Lowry et al., 2008a; see Fig. 1). During
the following decades, botanists, like their zoological counterparts,

Strength
Nature of barrier Effect M.lewisii ×
M.cardinalis
Seeds, seedlings, or adults
Niche differentiation
(ecogeographic 0.587
populations can be isolated in
isolation)
space as a result.
prepollination

Adults of two species have

nonidentical timing of
Phenological isolation na
reproduction

Adults of two species differ in

Pollinator
decreased pollinator
specialization
movement between the two 0.950
species.

reduced probability of reaching

Pollen-stigma
interactions & pollen pollen. This may lead to 0.958
competition reduction or absence of hybrid
seed
postpollination

Hybrid ovules are less likely to

mature than ovules formed by
Hybrid seed formation 0.405

Hybrid seeds are less likely to

germinate and survive than
F1 viability 0.203; –1.392
parental seeds

Hybrid plants have reduced

pollen and / or ovule fertility
F1 fertility 0.662
compared parental plants

Later generation hybrids have

F2 fertility & viability reduced viability or fertility na
compared to parental plants

Fig. 1 Barriers contributing to reproductive isolation in plants. Barriers are listed in the order in which they act. Barriers shown without shading are extrinsic and
require genetic differences in the two species, as well as environmental differences. Barriers shown with dark grey shading are intrinsic and act whether or not
environmental differences are present. Barriers with light grey shading are sometimes intrinsic but at other times involve environmental differences. The
estimated strength of each individual reproductive barrier in isolating Mimulus lewisii (bottom) from M. cardinalis (top) is given (data from Lowry et al.,
2008a). na, not available. Photos courtesy of J. Sobel.

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970 Review Tansley review
regularly tested for crossing barriers in glasshouse studies and
searched for chromosomal differences that might contribute to
them. However, studies of reproductive isolation in plants often
emphasized different aspects than those in zoology. The immobil-
ity of plants and environmental patchiness made ecotypes –
populations within species adapted to different habitats – an
important aspect of studies of plant speciation (Turesson, 1922;
Clausen et al., 1940; Lowry, 2012). Likewise, the early recognition
of chromosome doubling in plants (Winge, 1917) and its ubiquity
led generations of botanists to examine the role of polyploidy in
plant speciation (Stebbins, 1950; Grant, 1981). For much of the
past century, botanists were also more willing to consider a creative
role for hybridization in speciation (Anderson, 1949; Arnold &
Hodges, 1995) than their zoological counterparts.
The origins of reproductive isolation remain a central feature of
modern speciation studies in plants. A powerful rationale for this
research program lies in the strong but imperfect correspondence
between the strength of reproductive isolation and taxonomic
species in plants: when measured by postpollination reproductive
isolation, nearly 70% of taxonomic species represent reproductively
independent lineages (Rieseberg et al., 2006), a figure similar to that
found in animals. Despite our emphasis in this review on the roles of
gene flow and reproductive isolation in speciation, we recognize that
other evolutionary forces such as common descent, stabilizing and
parallel selection, and genetic constraints contribute to species
cohesion. However, selection acts most strongly on individuals, and
common descent and genetic constraints contribute to lineage
cohesion at all taxonomic levels. By contrast, gene flow and
reproductive isolation are population- and species-level phenomena
and are therefore justifiably the focus of most studies of speciation.
Here, we review the origins of reproductive isolation in plants,
considering evidence from intraspecific processes such as local
adaptation and partial reproductive incompatibility between
populations, as well as data on reproductive barriers between
well-established species. Our review is not intended to be
comprehensive; we focus on results from recent case studies, as
well as topics that have not been thoroughly reviewed elsewhere.
II. Reproductive barriers in plants
Reproductive barriers are typically classified according to when
they occur and/or whether their expression is dependent on the
environment (Fig. 1). We find the sole use of a ‘prezygotic vs
postzygotic’ classification of reproductive barriers commonly
employed by zoologists to be inadequate for plants because one
of the most commonly reported reproductive barriers, reduced
hybrid seed set, can have pre- or postzygotic mechanisms (or both).
Thus, for the sake of precision, we also employ the terms pre- and
postpollination barriers.
Only recently have botanists quantified most reproductive
isolating barriers in a variety of plant systems using a standard set of
criteria (Ramsey et al., 2003; Fig. 1). Briefly, the contribution of a
barrier to reproductive isolation (RI) is the reduction in gene flow
due to the barrier for interspecific crosses compared with intraspe-
cific crosses. For example, if pollinators were 30% less likely to visit
flowers of a different colour than of the same colour, then the
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strength of this barrier’s contribution to RI would be 0.3. If a single
barrier resulted in a complete block to gene flow, its strength would
be 1.0 (Coyne & Orr, 2004; Sobel & Chen, 2014). Not
surprisingly, the number of plant taxa where all components of
reproductive isolation (Fig. 1; Table 1) have been measured is small,
albeit growing. When Lowry et al. (2008a) reviewed reproductive
barriers, they found no species with information on all of the
relevant barriers, with ecogeographic barriers estimated for just two
of 19 case studies (see later). Also, certain genera were overrepre-
sented. Of the 19 cases reviewed, 10 were from Iris, Mimulus and
Helianthus. Six years later, we surveyed 124 studies that cite Lowry
et al. (2008a) and found 13 additional cases, representing 11 new
genera, where isolation strength had been quantified for a
minimum of three reproductive barriers (Table 1). Two of these
studies calculated the strength of ecogeographic isolation.
Data collected so far demonstrate that plants vary in patterns of
RI, although the total number of taxa studied remains small. Lowry
et al. (2008a) reported that prepollination barriers were often very
strong in plants and contributed more to total reproductive isolation
than postpollination barriers, and studies published in the last 6 yr
support this conclusion. For example, two taxa of closely related
deceptive Chilloglottis orchids in Australia were strongly isolated by
pollinator behaviour (RI > 0.8), likely due to differences in floral
scent cues (Whitehead & Peakall, 2014), whereas postpollination
barriers were very weak or negative. However, in some cases
postpollination barriers were strong as well: pollen competition led
to strong prezygotic barriers between Trillium camschatcense and
T. tschonoskii (RI > 0.95), and all F1 hybrids with T. tschonoskii as a
maternal parent failed to reach maturity (Ishizaki et al., 2013).
Although most studied barriers occur between species, crosses
between populations have been shown to result in male sterility in
several cases (Vickery, 1978; Martin & Willis, 2010; Scopece et al.,
2010; Pinheiro et al., 2013; Twyford et al., 2014). It would appear
that polymorphic barriers within species can be identical to the
barriers observed between species (e.g. hybrid necrosis and male
sterility), thus raising the possibility that the same evolutionary forces
are at work in the origins of barriers within and between species.
1. Associations between barriers: local adaptation and
extrinsic hybrid viability
When parents are adapted to different environments, hybrids
usually express intermediate trait values and so have reduced fitness
in the parental habitats (Rice & Hostert, 1993; Rundle &
Whitlock, 2001). This should be most apparent in cases with
greater levels of differentiation between habitats. Pure migrants
would have the lowest probability of establishing in the alternative
habitat, leading to an expected correlation between immigrant
inviability and extrinsic hybrid inviability. However, hybrid vigour
due to complementation of common deleterious alleles in each of
the two populations may disguise this pattern (e.g. Lowry et al.,
2008b). In seven reciprocal transplant experiments where both
parental and hybrid fitness were quantified, two showed weak
extrinsic reproductive isolation (Iris), one showed increased fitness
in hybrids (Mimulus), and four (Senecio, Artemesia, Oenathe and
Ipomopsis) showed strong immigrant inviability and extrinsic
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Table 1 Recent systems for which three or more reproductive isolating barriers have been estimated

Species Barriers estimated Patterns Citation

Antirrhinum valentinum
and A. controversum
Castilleja miniata,
C. rhexiifolia, C. sulphurea
Ceanothus roderickii and
C. cuneatus
Centaurium littorale and
C. erythraea
Chiloglottis valida and
C. aff. Jeanesii
Clarkia xantiana subspecies
Costus allenii and
C. villosissimus
Orchis mascula and
O. pauciflora
Orchis italica and
O. papilionacea
Petunia integrifolia and
P. axillaris
Quercus robur, Q. petraea,
Q. pubescens, Q. pyrenaica
Senecio lautus ecotypes
(dune, headland)
Trillium camschatcense and
T. tschonoskii
Phenologic, pollinator, pollen–stigma, F1
viability, F1 pollen fertility, F2 seed viability
Pollinator, F1 fruit and seed, F1 viability
Phenologic, pollinator, pollen–stigma, F1
viability
Phenologic, pollinator movement, pollen
competitition, F1 viability, F1 fertility
Pollinator, postpollination fruit set, F1
viability
Ecogeographic, phenologic, pollinator
behaviour, pollen–stigma, F1 viability, F1
fertility
Pollinator, floral mechanical, pollen
competition
‘Prepollination’, Pollen–stigma, pollen
competition, F1 viability, F1 pollinator
attraction
Phenological, pollinator behaviour,
pollen–stigma, fruit set
Ecogeographic, pollinator, pollen–stigma,
pollen competition, F1 viability, F1
fecundity
Pollen–stigma, pollen competition, hybrid
seed production, F1 viability
Phenological, immigrant inviability, hybrid
seed set, F1 viability, F1 fecundity
Phenological, selfing rates, pollen
competition, hybrid viability
Very strong RI due to pollinators
& G€
Carrio uemes (2014)
Varied: strong RI due to pollinator in one pair, Hersh-Green (2012)
strong F1 viability in another
Weak prezygotic RI for those barriers analysed Burge et al. (2013)
(but soil-specific habitat usage); selection
against hybrids on parental soils
Pollen competition was the strongest Brys et al. (2013)
barrier isolating selfing C. littorale from
outcrossing C. erythreae, where hybrid
fitness was the strongest barrier reciprocally
Strong isolation due to pollinator, but Whitehead &
postpollination barriers weak (heterosis). Peakall (2014)
Very strong phenological, pollinator Runquist et al. (2014)
barriers; postzygotic weak but present
Barriers asymmetric, with different barriers Chen (2013)
important for each species
‘Prepollination’ RI was strongest Scopece et al. (2013)
Pollen tubes failed on other species Pellegrino et al. (2010)
Strong ecogeographic, pollinator, Dell’Olivo et al. (2011)
pollen–stigma. Weak postzygotic RI
Barriers were often asymmetric. Pollen– Abadie et al. (2012);
stigma and pollen competition often Lepais et al. (2013)
stronger than postzygotic barriers
Moderately strong prezygotic barriers Melo et al. (2014)
(immigrant inviability, phenological);
postzygotic selection against F1s in field.
No intrinsic postzygotic RI
Pollen competition was a strong prezygotic Ishizaki et al. (2013)
barrier; postzygotic barriers included
complete hybrid inviability in one
direction, complete hybrid sterility in other

RI, reproductive isolation.

postzygotic isolation. However, immigrant inviability was often
stronger than extrinsic hybrid inviability (Table 2).
The origins of reproductive isolation must be approached from
multiple angles, including mapping the geographic context and
tracing the underlying evolutionary forces. Doing the latter will
often require knowledge of the genetic basis of the reproductive
barriers. In addition, we must understand the order in which
reproductive barriers arise, and the tempo of their evolution.
Although previous generations often examined these different
aspects individually, studying either ecological processes of prep-
ollination isolation or the genetic basis of postpollination barriers,
here we mirror contemporary approaches to studying speciation
that increasingly combine the two.
III. Geographic patterns and the origin of reproductive
isolation
The geography of the speciation process affects the degree of gene
flow that occurs between the diverging populations, the scale and
pattern of local adaptation, and the nature of the selective forces at
work. In particular, gene flow reduces differentiation between
populations, and is thought to make the process of speciation less
likely (Bank et al., 2012; Servedio et al., 2013; but see Abbott et al.,
2013). The effect of distance on gene flow depends on the mode of
pollen and seed dispersal: bird or wind-dispersed species require
much greater geographic distances to achieve the same isolation
that 100 m achieves for fly-pollinated species. Estimates of gene
flow in plants suggest that even widely distributed species
experience moderate gene flow (the average of effective population
size times the migration rate, Nem = 1.82  0.28; Morjan &
Rieseberg, 2004). This suggests that gene flow is often sufficient to
keep nearby populations from differentiating in the presence of
weak natural selection (i.e. when the reduction in fitness of the less-
fit genotype, s, is smaller than the migration rate, m; Morjan &
Rieseberg, 2004; Ellstrand, 2014). Although the increasing
availability of genetic data has made it easier to estimate m, we
still have a relatively few estimates spanning the genome and which
take ancestral polymorphism into account, and our ability to

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Table 2 The strength of extrinsic prezygotic (immigrant inviability in field (Savolainen et al., 2006). At the last glacial maxima, the island
transplants) and extrinsic postzygotic (hybrid inviability in field transplants) and a near neighbour would have covered c. 900 km2, yet up to 11
reproductive isolation. Senecio and Oenanthea barriers were quantified as in
Lowry et al. (2008a)
different plant species originated within this narrow geographic
region (Papadopulos et al., 2011). Most of these events likely
Species Type Prezygotic Postzygotic Reference involved ecological shifts: in Howea palms, the two sister-taxa occur
on different soils, whereas in Metrosideros two sister-taxa have
Artemesia Basin 0.727 0.909 Lowry et al. different (albeit overlapping) altitudinal distributions and differ-
tridentata (2008a,b)
ences in habitat. None of these cases involves genome duplications.
Mountain 0.973 0.636 Inferences regarding geographical patterns of speciation on
continents make use of contemporary species distributions but
Ipomopsis aggregata 1 0.247 Lowry et al. require caution. Ecogeographic isolation, whether in the past or the
(2008a,b)
present, can be defined as the ‘proportion of geographic isolation
tenuituba 0.923 0.308 that results from genetically based ecological differences between
taxa’ (Sobel, 2014; p. 565). It is quantified by estimating the extent
Iris fulva 0.061 0.03 Lowry et al. of shared and unshared suitable habitat, but there are several
(2008a,b)
challenges to doing this. There are questions of spatial scale: if
hexagona 0.083 0.056 plants have ranges that overlap at large geographic scales but not on
fine scales, how should range overlaps be quantified? For example,
Iris fulva 0.119 0.045 Lowry et al. plant species that are specialists on serpentine soils may be
(2008a,b)
surrounded by sister taxa on nonserpentine soils (e.g. Layia
brevicaulis 0.052 0.138 discoidea and L. glandulosa; Anacker & Strauss, 2014). Determin-
ing the appropriate spatial scale depends on knowledge of pollen
Mimulus Coast 0.874 1.8 Lowry et al. movement and seed dispersal, parameters rarely estimated when
guttatus (2008a,b)
studying the evolution of reproductive isolation in natural
Inland 0.999 0.233 populations (but see Lindtke et al., 2014). Different studies have
chosen different spatial scales: in Clarkia, populations separated by
Senecio Dune 0.78 0.37 Melo et al. 1 or 3 km were viewed as isolated (Runquist et al., 2014), whereas
lautus (2014)
in Petunia 20 km set the threshold (Dell’Olivo et al., 2011).
Headland 0.54 0.19 A second challenge concerns incomplete sampling. For a few rare
species, we may have complete knowledge of their occurrence, but
Oenanthe conioides 0.56 0.24 Westberg for most species recorded occurrences represent a small subset of
et al. (2010)
their range. Recent advances in modelling species’ distributions
aquatica 0.66 0.44 hold out the possibility that incomplete sampling can be partially
a resolved through models of species’ ranges using soil and climate
Data estimated from survivorship curves in Westberg et al. (2010).
data (e.g. Grossenbacher et al., 2014; Sobel, 2014). Modelled
ranges can then be compared with each other to determine the
estimate m in the past is weak (Strasburg & Rieseberg, 2011). As a degree of overlap, and so the degree of ecogeographic isolation. The
consequence, geographic patterns between sister taxa are frequently effect of sampling can be estimated by building several models, each
used in concert with genetic data to infer prior gene flow. That is, using a subset of the distribution data, and comparing their
species that have diverged in the presence of gene flow are expected similarity. Finally, there are challenges in making inferences about
to be highly similar genetically except at loci associated with past patterns from contemporary distributions. If species have
reproductive isolation. By contrast, allopatric species should overlapping ranges in the present, this might have arisen postdi-
exhibit less variation in genetic distance across the genome (e.g. vergence: as reproductive isolation evolved, the two species might
Roda et al., 2013). have been geographically isolated, and only later shifted or
expanded their ranges to the present pattern of overlap. Likewise,
some populations might have started to diverge when geograph-
1. Geographic relationships among sister-pairs
ically isolated, but fused once their ranges began to overlap.
How often do sister-species arise in close geographic proximity? Despite these challenges, several recent studies of distributions of
Botanists have accepted that sympatric speciation is likely to be sister taxa are useful in understanding the geography of the origins
common in plants due to characteristics such as self-pollination and of reproductive isolation. In sister-species pairs in the Cape Floristic
polyploidy (Stebbins, 1950; Grant, 1981). However, well-sup- Province, 57% showed partial range overlap, and 10% complete
ported generalizations about the geographic patterns of speciation overlap. Eighty percent showed at least a partial ecological shift in
have been lacking. Several recent studies have made use of well- habitat, which could include differences in fire frequency, pollin-
resolved phylogenies at the genus level to address this question. One ators, flowering time or soils (Van der Niet & Johnson, 2009). A
of the most compelling studies demonstrates that speciation has similar study examining North American species of Mimulus found
occurred within the narrow confines of Lord Howe Island that 80% of sister-species pairs had partially or completely

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overlapping ranges, and nearly all sister-species showed diverged
ecological niches (Grossenbacher et al., 2014). Likewise, a study
conducted on plants within the California Floristic Province, with
Mimulus providing 12 of 71 pairs, found a high degree of range
overlap in sister-species pairs – at least 80% showing at least partial
sympatry. Ninety-three percent showed some ecological shift, and
65% showed a complete shift in at least one ecological trait. Shifts in
climate or soil were most common (Anacker & Strauss, 2014).
Finally, younger sister-species pairs had greater asymmetry in range
sizes than older pairs in both the California Floristic Province
(Anacker & Strauss, 2014) and in Mimulus (Grossenbacher et al.,
2014). Whether this last pattern is due to higher rates of extinction
for species with the smallest ranges or later range expansion is
unclear.
The geographic patterns of speciation noted here suggest that
although geographic isolation might be limited during the
speciation process – divergent species are in close geographic
proximity – some degree of ecological isolation is a common
element. Although the level of gene flow present at any point in the
process remains uncertain, divergence due to differential selection
in the face of gene flow is likely to be a common scenario in the
origins of reproductive isolation in plants.
IV. Genetic changes and evolutionary drivers behind
reproductive isolation
Our understanding of the evolutionary forces that underlie the
origins of reproductive barriers – mutation, natural selection, and
genetic drift – has been greatly enhanced through investigations of
the genetic basis of barriers, particularly for postpollination barriers
(Fig. 1). Therefore, it is appropriate to consider both the
evolutionary drivers of reproductive barriers and their genetic basis
when evaluating the causes of reproductive isolation. As will be
evident below, in some cases (e.g. chromosomal rearrangements)
we have a clearer understanding of the genetic changes involved but
Table 3 Role of natural selection in the origins of reproductive isolating
barriers in plants
Example
Natural selection clearly understood
Adaptation to edaphic Mimulus on high-metal soil (Copperopolis;
conditions Wright et al., 2013); adaptation to
serpentine soils in Collinsia (Moyle et al.,
2012); Senecio in sand dunes and rocky
headlands (Melo et al., 2014)
Adaptation to wind Gilia inland vs coastal (Nagy, 1997; Nagy
& Rice, 1997)
Adaptation to disease Necrosis (Bomblies, 2010)
Reinforcement Phlox (Hopkins & Rausher, 2012)
Role of natural selection not well understood
Genome duplication Chamerion (Husband & Sabara, 2004)
(polyploidy)
Chromosomal translocations Mimulus (Stathos & Fishman, 2014)
Natural selection potentially absent
Gene duplication and Oryza (Ouyang & Zhang, 2013)
differential silencing
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more limited understanding of the evolutionary drivers, whereas in
other cases (e.g. aspects of local adaptation) the reverse holds true
(Table 3). Although our knowledge is preliminary, the evidence so
far suggests that mutation (e.g. polyploidy) and natural selection
might be the main drivers of plant diversity. Before reviewing this
evidence, we will briefly outline the roles of mutation, natural
selection and stochastic processes in speciation.
The mutations responsible for reproductive isolation occur at a
variety of scales, from single nucleotide changes that affect protein
structure or expression, to gene duplications, chromosomal
rearrangements and genome duplication (polyploidy). The con-
tribution of a single mutation to total reproductive isolation
depends on both the time of origin and its absolute effect on
reproductive isolation (Schluter & Nosil, 2011). As such, a
mutation with a very strong effect on reproductive isolation (e.g.
90% infertility) might contribute only a small relative increase in
reproductive isolation if it evolved late during the speciation process
(e.g. increasing total RI from 0.95 to 0.98). Although new species
rarely arise as a consequence of a single mutation, a major exception
is whole genome duplication, where a single macromutational
event can create strong isolation with parental populations almost
instantaneously. Other common mutations with a strong absolute
effect on reproductive isolation are chromosomal rearrangements
that through their effects on proper meiotic segregation can often
reduce fertility in hybrids.
Biologists often consider the role of genetic drift affecting allelic
frequencies in a population, but other stochastic processes can also
affect the identity and order in which mutations arise in a
population (Werth & Windham, 1991; Lynch & Force, 2000;
Schluter, 2009). Even when reproductive isolation is driven by
natural selection, historical contingencies such as differences in the
identity and order in which advantageous mutations arise in
geographically isolated populations can lead to reproductive
isolation – a process known as ‘mutation-order speciation’
(Schluter, 2009). Mutation limitation could thus be a significant
contributor to variation between allopatric populations, making it
a potentially important force during the early stages of the
speciation process.
Genetic drift cannot be ruled out as a factor in speciation and
almost certainly contributes. However, the time to fix alleles via
drift is relatively slow: 4Ne generations for neutral alleles. For many
cases where we have estimates of timescales and population sizes,
drift is simply too slow to explain the origins of reproductive
isolation (Sambatti et al., 2012). Therefore, it is probable that most
reproductive barriers evolve as a consequence of selection. Some
barriers likely originate as a direct product of selection, such as
ecogeographic isolation, immigrant inviability, phenological iso-
lation, pollinator isolation and ecological selection against hybrids.
Alternatively, reproductive barriers may be incidental byproducts
of selection on linked loci or on loci with pleiotropic effects. For
example, phenological isolation could be a byproduct of adaptation
to serpentine soils, or it could arise from an independent set of
genetic changes that were favoured by natural selection. A detailed
genetic analysis of the alleles responsible for barriers should help
distinguish these hypotheses, particularly when traits are correlated
and both seem to contribute to reproductive isolation.
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1. Genetic basis of reproductive isolation Prepollination reproductive isolation: flower colour and pollen–
pistil interactions Our knowledge of the genetic basis of plant–
Studying the genetics of reproductive barriers can shed light on
pollinator interactions is considerably more advanced, particularly
several aspects of their origins, as well as on their effectiveness as
for flower colour. One of the best-studied systems is the sister-
reproductive barriers. For instance, at the level of the genome,
species pair, Mimulus lewisii and M. cardinalis. The two differ in
barriers with a complex genetic architecture will confer greater
floral shape, colouration and pollinator visitation. A quantitative
genomic isolation in the face of ongoing gene flow than will traits
trait locus, yup, explains a great deal of variation in the carotenoid
with simple architectures (Barton & Hewitt, 1985). The genetic
(yellow) pigment concentrations between the two species (Brad-
basis of reproductive isolation can also be useful in testing
shaw & Schemske, 2003). A gene responsible for a large amount of
predictions of speciation theory such as the ‘snowball effect,’ in
the variation in anthocyanin (pink) pigment concentrations, ROI1,
which hybrid incompatibilities are predicted to accumulate at a
has been confirmed as well (Yuan et al., 2013). These two genes
faster than linear rate (Orr, 1995). Information about the identity
alter flower colour and pollinator behaviour, and so contribute to
and function of speciation genes can inform us about the traits and
the single most important reproductive isolating barrier between
evolutionary forces underlying reproductive isolation, particularly
the species (Ramsey et al., 2003). The evolutionary hypothesis in
for postpollination barriers. Finally, knowledge of the overall
this system is that differences in pollinator availability in different
genetic architecture of reproductive isolation allows us to ask
habitats led to divergent floral traits being favoured by natural
questions about the genomic context of genes involved in
selection. Evidence in Mimulus aurantiacus is consistent with this
reproductive isolation, such as how chromosomal differences affect
idea (Streisfeld et al., 2013).
the origins of reproductive isolation and how genetic differences
Although pollen–pistil interactions can provide extremely strong
accumulate over time. In the sections that follow, we examine the
reproductive barriers (Lowry et al., 2008a; Sambatti et al., 2012),
genetic basis and evolutionary drivers for different reproductive
their genetic bases have been studied much less than flower colour.
barriers, moving from prepollination barriers to postpollination
In the genus Solanum, evidence suggests that some of the proteins
barriers.
involved in self-incompatibility also function in reproductive
isolation. By introducing functional self-incompatibility proteins
Prepollination reproductive isolation: local adaptation One of
into self-compatible tomato, it was possible to create a pollen–pistil
the most exciting developments of the past decade has been progress
barrier where one had not previously existed (Tovar-M
endez et al.,
toward understanding the genetic basis of local adaptation. The
2014). It is possible that the same evolutionary events that led to
adaptation of plants to different environments has been understood
mating system shifts may have affected pollen–pistil barriers –
as an important reproductive isolating barrier – although often
although in this case potentially weakening the barrier in the case of
partial – for over 90 yr (Turesson, 1922; Stebbins, 1950; Sobel
the selfing species. In some instances, pollen–pistil interactions
et al., 2009). Local adaptation is likely a complex phenomenon,
create absolute barriers to cross-species pollination (e.g. Pellegrino
potentially involving evolution in response to different soils,
et al., 2010), but in many others reproductive isolation is
temperatures, competitors, pathogens and herbivores. However,
heightened through competition between intra- and interspecific
only one or a few aspects of the genetics of local adaptation have
pollen, a phenomenon known as conspecific pollen precedence
been examined in any given species thus far. In the past decade,
(Fig. 2). The evolutionary forces leading to conspecific pollen
quantitative trait loci increasing fitness in one environment have
precedence are often unclear, and identification of the loci involved
been identified in many taxa including Arabidopsis, Boechera,
may be necessary to elucidate the evolutionary forces.
Helianthus, Mimulus and Trifolium (Lexer et al., 2003; Weinig
One of the few systems in which progress has been made toward
et al., 2003; Gardner & Latta, 2006; Hall et al., 2010; Kooyers &
understanding the genetic basis of conspecific pollen precedence is
Olsen, 2012; Anderson et al., 2013; Des Marais et al., 2013; Oakley
et al., 2014). Traits examined include salt tolerance, cold tolerance,
(a) (b) (c)
flowering time, cyanogenesis and growth. In several cases, exper- Empty seed Mixed
iments tested for trade-offs in different habitats. Intriguingly, Fertilised seed

relatively few trade-offs were identified, suggesting that many of the

alleles underlying local adaptation may be favoured in one
environment and neutral in another (Anderson et al., 2013; Oakley
et al., 2014). The puzzling lack of trade-offs observed so far suggests
that generalist genotypes might be possible, as the alleles leading
high fitness in one environment need not lead to low fitness in other 100 100 0 0 50 50
environments. Numerous genes associated with adaptation in
natural populations have been cloned and characterized in 0 0 50 70 10 5
Arabidopsis or in crop–wild relatives. We expect that field
Fig. 2 Conspecific pollen precedence. (a) Intraspecific and (b) interspecific
experiments testing the fitness effects of alternative alleles (as
fecundity create baseline expectations for the proportion of hybrid seed
opposed to QTL) underlying local adaptation will be increasingly resulting from mixed pollen loads. However, in many cases, fewer hybrid
important (e.g. cbf2 locus underlying free tolerance in Arabidopsis; seeds are formed than expected due to competitive interactions between
Oakley et al., 2014). intra- and interspecific pollen (c).

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Mimulus, where analyses of transmission ratio distortion in progeny
derived from interspecific pollen mixtures implies that at least eight
genomic regions contribute to precedence (Fishman et al., 2008).
The precise loci and evolutionary forces involved remain unknown.
Another study in Mimulus suggests an alternative route to address
these questions. Aagaard et al. (2013) identified pollen proteins
expressed in the pistil by labelling the pollen with 15N, allowing the
pollen to germinate and grow down the pistil, then identifying 15N-
labelled proteins using mass spectrometry. The protein sequences
were then used to identify > 2000 genes, which were in turn screened
for signatures of selection. Several genes were identified with
elevated rates of molecular evolution and signatures of natural
selection, although the selective pressures remain unclear.
Prepollination barriers: Reinforcement – natural selection against
costly hybridization Wallace argued that selection would favour
the evolution of prezygotic barriers when postzygotic barriers
already existed, as individuals wasting fewer gametes would have
increased fitness (Wallace, 1912). This process is referred to as the
‘Wallace effect’ (Grant, 1966) or reinforcement (Dobzhansky,
1940), with the latter term becoming increasingly dominant in
recent years (Servedio & Noor, 2003). Although reinforcement was
initially viewed by Dobzhansky (1940) as a mechanism for
completing speciation, this seemed unlikely because selection for
reinforcement would weaken as hybridization rates decline. Also,
reinforcement alleles would only be favoured in areas of sympatry,
and so would be unlikely to spread across the range of species with
partially allopatric distribution patterns (Servedio & Noor, 2003;
Ortiz-Barrientos et al., 2009). Despite these concerns, theoretical
and experimental evolution studies indicate that reinforcement can
occur as a result of selection against the production of hybrids with
low viability or fertility, or from mating costs associated with
hybridization such as wasted gametes and stigma clogging
(Servedio & Noor, 2003; Ortiz-Barrientos et al., 2009). In
addition, considerable empirical support has been developed for
the presence of reinforcement in animals (Servedio & Noor, 2003),
although evidence for reinforcement in plants is less compelling: of
12 studies listed by Hopkins (2013) only one satisfies her four
criteria. This fully developed case comes from an examination of
Phlox drummondii and P. cuspidata (Hopkins & Rausher, 2011,
2012, 2014). Hybrids between the two species have decreased
fitness due to intrinsic postzygotic incompatibilities. In areas where
the two species are allopatric, both have light blue flowers, whereas
in areas of sympatry, P. drummondii has dark red flowers. Two
unlinked loci involved in the regulation of the anthocyanin
biosynthetic pathway underlie these changes (Hopkins & Rausher,
2011). Experimental arrays decoupling colour from intensity
demonstrated that the dark red flowers decrease the probability of a
pollinator moving pollen between the two species, reducing the rate
of formation of low fitness hybrids (Hopkins & Rausher, 2012).
Although the fitness advantage of the red colour, if any, remains
uncertain, that of the dark colour is clear.
Another example of reinforcement involves a diploid–tetraploid
complex in fireweed (Chamerion). Reinforcement is predicted to
evolve in this situation because there is postzygotic isolation
between diploids and tetraploids, and frequent areas of sympatry or
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parapatry. Experiments using diploid, natural tetraploid and
synthesized tetraploid Chamerion angustifolium reveal a pattern
that is compatible with reinforcement. In mixed-pollen load
experiments, established tetraploid plants are less likely to be
fertilized by pollen from diploid plants than are neotetraploid
plants (Baldwin & Husband, 2011). As with many pollen–pistil
interactions, the genetic basis is uncertain. The Chamerion study
represents an example in which conspecific pollen precedence
appears to have evolved to limit maladaptive hybridization.
Previously, most attention has focused on visible, but potentially
costly, reproductive barriers such as temporal, pollinator and
mating system isolation (Hopkins, 2013). Cryptic barriers such as
conspecific pollen precedence provide a less costly means for
reinforcement to evolve (Lorch & Servedio, 2007) and are worthy
of greater attention in future studies.
Similarities and differences in the genetics and evolutionary
drivers of reproductive isolation The genetics and evolutionary
drivers of prepollination isolation will likely differ from that of
intrinsic postzygotic isolation because the fitness effects of alleles
underlying the former depend mainly on the environment
(G 9 E), whereas those underlying the latter depend largely on
genetic background (G 9 G). This leads to a predicted genetic
architecture in which intrinsic postzygotic barriers derive from
deleterious interactions between two or more loci, whereas loci
underlying prepollination barriers act mainly independently.
These predictions have been largely confirmed in case studies
(reviewed in Rieseberg & Blackman, 2010).
Three distinct hypotheses exist to explain why alleles contrib-
uting to postzygotic RI between populations become common
within a population. The alleles might become common due to
linkage to alleles favoured by natural selection or might be the same
alleles with pleiotropic effects. Or, the alleles leading to postzygotic
RI might fix within a population due to drift. Later we focus on the
genetic evidence supporting these hypotheses in different cases.
Associations between reproductive isolating barriers: local adap-
tation and postzygotic isolation If intrinsic postzygotic isolation
arises as a byproduct of natural selection, then we might also expect
an association between local adaptation and postzygotic isolation.
We tested for this association by drawing on reviews of postzygotic
isolation (Rieseberg et al., 2006), local adaptation (Hereford,
2007) or parallel speciation (Ostevik et al., 2012). In each case
where either local adaptation or intrinsic postzygotic isolation had
been established, we searched the ‘Web of Science’ (http://
webofscience.com/) to locate information on other reproductive
barriers. However, we found only 13 cases with documented local
adaptation where results of crossing studies had been published
(Supporting Information Table S1). Of these, five (38%) showed
evidence of postzygotic incompatibilities. Thus, at this time, the
evidence that local adaptation is a key driver of the development of
other reproductive barriers is weak, but a few cases demonstrate
how it might occur.
In crosses between some varieties of rice, and between some
populations of Arabidopsis thaliana (reviewed in Bomblies, 2010),
the hybrid offspring show low viability. The necrosis is similar to
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that seen following a disease response. This reproductive barrier (a) Adult genotypes (b) Ancestral population
(which exists in low frequency) is therefore hypothesized to be a A A A A A a A A A A A A
byproduct of natural selection that favoured the increase in B B B B B b B B B B B B
frequency of a disease resistance allele in one of the populations. In C C c c C c C C C C C C
that genetic background, no damaging interactions occurred with
other alleles. However, the disease resistance allele interacts Ovules Divergence and gene silencing
negatively with alleles found in the other population, leading to A A A a A Population 1 Population 2
B B B b B
an autoimmune response that damages hybrids (reviewed in A A e e
C c C c c d d Hybridisation B B
Bomblies, 2010). In this case, it appears that one element of local C C f f
Aborted Aborted
adaptation – disease resistance – has a pleiotropic effect on
postzygotic isolation. Outcome of Killer system F1 hybridization ovules
A second case illustrates connections between local adaptation
A A A A A A A e A A
and postzygotic isolation, but where linkage rather than pleiotropy B B B B B B B d d B
is involved. Copper mining in the foothills of California left soils C C C C C C C f f f
with toxic copper concentrations. Although most of the surround- Aborted
ing Mimulus guttatus were unable to survive under these conditions, Fig. 3 Three tightly linked loci form a killer–protector system. (a) Two alleles,
a distinct population evolved that could tolerate copper and which A and B, together form a ‘killer’ system that leads to the abortion of ovules
were reproductively isolated from a Welsh population. Initial without the protector allele C (red band). This will favour chromosomes with
efforts found that the two traits – copper tolerance and reproductive the killer system, which will increase in the ancestral population. (b) After
divergence, parts of the killer–protector system are silenced by mutation in
isolation – were inherited together (Macnair & Christie, 1983).
each of the descendant populations. The loss of the protector allele C in
However, either pleiotropic effects of copper tolerance (where the population 2 has no fitness cost because the killer allele A has also been
same genetic change caused copper tolerance and hybrid lethality) silenced (yellow bands). However, hybrids between the two populations
or tight linkage could have been at work. Recent work (Wright recreate a functional killer system, so ovules without the protector allele are
et al., 2013) separated the two traits, demonstrating that linkage aborted, leading to decreased hybrid fertility (Ouyang & Zhang, 2013).
(< 0.32 cM: fewer than 0.32% of offspring of heterozygotes were
recombinants) rather than pleiotropy was key. In this case it appears subsp. indica), F1s have low female fertility. The genetic basis of
that the copper tolerance allele was linked to an allele leading to this loss of hybrid fertility is complex, involving three linked loci.
hybrid lethality. As M. guttatus moved onto the high copper soils, Two of the loci result in a ‘killer’ phenotype that leads to
natural selection led to an increase in the copper tolerance allele and preferential abortion of ovules lacking a ‘protector’ allele at a third
to the tightly linked incompatibility allele in a process known as locus (Fig. 3). Neither subspecies has a functional ‘killer’ phenotype
hitchhiking. Thus, postzygotic isolation arose as a byproduct of due to deletions in one of the two required loci in each subspecies. If
natural selection favouring copper tolerance due to linkage rather the intact ‘killer’ phenotype evolved with a functional ‘protector’
than pleiotropy. allele, then this linked set of loci could have been favoured by
A third case with apparent links between local adaptation and natural selection in an ancestral population. In heterozygotes,
postzygotic isolation is suggestive, but has yet to see a detailed ovules lacking the ‘killer’ package would have been eliminated,
genetic dissection. Collinsia sparsiflora occurs in the hills of leading to increased frequency of the ‘killer’ in the population. In
California’s coast range, with populations on and off serpentine this scenario, the original machinery of reproductive isolation
soils. In the field, ecotypes are strongly adapted to their home would have evolved before the split between the two subspecies,
environment. Both ecotypes are viable in the glasshouse, but show rising to fixation in the ancestor of the two. Subsequent evolution in
reduced hybrid fertility (Moyle et al., 2012). It is not clear whether the two subspecies silenced different parts of the ‘killer/protector’
this postzygotic isolation originated from the adaptation to package, leading to decreased fertility in hybrids in which some
the two environments, or if it is due to independent genetic ovules will lack the protector allele (Ouyang & Zhang, 2013). This
differences. scenario implicates natural selection favouring a selfish genetic
element (the linked killer–protector package) followed by potential
Postpollination barriers potentially arising from genomic con- nonadaptive differentiation in the two subspecies.
flicts In the past decade, a number of genes leading to postzygotic Cytoplasmic male sterility is a postzygotic reproductive barrier
reproductive isolation have been identified and found to have observed in multiple species (Scopece et al., 2010). In theory, an
evolved due to natural selection. However, in several cases, the initial mutation in a mitochondrial protein leads to pollen sterility
natural selection appears to have come from genomic conflicts and an increase in seed production (Fig. 4). The increased seed set
rather than adaptation to the external environment (Presgraves, can lead to the mutation becoming more common in the
2010). Genomic conflicts can occur when one gene within an population. If a nuclear restorer allele arises, it too can become
individual can obtain a fitness advantage at the expense of other common in the population, eliminating the male sterility.
genes within the same organism. However, some crosses between populations (or species) result in
One example in rice shows how reproductive isolation might hybrids that are male sterile due to a mitochondrial sterility allele
arise as a byproduct of genetic conflict. In many crosses between from one species and a missing restorer allele from the other. As
two subspecies (Oryza sativa subsp. japonica and O. sativa with the ‘killer’ system discussed earlier, the cytoplasmic male

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Pistil
(a) Anther Ovules (b)

Floral
phenotype

Nuclear
genotype

Mitochondrial
genotype S S S S

(d) (c)

r × r r r

S S S

(e) (f)

Fig. 4 Cytoplasmic male sterility (cms). (a) A

mitochondrial mutation leading to male
sterility (shown by loss of anthers) and
increased seed number will spread in a
population (b) but will not fix. A nuclear
mutation that restores male fertility will spread r × r r
to fixation (c). (d) Crosses between
populations may lead to fertile F1s (e) but some S S S S
male sterile F2s (f).

sterility alleles increase in frequency due to natural selection

Postpollination barriers: chromosomal rearrangements Chro-
independent of the external environment.
mosomal rearrangements can contribute to reproductive isolation
through their effects on effects on hybrid fertility, as well as by
Postpollination barriers: differential silencing The preceding
reducing interspecific recombination and gene flow (e.g. Lowry &
examples have examined how postzygotic isolation might arise due
Willis, 2010). The kinds of rearrangements that most commonly
to natural selection (Table 3). Stochastic processes may also be
contribute to isolation in plants are inversions, which alter the order
involved. In one case, a portion of F2s created by crosses between
of loci within a chromosome, as well as translocations that move
two subspecies of rice were found to suffer from pollen infertility.
genes to a different chromosome (Grant, 1975). Large transloca-
Mapping of the genes involved found that one of the two copies at a
tions have the greatest impact on the fertility of hybrids, with 50%
duplicated locus had been reciprocally disrupted in each of the two
of gametes expected to carry deletions or duplications of whole
subspecies (Fig. 5). Pollen grains with loss-of-function alleles at
chromosome arms (or portions of arms). In addition to their effects
both paralogs failed to germinate (Ouyang & Zhang, 2013). This
on fertility, translocations reduce recombination rates near
case is a potential candidate for stochastic processes leading to
centromeres or chromosomal breakpoints due to mechanical
reproductive isolation: it is possible that the mutations leading to
interference with pairing and crossing over. The fertility effects of
gene silencing in each subspecies were neutral, or even if beneficial,
chromosomal inversions are less severe, although for large inver-
that silencing the alternate paralog would have equivalent effects.
sions up to 50% of gametes can be inviable due to recombination
However, various scenarios involving natural selection are also
within the inversion resulting in unbalanced gametes. However,
consistent with the evidence here, including hitchhiking or
inversions have little or no effect on fertility if recombination is
different fitness effects of silencing in each genetic background
reduced. This can occur if the inversion is small, occurs in a low
(Muir & Hahn, 2015).

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(a) Ancestor recombination region of the genome, or if compensatory mech-

AABB (c) Divergence and silencing anisms evolve that reduce recombination directly, such as that seen
AaBB AABb Population 1 Population 2 in Dipterans (Coyne et al., 1993) or deer mice (Hale, 1986).
Population 1 Population 2
aaBB AAbb
Because most recombinant gametes will be inviable, effective
a a A A
recombination rates are expected to be low within inversions (and
AaBb near inversion breakpoints) regardless of whether or not such
Hybrids Ch 1 Ch2 Ch 1 Ch2
compensatory mechanisms exist. Reduced recombination can
(b) Initial population contribute to the evolution of reproductive barriers by facilitating
the accumulation of hybrid incompatibilities or other key repro-
A A a a A A
Hybridisation
ductive barriers traits in the presence of gene flow (Fig. 6; Noor
et al., 2001; Rieseberg, 2001; Navarro & Barton, 2003). A clear
Ch 1 Ch 2 Ch 1 Ch 2 Ch 1 Ch 2 example can be found in Mimulus where a chromosomal inversion
polymorphism controls flowering time and other trait differences,
Gene duplication Ch 1 Ch2
F1 hybrid pollen as well as multiple reproductive barriers between annual and
a
perennial ecotypes of M. guttatus (Lowry & Willis, 2010).
A A a A a A A
Because rearrangements are expected to harbour genic incom-
patibilities (Navarro & Barton, 2003), it can be challenging to
Ch 1 Ch 2
Sterile Fully functional distinguish between the effects of chromosomal rearrangement
Fig. 5 Postzygotic isolation as a result of differential silencing. (a) The itself on F1 fertility and the effects of genes contained within
traditional Dobzhansky–Muller model for the evolution of genetic rearrangements. Inducing polyploidy in a low-fertility F1 provides
incompatibilities. Two related populations evolved new mutations that a simple test of whether decreased F1 fertility is due to chromosomal
interact with low fitness in hybrids but retain functionality within each or genic factors (Dobzhansky, 1933). In some cases, such as in
population. Differential silencing of duplicated loci provides one example of a
hybrids between Mimulus cardinalis and M. lewisii, inducing
Dobzhansky–Muller incompatibility. (b) A locus was initially duplicated
before population divergence (c). During the course of divergence, each polyploidy restored hybrid fertility, suggesting that it was difficul-
population silenced one of the two duplicates (lower case alleles). F1 hybrids ties in meiotic pairing, rather than the effects of genes within the
would produce some pollen lacking functional alleles, leading to pollen rearranged segments that were responsible for the partial sterility of
sterility. diploid hybrids (Stathos & Fishman, 2014).
The mechanisms by which chromosomal rearrangements affect
(a) Habitat 1 Habitat 2 hybrid sterility are fairly clear. However, many questions about
their origins and establishment remain. If chromosomal rearrange-
A A A A a a a a ments lead to decreased fitness in heterozygotes (underdominance)
B B B B b b b b due to meiotic difficulties, how would the rearrangements initially
become common in a population? Early theories suggested that
(b) (c) very small populations might allow rearrangements to become
With inversion in P1 Without inversion in P1 common due to genetic drift, but the effective population sizes of
Parental
most plant species are too large to support such a hypothesis
F1 hybrid F1 hybrid Parental
(Whitney et al., 2010). Alternatively, if a rearrangement occurs
B a B B A a A A
with a mutation with strong positive effects on fitness, then the net
× × fitness of the combination could be neutral or positive despite the
A b A A B b B B
deleterious effects of the rearrangement on meiosis. Although the
co-occurrence of a favoured mutation and a chromosomal
Offspring in habitat 1 Offspring in habitat 1 rearrangement is likely to be rare, rearrangements could also be
favoured if they reduce recombination between locally adapted
alleles in a heterogeneous environment (Kirkpatrick & Barton,
B BB B A A A A
2006). This scenario seems more plausible, at least for inversions,
A AA A B B B b because selection would act on the cumulative fitness effects of the
linked alleles. Models of recombination, selection and gene flow are
Fig. 6 Chromosomal rearrangements facilitate the evolution of reproductive
complex and an active area of research, but beyond the scope of this
isolation in the presence of gene flow. (a) Loci A and B are linked and allele A
is favoured in one habitat but not another. Locus B controls flower colour. review (see Ortiz-Barrientos et al., 2002; Butlin, 2005; and
Gene flow from the second habitat may introduce the linked alleles a and b Seehausen et al., 2014, for perspectives on this topic).
from habitat 2 into habitat 1. Allele A will remain common in habitat 1 Rearrangements can also establish through association with a
because of natural selection favouring it over allele a. (b) If loci A and B are selfish genetic element, such as a pollen killer that biases the
within an inversion, then recombination will be reduced, and the selection on
outcome of meiosis to make it more likely that the chromosome
allele A will maintain allele B as well, and with it increased reproductive
isolation. (c) However, without an inversion, occasional recombination, even with the selfish element ends up in the next generation (White,
if rare, may lead to allele b introgressing, reducing the reproductive isolation 1978). However, this hypothesis seems unlikely to account for the
caused by allele B. high rate of karyotypic evolution seen in plants (Levin, 2002)

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(a) pq 1/4
BC
A a A a
Time Bc
B b B b bC
C c C c
D d D d bc
(b) pq 1/4
BC
A a A a
Time Bc
B b B c
C c C b bC
D d D d
cb
Fig. 7 Meiotic drive. (a) Two homologous chromosomes will undergo
crossing over during meiosis, creating recombinant chromatids (shown at left
by Bc and bC). In the absence of meiotic drive, the frequencies of the
chromatids in the ovules will be determined by their frequency after crossing
over. (b) If meiotic drive is present, some chromatids will be more likely to
appear in the ovules than would be expected from their frequencies after
crossing over. In this hypothetical case, a rearrangement leads to a higher
frequency of the cb haplotype.
because of the required concurrence of two uncommon events (as
with the favourable mutation hypothesis). On the other hand, if the
rearrangement itself is the cause of drive (i.e. female meiotic drive;
Fig. 7) then the hypothesis becomes more credible (Coyne, 1989).
An advantage in meiosis could arise, for example, if there was a
change in centromeric position or size (Chm
atal et al., 2014). In
support of this hypothesis, drive-like behaviour was reported for six
of 10 newly arisen translocations in common sorrel (Rumex acetosa;
Wilby & Parker, 1988). A theoretical advantage to female meiotic
drive as a selective explanation for the fixation of chromosomal
rearrangements is that it could enable their spread across the entire
range of a species rather than being limited to populations
experiencing particular environmental conditions.
Postpollination reproductive isolation: polyploidy In the case of
polyploidy, a single macromutation can generate a sterility barrier.
For a novel tetraploid arising from a diploid progenitor, hybrids
between the two will result in triploid offspring that may be
inviable due to poor endosperm development: seed set for
tetraploid 9 diploid crosses is highly reduced (80%+; Ramsey &
Schemske, 1998), although the fertility reduction in some taxa is
less severe (e.g. Chamerion, with a 45% reduction; Husband &
Sabara, 2004). Surviving triploids will often have decreased fitness
due to failures in meiosis: in Chamerion, the triploid fitness was
11% of the diploids (Husband & Sabara, 2004). Although the
genetics underlying reproductive isolation is less mysterious for
polyploidy compared with other speciation processes, the origin
and establishment of new polyploid plants is not fully understood.
As with other mutations that contribute to reproductive isolation,
a novel polyploid plant will initially be rare and so may suffer
frequency-dependent declines in fertility (Levin, 1975; Baack,
2005). The factors that allow for polyploid establishment in the
face of this minority cytotype disadvantage have been the subject
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of extensive theoretical exploration (see Oswald & Nuismer,
2011, and references therein) but more limited empirical
investigation. Although increased fitness or altered niches could
help explain the establishment of novel polyploid plants,
empirical tests have been limited and have yielded mixed results.
Even if contemporary polyploid taxa differ in habitat usage from
their diploid progenitors, it is unclear if these differences arose
with the initial chromosome doubling or after establishment.
Although some recent investigations of novel polyploid plants
(natural and/or synthetic) report evidence for ecologically relevant
changes are due to chromosome doubling (Ramsay, 2011;
reviewed in Ramsey & Ramsey, 2014), others do not (Glennon
et al., 2014).
V. The timing of reproductive barriers
Ideally, we would be able to trace the origins of reproductive
isolation from the first isolating barriers through to the present. For
most species, we are far from this goal. In the case of polyploid
speciation, it is clear that postzygotic reproductive isolation is an
initial barrier, but how rapidly other barriers accumulate (e.g.
habitat shifts) remains unclear. For most taxonomic species that
have originated without genome duplications, the pattern of barrier
origins is uncertain. How often do ecogeographic shifts initiate
speciation? Do pollen–stylar interactions drive the early (sexual
selection) or late (reinforcement) stages of speciation? Do barriers
arise sequentially or are they sometimes simultaneous?
Data on the timing of reproductive isolation often are collected
in the context of comparing reproductive barriers among species
that vary in divergence times. Genetic distance between species is
used as a measure of time since divergence: species with low
genetic distances are seen as more recently diverged, whereas those
with greater genetic distances diverged in the more distant past.
This measure of divergence may lead to biases because pairs of
species with incomplete reproductive isolation may have low
genetic distances due to ongoing introgression, even if the initial
barrier originated in the distant past. In addition, the lower rates
of molecular evolution observed in woody plants compared with
herbaceous plants (Gaut et al., 2011) call for caution in the use of
genetic distance as a proxy for time. Our ability to establish the
timing of barrier origination is constrained because we only have
partial evidence for many species. None of the available datasets
include ecogeographic isolation, whereas postzygotic barriers as
measured by hybrid pollen fertility remain the most common.
1. Patterns of reproductive isolation over time
The timing of the origins of reproductive isolation has been
examined in relatively few genera: Silene, Glycine and Streptanthus
(Moyle et al., 2004), Fragaria (Nosrati et al., 2011), Helianthus and
Madiinae (Owens & Rieseberg, 2014), orchids (Scopece et al.,
2007) and Coreopsis (Archibald et al., 2005). In several cases – food-
deceptive orchids, Silene, Coreopsis, Helianthus and Madiinae –
reproductive isolation is positively correlated with genetic distance,
that is a ‘clock-like’ accumulation of RI. In Coreopsis, Helianthus
and Madiinae, reproductive isolation accumulates faster in annuals
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than in perennials, possibly due to an increased frequency of
chromosomal rearrangements in annuals. The very limited avail-
able data restrict our ability to generalize. However, a few contrasts
are possible. For the 15% of angiosperm and 35% of fern speciation
events (Wood et al., 2009) that involve chromosome doubling,
strong postzygotic isolation likely preceded any prezygotic isolation
(Glennon et al., 2014). Although animals typically show a clock-
like accumulation of RI, particularly postzygotic, plants clearly
show more diversity. Polyploidy may explain some of the difference
in plants, but not in others (e.g. Streptanthus; Moyle et al., 2004).
Theory predicts that postzygotic incompatibilities should
increase faster than a linear rate with time if they are due to
interactions between loci (Orr, 1995; Wang et al., 2013). In the
model, ancestral alleles are assumed to not create hybrid incom-
patibilities: any allele that did would lead to decreased fitness and so
would be removed from the population. As mutations fix in each of
two diverging populations, each derived allele represents another
possible incompatibility between the two populations, leading
postzygotic incompatibilities to ‘snowball’ with time (Orr, 1995).
Even though the methods for analysing the accumulation of
mutations leading to intrinsic postzygotic isolation continue to
evolve (St€adler et al., 2012; Wang et al., 2013), the larger obstacle
lies in our lack of large-scale comparative genetic studies of hybrid
incompatibilities. Research on the genetics of reproductive isola-
tion between tomato species has begun to fill this gap in plants.
Regions associated with RI have been identified and mapped onto
the phylogeny of several of these species (Sherman et al., 2014),
thus allowing the study of the timing and accumulation of
quantitative trait loci responsible for hybrid seed sterility and
hybrid pollen sterility between Solanum species (Moyle &
Nakazato, 2010). Although the dataset is small, loci underlying
traits for hybrid seed set and hybrid pollen fertility appear to evolve
faster than linear with time, as predicted by theory.
VI. Synthesis and future directions
The past decade has seen considerable progress in our understand-
ing of the origin and importance of different reproductive barriers.
We now know that reproductive isolation is pervasive yet imperfect
across most closely related plant species. The rise of genus-level
phylogenies for many taxa has enabled the conclusive identification
of sister taxa and with this assessments of geographic and temporal
patterns of speciation. We now know that postzygotic reproductive
barriers sometimes accumulate in a clock-like way in plants, but not
in all cases, and that barriers accumulate differently for annuals
compared with perennials. We know that sister taxa usually show
some ecological divergence, but that early stages in speciation in
many cases likely involved some gene flow. We have some
knowledge of the genetic basis of several reproductive barriers,
including elements of local adaptation, flowering time, flower
colour and some postzygotic barriers. However, many important
questions remain. In no particular order of importance, we provide
a top-10 list below:
(1) To what extent are the patterns described here general across
plant taxa? Taxonomic sampling has been relatively narrow so far,
and broader sampling is urgently needed on all aspects of
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reproductive barriers in plants. We know very little about the
origin of reproductive isolation barriers for mosses, trees and
tropical taxa, compared with temperate forbs (but see McDaniel
et al., 2008; Lepais et al., 2013; and Chen, 2013).
(2) What is the order in which different reproductive barriers
arise? And does this answer depend on phylogeny, mating system or
life history? Surprisingly, outside polyploidy, we still cannot answer
these questions. We need additional data examining prezygotic
isolating barriers for both recent and more diverged species.
(3) When do stochastic processes have a role in the evolution of
reproductive isolation? Contingency may play an unexpectedly
important role in the development of hybrid incompatibilities
because of gene and genome duplication, and potentially arbitrary
patterns of gene loss. On the other hand, sampling drift is probably
too slow to contribute significantly to the evolution of isolation in
most plant lineages, although it might partially explain polymor-
phism in postzygotic reproductive barriers. One potential excep-
tion is in selfing lineages, where the relative importance of sampling
drift vs selection remains to be determined.
(4) How are chromosomal rearrangements established? Do they
contribute to isolation most importantly through their direct
effects on hybrid fertility or their effects on recombination? We
hypothesize that evolution in heterogeneous environments
accounts for the establishment of most rearrangements that
segregate within species, but that the fixation of strongly under-
dominant rearrangements between species will likely require female
meiotic drive. A research program that compares intra- and
interspecific variation in chromosomal rearrangements, their
fertility effects and their association with centromeric repeats
would likely resolve this question.
(5) What are the relative roles of natural and sexual selection?
Given the potential for strong pollen competition and female
choice in plants, we predict that sexual selection plays a significant
role (e.g. Gossmann et al., 2014). We call for studies of conspecific
pollen precedence to relate their results to sexual selection. What are
the mechanisms of conspecific pollen precedence? Is it mainly due
to differences in male–male competition or to female choice? Is
there a molecular signal of pollen competition or female choice?
(6) How important is reinforcement? There currently are few well-
documented cases in plants, but we suspect this is because botanists
have focused on visible and potentially costly reproductive barriers
rather than cryptic barriers, such as pollen–stylar interactions. Does
reinforcement require an allopatric phase? Can it be driven by
ecological selection against hybrids? Lastly, is there a genomic
signature of reinforcement (e.g. Andrew & Rieseberg, 2013)?
(7) Under what conditions does the genetic architecture of
reproductive isolation predict patterns of genomic divergence in
genome scans? In theory, regions of the genome responsible for
reproductive isolation should be the only ones showing strong
genetic differentiation in the presence of gene flow. However, in
allopatric species pairs, as well as those with complete reproductive
isolation, natural selection may also create heterogeneous genomic
divergence (Cruickshank & Hahn, 2014). Tests of this prediction
may be facilitated by examining diverging populations with
documented gene flow while considering the recombination
landscape.
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New Phytologist Ó 2015 New Phytologist Trust
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Phytologist
(8) What is the role of genetic conflict in the evolution of
reproductive barriers in plants, and for which barriers is conflict
likely to be important? Several lines of evidence suggest that
conflicts within genomes (e.g. female meiotic drive) and between
genomes (male cytoplasmic sterility) may be important factors in
the origin of reproductive isolation. Increased understanding of the
genetics of female meiotic drive, and dissecting cases with
chromosomal rearrangements in an effort to detect drive will be
crucial.
(9) Do hybrid incompatibilities snowball? Although there is some
early evidence in favour of the snowball hypothesis in tomato and
Drosophila, data are required from a larger number of species pairs
within these two genera to more confidently establish this pattern.
Also, these kinds of studies need to be expanded to other plant
systems if we are to understand the genetics of intrinsic postzygotic
reproductive isolation across kingdoms.
(10) To what extent does ecology drive postzygotic isolation in
plants? Does local adaptation lead directly to the evolution of
intrinsic reproductive isolation? Are fitness effects within species
correlated with their effects on RI between species? A research
program where all barriers are quantified and their genetic basis is
elucidated will help in achieving this goal.
All of these questions assume that reproductive isolation plays a
key role in creating the phenotypic gaps we see between species and
in plant diversification. Although we have argued strongly in
favour of this view in the Introduction section, greater validation of
this basic assumption would provide a stronger rationale for the
focus of many students of speciation (and this review) on the
evolution of reproductive isolation. The kinds of studies that could
provide additional validation range from comparative analyses of
how phenotypic variation is partitioned in sexual vs asexual
lineages to studies of how different barriers (and barrier strength)
affect diversification rates, to investigations of the relationship
between barrier strength and the partitioning of phenotypic
variation within and among co-occurring species. These kinds of
studies, in combination with those put forward in our top 10 list,
would allow us to better evaluate the importance gene flow and
reproductive isolation relative to other forces in shaping pheno-
typic variation and in facilitating the formation and maintenance
of species. And, ultimately, these kinds of studies might provide a
fully satisfactory answer to the question that puzzled Darwin
(1859, p. 171):
‘. . . why, if species have descended from other species by insensibly fine
gradations, do we not everywhere see innumerable transitional forms?
Why is not all nature in confusion instead of species being, as we see them,
well defined?’
Acknowledgements
We are grateful to Mark Rausher and two anonymous reviewers for
their helpful comments; to David Lowry, Ken Whitney, Maddie
James and Henry Arenas-Castro for useful comments on previous
versions of the manuscript; and to Dolph Schluter, Mike Whitlock,
Amy Angert and Sally Otto for valuable discussions on speciation.
D.O-B. is funded by grants from the Australian Research Council,
Ó 2015 The Authors
New Phytologist Ó 2015 New Phytologist Trust
Tansley review Review 981
and L.H.R by the US National Science Foundation and the Natural
Sciences and Engineering Research Council of Canada.
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Supporting Information
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Table S1 Studies of locally adapted sister taxa where intrinsic
postzygotic reproductive isolation was quantified
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