Sympatric of wild emmer wheat driven by ecology and chromosomal rearrangements

Hongwei Wanga,1,2, Huayan Yinb,1, Chengzhi Jiaoc,1, Xiaojian Fanga,1, Guiping Wanga,1, Guangrong Lid, Fei Nia, Penghuan Lia, Peisen Sua, Wenyang Gea, Zhongfan Lyua, Shoushen Xua, Yanhong Yanga, Yongchao Haoa, Xinxin Chenga, Jinxiao Zhaoa, Cheng Liua,3, Fengfeng Xuc, Xin Maa, Silong Suna, Yan Zhaoa, Yinguang Baoa, Cheng Liue,4, Jingjing Zhangf, Tomas Pavlicekg, Anfei Lia, Zujun Yangd,1,2, Eviatar Nevog,2, and Lingrang Konga,2

aState Key Laboratory of Crop , Shandong Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, 271018 Tai’an, China; bCollege of Agronomy, Qingdao Agricultural University, 266109 Qingdao, China; cNovogene Bioinformatics Institute, 100083 Beijing, China; dCenter for Informational Biology, School of Science and Technology, University of Electronic Science and Technology of China, 610054 Chengdu, China; eCrop Research Institute, Shandong Academy of Agricultural Sciences, 250100 Jinan, China; fSchool of Life Sciences, Zhengzhou University, Zhengzhou 450001, China; and gInstitute of , University of Haifa, Haifa 3498838, Israel

Contributed by Eviatar Nevo, December 27, 2019 (sent for review November 26, 2019; reviewed by Eugene V. Koonin, James Shapiro, and Alan Robert Templeton) In plants, the mechanism for ecological (SS) is nature by the examples in animals (e.g., resource competition, little known. Here, after ruling out the possibility of secondary , etc.) (5, 7, 8). In plants, which are stressed contact, we show that wild emmer wheat, at the microclimatically constantly by a diversity of biotic and abiotic stresses, ecological divergent microsite of “Evolution Canyon” (EC), Mt. Carmel, Israel, divergence was presumed essential in driving speciation. How- underwent triple SS. Initially, it split following a bottleneck of an ever, accepted examples of ecological SS are still limited in ancestral population, and further diversified to three isolated pop- plants, and evolutionary mechanisms for such speciation have ulations driven by disruptive . Remarkably, two not been well established. One compelling case is that of sister postzygotically isolated populations (SFS1 and SFS2) sympatrically of palm (Howea) that seem to have split on a tiny iso- branched within an area less than 30 m at the tropical hot and dry lated island in the Tasman Sea, possibly driven by divergent soil savannoid south-facing slope (SFS). A series of homozygous chro- preference, yet any underlying cellular and genetic mechanisms

mosomal rearrangements in the SFS1 population caused associated with this split have not been demonstrated (9). EVOLUTION sterility with the SFS2 population. We demonstrate that these two populations developed divergent adaptive mechanisms against Significance severe abiotic stresses on the tropical SFS. The SFS2 population evolved very early flowering, while the SFS1 population alterna- In plants, which are stressed constantly by diverse abiotic and tively evolved a direct tolerance to irradiance by improved ROS biotic stresses, ecological divergence is substantial in driving scavenging activity that potentially accounts for its evolutionary speciation, yet its demonstration under is still limited fate with unstable status. Moreover, a third prezygoti- in plants and needs elaboration. By analyzing wild emmer cally isolated sympatric population adapted on the abutting tem- wheat at the Evolution Canyon microsite, a hot spot of sym- perate, humid, cool, and forested north-facing slope (NFS), patric speciation, we highlighted how disruptive ecological separated by 250 m from the SFS wild emmer wheat populations. selection drives primary sympatric speciation in situ. Natural The NFS population evolved multiple resistant loci to fungal dis- selection, overruling the homogenizing effects of , eases, including powdery mildew and stripe rust. Our study illus- split the metapopulation of wild emmer wheat into three re- trates how plants sympatrically adapt and speciate under disruptive productively isolated populations. Each population evolved ecological selection of abiotic and biotic stresses. unique evolutionary trajectory with divergent adaptive strat- egies, including fungal disease resistance, irradiance tolerance, sympatric speciation | wild emmer wheat | Robertsonian translocation | and divergent flowering time. Our work provides a model for abiotic stress | biotic stress plant adaptive ecological sympatric speciation under biotic and abiotic stressors. nderstanding the mechanisms underlying speciation, the Usplitting of a group of interbreeding populations into two Author contributions: H.W., E.N., and L.K. designed research; H.Y., G.W., G.L., P.L., P.S., reproductively isolated groups, has been a major focus of evo- W.G., Z.L., S.X., Y.Y., Y.H., X.C., J. Zhao, C.L.3, X.M., Y.B., A.L., and Z.Y. performed research; 4 lutionary biology (1). In the widespread process known as C.L. , J. Zhang, and T.P. contributed new reagents/analytic tools; C.J., X.F., F.N., F.X., S.S., and Y.Z. analyzed data; and H.W. wrote the paper. “,” populations that become geographically Reviewers: E.V.K., National Institutes of Health; J.S., University of Chicago; and A.R.T., isolated experience selective pressures and/or that Washington University in St. Louis. ultimately give rise to two reproductively isolated new species (2). The authors declare no competing interest. In contrast, there are dramatically fewer known instances of spe- ciation occurring without geographical isolation, a concept known Published under the PNAS license. as sympatric speciation (SS) proposed by Darwin (2–4). It is Data deposition: The resequencing and SNP data generated in this work are deposited in the Genome Sequence Archive in the BIG Data Center, Beijing Institute of Genomics, generally agreed that an instance of SS must be substantiated with Chinese Academy of Sciences, under the accession no. CRA001873. evidence showing that the relevant species occur in the same 1H.W., H.Y., C.J., X.F., G.W., and Z.Y. contributed equally to this work. geographic location, are in , and display a 2To whom correspondence may be addressed. Email: [email protected], sister relationship (3). Claims of SS are bolstered by evidence that [email protected], [email protected], or [email protected]. any earlier periods of allopatric speciation are unlikely (5). Major 3State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Col- questions about SS typically center on how reproductive isolation lege of Agronomy, Shandong Agricultural University, Tai’an, 271018 Shandong, China. has arisen in sympatry and on the nature of the selection pressures 4Crop Research Institute, Shandong Academy of Agricultural Sciences, 250100 Jinan, and genetic drift that has driven the speciation (6). China. An increasing number of mathematical models have been This article contains supporting information online at https://www.pnas.org/lookup/suppl/ established to sustain the plausibility of SS, often including the doi:10.1073/pnas.1920415117/-/DCSupplemental. influence of that is particularly validated in

www.pnas.org/cgi/doi/10.1073/pnas.1920415117 PNAS Latest Articles | 1of9 Downloaded by guest on September 27, 2021 Guided by the widely held speculation that divergent selection incipient SS is under way at EC-I between populations present via microcosmic ecological differences are likely essential in fa- on the SFS or NFS. cilitating SS, a long-term research project since 1990 in Israel has Here, seeking to apply modern genome-scale technologies to enabled tracking of evolutionary histories at a microscale by examine potential speciation events in tetraploid wheat pop- designating four natural laboratories: the so-called “Evolution ulations at EC-I, we collected 47 accessions of wild emmer wheat Canyon” microsites (EC-I to EC-IV). At each of these EC sites, (Triticum dicoccoides Koern), the progenitor of bread wheat, there are sharp ecological contrasts within minuscule interslope from EC-I. Using genomics, cytogenetics, analysis of hybrid distances (6). The EC-I microsite comprises twin slopes with sterility patterns, and both observational and hypothesis-driven extremely sharp ecological contrasts: the south-facing slope confirmatory studies addressing observed divergence for eco- (SFS) and north-facing slope (NFS) are only separated by an logically important traits, we assessed the possibility of SS of wild average of 250 m, but the SFS receives 200 to 800% higher solar emmer wheat at EC-I. radiation than the NFS, resulting in a substantially hotter and Results drier slope (Fig. 1A). In contrast, the higher relative humidity Three Distinct Populations of Wild Emmer Wheat Have Diverged and reduced solar radiation of the NFS results in cooler and within EC-I following a Recent Population Bottleneck. To apply wetter conditions (10). These dramatic differences in microcli- modern high-resolution genetic analysis methods to the excep- mates in extremely close proximity have enabled very high spatial tionally diverse populations of species known to exist at the EC-I resolution studies unfolding the influence of environmental site, we collected 47 accessions from the tropical hot and dry gradients on fitness-related phenotypic traits, and offers one of savannoid south-facing slope (SFS) and abutting temperate, the best available opportunities to study the interplay between humid, and cool forested north-facing slope (NFS) locations of , , and further speciation. In past this site, within a transect of only 300 m. Notably, most of these decades, a variety of population-based analyses with five model accessions were collected from altitudes above the midlevel of organisms [Bacillus simplex (11), Drosophila melanogaster (12), each slope (stations 1, 2 on SFS; stations 6, 7 on NFS). We also spiny mice Acomys cahirinus (13), wild barley Hordeum sponta- obtained materials from the close vicinity around EC-I (e.g., neum (14), and sawtoothed grain beetles Oryzaephilus sur- ∼3.5 km from Beit Oren, ∼13 km from Bat Shlomo, and ∼30 km inamensis (15)] have marshaled evidence to strongly suggest that from Afula) and from surrounding countries to help determine

ABpopulation EC-I S 7 1 1 6 2 NFS WEW 5 3 2 4 3 4 5 S WEW 6 7 8 9 10 11 12 13

CD 4.16e-05 4.94e-05 ） 4.5 4 200 SFS1 pop.-SFS2 pop. 4 SFS2 pop.-NFS pop. 3.5 150 3 2.5 2 100 1.5 SFS1 pop. 50 1 SFS2 pop. 0.5 NFS pop. Effective population size (x10 0 Frequency of ortholog gene 0 3 4 5 10 10 10 0 2e−05 4e−05 6e−05 8e−05 1e−04 -9 Years (g=1,u=6.5x10 ) Ks

Fig. 1. Population history of wild emmer accessions in EC-I. (A) The geographic distribution of the emmer wheat accessions used in this study. The green circles represent the collection location outside EC-I. The red circle represents the location of EC-I. The arrows represent the collection stations on each slope. (B) of 168 wild emmer wheat accessions by neighbor-joining method with 1,000 bootstraps. The arabic notation following the collection location in Israel is shown in the map. (C) Inferred historical population sizes by pairwise sequential Markovian coalescent (PSMC) analysis. Three typical samples from each population were shown. The lower x axis gives time measured by pairwise sequence divergence, and the y axis gives the effective population size measured by the scaled rate. (D) Distribution of Ks values for 12,352 one-to-one orthologous gene sets between each population. The full-length transcripts from one representative sample from each population (SFS1-7, SFS2-14, and NFS-8) was employed for Ks evaluation.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1920415117 Wang et al. Downloaded by guest on September 27, 2021 the population structure of the materials we collected from EC-I analyses based on the data from the 55K Affymetrix Wheat (Fig. 1A and Datasets S1). We first determined which of the GeneChip: e.g., the SFS1, SFS2, and NFS populations sequen- SNPs present on the 55K Affymetrix Wheat GeneChip would be tially diverged and then further diversified (SI Appendix, Figs. S4 suitable for analysis of these wild emmer wheat accessions using and S5). In order to reconstruct the population history of these BLAST analysis of the chip probe sequences against the emmer three lineages, haploid pairwise sequential Markovian coalescent reference genome; a total of 30.6K SNPs were suitable for use (PMSC) analysis was conducted to infer the branching times and (e.g., SNPs having no more than two hits in the A and B sub- inbreeding effective population size (Ne) of the three pop- genomes). These SNPs were used to construct a neighbor-joining ulations of accessions collected from the EC-I microsite (17). phylogenetic tree (Fig. 1B). This analysis revealed that, consistent with the phylogeny, these Most of the wild emmer accessions collected from Israel were three populations have branched from the same ancestral pop- clustered together in the tree, and the accessions collected from ulation (Fig. 1C). Turkey, Syria, and other locations likewise clustered together Furthermore, each of the three populations apparently expe- (Fig. 1 A and B). The terminal taxa of the phylogenetic tree rienced the same bottleneck recently (∼3,000 to 6,000 y ago if include three distinct subpopulations among the accessions col- using substitution rate for rice) (18). The pronounced contem- lected at EC-I: two populations from the SFS locations (“SFS1” poraneity of the ancestral population bottleneck and the pop- and “SFS2”) and one from the NFS location (“NFS”). These ulation splits suggests that genetic drift induced by a population three populations are all monophyletic, and were resolved as bottleneck or founder effect was likely a strong influence on the sister relationships that are more similar to each other than to initial divergence of these three populations at EC-I (19). After the other emmer accessions from surrounding areas (Fig. 1B). these splits in the wild emmer populations at EC-I, all three All of the accessions collected from NFS and SFS clustered only populations gradually recovered Ne, albeit to differing extents, their own populations in the phylogeny, without any migration or with the SFS1 population eventually having the largest pop- radiation, thus clearly indicating apparent reproductive isolation ulation size and the SFS2 population having the smallest (Fig. 1C and adaptive divergence of these populations primarily based on and SI Appendix, Table S2). the local environment (Fig. 1 A and B), which is important to To validate the divergence times inferred from the PMSC assess sympatric speciation. results, we used an alternative method of comparing the synon- To estimate the levels of shared ancestry of each population in ymous substitution values (Ks) of orthologous genes. Total RNA EC-I, we subjected the SNP data to an “admixture” model based from three accessions (SFS1-7, SFS2-14, and NFS-8) represen- on a maximum-likelihood method. When the K values were in- tative of each population was extracted and analyzed using both creased from 2 to 10 (optimal K value), the NFS, SFS2, and SFS1 the PacBio Sequel platform for single-molecule sequencing and EVOLUTION populations were sequentially separated from other populations, the Illumina HiSeq XTen platform for short reads. After iden- apparently suggesting the lineage splits of the SFS1, SFS2, and tifying the full-length reads from the PacBio data, the high-depth NFS populations (SI Appendix, Fig. S1). Similar results were also coverage (>100×) short read data were used for SNP correction obtained from analysis using the “fastStructure” model (SI Ap- (Materials and Methods). After mapping to the emmer reference pendix, Fig. S1). genome, a total of 12,352 one-to-one orthologous gene pairs with To further define the three populations within EC-I deriving high specificity were employed in the Ks and Ka analyses using from ancestral introgression or incomplete lineage sorting, we model averaging (MA) (20) (SI Appendix, Fig. S6). The SFS1 performed multiple iterations of ABBA-BABA D-statistic tests population and SFS2 population were estimated to have di- using the GeneChip data (16). When using the SFS1 population verged first (Ks = 4.94E-05), around 4,800 y ago based on the as Pop. 3 and the populations from Bat Shlomo or other areas in nucleotide substitution rate of rice (18); the divergence of the Israel as outgroups (SI Appendix, Table S1), no significant gene SFS2 population and the NFS population was estimated as flow or introgression was detected between the SFS1 population slightly later (Ks = 4.16E-05; Fig. 1D). These results support the and the NFS or SFS2 populations. Similarly, we did not detect estimates from the phylogenetic and PMSC analyses indicating any gene flow between the SFS1 population and the populations that the branching of these populations of wild emmer wheat in from surrounding areas (SI Appendix, Table S1). These results EC-I occurred recently. are in agreement with the phylogenetic relationship and the admixture analyses (Fig. 1B and SI Appendix, Fig. S1), support- The SFS2 and NFS Populations Display Prezygotic Reproductive ing independent adaptive branching of each population within Isolation, whereas the SFS1 Population Displays Postzygotic Barriers. EC-I rather than admixture or secondary introduction. To evaluate the level of genetic differentiation among populations, To further assess the and evolutionary history the population fixation statistics (Fst) were examined using the of the wild emmer population represented by the accessions same whole-genome SNP data set (Fig. 2A and SI Appendix, Fig. collected at EC-I, 16 accessions from the NFS population, 9 S7). Very high Fst values (>0.46) were distributed across the accessions from the SFS1 population, and 10 accessions from the whole genome, especially between the SFS2 population and NFS SFS2 population were subjected to whole-genome sequencing, populations, reflecting a large differentiation among the three with an average sequencing depth of 9.45 to 18.49×. The map- populations in EC-I (Fig. 2A and SI Appendix, Fig. S7 and Table ping rate for the different accessions was over 98%, thus cov- S3). Lower divergence was found around regions of heterochro- ering 89.29 to 92.89% of the 10.1-gigabase (Gb) assembly of the matin in several such as 3A, 6A, and 7B (Fig. 2A), emmer reference genome “Zavitan” (Datasets S2). After vali- but the average Fsts between each population still reached 0.468 dation by BQSR (base quality score recalibration) and VQSR for SFS1/SFS2, 0.479 for SFS1/NFS, and 0.634 for SFS2/NFS (SI (variant quality score recalibration), a total of 46,301,637 high- Appendix, Fig. S7 and Table S3), values much higher than in quality SNPs were identified and used in subsequent analyses previously studied emmer wheat populations (21). Note that lower (Materials and Methods provides characteristics of the genomic genetic diversity was found for the SFS2 population (π = 0.36 × − − distribution of these SNPs; SI Appendix, Fig. S2). The average 10 3) and NFS population (π = 0.52 × 10 3) as compared to the − SNP density along the genomes of these accessions was 4.36 SFS1 population (π = 0.98 × 10 3), findings consistent with the SNP/Kb, with 4.02 SNP/Kb for the A subgenome and 5.05 SNP/ aforementioned Ne estimates (Fig. 1C and SI Appendix,Fig.S7 Kb for the B subgenome (SI Appendix, Figs. S2 and S3). and Table S2). These SNPs were used to generate a phylogenetic tree and to We did not detect any apparent gene flow between each pop- conduct a population structure admixture and PCA analysis. This ulation using a TreeMix analysis based on a maximum-likelihood revealed similar results to those generated by the corresponding tree (SI Appendix,Fig.S8), similar to results from D-statistic

Wang et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 27, 2021 Chromosomal Rearrangements in the SFS1 Population Caused the Postzygotic Reproductive Isolation. Motivated by the unantici- pated finding that the SFS1 and SFS2 populations have in situ diversified as postzygotically isolated populations on SFS, a small area located within 30 m, we subsequently attempted to unravel the underlying mechanism using a cytogenetics approach. The karyotypic variation via fluorescence in situ hybridization (FISH) was examined for the emmer wheat accessions in this study using four probes [Oligo-pSc119.2, Oligo-pTa535, Oligo-(GAA)7, and Oligo-3A1; Materials and Methods]. The banding pattern gener- ated by these four probes allows identification of all individual chromosomes of hexaploid wheat (22), and here the emmer wheat showed similar banding patterns with the hexaploid wheat cultivar “Chinese Spring” (SI Appendix, Fig. S9 A and B), e.g., the Oligo-pSc119.2 shows a single band on the short arm of Chr. 5A (SI Appendix, Fig. S9B, green). Thus, the diagram of signal pattern was generated for identifying chromosomal polymor- phisms of the wild emmer accessions (SI Appendix, Fig. S9C). Three atypical banding patterns were observed in the SFS1 population, findings which suggested the possible occurrence of Robertsonian translocations that were not present in the SFS2 or NFS populations (Fig. 3A and SI Appendix, Figs. S10 and S11). Based on these FISH signals, we determined that some SFS1 individuals have experienced chromosomal breakage and refusion of chromosomes 2A and 5A, thereby forming two new linkage groups: 2AS.5AS/2AL.5AL (Fig. 3A). We also observed other translocations in the SFS1 population, including1BS.4BS/1BL.4BL and 2BS.3BL/2BL.3BS (Fig. 3A and SI Appendix,Fig.S11). All of the translocation events were homozygotes (SI Appen- Fig. 2. Genetic differentiation of wild emmer populations in EC-I. (A)Genetic dix, Fig. S10), indicating they have become stable during evolu- differentiation revealed by Fst across genome window between each pop- tion. A pattern of various genotypes with/without translocations ulation. (B) Progeny ratio of F1 hybrids between accessions of SFS1 population exists in the SFS1 population (Fig. 3B). First, none of the Robertsonian and the other two populations. A 100% ratio of progeny was supposed as the seed setting rate of three progeny seeds per spikelet, and more than 10 spikes translocations were identified for the SFS1-1, SFS1-2, and per sample were employed for seed setting rate calculation. SFS1-3 individuals, which show relatively ancestral positions on the phylogeny. Other structural changes of the genome could not be ruled out, as some pollen mother cells (8 to 12%) analyses using the GeneChip data (SI Appendix,TableS1). The of the hybrid (crosses with NFS or SFS2 individuals) showed genetic drift parameter for the SFS2 population and the NFS disordered bridges during meiosis anaphase I (Fig. 3 B, c1). population is higher (>0.2). This is consistent with their relatively Second, three genotypes with one or two translocation events small Ne, indicating that genetic drift has contributed to the fast were found in independent phylogenetic lineages. The lineages of diversification of these two populations (SI Appendix,Fig.S8and SFS1-4 and SFS1-5 only involve the 1BS.4BS/1BL.4BL trans- Fig. 1C). Consistent with the phylogeographic and phylogenetic location. In addition to this translocation, the lineages of SFS1-6 and SFS1-7 also harbor the 2BS.3BL/2BL.3BS translocation, and relationships, the results of Fst and TreeMix indicate that pre- the lineages of SFS1-8 and SFS1-9 harbor 2AS.5AS/2AL.5AL (Fig. zygotic reproductive isolation has developed between each pop- 3B). These results suggest that the 1BS.4BS/1BL.4BL trans- ulation, blocking gene flow among populations. location was the initial translocation type in the SFS1 population, To test whether any postzygotic reproductive isolation has after which the other two translocations evolved. developed, we selected several individuals from each population, In crosses with the normal chromosome accessions of the NFS conducted crosses with interpopulation materials, and monitored or SFS2 populations, we indeed found severe meiotic disorders the fertility of progeny. Hybrids between individuals from the in the hybrids, mainly tetravalent status at metaphase I, reflect- SFS2 and NFS populations produced normal fertile progeny ing a typical Robertsonian translocation (23). Consistent with the (Datasets S3). However, large-scale hybrid sterility was found FISH results, single tetravalent status (26 to 31%) in pollen when crossing SFS1 individuals with SFS2 individuals and with mother cells was observed in the SFS1-4 and SFS1-5 individ- NFS individuals. For self-pollination, all nine individuals from uals that harbor the 1BS.4BS/1BL.4BL translocation, while the SFS1 population produced a high ratio of progeny (here double tetravalent status (9 to 16%) was observed for the indi- defining “high” as 70%, as emmer wheat can generally produce viduals bearing two translocation events (1BS.4BS/1BL.4BL and two seeds per spikelet, presuming three seeds per spikelet is 2BS.3BL/2BL.3BS; Fig. 3 B and C). We did not find abnormal 100%; Fig. 2B). However, interpopulation hybrids from the SFS1 pairing or division during meiosis in hybrids from crosses be- individuals (SFS1-1, SFS1-2, and SFS1-3) only produced a ∼30 tween the SFS2 and NFS individuals, which all showed equal metaphase I division status (Fig. 3, c ). to 50% ratio of progeny when crossed with SFS2 and NFS in- 2 Thus, we have clear cytogenetic evidence that three Robertsonian dividuals (Fig. 2B). Further, the hybrids from SFS1-4 and SFS1-5 translocation events have evolved sequentially in the SFS1 pop- produced about a 20% ratio of progeny, and the hybrids from ulation. Moreover, we demonstrate that these homologous chro- ∼ SFS1-6 to SFS1-9 only produced a 2 to 15% ratio of progeny in mosome rearrangements gained in the course of evolution cause crosses with SFS2 and NFS individuals (Fig. 2B). The much re- meiotic disorders in hybrids from crosses between SFS1 individ- duced fertility of these hybrids shows that postzygotic isolation uals with SFS2 and NFS individuals. We also showed that the has formed between the SFS1 population and the other pop- extent of such meiotic disorders is elevated for hybrids from ulations, supporting the inference of SS of the SFS1 population. crosses with SFS1 individuals harboring multiple chromosome

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1920415117 Wang et al. Downloaded by guest on September 27, 2021 Fig. 3. Chromosome rearrangements in SFS1 population. (A) The representative for three typical Robertsonian translocations by FISH. (B) The pattern ge- EVOLUTION nome rearrangements for different individuals in SFS1 population. The paring and division events during meiosis of F1 are shown in the table as percentages. (C) The representative of paring and division events during meiosis of F1. The abnormal paring or division is shown by arrows.

rearrangement events. Collectively, these results establish a plau- EC-I in SFS-mimic conditions (with relatively mild abiotic stress sible cellular mechanism to explain the postzygotic isolation of to avoid excessive pressure on the NFS population; Materials and hybrid sterility we observed (Fig. 2B and SI Appendix, Table S3). Methods). The SFS2 population exhibited much earlier flowering times than the SFS1 population; the SFS2 plants also flowered Disruptive Ecological Selection Has Driven the Discrete earlier than NFS plants (Fig. 4A and SI Appendix, Fig. S13). of the SFS1, SFS2, and NFS Populations. To unravel the evolutionary These findings suggest that the SFS2 population has evolved an mechanism(s) underlying the three diverged populations in EC-I, early flowering strategy to cope with the latter harsh abiotic especially for the sympatric speciation of SFS1 at the site based stress of its environment on the south-facing slope. on chromosome rearrangements, we further examined the ad- The relatively late flowering status of the SFS1 population is aptation strategies of these populations of emmer wheat. Re- puzzling, and is apparently inconsistent with the SFS2-type ad- lating directly to the ecology of the humid NFS, all accessions in aptation to its local environment. We thus checked the SNP the NFS population displayed high resistance to most of the variation of FT1 loci, 34 genes of the CONSTANTS-like family, tested powdery mildew (Blumeria graminis f. sp. tritici) isolates and 10 PRR genes that have been reported to function in plant and to three leaf rust (Puccinia triticina) isolates, while most flowering regulation. An SNP in the CCT domain of the accessions from the SFS1 and SFS2 populations were highly CONSTANS-like gene CO6 (TRIDC7BG063900) had an amino susceptible to the tested pathogen isolates (Fig. 4A and SI Ap- acid change from Lys to Glu at position 321 in the SFS1 population; pendix, Fig. S12A). There were two patterns of powdery mildew this mutation was not detected in any non-SFS1 emmer acces- resistance within the NFS population (SI Appendix, Fig. S12A), sions in our study (SI Appendix, Fig. S14 A and B). CONSTANS indicating for resistant genes within the NFS can activate the transcription of the FT1 gene by binding to the population. CORE element in its promoter (25), and we confirmed a We constructed F2:3 populations by crossing the susceptible flowering-promoting function for CO6 in vivo: three indepen- parent of SFS2-12 with resistant parents of NFS-10 and NFS-2 dent transgenic Brachypodium lines overexpressing emmer CO6 independently. For NFS-10, representing resistance pattern 1 (SI flowered significantly earlier than wild type plants (SI Appendix, Appendix, Fig. S12A), the F2 populations displayed a segregation Fig. S14 F and G). ratio of observed number, 3:1, showing qualitative regulation by Biochemically, both yeast one-hybrid and gel-shift assays a single resistance gene. Bulked segregant RNA sequencing revealed that CO6 (321Lys) from the SFS2 and NFS populations (BSR-seq) revealed a single strong peak on the terminus of the can strongly bind to this CORE element (SI Appendix, Fig. S14 Chr. 4A long arm (SI Appendix, Fig. S12B), a newly evolved C–E), but that CO6 (321Glu) from the SFS1 population had resistance locus to our knowledge. However, the resistance sig- substantially reduced binding activity to the CORE element (SI nals found for the NFS-2 individual (representing pattern 2) had Appendix, Fig. S14D). Moreover, the binding affinity was re- multiple weaker peaks in BSR-seq analysis (SI Appendix,Fig.S12C), covered when position 321 of the SFS1 CO6 protein was mutated indicating multiple quantitative resistance loci. back to Lys (SI Appendix, Fig. S14D). Thus, we hypothesized that Ecological studies of EC-I have reported early flowering the 321Glu mutation of the CO6 gene can at least partially ac- for many plants on the SFS, with some SFS emmer count for the differences in flowering times between the SFS2 wheat plants showing flowering 1 to 2 mo earlier than those from and SFS1 populations that encounter the similar abiotic stress the NFS (6, 24). We grew all of the collected accessions from conditions of the SFS of EC-I.

Wang et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 27, 2021 AC NFS pop. SFS2 pop. SFS1 pop. Disease resistance Irradiance tolerance 1150 Early flowering biological process 1130 w

e cellular component ld 100 ** molecular function

ery mi 50 *** * Number of genes 0

nent regionvirion lar virion part heme binding kinase activity ion channel activitytetrapyrrole bindingperoxidaseantioxidant activity immuneactivity responsecellularextracellu compo adenylate nucleotidekinase activity kinase oxidoreductaseactivity activity phosphotransferase activity ubiquitin ligase complex responseactivation to oxidative of immune stress response anaphase promoting complex D D D D1 2 PRX-SFS1-Ox-1 PRX-SFS1-Ox-2 U/g ) PRX-SFS1-Ox-2 nce tolerance Flowering time Powd Prx activity Irradia PRX-SFS1-Ox B D D D

PRX-SFS1-Ox-1 PRX-SFS1-Ox-2 PRX-SFS1-Ox-2 XP-CLR content (umol/g )

Top 1 - Top 5 2 PRX-SFS1-Ox O

Fig. 4. Distinct adaptation mechanisms of wild emmer populations in EC-I. (A) Representative photograph of phenotypic variance of accessions from each population. (B) The distribution of the XP-CLR score along 14 chromosomes with 20-Kb sliding window. The genome-wide threshold was defined both by the top 5% and 1%, as marked in black dotted lines. (C) GO terms enriched by candidate regions selected in the SFS1 population. The top 20 terms with the most significant enrichment are shown. The GO items involved with antioxidant pathway are noted with asterisk. (D) of transgenic wheat over- expressing TaPRX1 under irradiance condition. The seedlings were subjected to high radiation with 30,000 Lux light for 1 wk and then photographed.

The SFS1 populations’ flowering phenotypes are apparently We detected that a total of 30 general ROS scavengers of PRX inconsistent with efficient adaptation to the local environment of encoding loci in the SFS1 population exhibited elevated selec- the SFS, but we found that the SFS1 population has evolved an tion signals (Datasets S5), in which nine PRX genes showed fixed alternative strategy for mitigating the harsh abiotic conditions: nonsynonymous in the SFS1 population (Datasets S6). higher tolerance to irradiance. We did not find significant differ- We then cloned the for these genes from the SFS1 and ences in drought tolerance among the three EC-I emmer pop- SFS2 populations and expressed them in fission yeast (Materials ulations when growing plants at 20 °C with a 5,000-Lux light at the and Methods). In vitro assays using the same concentrations of seedling stage (data not shown). The SFS1 plants had significantly the recombinant proteins indicated that three SFS1 PRX pro- higher tolerance when the light was elevated to 30,000 Lux, teins, encoded by TRIDC2AG012890, TRIDC7AG049310, and exhibiting reduced oxidative injury on leaves and consistently re- especially TRIDC5AG058230 (here named PRX-SFS1), showed − duced levels of superoxide radicals (O2 ) than the accessions from significantly improved oxidative activity for ABTS substrates, other populations (Fig. 4A and SI Appendix,Fig.S15), indicating suggesting that the SFS1 PRX-SFS1 has evolved improved an example of unique adaptive evolution for increased antioxidant ROS scavenging capacity compared to the SFS2 allele (SI Ap- activity. This result is in agreement with the fact that there is an pendix, Fig. S16). To test whether this gene is involved in oxidative average of 200 to 800% higher radiation on the SFS than that on stress tolerance in vivo, we overexpressed PRX-SFS1 in wheat the NFS (10). using Agrobacterium-mediated transformation and analyzed three To identify potential selective signals underlying this change, independent T3 lines in light stress conditions. Under light treat- we scanned genomic regions by comparing the SFS1 population ment at 30,000 Lux, the transgenic lines displayed apparently re- and the other two populations using a likelihood method (XP- duced leaf injury compared to wild type plants. We also detected CLR) (26). To lessen the problem of fixation due to genetic drift, significantly higher peroxidase activity and reduced superoxide − we pooled the populations of SFS2 and NFS and conducted radical (O2 ) content in the transgenic plants. selection analyses in comparison with the SFS1 population. With Collectively, these results clearly illustrate that the three the cutoff value of 5%, we subjected the genes in selective populations in EC-I have undergone distinct evolutionary trajec- sweeps to Gene Ontology (GO) enrichment analyses and found tories in their respective adaptations to the local environment that the selective genomic regions were extremely enriched for (disease resistance for the NFS population, early flowering for the genes with annotations relating to oxidative stress tolerance (Fig. SFS2 population, high irradiance tolerance for the SFS1 pop- 4 B and C and Datasets S4), such as the members of cytochrome ulation). These adaptive differences support the driving roles of P450 and peroxidases (PRX) (27) (Datasets S5), further sup- disruptive ecological selection in the branching and diversification porting the involvement of antioxidant pathways in the evolution of each population (Fig. 5). For the sympatric diversification of the of the SFS1 population. SFS1 and the SFS2 populations, the difference in flowering time

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1920415117 Wang et al. Downloaded by guest on September 27, 2021 NFS SFS populations (Fig. 3 B and C). We thus conclusively demonstrate a Biotic stress Abiotic stress case of primary SS and characterize molecular events explaining 3x103 the reproductive isolation that has enabled the origin of separate emmer wheat species at the same EC-I microsite.

• Late flowering Disruptive Ecological Selection Drives Sympatric Speciation of Wild

Gene 1 Emmer Wheat at EC-I. Theoretical models for SS often include 2x103 disruptive speciation mechanisms such as divergent habitat – Disease resistance preference, resource partitioning, and/or assortative mating (3 6, • – • Irradiance tolerance 12, 13, 29 31). Several animal cases, like African indigobirds, • Early flowering maggots, and fish, indicate that such diversifying Gene 2 – 3 • Chr. rearrangement selection-causing sympatric speciation models are plausible (5 9). 10 However, in sessile plants, which are stressed constantly by a di- Gene 3  Reproductive isolation versity of biotic and abiotic stresses and which cannot escape to refugia like animals, mechanistic evidence is lacking for ecolog- ical SS (9, 24). In the present study, our haploid pairwise se- 0 (yr) quential Markovian coalescent (PMSC) analysis clearly indicated NFS population SFS2 population SFS1 population that the branching of the SFS1, SFS2, and NFS populations Fig. 5. Sympatric model of wild emmer wheat at EC-I. occurred soon after the bottleneck of an ancestral population The blue dotted line indicates the extent of genetic drift. The red full lines less than 10,000 y ago (Fig. 1C), suggesting that genetic drift indicate postzygotic reproductive isolation, and the red dotted line indicates induced by a founder effect might have been involved in the prezygotic reproductive isolation. The yellow five-pointed stars indicate initial split of these three populations at EC-I (19). With rela- powdery mildew-resistant genes evolved, and the orange one indicates strip tively small population sizes (SI Appendix,Fig.S7), genetic drift rust-resistant gene. was involved in the further diversification of these populations (SI Appendix,Fig.S8), and may partially account for the rapid differentiation of genetic pool frequencies and geno- should have been the major prezygotic barrier against gene flow. mic components of each population at EC-I (SI Appendix,Fig. However, the accumulation of mutations in each population, es- S1) (32).

pecially the series of chromosome rearrangements that have oc- However, even with genetic drift, our data indicate that dis- EVOLUTION curred among SFS1 plants, have resulted in the final postzygotic ruptive ecological selection has driven wild emmer wheat to di- reproductive isolation of SFS1 from the other EC-I populations. It versify as reproductively isolated populations, adaptive to the is noted that the independent clustering in phylogeny, large ge- contrasting abiotic (SFS) and biotic (NFS) stresses at the EC-I nomic differentiation, lack of gene flow, and distinct adaptive microsite. Remarkably, on the same slope, the SFS2 population strategies for the NFS and SFS2 populations all suggest that they has adapted to the strong irradiance, heat, and drought by are prezygotically isolated in the local environment at EC-I, in- temporally advancing its reproductive development to occur dicating incipient sympatric speciation. during a time window with relatively weaker abiotic stress (Fig. 4A and SI Appendix, Fig. S13). The SFS1 population has, in Discussion contrast, developed much stronger irradiance tolerance over the Sympatric Speciation of Wild Emmer Wheat at EC-I. In plants, clear past several thousand years (Fig. 4A and SI Appendix, Fig. S15), evidence supporting sympatrically split species with reproductive with relatively late flowering (Fig. 4A and SI Appendix, Fig. S13). isolation and identifying the underlying mechanisms is still lim- On the opposite slope, the NFS population evolved pronounced ited. Here, we present evidence that wild emmer wheat, the resistance to plant fungal pathogens that thrive in humid con- progenitor of tetraploid and hexaploid wheats, is incipiently ditions (Fig. 4A and SI Appendix, Fig. S12). Thus, both the data sympatrically speciating at EC-I, a hot spot microsite where SS in this study and the previously identified cases of incipient SS from bacteria to mammals has been demonstrated (28). Beyond at EC sites repeatedly confirm that divergent selection can simply identifying interslope diversification, our data clearly overrule gene flow and genetic drift to drive a metapopulation support also the split of two postzygotically isolated populations of organisms into distinct isolated populations undergoing SS (SFS1 and SFS2) on the same tropical hot south-facing slope (6, 11–14, 33, 34). (SFS), within a transect of less than 30 m, beside a third incipient speciation on the opposite NSF. We successfully ruled out the The Evolution of Reproductive Isolation in Wild Emmer Wheat. One possibility of or introgression using genetic major question about sympatric speciation is understanding how diversity data from wild emmer wheat accessions collected from reproductive isolation has arisen in sympatry. In this study, the the EC-I microsite, its surrounding areas across Israel, and sur- fact that a series of sequentially formed chromosome rear- rounding countries, which represent the origin (North Israel) and rangements in the SFS1 population, which resulted in postzygotic distribution region of wild emmer wheat in the Near East Fertile barriers, helps rule out the possibility of secondary contact (Fig. Crescent (Fig. 1 A and B). All our phylogeographic, phylogenetic, 3B). It is highly unlikely that a genetically identical but different admixture, D-statistic, and PMSC analyses (Figs. 1 and 2 and SI Robertsonian translocation could simultaneously have migrated to Appendix, Fig. S1 and Table S1) provide evidence that the SFS1, EC-I from surrounding areas. Indeed, similar patterns of these SFS2, and NFS populations at the EC-I microsite in Mount chromosome rearrangements were not found for the accessions Carmel have diverged sequentially from a single ancestral pop- outside EC-I in the present study (data not shown) or in previous ulation, following a population bottleneck that occurred less than studies that screened karyotypes of wild emmer populations from 10,000 y ago (Fig. 1 C and D). the areas or countries surrounding EC-I (35, 36). In addition to prezygotic isolation detected between any of the For the chromosome rearrangements, an intriguing possibility three diverged populations at EC-I, postzygotic barriers by chro- is that the particular adaptive strategy of the SFS1 population mosomal rearrangements of Robertsonian translocations were may have facilitated the evolution of postzygotic reproductive unique to the SFS1 population (Fig. 3 A and B and SI Appendix, isolation. UV radiation is widely recognized as a major cause of Figs. S9–S11). This resulted in a large extent of hybrid steril- chromosome disruption (27). This is supported by the evidence ity when crossing was conducted with the individuals from other that the genes in the SFS1 accessions exhibiting selection signals

Wang et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 27, 2021 are particularly enriched for antioxidant activity (Fig. 4 B–D), e.g., selected three samples from each population with the highest depth to modified activity of peroxidases (Fig. 4D and SI Appendix, Fig. represent each population. Genotype calls for each position were made S15), highlighting that the SFS1 population has been subjected to using the SAMtools (v1.19) mpileup command, filtering for reads with strong selection pressure due to the high irradiance stress char- minimum base and mapping quality scores of 30. Occasional heterozygous acterizing its site positioned below a cliff (station 1), which accepts sites were dealt with by randomly sampling one allele. Population di- vergence time and effective population sizes were estimated by PSMC with a the highest irradiance at EC-I (SI Appendix,Fig.S17)(10). − mutation rate of 6.5 × 10 9 substitutions per site per year and a generation Extensive mathematical modeling (4, 29, 30, 37) and experi- time of one generation per year. mental studies, including those from the Evolution Canyon model (28), have provided evidence that evolutionary branching in situ Genome-Wide Selective Sweep Analysis. In order to identify selective sweeps in followed by sympatric speciation is not only feasible, but may be a the SFS1 population, we combined irradiance-intolerant populations (SFS1 and common model of the origin of species, substantiating Darwin’s NFS) in a single group to exclude the potential effect of genetic drift. We used a insight of SS. This is plausible since geologically, edaphically, cli- likelihood method (the cross-population composite likelihood ratio, XP-CLR) matically, and biotically divergent canyons abound across our with the following parameter: –w1 0.02 200 10000 –p0 0.95. To run XP-CLR, planet (28). Nevertheless, more evidence for completed SS with we assigned all SNPs to genetic positions based on 0.18 cM/Mb for the A and B reproductive isolation is desirable, since the frequency and po- subgenomes (26). Windows with the top 5% XP-CLR values were identified. tential universality of the SS model is still debatable (3). Our For candidate genes, Gene Ontology (GO) enrichment analysis was performed < < present study presents long-sought and unambiguously clear using the R program with the default parameters (P 0.05 and FDR 0.05). mechanistic evidence that in situ sympatric speciation has oc- curred among the wild emmer plants—the progenitor for culti- Cytogenetic Analysis. Seeds were germinated on moist filter paper in the dark at room temperature until the roots grew to 1.5 to 2.0 cm in length. The vated wheat—not only interslope but also intraslope in the SFS of excised root tips were then treated as described before (42). The synthesized Evolution Canyon. oligonucleotide probes Oligo-pSc119.2, Oligo-pTa535, and Oligo-(GAA)7 (Invitrogen Biotechnology) were 5′ end-labeled with 6-carboxyfluorescein Materials and Methods (6-FAM) for green or 6-carboxytetramethylrhodamine (Tamra) for red sig- Phylogenetic Tree and Population Structure Analyses. A total of 1,364,793 SNPs nals. Probe Oligo-3A1 (FAM-5′-AATAATTTTACACTAGAGTTGAACTAGCTCTA- in coding regions (exonic and intronic) were used for TAAGCTAGTTCA-3′) was used to distinguish signal size and location differ- analysis. To clarify the phylogenetic relationships from a genome-wide ences in chromosomes 3A and 7A. The probe solution (20 ng/μLin2× SSC perspective, an individual-based neighbor-joining tree was constructed using and 1× TE buffer, pH 7.0) was denatured for 5 min in boiling water and then the p-distance as implemented in TreeBeST (v1.9.2), with bootstrap values placed on ice. A 6-μL probe solution was used for each slide. The hybrid- determined from 1,000 replicates (38). The population genetic structure was ization was performed at 37 °C overnight in a humid chamber. After hy- examined using the program ADMIXTURE (v1.23) (39). A total of 35 accessions bridization, the slides were washed using 2× SSC and then mounted with were used to estimate the genetic ancestry, specifying a K ranging from 2 to 3. a Vectashield mounting medium containing 1.5 μg/mL 4,6-diamidino-2- A principal component analysis was also conducted to evaluate the genetic phenylindole (DAPI; Vector Laboratories). Images were captured with an structure of the populations using GCTA (40). First, a genetic relationship Olympus BX-51 microscope equipped with a DP-70 CCD camera. Particular matrix was obtained using the parameter “–make-grm”; then, the top three chromosomes were identified by comparing the signal pattern of probes principal components were estimated with “–pca3”. hybridized to hexaploid wheat according to ref. 22. Detection of Gene Flow Using TreeMix. Population relatedness and migration Data and Material Availability. The resequencing and SNP data generated in events were inferred using TreeMix (41). The 1,364,793 coding-region SNPs this work are deposited in the Genome Sequence Archive in the BIG Data were used to build a maximum-likelihood tree, with a window size of 2,000 SNPs. This was repeated 10 times. The tree with the lowest SE for the re- Center, Beijing Institute of Genomics, Chinese Academy of Sciences, under siduals was selected as the base tree topology. The population pairs with an the accession no. CRA001873. above-zero standard residual error were identified as candidates for admix- ture events, representing populations that the data indicate are more closely ACKNOWLEDGMENTS. This work was supported by National Natural related to each other than is demonstrated in the best-fit tree (41). TreeMix Science Foundation of China Grants (31520103911 and 31871610), National Key Research and Development Program Grant (2016YFD0100102-2), Trans- was then run using between one and six introduced migration events. When genic Special Item of China Grants (2016ZX08002003-002 and 2016ZX08009- three migration events were added, the residuals were much lower than for 003), Agricultural Variety Improvement Project of Shandong Province Grant the trees generated using other numbers of migration events. (2019LZGC010), and Shandong Provincial Natural Science Foundation Grant (ZR2017MC016). E.N. thanks the Ancell-Teicher Research Foundation of Ge- PSMC Analysis. PSMC was used to analyze the evolutionary demographic netics and for financial support of the Evolution Canyon changes of wild emmer wheat in “Evolution Canyon” (Mt. Carmel, Israel). We model.

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