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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). © 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 2501 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 © 2016 John Wiley & Sons Ltd 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 2502 A . M . K E N N E Y and A . L . S W E I G A R T 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 © 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 2503 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, © 2016 John Wiley & Sons Ltd 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 © 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 2505 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. References Aagaard JE, George RD, Fishman L, MacCoss MJ, Swanson WJ (2013) Selection on plant male function genes identifies candidates for reproductive isolation of yellow monkeyflowers. PLoS Genetics, 9, e1003965. Anderson E (1949) Introgressive Hybridization. John Wiley & Sons, New York. 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Zuellig MP, Kenney AM, Sweigart AL (2014) Evolutionary genetics of plant adaptation: insights from new model systems. Current Opinion in Plant Biology, 18, 44–50. A.M.K. and A.L.S. planned and designed the research. A.M.K. and A.L.S. performed experiments and analysed data. A.M.K. and A.L.S. wrote the manuscript. © 2016 John Wiley & Sons Ltd 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.