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Pleistocene climatic oscillations and the speciation history of an alpine endemic and a widespread arctic‐alpine plant

2012; Wiley; Volume: 194; Issue: 2 Linguagem: Inglês

10.1111/j.1469-8137.2012.04061.x

1469-8137

Hajíme Ikeda, Tor Carlsen, Noriyuki Fujii, Christian Brochmann, Hiroaki Setoguchi,

Lichen and fungal ecology

New PhytologistVolume 194, Issue 2 p. 583-594 Full paperFree Access Pleistocene climatic oscillations and the speciation history of an alpine endemic and a widespread arctic-alpine plant Correction(s) for this article Corrigendum Volume 210Issue 4New Phytologist pages: 1479-1479 First Published online: March 15, 2016 Hajime Ikeda, Hajime Ikeda Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba 305-0005, Ibaraki, JapanSearch for more papers by this authorTor Carlsen, Tor Carlsen Microbial Evolution Research Group, Department of Biology, University of Oslo, PO Box 1066 Blindern, NO-0316 Oslo, NorwaySearch for more papers by this authorNoriyuki Fujii, Noriyuki Fujii Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto, Kumamoto 860-8555, JapanSearch for more papers by this authorChristian Brochmann, Christian Brochmann National Centre for Biosystematics, Natural History Museum, University of Oslo, PO Box 1172 Blindern, NO-0318 Oslo, NorwaySearch for more papers by this authorHiroaki Setoguchi, Hiroaki Setoguchi Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto 606-8501, JapanSearch for more papers by this author Hajime Ikeda, Hajime Ikeda Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba 305-0005, Ibaraki, JapanSearch for more papers by this authorTor Carlsen, Tor Carlsen Microbial Evolution Research Group, Department of Biology, University of Oslo, PO Box 1066 Blindern, NO-0316 Oslo, NorwaySearch for more papers by this authorNoriyuki Fujii, Noriyuki Fujii Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto, Kumamoto 860-8555, JapanSearch for more papers by this authorChristian Brochmann, Christian Brochmann National Centre for Biosystematics, Natural History Museum, University of Oslo, PO Box 1172 Blindern, NO-0318 Oslo, NorwaySearch for more papers by this authorHiroaki Setoguchi, Hiroaki Setoguchi Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto 606-8501, JapanSearch for more papers by this author First published: 13 February 2012 https://doi.org/10.1111/j.1469-8137.2012.04061.xCitations: 25 Author for correspondence: Hajime Ikeda Tel: +81 29 853 8459 Email: ike@kahaku.go.jp AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary • Even in cases in which geographic isolation appears to have driven the speciation of regional endemics, range shifts during the Pleistocene climatic oscillations may also have influenced their evolutionary history. Elucidating speciation history can provide novel insights into evolutionary dynamics following climatic oscillations. • We demonstrated a sister relationship between the Japanese alpine endemic Cardamine nipponica and the currently allopatric, widespread arctic-alpine Cardamine bellidifolia (Brassicaceae) based on internal transcribed spacer (ITS) sequences and 10 other nuclear genes. Speciation history was inferred using demographic parameters under the isolation with migration model. • The estimated demographic parameters showed that the population size of C. nipponica was similar to that of C. bellidifolia and that gene flow occurred exclusively from C. nipponica to C. bellidifolia after speciation. • The inferred speciation history, which included gene flow, suggests that geographic barriers between the peripheral C. nipponica and the widespread C. bellidifolia were reduced during the Pleistocene. The asymmetric introgression implies that genetic isolation may have been involved in the speciation of C. nipponica. Our results suggest that even currently allopatric species may not have diverged solely under geographic isolation, and that their evolutionary history may have been influenced by Pleistocene range dynamics. Introduction Geographic isolation reduces opportunities for gene flow within a species. Therefore, genetic differences accumulate across the species’ range and may lead to allopatric speciation. Given that marginal populations of widespread species are more likely than other populations to become isolated, they can play an important role in speciation. Consequently, peripheral speciation might be a common way by which regional endemics originate. Population dynamics during the Pleistocene, however, had a large influence on evolutionary history. Species that avoided extinction expanded and retreated as a result of environmental changes during the Pleistocene, resulting in their current distribution and genetic structure throughout their range (Avise, 2000; Hewitt, 2000). A simple evolutionary consequence is that range contractions of widespread species have left isolated populations in marginal areas, leading to peripheral speciation of regional endemics. In this case, the population sizes of endemics would be much smaller than those of their ancestral and sister species, and a lack of migration would be observed after speciation (Conye & Orr, 2004). However, Pleistocene population dynamics, such as range expansion, contraction and/or population extinction, could blur distribution patterns and complicate the evolutionary history of a species. Indeed, it is now recognized that introgression may have occurred between species during past Pleistocene range expansions and contractions at times when they were sympatric (Kikuchi et al., 2010) and that, in general, the effects of historical hybridization between species that are currently allopatric might be more frequent than previously thought (Cronn & Wendel, 2004; Pelser et al., 2011). Thus, the speciation process cannot simply be inferred based on the current range of a species. A better understanding of the evolutionary history of regional endemics is likely to provide novel insights into the evolutionary dynamics of species during climatic oscillations. Here we assess the potential influence of Pleistocene population dynamics on the evolutionary history of a species through an examination of the speciation history of a Japanese alpine endemic, Cardamine nipponica (Brassicaceae). The high mountains in Japan range from 2000 to 3000 m in altitude and 35° to 44° in latitude, and harbor an alpine flora consisting of species that also occur in the Arctic (e.g. Empetrum nigrum, Diapensia lapponica, and Vaccinium vitis-idaea) as well as endemics that appear to be closely related to arctic-alpine species. Specifically, one-third of the Japanese alpine plant species are either endemic to this archipelago, but with closely related arctic species, or are themselves widely distributed in the Arctic (Shimizu, 1982, 1983). C. nipponica, a perennial, mostly autogamous herb with compound leaves (Kitakawa, 1999; H. Ikeda, pers. obv.), is a representative of such endemics. The narrow range of this endemic species is located at the periphery of the range of an apparently closely related, widespread arctic–alpine species, Cardamine bellidifolia, which also is perennial and mostly autogamous, but with simple leaves (Hultén & Fries, 1986; Brochmann & Steen, 1999). The current range of C. bellidifolia extends throughout the entire Circum-Arctic and into the high mountains of North America and East Asia (Fig. 1). Both species are diploid (2n = 16; Kučera et al., 2005; Warwick & Al-Shehbaz, 2006; Y. I. Iwatsubo, pers. obv.). The current distributions of C. nipponica and C. bellidifolia do not overlap, but C. bellidifolia extends southward to Sakhalin Island north of Japan (Fig. 1). Thus, one may reasonably hypothesize that the Japanese endemic C. nipponica is sister to the arctic-alpine C. bellidifolia and has diverged in the periphery of the range of this widespread species. However, a previous study inferred a nonsister relationship between these two species based on internal transcribed spacer (ITS) sequences (Carlsen et al., 2009). That study used DNA extracted from an old herbarium specimen of C. nipponica, and thus the risk of contamination or other errors during experimental procedures may have been high. Therefore, re-evaluating the phylogenetic position of C. nipponica and its relationship with C. bellidifolia would be worthwhile. Figure 1Open in figure viewerPowerPoint (a) Distribution ranges of Cardamine bellidifolia (light gray) and C. nipponica (dark gray). Black squares represent sampling sites for C. bellidifolia. (b) Detailed range (black dots) and sampling sites (dots with arrows) for the Japanese endemic C. nipponica. Detailed information on the localities is given in Supporting Information Table S1. In this study, we first re-examined whether the Japanese endemic C. nipponica is sister to the arctic–alpine C. bellidifolia. Initially, we reanalyzed the previously published as well as new ITS sequences of the genus Cardamine. Because phylogenetic relationships inferred between close relatives are sometimes inconsistent across genes as a result of incomplete lineage sorting and/or gene flow, multilocus analyses are necessary to determine a robust sister species relationship. Therefore, using several putative sister species identified in the ITS analysis, further phylogenetic analyses were conducted based on 10 other nuclear genes. Given that C. nipponica and C. bellidifolia were indeed identified as sister taxa in our study, we next explored their speciation history statistically using demographic parameters under the isolation-with-migration (IM) model (Nielsen & Wakeley, 2001; Hey & Nielsen, 2004). Based on these parameters, we attempted to evaluate the potential peripheral speciation between the sister species and infer the evolutionary influence of Pleistocene climatic oscillations. Materials and Methods Sample collection and DNA extraction One individual of C. nipponica from each of six populations from northern Japan and 12 populations from southern Japan was selected based on previous studies (Ikeda et al., 2008, 2009a,b, 2011), and previously extracted DNA or obtained sequences were used. These samples covered the entire range of the species and provide a random representation of its polymorphisms (Fig. 1; Ikeda et al., 2008). For C. bellidifolia, 20 individuals representing the entire distribution range were used (Fig. 1), and DNA was extracted from silica-dried leaf materials or leaf samples from herbarium specimens using the DNeasy Kit (Qiagen, Hilden, Germany). In addition, six and eight individuals of Cardamine alpina and Cardamine resedifolia, respectively, were included to test whether C. bellidifolia is a sister species of C. nipponica, and their DNA was extracted from silica-dried leaf materials using the DNeasy Kit (Qiagen). The detailed localities of the samples are shown in the supporting information (Supporting Information, Table S1). Our analyses included sequencing of multiple nuclear loci to reduce the bias found in single-locus genealogies as a result of incomplete lineage sorting and the effects of our relatively small sample size for each species. Cardamine glauca was selected as an outgroup for phylogenetic and population genetics analyses because this species is sister to the clade that includes C. bellidifolia L. (Carlsen et al., 2009) and C. nipponica Franch. et Savat. (see the Results section). The DNA of C. glauca was extracted from silica-dried leaf materials of eight individuals using the DNeasy Kit (Qiagen). Sequencing ITS in C. nipponica and phylogenetic analyses We sequenced the internal transcribed spacer (ITS) region from C. nipponica to reassess the phylogenetic position of this species. PCR amplification of ITS from C. nipponica was conducted according to a previous phylogenetic analysis (Carlsen et al., 2009). After gel purification with glass powder using GeneClean II (Bio 101, Vista, CA, USA), PCR products were sequenced directly from both directions using an ABI 3130 Genetic Analyzer (POP-7 polymer and 80 cm capillary; Applied Biosystems, Foster City, CA, USA). The sequences were aligned with those of other Cardamine species (88 species) from a database (Table S2) using Clustal X (Thompson et al., 1997). In total, 98 sequences were included, most of which had also been used in the previous phylogenetic study (Carlsen et al., 2009). The newly obtained sequences were deposited in the DDBJ (AB639120–AB639136). Rorippa indica and Rorippa islandica were used as outgroups (Table S2). A maximum-likelihood (ML) tree was estimated with TREEFINDER (Jobb et al., 2004) using the default setting. Different substitution models were applied to the 5.8S rDNA gene (TIM2 + G) and its flanking regions (ITS1 and ITS 2; GTR + G), which were estimated using the partition option of the software. The significance of branches was evaluated by 1000 bootstrap resamplings. In addition, the Bayesian tree was estimated using MrBayes, ver. 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003). Applying the partitioned models of substitutions, Markov chain Monte Carlo (MCMC) searches were run for 5 000 000 generations with four chains, and trees were sampled every 100 generations. Of the four chains, one was kept cold, and the other three were heated at a low-temperature setting (temperature = 0.02). After the first 50 000 trees were discarded as burn-in based on the stationarity of the likelihood values, a majority-consensus tree and posterior probabilities were obtained from the estimated distribution of trees. Because two replicate runs resulted in a consistent tree, posterior probabilities were summarized from the two runs. PCR and sequencing of multiple nuclear genes Polymerase chain reaction amplification of nuclear genes and sequencing procedures were conducted following a previous study (Ikeda et al., 2009a). PCR amplification was attempted for 10 loci, including six loci with previously reported primers (COP1, DET1, GA1, TFL1, CHS, and F3H; Kuittinen et al., 2002), three loci from portions of entire genes using previously designed primers (PHYA, PHYC, and CRY1; Table S3; Ikeda et al., 2009b; 2011), and one locus using primers designed specifically for this study (CO; Table S3). All PCR products were directly sequenced using an ABI 3130-avant Genetic Analyzer (POP-7 polymer and 36-cm capillary; Applied Biosystems). The entire sequences of the amplified PCR products were determined using both PCR primers for six loci (COP1, DET1, F3H, PHYA, PHYC, and CRY1). Sequences of portions of the amplified PCR fragments were determined using one of the PCR primers and an internal primer (Table S3) for four loci (CO, CHS, TFL1, and GA1), and these partial sequences were used for further analysis. In total, c. 5800 bp from 10 loci (491–750 bp per locus) were analyzed. These amplified loci are located in portions of the coding regions of genes (Kuittinen et al., 2002; Ikeda et al., 2009b, 2011), suggesting they were not tightly linked to one another. Polymorphisms were confirmed by sequencing several independent PCR products. As expected from the reported diploidy of all four species (2n = 16; Kučera et al., 2005; Warwick & Al-Shehbaz, 2006; Y. I. Iwatsubo, unpublished), we obtained unambiguous electropherograms, and no sequences had more than one heterozygous position. Therefore, alleles of each sample were visually determined. Sequences of each locus were aligned using the Auto Assembler software (Applied Biosystems). All new sequences were deposited in the DDBJ (AB607347–AB607800). The relationships among alleles at each locus were determined based on sequences excluding insertions and deletions (indels) using TCS1.06 (Clement et al., 2000). Multilocus analyses for determining phylogenetic relationships The phylogenetic relationships among C. nipponica, C. bellidifolia, C. alpina, and C. resedifolia were estimated using Bayesian approaches with the sequences of 10 nuclear loci. In total, 42 samples with no missing data for any loci were used. In addition, one sample representing the sequence of C. glauca was used as an outgroup for each locus. Because samples of C. glauca frequently had heterozygous indels, we failed to obtain unambiguous sequences of all loci from a single sample. Instead, each locus was sequenced from several samples, and the resulting sequences were used for the outgroup sequences. First, to assess to what degree the genetic composition of each individual was influenced by introgression, Bayesian clustering, which estimates clusters assuming Hardy–Weinberg equilibrium, was conducted based on combinations of alleles from multiple loci using STRUCTURE, ver. 2.1 (Pritchard et al., 2000; Falush et al., 2003). The probability of assigning individuals into clusters was estimated using an admixture model with correlated allele frequency using 250 000 generations, following a 100 000 generation burn-in period. The number of clusters (K) was set from one to seven, and 20 runs were repeated for each K. The symmetric similarity coefficient (SCC: H’) was calculated between all pairs of runs for the same K using CLUMPP (Jakobsson & Rosenberg, 2007). The final assignment of each individual was estimated as the mean overall configuration. In addition, the plausible number of clusters was evaluated according to the model value (ΔK ) based on the second-order rate of change of the likelihood function (Evanno et al., 2005). Because C. nipponica and C. bellidifolia are mainly autogamous, the assumption of Hardy–Weinberg equilibrium might be violated, which may lead to spurious signals of population substructure (Falush et al., 2003). Bayesian clustering was also implemented using the program InStruct (Gao et al., 2007), which extended STRUCTURE by eliminating the assumption of Hardy–Weinberg equilibrium, using the same settings as STRUCTURE. To construct the phylogenetic tree, concatenated data sets for 10 nuclear loci were analyzed using partitioned Bayesian and ML analyses. Each locus was a single partition with its own substitution model and unlinked parameters, with the exception of those used for trees. Substitution models for each partition were estimated using TREEFINDER (Jobb et al., 2004). The Bayesian tree was estimated using MrBayes, ver. 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003). MCMC searches were run for 1 000 000 generations with four chains, and trees were sampled every 100 generations. Of the four chains, one was kept cold, and three were heated at a low-temperature setting (temp = 0.005). After the first 100 000 trees were discarded as burn-in based on the stationarity of the likelihood values, a majority-consensus tree and posterior probabilities were obtained from the estimated distribution of trees. Because two replicate runs resulted in a consistent tree, posterior probabilities were summarized from the two runs. The ML tree was estimated with TREEFINDER (Jobb et al., 2004) using the default setting. The significance of each branch was evaluated by 1000 bootstrap resamplings. We conducted both Bayesian and ML analyses using two data sets that either did or did not include corresponding database sequences of Arabidopsis thaliana (ecotype = Columbia). When A. thaliana was included, only exon sites were used, whereas all sites were used for the data set without A. thaliana. In addition to the concatenated analyses, a species tree was estimated using a Bayesian hierarchical method implemented by BEST, ver. 2.3 (Liu, 2008). This approach employs coalescent theory across multiple loci to generate a posterior distribution of species trees and can represent the independent evolutionary history across loci (Liu et al., 2008). The same data sets used in the concatenated analyses were used for estimating the species tree, while all parameters, including those for trees, were unlinked. MCMC searches ran for 50 000 000 generations with eight chains, in which one was kept cold and seven were heated at a low-temperature setting (temp = 0.10). Trees were sampled every 1000 generations, and the first 5000 trees were discarded as burn-in based on the stationarity of likelihood values. A majority-rule consensus tree was obtained from the estimated distribution of species trees. Because two replicate runs resulted in consistent topology, the posterior probabilities of the species tree were summarized from the two runs. A species tree was solely obtained from data sets without A. thaliana. Summary statistics and neutrality tests The average number of pairwise nucleotide differences per site (π; Nei, 1987), the genetic divergence from C. glauca (KCg), and the minimum number of recombinations (RM) following the four-gamete test (Hudson & Kaplan, 1985) were calculated for C. nipponica and C. bellidifolia. To evaluate whether evolution at each locus follows a standard neutral model, Tajima’s D (Tajima, 1989) was estimated, and any deviation from neutral expectation was evaluated using 10,000 coalescent simulations. These summary statistics were estimated using DnaSP, ver. 5.0 (Librado & Rozas, 2009). Furthermore, the HKA test (Hudson et al., 1987) was conducted to evaluate all loci evolved under neutral equilibrium using the HKA program (http://lifesci.rutgers.edu/~heylab/HeylabSoftware.htm#HKA). Using the HKA test, we evaluated whether nucleotide diversities within a given species (i.e. C. nipponica or C. bellidifolia) followed expectations based on divergences from C. glauca. Demographic parameters of the IM model To determine speciation history, the demographic parameters of the IM model (population size: θ1, θ2, θA; migration rate: m1, m2; divergence time: t), which are expressed in terms of a mutation rate (μ), were calculated using the IMa program (Hey & Nielsen, 2007). The IM model assumes that an ancestral species with population size NA (= θA/4μ) split into two species at T (= tμ) yr before present (BP). After speciation, the descendants had distinct population sizes (N1 = θ1/4μ, N2 = θ2/4μ) and experienced constant migrations (m1, m2; migration rates per mutation event). Because the model assumed panmixia for each population, the estimated migration rates (m1, m2) represent gene exchange between sister species. The program IMa estimates probability density functions for parameters under the model and assesses the posterior probability densities for model parameters using MCMC methods (Hey & Nielsen, 2004, 2007). In addition, the likelihood of the nested models, such as the model without migration (m1 = m2 = 0) or that with equal population sizes (θ1 = θ2 = θA), was calculated using the estimated functions. In comparing the likelihood of the IM model with each nested model using the likelihood ratio test, the significance of the IM model, as well as the estimated demographic parameters, was evaluated. The IM model is based on several assumptions such as neutrality of loci, no recombination within loci, free recombination across loci, and no migrations from an unsampled third species to the focal species (Nielsen & Wakeley, 2001; Hey & Nielsen, 2004). However, a recent simulation study showed that estimated demographic parameters were quite robust to a violation of assumptions such as the population structure within species (Strasburg & Rieseberg, 2010), while a violation in the mode of speciation such as a change in migration rates resulted in a false estimation of demographic parameters (Becquet & Przeworski, 2009). In our study systems, all loci apparently did not violate the assumptions (see the Results section), and the putative other species, C. resedifolia and C. alpina, are restricted to central and southern European mountains and thus gene exchange with C. nipponica and C. bellidifolia appears unlikely. Running IMa involved two steps: M-mode and L-mode. First, functions of the model parameters were estimated in M-mode. After several preliminary runs using a wide range of prior probability densities (m1 = m2 = 20; q1 = 20; t = 20), demographic parameters were estimated by 5 × 107 MCMC steps following 500 000 burn-in periods with Metropolis coupling implemented using 25 chains under the geometric increment model (h1 = 0.925; h2 = 0.625) and with a narrower prior probability density (m1 = m2 = 2.5; q1 = 2.5; t = 5), from which 2.5 × 105 genealogies were saved. The M-mode run was repeated three times with different random seeds to check for convergence. Using the functions of model parameters estimated in three independent runs in M-mode, the marginal posterior distribution and the maximum likelihood estimates (MLEs) of demographic parameters were predicted by running IMa in L-mode. In addition, likelihood ratio tests were conducted in L-mode to evaluate whether the IM model fitted better than the nested models assuming unidirectional migration (m1 or m2 = 0) or equal population sizes (θ1 = θ2, θ1 = θA, θ2 = θA, or θ1 = θ2 = θA). Given that our sequence data had no sites with multiple recurrent mutations, an infinite site model of mutation was applied to all loci. To remove intralocus recombinations from loci with recombinations (COP1, DET1, and GA1), the longest blocks of sequences without recombinations were selected based on a four-gamete test (Hudson & Kaplan, 1985) and used for parameter estimation. The estimated divergence time and population sizes were scaled by the mutation rate for C. nipponica used in our previous study (8.67 × 10−9 substitutions per site yr–1; Ikeda et al., 2009a). For scaling population sizes, we assumed 1 yr per generation because both C. nipponica and C. bellidifolia can flower and fruit a few months after germination in the laboratory (H. Ikeda, pers. obv.). Results ITS phylogeny of the genus Cardamine including C. nipponica Internal transcribed spacer (ITS) sequences were obtained from all samples of C. nipponica except Cn_17, in which 10 distinct types were found among 615 bp sequences (Table S1). Although most branches of the ML tree were not well supported, all sequences of C. nipponica formed a well-supported monophyletic group with C. bellidifolia, C. alpina, and C. resedifolia except for the previously reported sequence (EU819349) that grouped with C. microzyga in agreement with the previous paper (Carlsen et al., 2009) (bootstrap, 100%; posterior probability, 1.00; Fig. 2). In our analysis, the C. nipponica-bellidifolia-alpina-resedifolia clade (bootstrap, 100%; posterior probability, 1.00) was sister to another well-supported monophyletic group including C. glauca and C. pancicii (bootstrap, 93%; posterior probability, 1.00). Notably, C. nipponica and C. bellidifolia formed a well-supported monophyletic group (bootstrap, 96%; posterior probability, 1.00), supporting a sister relationship between these two species. Furthermore, all our new C. nipponica sequences except ITS_10 formed a well-supported monophyletic group (bootstrap, 97%; posterior probability, 1.00), whereas ITS_10, which was detected in Shokanbetsudake (Table S1), grouped with C. bellidifolia (bootstrap, 52%; posterior probability, 0.99). Most of the C. nipponica samples from southern Japan (ITS_6–ITS_9) formed a monophyletic group (bootstrap, 99%; posterior probability, 1.00). Figure 2Open in figure viewerPowerPoint A maximum-likelihood phylogenetic tree of Cardamine based on internal transcribed spacer (ITS) data. Numbers along the branches indicate bootstrap values based on 1000 resamplings (>50%) and posterior probabilities of Bayesian analysis (> 0.5). Sister relationship between C. nipponica and C. bellidifolia inferred from other nuclear regions Sequences of the 10 nuclear genes (in total c. 5800 bp) were obtained from most samples of C. nipponica, C. bellidifolia, C. alpina, and C. resedifolia, and the alleles in each sample were determined. Although the alleles in each species have similar sequences, many individual alleles were shared among two or more of the species (Fig. S1). For example, C. bellidifolia shared alleles with other species at seven of the 10 loci. At five of these seven loci, C. bellidifolia shared alleles with C. nipponica, whereas it shared alleles at only one or two loci with C. alpina and C. resedifolia. Despite the occurrence of shared alleles, Bayesian clustering and the phylogenetic tree inferred from the concatenated data sets showed that the four species could be unambiguously distinguished from each other. In the Bayesian clustering implemented by STRUCTURE, highly consistent configurations of individual assignments were detected at K = 5 and 6 (H’ = 0.95 and 0.96, respectively; Fig. S2). In particular, the ΔK was the highest at K = 5, representing the most probable number of clusters (Fig. S2). At K = 5, the genetic composition of each species was unambiguously distinguished from that of the others (Fig. S2). C. nipponica was divided into two distinct clusters, one corresponding to northern Japan and the other to southern Japan (Fig. S2). These patterns of individual assignments were consistent with the results of the clusterings using InStruct (Fig. S3). The inferred phylogenetic relationships were mostly consistent between the Bayesian and ML approaches (topologies of the Bayesian analyses not shown). Whether A. thaliana was included or not, the phylogenetic analyses of the concatenated data sets consistently resolved three major clades: C. alpina, C. resedifolia, and C. nipponica–C. bellidifolia (Figs 3, S4; bootstrap, > 95%; posterior probabilities, 1.00). In addition, C. nipponica was consistently mono