DOI: 10.5061/dryad.dbrv15f08
title: Autopolyploidy-driven range expansion of a temperate-originated plant to pan-tropic under global change

description: Angiosperms are believed to have emerged initially in the tropics and
 expanded their distribution range polewards through diverse mechanisms,
 for example polyploidization-driven cold-tolerance evolution. Reversed
 expansion from temperate to pantropic climate through a
 polyploidization-driven shift in heat-tolerance remains largely unknown.
 Here, we found autopolyploidy in relation to the global expansion of
 Solidago canadensis from its temperate-climate native range in North
 American to hot-summer climate in an introduced range. Our
 cytogeographical study of 2062 accessions from 471 locations worldwide
 demonstrates that ploidy levels correlates negatively with latitude and
 positively with average temperature. An isotherm-dependent shift of the
 climate niches at the threshold of 20-24ºC between geo-cytotypes can be
 attributed mainly to autopolyploidy-driven differentiation of heat
 tolerance; only polyploids and not diploids are able to complete sexual
 reproduction, germinate and grow in the hot-summer climate of low
 latitudes. Ploidy-dependent fertility appears to play a key role in the
 hot-summer introduced range in the northern hemisphere through both
 pre-adaptation and rapid post-introduction adaptive evolution of delayed
 flowering and improved heat-tolerance during embryo development. MaxEnt
 model predicts continuous expansion of this weed under global change.
 These results provide new insights into the mechanisms governing
 autopolyploidy-driven backward range-expansion of plant species from
 temperate origins.
description: <strong>Field sampling</strong> We
 collected seeds and leaf samples from up to 21 (median = 5) individuals
 from 78 locations in the native (NA) ranges (64 from the US, 14 from
 Canada) and 72 locations in the introduced (IN) ranges (4 from Japan, 1
 from Russia, 3 from Europe, 1 from India, 63 from China), as well as two
 fields in China (Lianyungang, Jiangsu and Kunming, Yunnan), where the
 species was cultivated for sale as cut flowers (Supporting Information
 Table S1-3). At each site, sampling plots were separated by 10 m to
 minimize the chance of sampling multiple ramets from individual genets.
 For populations from India and 40 Chinese populations, leaf samples were
 collected from plants which were generated from field-collected rhizomes
 grown in a common garden. Leaf samples were silica-dried to allow DNA
 extraction for molecular identification of the species (ITS and psbA-trnH
 sequencing), and seed samples were air dried at room temperature prior to
 experiments. <strong>Molecular
 identification</strong> To confirm the correct
 identification of each of the 152 collected populations, we sequenced the
 ribosomal ITS (608bp) and psbA-trnH (213bp) intergenic spacer of up to
 five individuals from each population (Table S1, S2). To this end, we
 extracted total DNA from the dried leaves using a Plant Genomic DNA kit
 (DP305, TIANGEN BIOTECH CO., LTD, Beijing, China). The detailed
 information on primers and PCR reaction system and cycle conditions is
 presented in the Supplementary Information. The ribosomal ITS and
 chloroplast psbA-trnH were sequenced and edited using the DNAstar EdiSeq
 software. As the reference for alignment, we extracted the data for S.
 canadensis (ITS, HQ142590.1; psbA-trnH, KX214929.1) from GenBank and
 aligned them with the ITS and psbA-trnH sequences of our samples using the
 Clustal X 1.83 software. The ITS1, 5.8S and ITS2 gene boundaries and
 psbA-trnH were determined from ITS and psbA-trnH sequences of the Solidago
 genus in GenBank. To further verify the species identity of our samples,
 we conducted a BLAST search in the database of the National Center for
 Biotechnology Information (NCBI) to determine the species with the most
 similar identities (Table S1, S2). In addition, we built phylogenetic
 trees (Fig. S1, S2) for our samples with the ITS and psbA-trnH sequences,
 using the MEGA 4.1 Beta 3 software based on the principle of maximum
 parsimony method. We assessed branch support with 2,000 bootstrap
 replicates. <strong>Determination of ploidy
 level</strong> First, as a (diploid) reference
 for the ploidy-level determination, we assessed the exact chromosome
 numbers of the progeny from each of the 13 accessions/seed families of the
 Canadian population CA09 (Table S3, Fig. S3a), using mitotic root-tip
 squashes under a microscope. We grew up plants from each seed
 family/accession in the greenhouse, except for the Chinese and Indian
 populations for which field samples of maternal plants were used for
 ploidy examination. Fresh leaf material was held in self-sealing plastic
 bags containing silica and transported to the laboratory for ploidy level
 determination. We applied a modified flow cytometry method by Galbraith et
 al. (1983) for the determination of ploidy level. Briefly, 0.5 g fresh
 leaf tissue was chopped up with a razor blade in 2 ml of ice-cold
 Galbraith buffer (PH7.0, 45mM MgCl2, 30mM sodium citrate, 20mM
 4-morpholinepropane sulfonate, 0.5% w/v polyvinylpyrrolidone and Triton
 X-100 1 mg/ml) in a vitreous Petri dish held on a chilled brick. The
 resultant fine slurry was filtered through a 50μm microfilter into 2 ml
 polystyrene tubes and incubated at 4 oC for 5min. The final filtrate was
 treated with 10 μL/ml of RNase A for 10 min at 4°C. Then, 200 μL/ml of a
 propidium iodide (PI) staining solution was added and incubated at 4°C for
 30 min in darkness. The fluorescence strength of PI-DNA conjugates was
 measured by flow cytometric (BD FACS, USA) using a 488 nm laser and 590/40
 emission filter (Arumuganathan & Earle 1991). The diploid CA09
 population (Table S3) was used as an external reference. Flow-cytometry
 data acquisition and analysis was done with the software FloMax version
 2.0 (Quantum Analysis, Münster, Germany). Only histograms with CVs smaller
 than 10% and with a minimum peak height of 50 particles were accepted.
 Other histograms were discarded and corresponding samples were designated
 no signal (Suda and Travnicek 2006). To ensure the measurement quality,
 the external reference material (CA09, the fluorescence intensity scale at
 position 50, Fig. S3a) were measured and analyzed at regular intervals in
 the analysis process to verify the corresponding position of the peak of
 new samples that indicates their ploidy levels. Accordingly, each sample
 was identified as diploid (2x), tetraploid (4x) or hexaploid (6x). Based
 on the combination of cytotype (2x/4x/6x) and geographical range (native
 [NA] / introduced [IN]), we assigned each accession to one of the
 following six geo-cytotypes: NA2x, NA4x, NA6x, IN2x, IN4x or IN6x. As some
 of the 152 populations included more than one cytotype, the total number
 of population × cytotype combinations was 233. We also
 extracted data on chromosome numbers of 321 S. canadensis populations with
 known locations from ISI-indexed journals, books and databases of
 cytological indices (Index of Plant Chromosome Numbers [IPCN],
 International Organization of Plant Biosystematics [IOPB] Chromosome
 Number Reports). Detailed information about the published ploidy records
 are listed in Table S4. In total, our study includes information on the
 1172 newly collected accessions/seed families for which we determined
 ploidy (Table S3) and 890 previously published ploidy records in 471
 locations worldwide (Table S4). We used these data points to create a
 cytogeographical map in ArcGIS 10.0 (Environmental Systems Research
 Institute, Redlands, California, USA). For each of the 471 locations, we
 extracted the mean July temperature of each sample site using ArcGIS 10.0.
 Using the temperature of the hottest month (July) in the northern
 hemisphere, we try to guide the hypothesis that the thermotolerance
 evolution of S. canadensis may occur in populations with different ploidy
 levels. A Tukey test was used to detect differences among the different
 geo-cytotypes separated by the latitude distribution and mean July
 temperature of sample site. To examine whether the latitude distribution
 and mean July temperature were affected by geocytotypes, we used two-way
 ANOVA, with explanatory variables being origin (native, introduced),
 ploidy level (diploid, tetraploid, hexaploid) and their
 interaction. <strong>Seed germination at
 different temperatures</strong> Since not all 150
 populations had enough seed, which were collected in field, only 30 native
 and 56 introduced cytotype populations representing all geo-cytotypes were
 tested in this experiment (Table S3 E1). We bulked the seeds from up to
 five accessions (if available) from each population, and tested their
 germination under two temperature regimes with a temperature of 25°C
 (12h/12h, day/night) and an extreme temperature of 39°C (12h/12h,
 day/night), respectively (the two temperatures were based on Li, 2011). A
 total of 150 seeds from each population were divided into three sets as
 three replicates. The 50 seeds were placed on wet filter paper in
 petri-dish (diameter = 9 cm) and kept in the growth chamber (2 m long ×
 0.8 m wide × 0.8 m high; Ningbo Jiangnan Biological Instrument factory,
 Zhejiang, China). The photon flux density was 250 μmol m−2 s−1, with an
 85% relative humidity (RH) in the chamber. The germination process was
 observed every day lasted for 15 days, during which the filters were kept
 wet. Petri-dishes were randomly distributed in chambers and their position
 was changed every day. Seeds infected by fungi or bacteria were removed
 and not considered for calculations, but the same number of new seeds were
 added. A seed was considered as germinated if the radicle grew longer than
 seed length. The seed germination inhibition rate was calculated using the
 following equation: Inhibition rate = [(a-b)/a] × 100%, with “a” and “b”
 representing mean germination rate of population at 25°C and 39°C. One-way
 ANOVA was used for data analysis with Duncan’s parametric tests being
 applied to compare the differences of the germination inhibition rate
 among geo-cytotypes. <strong>Common garden
 comparative experiments</strong> To examine
 ecological adaptation and reproduction of the different geo-cytotypes, two
 comparative common garden experiments were conducted in the introduced
 range (IRCG) at Nanjing Agricultural University (32º02′N, 118º50′E;
 Nanjing, Jiangsu, China) and in the native range (NRCG) at the University
 of Georgia (33º56′N, 83º22′W; Athens, GA, USA), respectively. The IRCG
 experiments started on December 3, 2011 and December 5, 2012, while NRCG
 ones did not begin until December 10, 2012. At IRCG, the mean annual
 rainfall was 1090.7 mm, annual mean temperature was 15.6°C, mean max and
 min temperatures in January were 7.2°C and -0.7°C, and mean max and min
 temperatures in July were 32.2°C and 24.9°C. A total of 507 accessions
 propagated from the 30 native and 56 introduced populations were used in
 IRCG (population details in Table S3 E1). At NRCG, mean annual rainfall
 was 1250.0 mm, annual mean temperature was 15.9°C, mean max and min
 temperatures in January were 13.9°C and 1.4°C, and mean max and min
 temperature in July were 32.8°C and 20.8°C. A subset of plants from 26
 populations, including all geo-cytotypes, was grown in NRCG (population
 details in Table S3 E2). At both common gardens, seeds from up to 5
 maternal plants of each population were germinated in seedling trays
 filled with a 1:2 mixture of soil and sterilized compost in a greenhouse
 (18-25oC) in December. To minimize maternal effects associated with the
 quality of sampled seeds, at least 30-day-old seedlings with similar
 heights, basal diameters and leaf numbers were selected from each maternal
 plant per population and individually transplanted into 11-cm-wide,
 8-cm-deep, 1 L plastic pots containing the same growing medium. Potted
 plants were randomly placed in a glasshouse supplemented with artificial
 light for 16 h during the day and watered as needed, usually every 2-4
 days. Each accession was grown individually outdoors in a 5-L plastic pot
 filled with the same growing medium in March (three month after planting,
 the daily average temperature was above 10 oC). Trays and pots were
 reassigned randomly to new positions weekly. Pot management in two common
 gardens was the same. To determine heat and frost
 tolerance with different geo-cytotypes (Table S3 E1), seedlings of S.
 canadensis accessions were transplanted to 1-L plastic pots filled with a
 1:2 mixture of soil and sterilized compost 30 days (January 2, 2013) after
 sown in the greenhouse at IRCG. Two months later (March 3, 2013), the
 plants were divided into three sets for different experiments.
 The first set of 516 plants (six offspring individuals of 1 to 6
 accessions from each population) was used to determine the heat tolerance
 (beginning on March 3, 2013). Plants were kept with saturated soil
 moisture and subjected to 45±1°C for 10h in a growth chamber (E-36HO,
 PERCIVAL, U.S.), and assessed leaf heat damage. Seedlings were assessed
 according to a heat-damage index: 0) for healthy and normal seedlings; 1)
 when the tip of leaves was slightly burned; 2) 1-2 leaves were wilted; 3)
 3-4 leaves were wilted; 4) &gt; 4 leaves were wilted; and 5) all
 leaves wilted. Heat-tolerance index (HI) was calculated as 1-Σ[(number of
 seedling corresponding grade) / ( maximum grade total number of
 seedling)]. The second set of 774 plants (nine
 offspring individuals of 1 to 3 accessions from each population) was used
 to examine physiological responses to high temperature (beginning on March
 5, 2013). The 3rd to 5th leaves from the shoot apex of plant were sampled
 and immediately frozen in liquid nitrogen and stored at -80°C until use
 after exposing to 45±1°C for 0, 2 and 4h, respectively (room temperature
 was about 28°C). The reactive oxygen species (ROS) levels (i.e. H2O2
 content), activities of antioxidant enzymes including Cu/Zn superoxide
 dismutase activity (Cu/Zn-SOD), peroxidase activity (POD), catalase
 activity (CAT), glutathione reductase activity (GR), contents of
 non-enzymatic antioxidants (ASA), and osmoregulatory substances (i.e.
 soluble proteins) were assayed using commercial reagent kits purchased
 from Nanjing Jiancheng Bioengineering Institute (Nanjing,
 China). The third set of 430 plants (five offspring
 individuals of 1 to 5 accessions from each population) was used to
 estimate freezing tolerance (beginning on March 9, 2013). We examined the
 relative PIABS (performance index on absorption basis) using
 chlorophyll-fluorescence measurements (Pocket-PEA, Hansatech Instruments
 LTD., King’s Lynn, Norfolk, PE32 1JL. UK) (Xie et al. 2015). The initial
 PIABS was measured after 30 min dark adaptation at room temperature. The
 upper, middle and lower parts of both sides of veins in each leaf (the
 fifth mature leaf) were measured. The freezing PIABS was measured at the
 same part of each leaf after the exposure of detached leaves to -8°C for
 30 minutes. The relative PIABS = freezing PIABS/initial PIABS (Xie et al.
 2015). The relative PIABS represents the freezing tolerance of S.
 canandensis. One-way ANOVA was used to analyze the
 differences of heat-damage index, freezing tolerance (relative PIABS),
 inhibition rate of germination, ROS level, SOD, POD, CAT, GR, ASA, and
 soluble proteins among different geo-cytotypes. Duncan’s test was applied
 when assumptions of normality and homogeneity of variance were fulfilled;
 otherwise non-parametric ANOVA (Dunnett’s C test) would be used. In
 addition, to confirm the effect of environmental temperature factors on
 plant heat tolerance, we used canonical correspondence analysis (CCA) to
 assess the relationship between “species factors” and “environmental
 factors” as implemented in CANOCO for Windows 4.5 (Ter Braak &
 Smilauer 2002). For species factors, we included the heat-damage index,
 ROS level (H2O2 content) and antioxidative parameters. For environmental
 factors, we included latitude and six bioclimatic variables, including
 annual mean temperature (bio 1), isothermality (bio 3), minimum
 temperature of coldest month (bio 6), mean temperature of driest quarter
 (bio 9), mean temperature of warmest quarter (bio 10) and mean temperature
 of coldest quarter (bio 11). Furthermore, because “species factors” also
 were significantly affected by “ploidy level”, we also included “ploidy
 level” as an environmental factor. To examine
 reproductive biology, we kept S. canadensis plants in both common gardens,
 with 860 pots of plants involved in IRCG (March 30, 2012 and 2013; Table
 S3 E1) and 260 pots in NRCG (April 4, 2013; Table S3 E2). In both common
 gardens, we assessed flowering phenology (onset, peak and end of
 flowering) and the number of viable seeds produced per plant. The latter
 was based on the number of seeds per plant and the germination rate at
 25°C for each population (the details are described as above). One-way
 ANOVA was used to analyze the data for first flowering dates, flowering
 duration and number of viable seeds among the six geo-cytotypes. Duncan’s
 test or Dunnett’s C test was applied to separate means following the
 principle described above. An additional set of
 measurements was taken only in IRCG in 2012. Because plants from some of
 native-range North American populations failed to produce viable seeds, we
 tested for pollen viability (Table S3 E5), stigma receptivity (Table S3
 E6), germination rates of pollen grains on stigmas (Table S3 E7) and
 pollen-tube growth (Table S3 E7) for these native populations. As S.
 canadensis is self-incompatible and the different cytotypes are not
 cross-compatible (Yao 2014), we always used pollen and stigmas from
 different plants of the same cytotype in a population. We also recorded
 the mean and maximum daily temperatures during embryo development of
 plants from native populations to assess the relationship between embryo
 development and high temperatures (Table S3 E3). To
 assess the impact of high temperatures on seed sterility more accurately,
 additional measurements were taken also in IRCG in 2014 by transferring
 offspring of 48 maternal plants from 16 populations (Table S3 E4) that did
 not produce viable seeds in IRCG to a climatic chamber (Room C3006 in
 Collage of Life Science, Nanjing Agariculture University) with a
 temperature regime of 24/18°C (12h/12h, day/night, the photon flux density
 was 250 μmol m−2 s−1) before flowering in July 2014. These populations
 included four NA2x, four NA4x, four NA6x and four IN2x populations. In
 addition, offspring of 24 maternal plants from 4 populations each of IN4x
 and IN6x that could produce viable seeds in IRCG were also transferred to
 the climatic chamber with a temperature regime of 36/30°C (12h/12h,
 day/night) before flowering in Oct. 2014 (Table S3 E4). An identical set
 of plants that included the above 24 populations was also prepared and
 placed outdoors in the common garden to serve as controls. The daily
 management of all experimental plants was the same to ensure their normal
 and comparable growth. Plants belonging to the same cytotype and grown
 under the natural and controlled conditions were artificially pollinated
 with the pollen from each other. Seeds per plant were harvested when
 mature and the number and germination rate of well-developed seeds were
 determined and used to estimate the number of viable seeds.
 <strong>Potential distributions of the different
 cytotypes</strong> To predict the potential
 distribution of S. canadensis under the current and potential future
 (2050) climate, based on a climate-change scenario, we used MaxEnt
 climatic niche models (Young and Evangelista 2011) and ArcGIS software.
 For these models, we used 5354 occurrence data of S. canadensis, including
 new data from our own research (n=150) (Li 2011, Lu et al. 2020), data
 from the literature (n=321) (Table S4), records downloaded from the Global
 Biodiversity Information Facility (GBIF; n=4784) and additional records
 from Chinese literature (n=101). We used MaxEnt Jackknife tests (test gain
 &gt; 0.7) to select 9 key bioclimatic variables (Elith et al. 2006)
 out of the 19 variables available in WorldClim (http://www.worldclim.org)
 (Table S5, Fig. S8). The nine bioclimatic variables are annual mean
 temperature (bio 1), isothermality (bio 3), minimum temperature of coldest
 month (bio 6), mean temperature of driest quarter (bio 9), mean
 temperature of warmest quarter (bio 10), mean temperature of coldest
 quarter (bio 11), precipitation of driest month (bio 14), precipitation of
 driest quarter (bio 17) and precipitation of coldest quarter (bio 19). The
 WorldClim Current Conditions dataset (bioclimatic data averaged across the
 period 1950-2000) at a spatial resolution of 10 minutes was used to model
 the current climatic niche of S. canadensis. To forecast the potential
 future suitable ranges for infestation, we used the climate dataset of
 WorldClim for the year 2050 based on the Representative Concentration
 Pathway (RCP) scenario 4.5 (http://tntcat.iiasa.ac.at:8787/RcpDb). It is a
 moderate scenario where the total radiative forcing stabilizes before 2100
 (Dyderski et al. 2018). For the predictions of the
 climatic niche modelling, occurrence data for S. canadensis was randomly
 divided according to the algorithms of the MaxEnt software into training
 data (75% of occurrence points) for model prediction and test data (25% of
 occurrence points) for model validation. All additional parameters were
 set to their default values. A receiver operating characteristics (ROC)
 curve plot was built, and the scores of the area under the curve (AUC)
 indicated a good (AUC values at 0.8–0.9) to excellent (AUC values of
 0.9–1.0) model fit (Fig. S9). The forecasted climatic suitability of
 different areas around the globe was classified into five categories using
 natural segmentation method based on clustering analysis in ArcGIS
 software: unsuitable (0–0.2327), low suitable (0.2327–0.5), margin
 suitable (0.5–0.6), suitable (0.6–0.7) and high risky suitable (0.7–1.0)
 (Kigen et al. 2019). <strong>Supporting
 Information Materials and Methods</strong>
 <strong>Molecular identification</strong>
 A pair of primers (upstream: 5'-CGTAACAAGGTTTCCGTAG-3',
 downstream: 5'-TTATTGATATGCTT AAACTCAGCGGG-3') were used to
 amplify ITS rDNA gene and another pair of primers (upstream:
 5’-CCGCCCCTCTACTATTATCTA-3’, downstream: 5’-TCTAGACTTAGCAGCTATTG-3’) were
 used to amplify psbA-trnH intergenic spacer. Polymerase chain reaction
 (PCR) was carried out in 50μL containing 2μL of 10μmol/L primers,
 10×buffer 5μL, 25mM Mg2+ 4μL, 2.5mmol/L dNTPs 4μL, 50ng/μL DNA
 templates1μL and 5U Taq DNA polymerase 0.5μL. The PCR cycle was 95°C for 4
 min followed by 30 cycles of 95°C for 30s, 52°C for 60s, 72°C for 60s, and
 a final extension step at 72°C for 7 min. PCR products were separated on
 1% agarose gels and stained with ethidium bromide. The PCR products were
 purified and sequenced. <strong>Common garden
 sites and materials</strong> Two pot common
 garden greenhouse pot experiments were established at Nanjing Agricultural
 University [Nanjing, Jiangsu, China (32º2’N, 118º50’E), December 2011] and
 University of Georgia [Athens, GA, USA (33º56’N, 83º22’W), December 2012].
 Seeds from all maternal plants of 86 populations were germinated in
 seedling trays filled with 1:2 mixture of soil and sterilized compost in
 the introduced common garden. For all of the populations at least five
 individual plants were originally collected each sampling plot but a few
 populations only included less than five individuals each cytotype through
 detection of ploidy. The populations with less individuals, all
 characterized according to cytotype by detection of their ploidy, were
 considered in the experiment and their lesser number did not significantly
 affect the obtained results according to statistical tests (Supporting
 Data 1). <strong>Measurement of physiological and
 biochemical characteristics associated with seedling
 thermotolerance.</strong> The activity of Cu/Zn
 superoxide dismutase (Cu/Zn SOD, EC.1.15.1.1) was measured by test Kit
 (Nanjing Jiancheng Bioengineering Institute, China; A001-4). The activity
 of peroxidase (POD, EC 1.11.1.7) was .measured by Guaiacol method (Li et
 al. 2013). Catalase activity (CAT, EC.1.11.1.6) was .measured by test Kit
 (Nanjing Jiancheng Bioengineering Institute, China; A007-1-1) (Yong et al.
 2008). Glutathione reductase (GR, EC.1.6.4.2) was measured by test Kit
 (Nanjing Jiancheng Bioengineering Institute, China; A062-1-1) (Li et al.
 2017). Protein content was detected by the method of coomassie brilliant
 blue (Deng 2013). The H2O2 concentration was detected by monitoring the
 absorbance of the titanium- peroxide complex at 415 nm, following the
 method of Patterson (Brennan & Frenkel 1977).
 <strong>Reproductive traits</strong>
 Growth of all potted plants was regularly monitored in both the
 native and introduced common gardens. Flowering phenology was assessed
 after onset of flowering (first flower head open), peak flowering
 (&gt;50% flower head open) and end of flowering (&gt;50% flower
 heads closed); in each plant flowering dynamics was recorded for every
 shoot (capitulescence) at 3-d intervals. The duration of flowering per
 plant (days between onset and end of flowering) was determined.
 Infructescences of all flowering individuals were bagged before seeds
 matured and dispersed. After seed matured, infructescences per plant were
 harvested, dried naturally and weighed. The number of seeds of each plant
 was estimated from the average 500-seed weight of five samples per
 population. The number of viable seeds was estimated by the number of
 seeds and the germination rate at 25°C per population. Five individuals
 per population were recorded and measured (Table S3 E1, E4).
 <strong>Cross-compatibility
 experiments</strong> A group of seedlings
 representative of the six geo-cytotypes was sown every 40 days (beginning
 Jan. 15, 2012) to ensure synchrony of their flowering periods. Two plants
 matching in flowering period from the six geo-cytotypes were selected at
 the early flowering stage for reciprocal crossings. The
 positive and reverse crosses were made in all possible combinations
 between paired individuals belonging to the six different geo-cytotypes
 (three each from native and introduced ranges). Three populations were
 selected from each cytotype except for single introduced diploid (Table S3
 E7). For each cross, three individual plants from each population were
 used; each one was considered a replication. Additionally, two kinds of
 artificial self-pollinations were defined as controls: those of different
 individuals within each cytotype and those of different florets of same
 individual.The same procedure was used to select the individuals for
 artificial self-pollination.  For same individual, artificial
 self-pollinations were made between different clonal shoots from a
 maternal plant, different capitula on the same shoot and different tubular
 florets within a single capitulum. The bisexual tubular
 florets in the capitulum were carefully removed and the emasculation
 capitula were isolated with paper bags. After 5-7 days, plants were
 artificially pollinated when the stigma of ligulate flowers grew out of
 the petals and showed a “Y” shape. The pollen of selected male parents was
 collected with a brush and applied to the column head of the emasculation
 female parent and the paper bags were replaced quickly after pollination.
 Capitula were collected at 1h, 2h, 4h, 8h, 24h, 48h, and 72h after
 pollination (Fig. S5d), and thereafter gathered every 3 days, fixed with
 Carlo fixation liquid for further observation of pollen tube growth and
 embryo development. The mature seeds from each cross-combination were
 carefully harvested and their germination was tested at 25°C.
 At the full-bloom stage of the S. candensis, six capitula from
 each of five populations per geo-cytotype were selected to determine their
 pollen vitality with the benzidine method (King 1960) (Table S3 E5).
 Briefly, new blooming capitula, collected between 9:00 and 11:00hrs in a
 sunny day, were placed into concave slides, followed by the addition of
 reagent I (0.5% biphenyl 10ml, 0.5% alpha naphthol 10ml, 0.25% sodium
 carbonate 10ml) and 0.3% hydrogen peroxide. Capitula were mashed and the
 mixture was transferred into a 1.5ml centrifuge tube and incubated for 50
 min at 30°C. Stained pollen grains were observed and photographed with a
 microscope (Olympus BX53). Vigorous pollen grains stained black while the
 undynamic pollen grains stained yellow. Ten visual fields of each slide
 were randomly selected for assessment. To determine
 stigma receptivity, capitula were also collected as described before
 (Table S3 E6). The florets were placed into concave slides, followed by
 the addition of the staining benzidine-hydrogen peroxide solution (1%
 benzidine: 3% hydrogen peroxide: H2O = 4:11:55 volume ratio), and observed
 under the same microscope. Vigorous stigma stained blue and produced a
 large number of bubbles. Nine florets per geo-cytotype were observed and
 counted. Additional capitula were soaked in 70% ethanol for 30 min and for
 additional 30 min in 90% ethanol after fixation with Carnoy's fluid
 for at least 24 hour. Then, the capitula were preserved in 70% ethanol.
 Before observation, capitula were rehydrated sequentially in 50%, 30%, 10%
 ethanol and distilled water for ten min each, softened in 6 mol/L NaOH for
 eight hours, washed three times with distilled water and stained with 0.1%
 aniline blue for 12 hours. The number of pollen grains germinating on the
 stigma and pollen tubes growing into the ovary were counted under the
 fluorescence microscope (Carl Zeiss, Axio Observer Z1), after making
 microscopic slides of tissue samples. Thirty single flowers of each cross
 combination were observed and counted. After artificial
 pollination, capitula were sampled and fixed in formalin–acetic
 acid–alcohol (Berlyn & Miksche 1976) (FAA) at different embryonic
 developmental stages. Before observation, capitula were immersed orderly
 in 50% alcohol, 30% alcohol, 10% alcohol and distilled water every ten
 min. Individual flowers were carefully separated from capitula with
 forceps and immersed and softened in 2.5 mol·L-1 NaOH solution for six
 hours, and washed three times with distilled water. The single flowers
 were carefully placed on a slide and stained by aniline blue - lactic acid
 - phenol dye solution (Hu 1994) for five min. Before dropping 40% glycerol
 on individual flowers, redundant dye solution was absorbed by filter
 paper. Single flowers were covered with slide cover slips. The number of
 normal embryos at the different development stages was counted under the
 light microscope (Olympus BX53) (Table S3 E3). The normal development and
 abortion process of embryo is illustrated in Fig. S6: After pollination,
 the developing proembryo could be observed within 24 hours, globular
 embryos, the spherical embryo formed in 3~5 days after pollination (DAP),
 the heart-shaped embryo formed in 6~8 DAP, the torpedo-shaped embryo
 formed in 20~25 DAP and gradually matured. However, in the case of the
 following, we considered to be abnormal: 1, the development of embryos is
 seriously lagging. 2, the periphery of the embryo is transparent and the
 embryo atrophy or cluster. 3, the embryo is severely atrophied and only
 some small irregular clusters can be observed inside the peel. 4, only
 pericarp, no embryos and clusters can be observed inside the
 peel. <strong>Prediction of potential
 geographical distribution of S.
 canadensis</strong>
 <strong>Occurrence data</strong>
 We searched published information on chromosome numbers of S.
 canadensis in relation to location in ISI-indexed journals, books and in
 cytological indices [Index of Plant Chromosome Numbers (IPCN);
 International Organization  of Plant Biosystematics (IOPB) Chromosome
 Number Reports] (Table S4) and sampled wild populations in the native and
 introduced range (Table S3).
 <strong>Environmental variables, isotherms and processing
 of environmental data</strong> The “Current
 Conditions” (Current Conditions 1950~2000) bioclimatic dataset was used to
 extract the current ecological niche of S. canadensis. For simulation of
 the future suitable region, we used the future climate dataset in the
 RCP4.5 scenarios (http://tntcat.iiasa.ac.at:8787/RcpDb) provided by the
 Coupled Model Intercomparison Project (CMIP). The RCP 4.5 is developed by
 the MiniCAM modeling team at the Pacific Northwest National
 Laboratory's Joint Global Change Research Institute (JGCRI). It is a
 stabilization scenario where total radiative forcing is stabilized before
 2100 by employment of a range of technologies and strategies for reducing
 greenhouse gas emissions and has a higher priority. We
 simulated the distribution of suitable region in the future climate change
 scenarios based on the current ecological niche of S. canadensis. The
 future climate dataset of RCP4.5 was used to drive the ecological niche
 modeling, before that it should be treated with the following: (1) the
 value of each environmental variable corresponding to all records in the
 current climate data layer was extracted by the spatial data processing
 tools of ArcGIS, (2) the value was assigned a value of background pixels
 at corresponding position in the future climate data layer.
 <strong>Data processing</strong>
 Occurrence data of S. canadensis was randomly divided according
 to the algorithms of the software (MaxEnt) itself into training data (75%
 of occurrence point data used for model prediction) and test data (25% of
 occurrence point data used for model validation) with all additional
 parameters set as default. A receiver operating
 characteristics curve (ROC) plot was built by plotting the sensitivity
 values and the false positive fraction for all available probability
 thresholds (Manel et al. 2001) (Fig. S9). The area under the curve (AUC)
 was a measure of the area under the ROC, ranging from 0.5 to 1.0. The
 higher the AUC score, the better the model is at predicting
 presence/absence, indicating the environmental variables have high
 correlation with the predicted distribution of species, and prediction of
 the model is of high validity. The predicted model can be invalid (AUC
 value is lower than 0.6), poor (AUC value is 0.6–0.7), fair (AUC value is
 0.7-0.8), good (AUC value is 0.8–0.9) and excellent (AUC value is 0.9–1.0)
 (Swets 1988). Using a heuristic estimation during training of the model
 and by means of a jackknife test, the importance of each environmental
 variable in the model was evaluated (Fig. S8). The
 model was visually exhibited in ArcGis (version 10.0) to produce a single
 final distribution map. Different map colors corresponded to different
 fitting indices in the potential distribution map. The analysis base map
 was obtained from the National Fundamental Geographic Information System
 (http://nfgis.nsdi.gov.cn/).
description: There are some missing values in the dataset:
 1.) Six missing values will be finded in antioxidative parameters
 of cytotypes among native and invasive <i>S. canadensis</i>
 after heat exposure (45 ºC for 0, 2 and 4 h respectively) （File:
 Heat_tolerance.xlsx，Sheet: antioxidative parameters）
 2.)  In the parts of cross experiment, 29 replications were
 collected in some combinations，which should be 30 replications in plan
 (Fig. S5h and S 6a, b, c). Only 19 replications of the "IN6x X
 NA4x" ombinations were collected in the observation of germinated
 pollen grains on stigmas (Fig S 6a) and  11 replications of the "IN4x
 X NA4x" ombinations were collected in the observation of pollen tubes
 entering into the ovaries (Fig S 6b). 3. ) ITS
 sequences and psbA-trnH intergenic spacer sequences of 152<em> S.
 canadensis</em> populations were submit the data in a format of
 ".txt". You could read and align them using MEGA 4.1 Beta 3
 software.