Download Reproductive isolation and introgression between sympatric

Molecular Ecology (2016) 25, 2499–2517
doi: 10.1111/mec.13630
GENOMICS OF HYBRIDIZATION
Reproductive isolation and introgression between
sympatric Mimulus species
A M A N D A M . K E N N E Y * † and A N D R E A L . S W E I G A R T *
*Department of Genetics, University of Georgia, Athens, GA 30602, USA, †Department of Biological Sciences, St. Edward’s
University, Austin, TX 78704, USA
Abstract
Incompletely isolated species provide an opportunity to investigate the genetic
mechanisms and evolutionary forces that maintain distinct species in the face of ongoing gene flow. Here, we use field surveys and reduced representation sequencing to
characterize the patterns of reproductive isolation, admixture and genomic divergence
between populations of the outcrossing wildflower Mimulus guttatus and selfing
M. nasutus. Focusing on a single site where these two species have come into secondary contact, we find that phenological isolation is strong, although incomplete, and
is likely driven by divergence in response to photoperiod. In contrast to previous field
studies, which have suggested that F1-hybrid formation might be rare, we discover patterns of genomic variation consistent with ongoing introgression. Strikingly, admixed
individuals vary continuously from highly admixed to nearly pure M. guttatus, demonstrating ongoing hybridization and asymmetric introgression from M. nasutus into
M. guttatus. Patterns of admixture and divergence across the genome show that levels
of introgression are more variable than expected by chance. Some genomic regions
show a reduced introgression, including one region that overlaps a critical photoperiod
QTL, whereas other regions show elevated levels of interspecific gene flow. In addition, we observe a genome-wide negative relationship between absolute divergence
and the local recombination rate, potentially indicating natural selection against M. nasutus ancestry in M. guttatus genetic backgrounds. Together, our results suggest that
Mimulus speciation is both ongoing and dynamic and that a combination of divergence
in phenology and mating system, as well as selection against interspecific alleles,
likely maintains these sympatric species.
Keywords: flowering time, hybridization, introgression, Mimulus, reproductive isolation, speciation
Received 21 January 2016; revision received 18 March 2016; accepted 22 March 2016
Introduction
The question of whether a group of organisms is sufficiently similar to be called a single species or has accumulated a enough variation to justify splitting it into
two has fascinated biologists since before Darwin. In
the early twentieth century, Dobzhansky and Mayr
emphasized the importance of reproductive isolation
for speciation, arguing that the cessation of gene flow
positions diverging taxa along distinct evolutionary
tracks (i.e. the Biological Species Concept [BSC],
Correspondence: Amanda M. Kenney, Fax: 512 448 8764;
E-mail: [email protected]
© 2016 John Wiley & Sons Ltd
Dobzhansky 1937; Mayr 1942). But almost since its
inception, evolutionary biologists have objected to a
strict view of the BSC, pointing to many examples of
species pairs that remain separate despite a considerable gene flow (e.g. Anderson & Hubricht 1938; Epling
1947; Valentine 1948; Anderson 1949; Arnold 1997;
Mallet 2008). Indeed, it has long been recognized that
species boundaries might best be described as semipermeable (see Harrison & Larson 2014) with certain genomic regions being exchanged readily between species
and others resisting introgression (Barton 1979; Barton
& Hewitt 1981; Harrison 1990; Wu 2001).
Naturally hybridizing species offer a unique chance
to observe the functional effects of divergent gene
2500 A . M . K E N N E Y and A . L . S W E I G A R T
combinations in situ. This is because hybrid genomes
are mosaics that reflect different evolutionary processes
(Barton & Hewitt 1981). On the one hand, alleles that
are not disfavoured in hybrids or pure species (i.e. neutral or conditionally neutral) should spread easily
throughout hybridizing populations, even with only a
small amount of interspecific gene flow (e.g. Sambatti
et al. 2012). In contrast, alleles that disrupt the development or reproduction of hybrids – either because of
genetic incompatibilities (intrinsic postzygotic isolation)
or because of ecological maladaptation (extrinsic
postzygotic isolation) – are not expected to introgress
between species (e.g. Payseur et al. 2004). An additional
possibility is that alleles might actually flourish in
hybrid backgrounds (e.g. Kim & Rieseberg 1999; Martin
et al. 2006; Dasmahapatra et al. 2012). The genomic composition of a hybridizing population, then, represents a
balance between migration of alleles at neutral/universally advantageous loci and selection against hybrids
that carry incompatibilities or disfavoured alleles at loci
for local adaptation (Barton & Hewitt 1985).
Studies that have quantified genome-wide introgression between closely related but ecologically divergent
species have frequently discovered variable rates of
gene flow among loci (Rieseberg et al. 1999; Nolte et al.
2009; Gompert et al. 2010; Teeter et al. 2010; Ellegren
et al. 2012, Renaut et al. 2013; Larson et al. 2014; Delmore et al. 2015). Intriguingly, regions of reduced introgression sometimes coincide with QTLs or candidate
genes for reproductive isolation and/or local adaptation
(Carling & Brumfield 2009; Janousek et al. 2012; Via
et al. 2012; Larson et al. 2013). However, in other cases,
there is little correspondence between regions identified
as highly differentiated in genome scans and regions
containing QTLs for phenotypic differences (Yatabe
et al. 2007; Eckert et al. 2010). This mismatch might simply reflect an insufficient power of genome scan data to
detect QTL effects. Alternatively, it may suggest that
the phenotypes measured in QTL studies do not always
contribute to reproductive isolation in natural populations. If, for example, local adaptation is often caused
by conditionally neutral alleles (Hall et al. 2010; Fournier-Level et al. 2011; Leinonen et al. 2013), these loci
may not remain divergent when populations come into
secondary contact.
In addition to understanding the effects of individual
loci on reproductive isolation between diverging taxa, it
is important to determine whether particular genomic
features facilitate speciation in its early stages. For
example, chromosomal rearrangements, which often
suppress recombination when heterozygous, might
allow the accumulation of genes that contribute to isolation, even in the face of hybridization (Noor et al. 2001;
Rieseberg 2001; Navarro & Barton 2003). Indeed, a
number of studies have found higher levels of genetic
differentiation inside than outside chromosomal rearrangements between recently diverged species (Michel
et al. 2006; Noor et al. 2007; McGaugh et al. 2012; Barb
et al. 2014; Lohse et al. 2015). Even in the absence of
rearrangements, interspecific gene flow might be
reduced in regions of relatively suppressed recombination due to selection against alleles that are disfavoured
when introduced into the sister species (Nachman &
Payseur 2012). Consistent with this expectation, several
studies have found that genomic regions with elevated
divergence occur near centromeres and telomeres
(Turner et al. 2005; Carneiro et al. 2009; Ellegren et al.
2012) or coincide with genetic markers that experience
lower recombination rates (Geraldes et al. 2011; Renaut
et al. 2013). However, because most of these studies
have used relative measures of genetic differentiation
(e.g. FST), this pattern might be driven by a reduction in
diversity within species due to linked selection rather
than by gene flow between species (Noor & Bennett
2009; Nachman & Payseur 2012; Cruickshank & Hahn
2014). In a handful of recent investigations that have
examined the relationship between recombination rate
and absolute divergence (the latter is independent of
diversity within species), there is mixed support for the
idea that regions of suppressed recombination facilitate
the build-up of reproductive isolation during ongoing
gene flow (Nachman & Payseur 2012; Brandvain et al.
2014; Feulner et al. 2015; Janousek et al. 2015).
In this study, we focus on Mimulus guttatus and
M. nasutus, two closely related (2n = 28) species with
both strong reproductive isolation and ongoing introgression. Natural populations of M. guttatus are abundant throughout much of western North America and
occupy diverse ecological habitats. The distribution of
M. nasutus overlaps broadly with that of M. guttatus,
but its range is more restricted. In regions where the
two species co-occur, locally sympatric and allopatric
populations are common. Mimulus guttatus is self-compatible, but predominantly outcrossing, with large, beepollinated flowers, whereas M. nasutus is a selfer with
reduced, mostly closed flowers. The two species are
reproductively isolated by differences in floral morphology associated with their distinct mating systems, flowering phenology and pollen–pistil interactions (Kiang &
Hamrick 1978; Martin & Willis 2007; Fishman et al.
2014a), as well as by hybrid incompatibilities (Vickery
1978; Sweigart et al. 2006, 2007; Case & Willis 2008;
Martin & Willis 2010; Sweigart & Flagel 2015). In one
particularly detailed study of reproductive isolation
between sympatric M. guttatus and M. nasutus, premating barriers including the differences in mating system
and flowering time limited the opportunity for
hybridization to less than 1% (Martin & Willis 2007).
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Nevertheless, despite this strong reproductive isolation,
at the genome level, there is clear evidence of both historical and ongoing introgression (Sweigart & Willis
2003; Brandvain et al. 2014). A key question, then, is
how Mimulus species are maintained in the face of this
gene flow.
Here, we investigate the interplay between species
divergence and ongoing introgression in sympatric
Mimulus species. We focus on populations of M. guttatus and M. nasutus that co-occur at Catherine Creek
(CAC), a site in the Columbia River Gorge where we
recently discovered evidence for substantial genomic
introgression between species (Brandvain et al. 2014).
Our previous work also suggests that reproductive isolation at CAC is driven, at least in part, by divergence
in flowering phenology: using a wild-collected line of
each species, we showed that two major genetic loci
contribute to the differences in flowering response to
critical photoperiod (Fishman et al. 2014a). However,
because both of these previous studies relied on a single
line each of M. guttatus (CACG6) and M. nasutus
(CACN9), our understanding of reproductive isolation
and genomic variation at the population level has been
somewhat limited. Here, we perform a field study to
characterize a key component of premating isolation –
flowering asynchrony – and to investigate the potential
for hybridization. Surprisingly, and in contrast to a previous study of sympatric M. guttatus and M. nasutus in
which only pure species were observed (Martin & Willis 2007), we discovered a large number of morphologically intermediate individuals at the CAC site, which
we initially categorized as putative hybrids. Our subsequent analyses show that these individuals are also
intermediate in flowering phenology and genomic composition, confirming their hybrid status. In light of this
finding, we examine whether the patterns of shared
genomic variation are driven primarily by the presence
of early-generation hybrids or instead by ongoing introgressive hybridization. Finally, we investigate how patterns of introgression across the genome are affected by
loci that contribute to reproductive isolation and by
variation in recombination. Our findings provide key
insights into the genetic mechanisms and evolutionary
processes that maintain species in sympatry.
Materials and methods
Study populations
We investigated phenological isolation and the patterns
of hybridization between M. guttatus and M. nasutus at
a sympatric site near Catherine Creek (CAC) in the
Columbia River Gorge National Scenic Area of southern
Washington. At this site, M. guttatus and M. nasutus
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grow along ephemeral seeps and streams that run
downhill through a rocky, sloping meadow. The two
species occur in somewhat distinct microhabitats, but
there is a good deal of spatial overlap. In general,
M. guttatus grows in areas of pooled water that stay
wet throughout spring and into summer, whereas
M. nasutus grows in clumps of moss located in flowing
water on rocks that dry out in early May. Greater numbers of M. guttatus tend to occur in northern parts of
the meadow, with M. nasutus predominating to the
south. However, the microhabitats are patchy, and the
two species often grow very close together (within several metres or even adjacent to one another). Additionally, there are microhabitats that appear intermediate in
terms of water availability and persistence. In such
areas, we discovered both M. guttatus and M. nasutus,
along with large numbers of morphologically intermediate individuals that were difficult to classify as belonging to either species. These individuals had calyces
typical of M. nasutus (elongated upper lobe, especially
while in fruit) and larger flowers resembling M. guttatus; some had leaf serrations more characteristic of
M. nasutus leaves. Although there have been almost no
previous reports of putative hybrids between M. guttatus and M. nasutus in nature (Vickery 1964; Kiang &
Hamrick 1978; Martin & Willis 2007; but see Munz
1959), we hypothesized that these intermediate individuals at CAC might be the products of ongoing
hybridization. To investigate this possibility, we performed a series of phenotypic and genetic analyses (described below).
Flowering phenology
In the spring/summer of 2012, we characterized flowering phenology of M. guttatus, M. nasutus and putative
hybrids at the CAC site along two natural transects,
each of which follows an ephemeral rocky stream
through the meadow (hereafter referred to as stream 1
and stream 2). We set up ten 0.5-m2 square plots along
each transect for a total of 20 plots (Fig. 1). Based on
the visual assessment of species composition, we classified each plot as belonging to one of three categories:
M. guttatus, M. nasutus or mixed. Plots identified as
‘mixed’ contained putative hybrids and/or combinations of both M. guttatus and M. nasutus. We located
plots haphazardly where Mimulus plants were growing,
with an effort to represent the natural variation in habitat and species composition. Casual observations indicated that stream 1 contained more M. nasutus
individuals relative to M. guttatus than stream 2. Beginning on April 23, we recorded the number of open
flowers within each plot every day to every other day.
On a previous visit to the field site, we observed that
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M. nasutus began flowering during the first week of
April. By the time flowering phenology recording
began, M. nasutus was in peak bloom and putative
hybrids had just begun flowering; therefore, our survey
missed the early distribution of M. nasutus’ phenology.
We note that this sampling scheme was designed to
document the phenology of the plants present, not to
quantify the density or the number of M. guttatus vs.
M. nasutus plants growing at the site. For each plot, we
measured mean flowering date (weighted by the number of flowers each day). We then used two-way ANOVAs
to determine the effect of plot type (M. guttatus, M. nasutus or mixed), transect (stream 1 or stream 2) and
their interaction on mean flowering date. Statistical tests
were performed in JMPâ Pro 11.0.0 (Cary, NC, USA).
Growth chamber experiment
To investigate the effect of photoperiod on flowering
for M. guttatus and M. nasutus at Catherine Creek, we
grew individuals in two separate growth chamber treatments with 12- or 16-h day lengths. Note that M. guttatus and M. nasutus accessions vary in the ability to
flower under short photoperiods (10–12 h), but almost
all accessions initiate flowering under 16-h days (Friedman & Willis 2013). Therefore, we chose 12- and 16-h
day lengths (corresponding to March 18 and June 20 at
Catherine Creek) to test for divergence in response to
critical photoperiod, not to quantify photoperiod
requirements per se. These same day lengths were used
in our previous study demonstrating that flowering
time divergence is largely due to two major critical photoperiod QTLs (Fishman et al. 2014a). For M. guttatus,
we used 11–23 individuals from each of five inbred
maternal families, and for M. nasutus, we used 23 individuals from each of two inbred maternal families (for
a total of 142 plants per treatment). The M. guttatus
families originated from plots 1.7, 1.9, 1.10 and the area
between 1.7 and 1.9/1.10, and M. nasutus families came
from plot 1.3 and the area around plots 1.1 and 1.2 (see
Table 1 and Fig. 1). To minimize maternal effects, samples from Catherine Creek originated as seeds that had
been subjected to at least one generation of self-fertilization at the University of Georgia greenhouse. Seeds
were planted into 2.5” pots containing Fafard 3b potting
mix (Sun Gro Horticulture, Agawam, MA, USA), chilled
for 7 days at 4 °C to promote germination and then
moved to University of Georgia Conviron (Winnipeg,
MB, CAN) growth chambers with lights set to 12- or
16-h days. Seedlings were transplanted into 96-well flats
after germination. Positions within each flat were randomized. Plants were bottom-watered daily and temperatures were maintained at 22 °C during the day and
at 16 °C during night. We monitored individuals for
Table 1 Mean flowering and genomic composition of naturally
occurring M. guttatus, M. nasutus and hybrids
Plot
Species†
Mean flowering
date‡
Prop. M. guttatus
ancestry§
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
1.10
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
N
N
N
N
H
H
G
N
G
G
N
N
G
G, H, N
G
G
G/H
G/H
G/H
G
2.3
3.0
3.4
4.8
8.4
15.2
44.6
5.8
42.9
44.3
5.2
5.9
22.1
10.7
25.5
24.6
16.9
17.1
17.1
18.5
—
—
—
0.00
0.69
0.76
1.00
—
—
—
—
—
1.00
0.99
0.99
0.99
0.99
0.95
0.92
0.99
(2.3)
(0.2)
(0.4)
(0.6)
(3.0)
(8.6)
(8.6)
(0.9)
(8.5)
(8.9)
(0.7)
(1.0)
(4.4)
(2.4)
(4.9)
(4.8)
(3.3)
(3.4)
(3.4)
(3.5)
(0, 2)
(0.118, 7)
(0.027, 10)
(0, 2)
(0.001,
(0.009,
(0.006,
(0.007,
(0.003,
(0.017,
(0.018,
(0.005,
7)
2)
7)
7)
3)
5)
7)
9)
†
Species designations are a priori identifications. N = M. nasutus, G = M. guttatus, H = hybrid. Commas indicate a mix of
types; slashes indicate uncertainty in identification.
‡
Mean flowering date weighted by the number of flowers on
each day (Julian, April 23 as the starting date). Standard errors
in parentheses.
§
Values from STRUCTURE analyses, see text. In parentheses are
standard errors and sample sizes.
date of first flowering. If plants did not flower 8 weeks
after germination, we recorded them as nonflowering.
For each species, we determined the proportion of individuals that flowered under each light treatment.
DNA extraction, library preparation and sequencing
To examine the genomic variation within and between
Mimulus species at Catherine Creek, we harvested leaf
and/or flower bud tissue from 96 wild individuals
across both stream transects (Table S1, Supporting
information, note that the final number of individuals
analysed was <96 due to various quality issues). We
chose individuals haphazardly from within the 20 study
plots, attempting to sample the range of phenotypic
and phenological variation present. Note that seven of
the 96 individuals were located just outside the study
plots (<2 m away). We placed tissue samples on dry ice
until freezing at 80 °C. We isolated genomic DNA
using a standard CTAB/chloroform extraction protocol
as described in Holeski et al. (2014). Following extraction, we quantified DNA using the Quant-iT PicoGreen
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Fig. 1 Diagram of field site and study
plots. Each square represents a 0.5 m2
plot of M. guttatus (red), M. nasutus (yellow), or mixed (grey) individuals. Note
that mixed plots contained combinations
of plants identified as putative hybrids,
M. guttatus, and/or M. nasutus.
dsDNA Assay kit (Thermo Fisher Scientific, Waltham,
MA, USA) and diluted each sample to 5 ng/lL.
We generated a genomic library for genotyping the
96 wild-collected individuals by using the multiplexed
shotgun genotyping (MSG) method of Andolfatto et al.
(2011). In a 96-well plate, we digested 50 ng of genomic
DNA (15 U MseI; New England Biolabs, Ipswich, MA,
USA) for each sample in 20-lL reaction volume for 3 h
at 37 °C followed by heat inactivation at 65 °C for
20 min. Next, using 48 unique barcoded adapters, we
formed two sets of individuals (each set with 48 individuals carrying barcodes 1–48). We added 5 pmol of
the barcoded adapters to each well and performed ligation reactions using 1200 U of T4 DNA ligase (New
England Biolabs) in total volumes of 50 lL at 16 °C for
3 h followed by heat inactivation at 65 °C for 10 min.
We precipitated DNA in each well by adding 5 lL of
3M sodium acetate and 50 lL of isopropanol. We then
pooled each set of 48 individuals into a single tube,
added 1 lL of glycogen, and incubated overnight at
4°C. After resuspending each of the two samples in
100 lL TE, we purified them using a 1.5 volume of
Agencourt AMPure beads (Beckman Coulter, Brea, CA,
USA). Following purification, the Georgia Genomics
Facility size-selected fragments of 375–425 bp using a
QIAquick Gel Extraction kit (QIAGEN, Germantown,
MD, USA). For each of the two sets of 48 individuals,
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we then performed 18 cycles of PCR using a Phusion
High-Fidelity PCR master mix (New England Biolabs)
and one of two unique ‘index’ primers that bind to
common regions in the barcoded adapters. Following
this PCR amplification, each of the 96 CAC individuals
carried a unique barcode/index identifier. We performed clean-up on each of the sets using a 0.8 volume
of Agencourt AMPure beads, resuspended in 40 lL
QIAGEN EB buffer, and quantified the DNA using a
Qubit fluorometer (Thermo Fisher Scientific). We combined the two indexed sets into one library of 96 individuals at a final concentration of 15 lM.
We sent our library of 96 wild-collected individuals
to the Genomic Services Lab at HudsonAlpha (Huntsville, AL, USA) for sequencing. Sequencing was performed in a single lane on an Illumina Hiseq 2000 for
single-end, 50-bp reads.
Genome sequence alignment and variant detection
Before sequence processing, we trimmed adapter and
low-quality sequences using Trimmomatic (Bolger et al.
2014). We de-multiplexed and removed the 6-bp barcode
sequence from the reads in each library using Stacks
(Catchen et al. 2011). We then aligned the resulting 44bp single-end reads for each individual to the M. guttatus v2.0 reference genome (Hellsten et al. 2013) using
2504 A . M . K E N N E Y and A . L . S W E I G A R T
Burrows-Wheeler Aligner (bwa, Li & Durbin 2009) with
a minimum alignment quality threshold of Q25 (filtering
performed using SAMTOOLS, Li et al. 2009). To allow for
the variation in SNP density, we set the number of
allowed sequence differences per read (-n) to 4. To minimize SNP errors around insertion/deletion polymorphisms, we performed local realignment for all samples
simultaneously using the Genome Analysis Tool kit
(GATK, McKenna et al. 2010; DePristo et al. 2011).
We produced a set of high-quality SNPs and invariant
sites using the GATK Unified Genotyper (McKenna et al.
2010; DePristo et al. 2011). Individual coverage per read
is low in libraries produced from frequent-cutter restriction enzymes like MseI. Therefore, to increase our confidence in variant calls and to obtain genotypes from
parental reference lines for use in local ancestry assignment (see below), we called sites for wild CAC individuals simultaneously with a panel of previously
resequenced Mimulus accessions (M. guttatus: N = 13,
M. nasutus: N = 5, see Table S2, Supporting information).
Included among these ‘parental reference lines’ were
two high-coverage lines collected from Catherine Creek:
CACG6 (M. guttatus) and CACN9 (M. nasutus). Wholegenome sequence generation and alignment details for
these Mimulus accessions are described in Brandvain
et al. (2014).
Following site calling with the GATK, we performed
filtering steps using the PYTHON (www.python.org) module PYVCF (https://github.com/jamescasbon/PyVCF) in
conjunction with custom PYTHON scripts. SNPs were
excluded if they had site quality scores <40.0. Invariant
sites were excluded if they had MQ scores <30. For all
sites, individual CAC genotypes were only kept if they
had a minimum depth of five reads. Sites were
excluded that did not have a minimum number of CAC
individuals called (see below for the minimum required
for each downstream analysis). For each parental reference line, called genotypes were limited to sites with a
minimum depth of five reads and a maximum depth of
the mean plus standard deviation in coverage for that
line. In addition, heterozygous parental genotypes were
treated as missing data. Sites were excluded that did
not have a minimum number of parental reference
genotypes calls (see below for the minimum required
for each downstream analysis). Additional filtering of
sites was performed as needed using MICROSOFT EXCEL
(Microsoft, Redmond, WA, USA).
After initial quality assessments, we removed 21 of the
wild-collected CAC individuals from our data set. Four
samples were contaminated during library preparation;
these samples were not included in GATK site/SNP calling. The remaining samples were included in the GATK
site/SNP calling, but removed from subsequent analyses.
Of these, five samples were putatively identified as the
allopolyploid species Mimulus sookensis based on the
genotypes of their progeny. Hybridization between
M. guttatus and M. nasutus has generated multiple, independent origins of M. sookensis (Sweigart et al. 2008;
Benedict et al. 2012; Modliszewski & Willis 2012). Specifically, as part of a related study, we discovered that the
progeny of six CAC individuals (and one individual for
which we did not have progeny) had identical heterozygous genotypes at multiple, intron-spanning, lengthpolymorphic markers (between two and eight markers
genotyped per individual; A. M. Kenney and A. L. Sweigart, unpublished data), a pattern observed in known
M. sookensis individuals (Sweigart et al. 2008; Modliszewski & Willis 2012). We note that M. sookensis is morphologically similar to M. nasutus (Benedict et al. 2012),
and these six individuals were restricted to three M. nasutus plots and appear to be relatively rare at Catherine
Creek. Therefore, it is unlikely that any of the individuals
we identified as hybrids were undetected M. sookensis.
Four samples were individual duplicates; we kept the
four with higher read coverage. Finally, eight individuals
were removed due to relatively low read coverage. The
final number of wild-collected CAC individuals included
in the analyses below is 75.
Genetic clustering analysis
To estimate the ancestry proportion of each CAC individual, we used the admixture model in STRUCTURE
(Pritchard et al. 2000). For this analysis, we used only
SNPs with a depth of coverage between 5 and 65 reads.
To increase confidence in SNP calls, we included only
sites for which at least one M. guttatus and one M. nasutus parental reference line were also called (with only
invariant and biallelic sites allowed). Additionally, we
filtered sites so that at least 60% of the CAC samples
were genotyped at each SNP. These filtering steps
resulted in a set of 3253 biallelic SNPs across the genome
(CAC individual coverage ranged from 30.1% to 96.9%
of sites genotyped). We ran STRUCTURE for K-values 1–6,
with ten MCMC replicates at each K. Each replicate run
included a total of 75 000 iterations, with the first 25 000
discarded as burn-in. We had STRUCTURE infer alpha, the
average admixture proportion, and used an initial value
of 0.5. Analysis with STRUCTURE HARVESTER (Earl and vonHoldt 2012), which uses the Evanno method (Evanno
et al. 2005), revealed K = 2 to be the most likely number
of clusters. We combined the ten runs at K = 2 using
CLUMPP (Jakobsson & Rosenberg 2007).
Detecting introgression
To characterize patterns of introgression across the
Mimulus genome, we used two complementary
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methods: (i) identification of ancestry across chromosomes with the hidden Markov model approach of HAPMIX (Price et al. 2009) and (ii) measurement of absolute
pairwise divergence (dxy) between the wild-collected
CAC individuals and a high-coverage parental reference line for M. nasutus (CACN9).
We assessed patterns of introgression using the program HAPMIX, which relies on haplotype information
(from SNPs only) from parental reference lines of
each species (Table S2, Supporting information) to
inform local ancestry inference in admixed individuals
(Price et al. 2009). One advantage of using a haplotype
approach to detect introgression in this system is that
linkage disequilibrium (LD) decays very quickly in
M. guttatus compared to M. nasutus (Brandvain et al.
2014). Extensive LD in M. nasutus should facilitate the
accurate detection of true introgression vs. ancestral
polymorphism, the latter of which is not expected to
generate long, extended blocks of shared variation.
For each chromosome, we only included SNPs with
genotypes for at least 20% of the CAC individuals, as
well as three allopatric M. guttatus and two M. nasutus
parental reference lines. Polymorphism was required
among the parental references lines, but not the CAC
individuals (polymorphism at the latter depends on
which individuals were sequenced at a particular
site).
Because HAPMIX uses genetic distance between SNPs
to inform switches in local ancestry, we used recombination rates (cM/Mb) from an integrated genetic map
(described in Brandvain et al. 2014) to give each site a
proportional genetic position. Before computing recombination rates, we thinned markers from the genetic
map to an intermarker distance of at least 100 kb. We
removed sites from the SNP data set that were beyond
the physical boundaries of the genetically mapped
markers, as well as regions on four chromosomes with
significant discrepancies between the genetic and physical maps. These filtering steps resulted in a set of 66795
biallelic SNPs across the fourteen chromosomes of the
M. guttatus v2.0 reference genome. We used the program FASTPHASE (Scheet & Stephens 2006) to impute
missing data in the parental reference lines (individual
coverage initially ranged from 67.7% to 99.5% of SNPs
per chromosome among the 14 parental individuals and
14 chromosomes). For the wild-collected CAC individuals, individual coverage ranged from 8.1% to 94.4% of
SNPs genotyped per chromosome among all 75 individuals and 14 chromosomes.
We ran HAPMIX using the LOCAL ANC + ANC_INT_
THRESH mode, with a threshold of 0.75. We set theta
(the expected average admixture proportion) to 0.80
and lambda (the number of generations since admixture) to ten. Additional runs with lambda set to 100
© 2016 John Wiley & Sons Ltd
produced qualitatively similar results. Using the raw
output, we computed the average ancestry for
each individual in 50-kb windows across the 14 chromosomes. Then, to quantify population-level patterns of
introgression, we computed average ancestry for each
50-kb window among all M. guttatus and admixed CAC
individuals (N = 68, see Table S1, Supporting information; 3333 windows 50-kb windows were included in
our analyses; these windows had at least one SNP in
the HAPMIX analysis; note that ancestry was assigned for
a minimum of 44% of the 68 individuals in all these
windows). To generate a null distribution of average
ancestry, we randomly shuffled 50-kb windows within
each individual (preserving its overall genomic makeup) and then calculated the population average ancestry
per window. We repeated this permutation 5000 times
using a custom script in R (RC Team, 2015). We compared observed values of average ancestry across the
genome to the permuted top 97.5% and bottom 2.5%
values, respectively.
As a complementary estimate of introgression across
the genome, we also examined absolute pairwise divergence (dxy) between the parental line M. nasutus
(CACN9) and each of the wild CAC M. guttatus or
admixed individuals (N = 68, see Table S1, Supporting
information). We chose to include dxy as a second, complementary analysis to see whether alternative methods
yield consistent results. For all 68 pairwise comparisons,
we computed average dxy in 50-kb windows using custom scripts in Python. We note that our approach of
using 50-kb windows of divergence to CACN9 should
also facilitate our ability to identify regions of introgression vs. short segments containing old, incompletely
sorted variation due to extensive LD in M. nasutus. Biallelic SNPs and invariant sites were filtered as described
above. Additionally, we included only sites with genotypes for at least one wild CAC individual, as well as
two M. guttatus and two M. nasutus (including CACN9)
reference lines. These filtering steps resulted in a total
of 10533373 sites across the fourteen chromosomes of
the M. guttatus v2.0 reference genome. Due to the nature of our low-coverage data, different individuals were
genotyped at different sites, but many individuals were
represented in most 50-kb windows (4214 50-kb windows were included in our analyses; these windows
had an average of at least 40 sites genotyped for at least
40% of the 68 individuals). To examine interspecific
divergence at the population level, we averaged values
of dxy for each 50-kb window among the 68 comparisons. Finally, we generated a null distribution of dxy by
randomly shuffling 50-kb windows within individuals
and performing 5000 permutations as above to obtain
the top 97.5% and bottom 2.5% permuted average
divergence per window.
HAPMIX
2506 A . M . K E N N E Y and A . L . S W E I G A R T
Correlation between divergence and recombination
To compare the flowering curves of naturally occurring
M. guttatus, M. nasutus and morphologically intermediate individuals (i.e. putative hybrids), we combined
data from all plots within each of the two stream transects (Fig. 2). At both streams, M. nasutus initiated and
finished flowering earlier than M. guttatus (note that
M. nasutus began flowering prior to our survey). In general, flowering times were intermediate for plots with
putative hybrid individuals and/or combinations of
both species. For each of the 20 study plots, we also
measured mean flowering date (Table 1) and used a
two-way ANOVA to compare values at the two stream
transects. There was an effect of plot type with M. nasutus plots flowering earliest, mixed plots flowering at
intermediate times and M. guttatus plots flowering latest (F = 25.8, P < 0.0001). Additionally, there was a significant effect of transect, with stream 2 flowering
earlier than stream 1 (F = 18.8, P = 0.0007). We also
detected a highly significant plot type x stream interaction (F = 45.9, P < 0.0001). This interaction appears to
be driven primarily by M. guttatus, which flowered
much later in stream 1 than in stream 2, and may be
due to a combination of genetic and environmental
effects. All three M. guttatus plots from stream 1
retained moisture longer in the season than other plots.
The M. guttatus individuals in plot 1.7 were similar in
size to the M. guttatus individuals in stream 2. In
400
300
200
100
(b)
1
16
33
49
66
B
Flowering Day
M. guttatus
Mixed
300
400
0
Mean no. of flowers per plot
M. nasutus
200
M. nasutus
100
Natural flowering phenology
M. guttatus
Mixed
0
Results
(a)
Mean no. of flowers per plot
To investigate how the patterns of introgression across
the genome are affected by the variation in recombination, we calculated the nonparametric Spearman correlation between absolute pairwise divergence (dxy, the
mean number of pairwise sequence differences between
CACN9 and each of the wild-collected M. guttatus and
admixed individuals) and recombination in 100-kb windows using R (Team R Core 2015). For this analysis, we
filtered windows to include only those with an average
of at least 80 sites genotyped for at least 40% of the
68 M. guttatus and admixed individuals. We used
smoothened recombination rates described in Brandvain
et al. (2014).
Note that there are several chromosomal inversions
segregating within M. guttatus (Lowry & Willis 2010;
Fishman et al. 2014b; Lee et al. 2016) and one mapped
inversion between M. guttatus and M. nasutus (Fishman
et al. 2014b). However, we do not yet know whether
these or other inversions are present at Catherine
Creek.
1
16
33
49
66
4/23
5/8
5/25
6/10
6/27
Day, date
Fig. 2 Flowering phenology for M. guttatus (red), M. nasutus
(yellow), and mixed (grey) study plots. Points show average
flower number for each of the plot types beginning on Day 1
of the survey (April 23) and ending on Day 66 (June 27). Flowering in M. nasutus and in some mixed plots had already
begun prior to our survey. (a) Stream 1. (b) Stream 2.
contrast, many of the M. guttatus individuals in plots
1.9 and 1.10 seemed to be larger with more branches
and bigger flowers relative to the other plots. Moreover,
these phenotypic differences observed for M. guttatus in
plots 1.9 and 1.0 mirror some of the differences between
perennial and annual M. guttatus ecotypes (Lowry et al.
2008b), and it is possible both ecotypes exist at Catherine Creek.
Our previous work has suggested that flowering phenology differences between M. guttatus and M. nasutus
© 2016 John Wiley & Sons Ltd
Genomic composition
To investigate the potential for hybridization between
M. guttatus and M. nasutus at Catherine Creek, we
examined the genomic composition of wild-collected
individuals (N = 75) using the program STRUCTURE. Overall, ancestry proportions estimated by STRUCTURE were
consistent with our a priori species identifications for
each plot, with the two genetic clusters corresponding
to the two species (Table 1 and Table S1, Supporting
information). Approximately half of the individuals
were assigned almost exclusively to one of the two clusters (38 individuals with >99% ancestry to either cluster,
M. nasutus = 7, M. guttatus = 31), indicating clear
genetic divergence between species (Fig. 3, Table S1,
Supporting information). Strikingly, the remaining samples showed mixed ancestries, forming a continuous
spectrum of genetic variation from highly admixed
individuals (likely early-generation hybrids, based on
large blocks of alternating ancestry seen in Fig. S1, Supporting information) to late-generation hybrids/relatively pure M. guttatus (Fig. 3, Table S1, Supporting
information). All of these genetically intermediate samples were collected from field plots with large numbers
of morphologically intermediate individuals (Table 1,
especially plots 1.5 and 1.6), providing strong evidence
for historical and ongoing hybridization at Catherine
Creek. Additionally, the pattern of admixture is highly
asymmetric with ancestry proportions varying smoothly
towards M. guttatus, but not towards M. nasutus (note
© 2016 John Wiley & Sons Ltd
0.8
0.6
0.4
Ancestry proportion
0.2
0.0
at Catherine Creek might be explained, at least in part,
by genetic divergence at two major-effect QTLs for photoperiod response (Fishman et al. 2014a). However,
because this previous study was performed using only
one line each of M. guttatus and M. nasutus, we could
not determine whether individuals within each species
vary in their response to photoperiod. To investigate
this possibility, we examined flowering behaviour of
multiple CAC accessions in a common garden experiment with treatments of 12- or 16-h day lengths. We
found that the five M. guttatus lines never flowered
under 12-h days (% flowering = 0, SE = 0, N = 96, 5
lines with 11–23 individuals each), whereas two M. nasutus lines flowered approximately three-quarters of the
time (% flowering = 74, SE = 26.1, N = 46, 2 lines with
23 each). In contrast, under 16-h days, the same lines of
both species almost always flowered (M. guttatus: %
flowering = 91, SE = 9.1, N = 96; M. nasutus: % flowering = 100, SE = 0, N = 46). These results are consistent
with our observations of flowering phenology in the
field and provide strong evidence that sympatric Mimulus species are genetically divergent for response to critical photoperiod.
1.0
R E P R O D U C T I V E I S O L A T I O N I N S Y M P A T R I C M I M U L U S 2507
Individual
Fig. 3 Genomic composition at Catherine Creek. STRUCTURE plot
for wild-collected CAC individuals (N = 75) for the optimal
number of genetic clusters (K = 2), with ancestry proportion
(Q) on the y-axis. The plot is for 3253 genome-wide loci
grouped by species; M. guttatus cluster is in red and M. nasutus cluster is in yellow.
the sharp drop-off between pure M. nasutus and highly
admixed individuals). Consistent with earlier studies at
this and other sympatric sites (Sweigart & Willis 2003;
Brandvain et al. 2014), this pattern suggests that interspecific gene flow occurs primarily in one direction
from M. nasutus into M. guttatus.
Variation in introgression across the genome
To investigate genome-wide patterns of introgression
between M. guttatus and M. nasutus at Catherine Creek,
we used two complementary methods. First, we used
the hidden Markov model approach of HAPMIX (Price
et al. 2009) to infer the ancestry of chromosomal segments across the genomes of the wild-collected CAC
individuals by comparing them to parental reference
lines (see Materials and methods, Table S2, Supporting
information). Second, we estimated absolute pairwise
divergence (dxy) between M. nasutus (CACN9) and each
of the wild CAC individuals. Overall, genome-wide
estimates of introgression using both of these methods
were largely consistent with the individual admixture
proportions inferred from STRUCTURE (Fig. 3: compared
to Figs S1 and S2, Supporting information). CAC individuals identified as M. nasutus in STRUCTURE were composed almost exclusively of chromosomal segments
inferred as M. nasutus in HAPMIX (Fig. S1, Supporting
information), and showed very low divergence with
CACN9 (Fig. S2, Supporting information). Similarly, we
found that increasingly ‘pure’ M. guttatus individuals
had more and longer chromosomal segments inferred
as M. guttatus across their genomes (Fig. S1, Supporting
information). Both analyses also provide additional
2508 A . M . K E N N E Y and A . L . S W E I G A R T
1.0
Genome-wide mean ancestry
0.8
2.5%/97.5% genome-wide permuted ancestry
0.6
M. guttatus ancestry proportion
M. nasutus ancestry proportion
0.4
0.2
Chr 1
Chr 2
Chr 3
0
Region not included/missing
1.0
Average ancestry proportion
0.8
0.6
0.4
0.2
Chr 4
Chr 5
Chr 7
Chr 6
0
1.0
0.8
0.6
0.4
0.2
Chr 8
Chr 9
Chr 11
Chr 10
0
1.0
0.8
0.6
0.4
0.2
0
Chr 12
Chr 13
Chr 14
Chromosome, physical position
Fig. 4 Introgression across the genome inferred with HAPMIX. Each black point represents the average ancestry proportion of the
68 M. guttatus and admixed CAC individuals in a 50-kb window. Number of 50-kb windows with data = 3333, which corresponds
to 166.7 Mb, or 57% of the v2.0 M. guttatus sequence assembly for chromosomes 1–14. Plot regions are colored red and yellow to aid
in identifying regions with elevated and reduced M. guttatus ancestry. Genome-wide mean M. guttatus ancestry (0.841) is shown as a
solid blue line; 2.5 and 97.5 percentile values (0.775 and 0.914, respectively) from the permuted dataset are shown as dashed blue
lines. Black bars above chromosomes seven and eight indicate the positions of two critical photoperiod QTLs. Each panel represents
one chromosome. Large areas not included (shown in gray) on chromosomes 3, 8, 10, and 12 represent genomic regions for which
ancestry cannot be estimated due to problems with the genome assembly and unknown recombination rates. Distance between the xaxis ticks is 2 Mb, with the first tick at 1 Mb.
evidence that the majority of introgression is directional
from M. nasutus into M. guttatus.
Instead of focusing on individual introgression
events, we wanted to identify genomic regions with
particularly high or low rates of interspecific gene flow
at Catherine Creek. To do so, we estimated average
ancestry (Fig. 4) and divergence from M. nasutus
(Fig. 5) across the genome in 50-kb windows. Because
introgression is highly asymmetric (towards M. guttatus), we removed the seven CAC samples with >99%
ancestry assignment to M. nasutus (from STRUCTURE)
from both analyses. Among the remaining M. guttatus
and admixed CAC individuals (N = 68), the average
genome-wide proportion of M. guttatus ancestry (estimated by HAPMIX) was 0.841 (SE = 0.00246, N = 3333
50-kb windows) and the average dxy was 0.024
(SE = 0.00016, N = 4214 50-kb windows). We found
substantial variation in levels of introgression across the
genome with the two methods producing largely similar patterns (Spearman’s q = 0.411, P < 2.2e-16,
N = 3226 50-kb windows). Moreover, certain genomic
regions appear more or less resistant to introgression
than expected by chance: for both average ancestry
(Fig. 4 and Fig. S3, Supporting information) and absolute divergence (Fig. 5 and Fig. S4, Supporting information), approximately half of all 50-kb windows across
the genome had values above or below 95% of the windows from permuted data sets (produced by shuffling
the positions of 50-kb windows within individuals).
Note that permuting the data does not generate a prediction for a scenario of no introgression (which for
Fig. 4, for example, would be nearly 100% M. guttatus
ancestry for the 68 individuals across all the chromosomes). Rather, it generates a prediction for the null
© 2016 John Wiley & Sons Ltd
R E P R O D U C T I V E I S O L A T I O N I N S Y M P A T R I C M I M U L U S 2509
0.12
0.10
Chr 1
Chr 2
Chr 3
0.08
Genome-wide mean divergence
0.06
2.5%/97.5% genome-wide permuted divergence
0.04
0.02
0
0.12
0.10
Chr 4
Chr 5
Chr 7
Chr 6
Average divergence
0.08
0.06
0.04
0.02
0
0.12
2
0.10
0
Chr 8
Chr 9
Chr 10
Chr 11
0.08
08
0.06
06
0.04
04
0.02
02
0
0.12
0.10
Chr 12
Chr 13
Chr 14
0.08
0.06
0.04
0.02
0
Chromosome, physical position
Fig. 5 Introgression across the genome inferred using absolute divergence. Each black point represents average divergence of the
68 M. guttatus and admixed individuals vs. the M. nasutus line CACN9 in a 50-kb window. Number of windows with data = 4214,
which corresponds to 210.7 Mb, or 72% of the v2.0 M. guttatus sequence assembly for chromosomes 1–14. Genome-wide average dxy
(0.024) is shown as a solid blue line; 2.5 and 97.5 percentile values (0.018 and 0.031, respectively) from the permuted dataset are
shown as dashed blue lines. Black bars above chromosomes seven and eight indicate the positions of two critical photoperiod QTLs.
Each panel represents one chromosome. Distance between the x-axis ticks is 2 Mb, with the first tick at 1 Mb.
hypothesis that the variation in introgression across the
genome is randomly distributed. Our finding that some
regions show reduced or elevated introgression suggests that certain loci and/or genomic features might
prevent or facilitate gene flow between sympatric
Mimulus species.
In this system, we have strong candidate loci for
reproductive isolation at Catherine Creek, facilitating
direct investigations of how introgression levels are
affected in surrounding genomic regions. Results from
our field survey and common garden experiment suggest that genetic divergence in flowering time may be an
important barrier to interspecific mating. Furthermore,
our previous work has shown that two major-effect
QTLs – one on LG7 and one on LG8 – contribute to the
differences in photoperiod response between two inbred
lines of M. guttatus (CACG6) and M. nasutus (CACN9)
that originated from Catherine Creek (Fishman et al.
2014a). If these same two loci cause reproductive isolation in nature, we might expect to find a signature of
© 2016 John Wiley & Sons Ltd
reduced introgression in nearby genomic regions among
the sample of wild-collected M. guttatus and admixed
CAC individuals. Indeed, sites linked to the LG7 locus
show elevated M. guttatus ancestry (Fig. 4) and absolute
divergence with M. nasutus (Fig. 5) relative to most of
the genome, consistent with lower levels of introgression. The mean M. guttatus ancestry within the LG7
QTL interval (~3.157–3.987 Mb) is 0.965 (greater than
both the genome-wide and chromosome-wide 97.5%
permutation thresholds) and, strikingly, the LG7 QTL
interval overlaps with the region of highest M. guttatus
ancestry on LG7 (Fig. 4). Although divergence is more
variable throughout the genome, there is nevertheless a
reduction in introgression at the LG7 QTL compared to
much of the chromosome (QTL interval mean divergence = 0.028, LG7 mean divergence = 0.022; percentage
of 50-kb windows within LG7 QTL with divergence
<0.016 [the 2.5% threshold] = ~5.9%, percentage of 50-kb
windows on LG7 with divergence <0.016 = ~31.9%). In
contrast, the LG8 QTL, which spans a much larger
2510 A . M . K E N N E Y and A . L . S W E I G A R T
interval, shows no evidence of reduced introgression:
both M. guttatus ancestry (Fig. 4) and dxy (Fig. 5) are
similar to, or even below, the genome-wide averages.
Below, we discuss key differences in the phenotypic
effects and evolutionary histories of these two flowering
time loci that might explain their contrasting patterns of
introgression.
Along with the potential effects of individual loci on
introgression at CAC, we discovered a genome-wide
negative correlation between average absolute divergence from M. nasutus (i.e. dxy between CACN9 and
68 M. guttatus/admixed individuals) and the local
recombination rate (Spearman’s q = 0.219, P < 2.2e-16;
average dxy computed in 2068 100-kb windows, which
corresponds to 206.8 Mb or 70.4% of the v2.0 M. guttatus
sequence assembly for chromosomes 1–14). Additionally, we found a negative relationship between nucleotide diversity (p) and the local recombination rate within
species for both M. guttatus (Spearman’s q = 0.161,
P = 3.1e-14, sites required ≥ 4 individuals with M. guttatus ancestry ≥ 99% [from STRUCTURE], N = 2206 100-kb
windows) and M. nasutus (Spearman’s q = 0.162,
P = 8.9e-09, sites required ≥ 4 M. nasutus individuals,
N = 1241 100-kb windows). Note that this result cannot
be driven only by linked selection within species, which
is expected to produce a positive correlation between
nucleotide diversity and local recombination rate (Nachman & Payseur 2012; Cruickshank & Hahn 2014).
Instead, purifying selection in gene-rich regions (and
background selection at linked sites) might explain this
negative correlation within species: in Mimulus, gene
density is positively correlated with recombination
(Spearman’s q = 0.727, P < 2.2e-16, N = 2684 100-kb
windows) and we found a significant negative correlation between nucleotide diversity and gene density in
both M. guttatus (Spearman’s q = 0.226, P < 2.2e-16,
N = 2206 100-kb windows) and M. nasutus (Spearman’s
q = 0.177, P = 3.5e-10, N = 1241 100-kb windows).
Taken together, these results are consistent with a scenario in which selection against M. nasutus ancestry in
hybrid and M. guttatus genetic backgrounds reduces
introgression at linked sites.
Discussion
Recently diverged species that come into secondary
contact – particularly those still connected by some
degree of gene flow – provide an excellent opportunity
to investigate the evolution and maintenance of reproductive barriers in situ. Our field study at Catherine
Creek showed that natural flowering phenology differs
profoundly between the closely related outcrosser
M. guttatus and selfer M. nasutus. This difference seems
to be due in part to species divergence in photoperiod
response: in a controlled growth chamber experiment,
M. nasutus flowered readily under short days, whereas
M. guttatus required longer days. Surprisingly, despite
this and other potentially strong premating barriers
(e.g. mating system), we discovered substantial admixture between Mimulus species at Catherine Creek. Patterns of genomic variation indicate that these admixed
individuals range continuously from early-generation
hybrids to nearly pure M. guttatus. As found in previous studies of these species (Sweigart & Willis 2003;
Brandvain et al. 2014), introgression at Catherine Creek
is highly asymmetric with gene flow occurring primarily from M. nasutus into M. guttatus. We also discovered
that levels of introgression across the genome are more
variable than expected by chance. Whereas some genomic regions seem to remain distinct between species,
including one containing a critical photoperiod QTL,
others show elevated levels of gene flow that might
indicate adaptive introgression. Finally, our finding of a
genome-wide negative relationship between divergence
and recombination complements previous work (Brandvain et al. 2014) and suggests that natural selection
against M. nasutus ancestry in M. guttatus backgrounds
contributes to the maintenance of species in the face of
ongoing gene flow.
Divergence in flowering phenology between sympatric
species
Reproductive asynchrony is an important premating
isolating barrier between many species (e.g. Cruzan &
Arnold 1994; Quinn et al. 2000; Schemske 2000; Levitan
et al. 2004; Lowe & Abbott 2004; Savolainen et al. 2006;
Pascarella 2007; Murphy & Zeyl 2012). In this study, we
showed that M. guttatus and M. nasutus at Catherine
Creek are divergent for flowering phenology, a key contributor to reproductive isolation at other sympatric
sites where these species co-occur (Martin & Willis
2007). Across the M. guttatus species complex, there is
substantial variation in flowering behaviour, particularly in the critical photoperiod necessary for floral
induction (Friedman & Willis 2013). In general, M. guttatus populations and species with larger flowers transition more slowly from vegetative growth to flowering,
making them vulnerable to desiccation in what is often
a very short growing season (Hall & Willis 2006; Mojica
et al. 2012). Along this continuum, the small-flowered
M. nasutus tends to begin flowering earlier, occurring in
habitats that dry out sooner in the summer (Kiang &
Hamrick 1978). At Catherine Creek, where M. nasutus
usually grows in clumps of moss that dry out completely by early May, our common garden experiment
shows that this shift to earlier flowering is caused, at
least in part, by an ability to flower under much shorter
© 2016 John Wiley & Sons Ltd
R E P R O D U C T I V E I S O L A T I O N I N S Y M P A T R I C M I M U L U S 2511
day lengths than M. guttatus. Moreover, our previous
work has shown that this interspecific difference in
response to critical photoperiod is explained almost
entirely by two major QTLs – at least under controlled,
greenhouse conditions (Fishman et al. 2014a). An important question for future studies is whether these same
two QTLs also contribute to divergence in flowering
phenology in the field.
Despite clear interspecific differences in flowering
phenology at Catherine Creek, there is also substantial
phenotypic variation within species, as well as considerable overlap between the two Mimulus species (Fig. 2).
This variation is perhaps not surprising given that flowering in angiosperms is regulated by an integrated
genetic network responding not only to photoperiod,
but also to temperature, nutrient levels, the length of
winter (vernalization) and/or autonomous signals
(Andres & Coupland 2012). Strikingly, the two study
sites at CAC differed dramatically in flowering phenology, with the peak number of flowers for M. guttatus
occurring nearly a month earlier at stream 2 than at
stream 1. It is possible this difference is driven by plastic
responses to heterogeneity in water persistence and
other, unmeasured abiotic factors. However, genetic
variation in flowering time associated with microhabitat
may also underlie this difference. If so, this could reflect
variation among annual M. guttatus individuals, which
has been observed within a large population from central Oregon (Scoville et al. 2009), or possibly the presence of both annual and perennial M. guttatus ecotypes
(Lowry et al. 2008b). Alternatively, it is possible that the
variation in flowering between the two streams is
caused by different levels of introgression from M. nasutus (with M. guttatus individuals in stream 2 being more
introgressed than in stream 1). Although our population
genomic analyses did not include any individuals from
plots 1.9 and 1.10, the two samples from plot 1.7 were
among the least introgressed M. guttatus individuals.
Regardless of the cause, the phenological difference
between streams resulted in greater overlap in flowering
between hybrids and M. guttatus at stream 2, increasing
the potential for assortative mating and directional
introgression. Similarly, at a sympatric site in California
with evidence of ongoing asymmetric introgression
(Don Pedro Reservoir [DPR]; Sweigart & Willis 2003;
Brandvain et al. 2014), sites vary in their degree of phenological overlap between species, and the flowering
phenology of experimental F1 hybrids was shown to
overlap more with M. guttatus than M. nasutus (Martin
& Willis 2007). In stark contrast to CAC, however, there
are apparently few natural hybrids at the DPR sympatric site (none are reported in Martin & Willis 2007).
At Catherine Creek, particularly near stream 1, casual
observations suggest that hybrids occupy somewhat
© 2016 John Wiley & Sons Ltd
intermediate habitats in terms of water persistence.
Whether this is due to relative rates of formation in the
first place (i.e. interspecific crosses might occur in close
proximity to intermediate habitats with limited seed dispersal of hybrid offspring) or to hybrid fitness in intermediate vs. parental habitats is an open question. At
both CAC and DPR, longitudinal studies across years
that vary in seasonal rainfall might provide insight into
how local adaptation for flowering time differences
intersects with environmental heterogeneity, rates of
hybridization and patterns of introgression.
Historical and ongoing introgression
In many recently diverged plant taxa, premating reproductive barriers are thought to be particularly important
for speciation (Lowry et al. 2008a; Baack et al. 2015) and
Mimulus is no exception. In their detailed study of
reproductive isolation between M. guttatus and M. nasutus at DPR, Martin & Willis (2007) found that ecological
divergence associated with mating system reduced the
number of F1 hybrids produced to less than 1% of offspring from within-species crosses. Given such significant premating isolation, and the fact that no hybrids
were observed in the study at DPR (Martin & Willis
2007) or in classic accounts of Mimulus species’ distributions (Vickery 1964; Kiang & Hamrick 1978), rates of
natural hybridization between M. guttatus and M. nasutus have been assumed to be low. Along with strong
premating barriers (e.g. differences in flower morphology and flowering time), postmating isolation between
these species is also common (Vickery 1978; Sweigart
et al. 2007; Fishman et al. 2008; Martin & Willis 2010;
Sweigart & Flagel 2015). Cumulatively, then, it would
seem that reproductive isolation between M. guttatus
and M. nasutus is nearly complete. However, this view
has been somewhat at odds with population genetic
evidence for historical and ongoing introgression
between Mimulus species at sympatric sites (Sweigart &
Willis 2003; Brandvain et al. 2014), as well as their
repeated production of an allopolyploid species (Sweigart et al. 2008; Modliszewski & Willis 2012). And
although even a small amount of interspecific gene flow
can leave a significant signature of introgression across
the genome (Sambatti et al. 2012), our current results
suggest that, at least at Catherine Creek, there is more
hybridization between M. guttatus and M. nasutus than
previously appreciated.
But what are the consequences of this hybridization
at Catherine Creek? Our finding that admixture proportions vary continuously from approximately 60% to
nearly 100% M. guttatus ancestry suggests that postzygotic barriers between the two species are not complete.
Although intrinsic hybrid incompatibilities between
2512 A . M . K E N N E Y and A . L . S W E I G A R T
M. guttatus and M. nasutus are common, because most
involve recessive alleles (Sweigart et al. 2007, Martin &
Willis 2010; Sweigart & Flagel 2015), many hybrid genotypes (including F1 hybrids) are at least partially viable
and fertile. We have not yet tested for extrinsic postzygotic isolation between M. guttatus and M. nasutus, but
the pattern of introgression at CAC suggests that F1
and later-generation hybrids might often survive and
backcross to M. guttatus. In experimental crosses, reciprocal F1 hybrids between CAC M. guttatus and M. nasutus are generally equally viable and fertile (A. M.
Kenney and A. L. Sweigart, unpubl. results). The strong
asymmetry of interspecific gene flow is likely mediated
by the occasional visit of a pollinator to a small, slightly
open-flowered M. nasutus. In sympatric populations, we
expect that the majority of pollinator visits are to showy
M. guttatus flowers, so a visit to M. nasutus (or a
hybrid) will likely be preceded and followed by visits
to M. guttatus. Directional backcrossing is likely facilitated by F1 hybrid floral morphology, which more closely resembles that of the outcrossing M. guttatus
(Fishman et al. 2002), and by overlapping hybrid and
M. guttatus flowering phenologies. Alternatively, if
M. guttatus flowers are much more locally abundant
than M. nasutus when F1 hybrids are flowering, we
might predict that backcrossing to the more abundant
parent could generate the observed pattern of asymmetric introgression (Ellstrand & Elam 1993). To test this
idea, it will be important to quantify the relative abundance of M. guttatus, M. nasutus and hybrid flowers
during periods of phenological overlap. It is also possible that habitat isolation is less pronounced between
hybrids and M. guttatus (A. M. Kenney, personal observations), but additional field experiments are needed to
test this hypothesis. In the rare event that a pollinator
transfers pollen between an F1 hybrid and the highly
selfing M. nasutus (see Brandvain et al. 2014 for evidence of occasional gene flow in this direction), subsequent generations might suffer lower fitness due to
inbreeding depression, which can be substantial in
M. guttatus (Willis 1993).
Importantly, our STRUCTURE results provide further
support for the inference of historical introgression at
sympatric sites made from analyses of whole-genome
sequence data for two M. guttatus samples collected
from CAC and DPR (Brandvain et al. 2014). In this previous work, we argued that the distribution of introgression block size within a single M. guttatus CAC
individual was consistent with a large number of historical introgression events. Our current data set, which
includes a number of highly admixed individuals that
appear to be the result of relatively recent hybridization, suggests that interspecific gene flow at CAC has
continued right up to the present.
Variation in introgression across the genome and the
maintenance of species
A fundamental question for speciation is whether certain genetic mechanisms or evolutionary forces are capable of countering the homogenizing effects of historical
and ongoing gene flow. At Catherine Creek, directional
introgression from M. nasutus into M. guttatus seems to
be common, resulting in a mosaic of shared variation
across the genome. Furthermore, the pattern of introgression is not random; rather, our analyses show that
there are certain genomic regions that introgress more
or less than expected by chance. Regions with elevated
gene flow might be due to the spread of advantageous
alleles between species or simply to neutral introgression. On the other hand, genomic regions that remain
distinct despite ongoing introgressive hybridization
might provide clues about the genetic loci and/or genomic features that contribute to reproductive isolation.
Our previous finding that two major QTLs contribute
to species divergence in photoperiod response at CAC
(Fishman et al. 2014a) sets the stage for direct investigations of introgression at loci with putative effects on
reproductive isolation. Interestingly, we observed very
different patterns of genomic variation surrounding the
two key flowering time loci. Relative to the rest of the
genome, introgression was reduced at the LG7 locus, as
would be expected if it contributes to reproductive isolation in the wild. In contrast, introgression at the LG8
flowering time locus was somewhat elevated. One simple
explanation for this finding might be that the LG8 photoperiod QTL does not cause reproductive isolation
between Mimulus species in the field. A number of recent
studies in Arabidopsis and its relatives have identified different sets of flowering time QTLs in greenhouse vs. field
experiments (Brachi et al. 2010; Leinonen et al. 2013), suggesting that genes for flowering behaviour are highly
influenced by complex natural environments. Moreover,
the idea that locally adapted alleles should not introgress
between hybridizing species (Harrison 1990; Wu 2001;
Nosil et al. 2005) holds only if local adaptation is caused
by loci that show antagonistic pleiotropy (i.e. fitness
trade-offs across habitats with native alleles outperforming foreign ones). If instead local adaptation is caused by
distinct combinations of loci that are individually beneficial in one environment but effectively neutral in another
(i.e. conditional neutrality), introgression should not be
impeded, and we might predict asymmetric introgression of the conditionally favoured allele into the alternate
background at each locus. Perhaps, then, the LG7 locus is
antagonistically pleiotropic, whereas the LG8 locus is
conditionally neutral. Given the somewhat elevated
levels of introgression at LG8, one might even imagine
that early-flowering alleles from M. nasutus at this locus
© 2016 John Wiley & Sons Ltd
R E P R O D U C T I V E I S O L A T I O N I N S Y M P A T R I C M I M U L U S 2513
could provide an advantage in microhabitats prone to
seasonal drought, allowing M. guttatus to expand its
range. Going forward, field experiments at Catherine
Creek will be critical for determining the phenotypic
effects of these putative isolation loci in an ecologically
relevant context.
Differential introgression at the LG7 and LG8 loci
might also be explained by genetic background effects
in this diverse natural population and/or by distinct
evolutionary histories of the photoperiod loci themselves. Our previous study investigated the genetic basis
of flowering time using two inbred lines (M. guttatus:
CACG6, M. nasutus: CACN9) and a targeted mapping
approach, in which we genotyped F2 hybrids only at
markers flanking the LG7 and LG8 QTLs (these two loci
had recently been mapped in a distinct cross between
lines of M. nasutus and M. guttatus derived from two
allopatric populations in Oregon; Fishman et al. 2014a).
Together, the two QTLs explained 84% of the parental
difference in flowering response to photoperiod, but it is
entirely possible that additional modifier alleles segregate at Catherine Creek. Furthermore, different patterns
of introgression at the two photoperiod loci might also
be due to evolutionary history: whereas the LG7 earlyflowering allele appears to be specific to M. nasutus, the
LG8 QTL might share a common genetic basis with
flowering time QTLs mapped within M. guttatus (Friedman & Willis 2013; Zuellig et al. 2014). If the LG8 QTL is
already polymorphic within M. guttatus at CAC, perhaps introgression from M. nasutus introduces no novel
functional variants. We note, however, that because the
LG8 QTL is broad and includes several candidate genes,
identification of the underlying polymorphisms will be
necessary to test this hypothesis.
Along with the potentially important effects of individual loci like those on LG7 and LG8, we also found
evidence that the variation in local recombination rate
may contribute to heterogeneity in introgression levels
across the genome. Under a scenario of ongoing gene
flow between CAC Mimulus species, strong selection
against introgression in regions of suppressed recombination – due to linkage with maladaptive, heterospecific
alleles – might lead to concomitant increases in divergence. However, it is important to note that adaptive
evolution within each species might generate a similar
pattern, with higher levels of differentiation occurring
in low-recombination regions that are more likely to be
influenced by selective sweeps and/or background
selection (Noor & Bennett 2009; Nachman & Payseur
2012). To distinguish between these possibilities, we
examined absolute divergence (dxy) across the genome,
which, unlike relative measures of differentiation (e.g.
FST), is not influenced by linked selection within species
(see Cruickshank & Hahn 2014). Strikingly, we
© 2016 John Wiley & Sons Ltd
discovered a highly significant negative correlation
between absolute divergence and local recombination
rate, consistent with selection against the introgression
of M. nasutus ancestry in M. guttatus. In a recent investigation of genome-wide variation between the two species, Brandvain et al. (2014) found this same negative
relationship, despite using a very different data set
(whole-genome sequences from only a handful of individuals, including one sample each of CAC M. guttatus
and M. nasutus). Moreover, like in the Brandvain et al.
(2014) study, we did not find a positive relationship
between recombination and nucleotide diversity within
M. guttatus or M. nasutus, indicating that linked selection within species is not driving the observed patterns
of divergence at Catherine Creek. Instead, our results
suggest that ongoing gene flow – primarily from M. nasutus into M. guttatus – might play a major role in shaping the patterns of admixture across the genome.
Conclusions
The picture emerging from this study is one of Mimulus
speciation as an ongoing, dynamic process. Our detailed
look at Catherine Creek, a site where M. guttatus and
M. nasutus populations have come into secondary contact, has shown that hybridization can strongly impact
population genetic diversity. Nevertheless, it appears
that species are maintained in the face of this gene flow
by a number of barriers. In one direction (towards M. nasutus), introgression seems to be almost entirely prevented by divergence in traits related to mating system
and flowering phenology. In the other direction (towards
M. guttatus), introgression appears to be common, but
our population genomic analyses suggest that there is
also persistent natural selection against this influx of
M. nasutus ancestry. This pattern of selection is consistent
with the results from experimental crosses between
M. guttatus and M. nasutus, which show pervasive transmission ratio distortion in hybrid offspring, with a widespread bias towards M. guttatus alleles (Fishman et al.
2001, 2008). At least some of this distortion appears to be
caused by loci that influence pollen performance
(Aagaard et al. 2013), and M. guttatus alleles at such loci
are expected to have a competitive advantage over inbred
M. nasutus alleles (Brandvain & Haig 2005). Going forward, a key question is to what extent distortion against
M. nasutus alleles in a heterospecific genetic background
impedes introgression into M. guttatus at Catherine
Creek. Additionally, field experiments will provide an
important complement to the current study. For example,
by genotyping the progeny of experimental and/or natural hybrids, it will be possible to test directly whether
hybrids backcross exclusively to M. guttatus (as predicted) or instead to both species, as well as tease apart
2514 A . M . K E N N E Y and A . L . S W E I G A R T
the roles of floral morphology, phenology and species
abundance in shaping these patterns. In addition, planting out experimental hybrid genotypes, such as nearly
isogenic lines for the flowering time loci on LG7 and LG8,
or F2s segregating at a suite of vegetative and reproductive traits, will be critically important for understanding
both the genetic basis of assortative mating and the role
of natural selection in maintaining these Mimulus species.
Acknowledgements
We thank Michael Boyd, Rachel Hughes and Kevin Tarner for
expert greenhouse care. We also thank Taylor Gordy for greenhouse and laboratory assistance and Galen and Vicki Sweigart
for help in the field. We are grateful to Yaniv Brandvain, Lex
Flagel and Charles Hauser, who provided bioinformatics assistance that significantly improved the quality of this work. We
are also grateful to Yaniv Brandvain, Lila Fishman, Lex Flagel,
Dave Hall and Matt Zuellig for valuable discussions. We thank
Richard Abbott and four anonymous reviewers for their
thoughtful comments, which significantly improved the quality
of this manuscript. We also thank Brandon Campitelli, Tom
Juenger, Rachel Kerwin, John Lovell and Alice MacQueen for
helpful feedback on an earlier version of this manuscript. We
thank the University of Georgia Advanced Computing
Resource Center for expert computing advice. The Duke Genome Sequencing Core and the Joint Genome Institute generated
sequences used in this study. This work was supported by
funds from the University of Georgia Research Foundation and
from National Science Foundation Grant DEB-1350935 to A.L.S.
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A.M.K. and A.L.S. performed experiments and analysed
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Data accessibility
Whole-genome sequences for all parental lines can be
found on the NCBI SRA (see Table S2, Supporting
information for accession numbers).
Parsed (but raw/not filtered for quality) sequence
reads for all 75 CAC individuals are available on the
NCBI SRA under study accession SRP072552.
Phenotypic data are available on Dryad under doi:
10.5061/dryad.6p8p0.
Supporting information
Additional supporting information may be found in the online version of this article.
Fig. S1 Individual HAPMIX ancestry in 50-kb windows for all 75
individuals across the 14 chromosomes.
Fig. S2 Individual absolute divergence (dxy) in 50-kb windows
between the resequenced M. nasutus line CACN9 and all 75
individuals across the 14 chromosomes.
Fig. S3 Histogram of average ancestry as inferred by HAPMIX in
50-kb windows for 68 wild-collected M. guttatus and admixed
CAC samples.
Fig. S4 Histogram of average absolute divergence (dxy) in 50kb windows for pairwise comparisons between resequenced
M. nasutus line CACN9 and 68 wild-collected M. guttatus and
admixed CAC samples.
Table S1 Individual IDs, sample locations, and STRUCTURE
results for wild-collected Mimulus samples from the Catherine
Creek population.
Table S2 Information about the resequenced Mimulus accessions used in this study.