Phenotypic diploidization in plant functional traits uncovered by synthetic neopolyploids in Dianthus broteri

Journal of Experimental Botany, Vol. 72, No. 15 pp. 5522–5533, 2021 doi:10.1093/jxb/erab179 Advance Access Publication 28 April 2021 Abbreviations: ABS/CS, absorbed energy flux per leaf cross-section; AN, net photosynthetic rate; Ca, ambient CO2 concentration; Ci, intercellular CO2 concentration; DI/CS, dissipated energy flux per leaf cross-section; ET/CS, electron transport energy flux per leaf cross-section; ETRmax, maximum electron transport rate; FC, flow cytometry; Fv/Fm, maximum quantum efficiency of PSII; gm, mesophyll conductance; gs, stomatal conductance; PI, performance index; PPFD, photosynthetic photon flux density; RC/CS, reaction centres per cross-section; Rd, dark respiration; TR/CS, trapped energy flux per leaf cross-section; Vc,max, maximum carboxylation rate of Rubisco; WGD, whole-genome duplication. © The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH PAPER Phenotypic diploidization in plant functional traits uncovered by synthetic neopolyploids in Dianthus broteri JuanJoséDomínguez-Delgado*,, JavierLópez-Jurado, EnriqueMateos-Naranjo and FranciscoBalao Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apdo. 1095, 41080-Sevilla, Spain * Correspondence: [email protected] Received 22 September 2020; Editorial decision 20 April 2021; Accepted 26 April 2021 Editor: Greg Rebetzke, CSIRO Agriculture and Food, Australia Abstract Whole-genome duplication and post-polyploidization genome downsizing play key roles in the evolution of land plants; however, the impact of genomic diploidization on functional traits still remains poorly understood. Using Dianthus broteri as a model, we compared the ecophysiological behaviour of colchicine-induced neotetraploids (4xNeo) to diploids (2x) and naturally occurring tetraploids (4xNat). Leaf gas-exchange and chlorophyll fluorescence analyses were performed in order to asses to what extent post-polyploidization evolutionary processes have affected 4xNat. Genomic diploidization and phenotypic novelty were evident. Distinct patterns of variation revealed that postpolyploidization processes altered the phenotypic shifts directly mediated by genome doubling. The photosynthetic phenotype was affected in several ways but the main effect was phenotypic diploidization (i.e. 2x and 4xNat were closer to each other than to 4xNeo). Overall, our results show the potential benefits of considering experimentally synthetized versus naturally established polyploids when exploring the role of polyploidization in promoting functional divergence. Keywords: Chlorophyll fluorescence, colchicine-induced autopolyploidy, Dianthus broteri, functional traits, leaf gas exchange, phenotypic diploidization, photosynthesis. Introduction Polyploidy, the genomic condition resulting from wholegenome duplication (WGD), is a widespread phenomenon that is considered to be a major driving force in evolution and diversification of flowering plants (Chen, 2007; Soltis et al., 2009; Tank etal., 2015; Landis etal., 2018). Although WGD is usually accompanied by hybridization (i.e. allopolyploidy), pure WGD (i.e. autopolyploidy) per se can induce immediate changes at the cellular and phenotypic levels as a result of the dramatic changing of the relationship between gene copy number and other cellular components (Doyle and Coate, applyparastyle "fig//caption/p[1]" parastyle "FigCapt" Phenotypic diploidization in plant functional traits | 5523 2019). Newly formed polyploids (hereafter referred to as neopolyploids) usually show an increase of cell size in concert with the DNA content (‘nucleotypic effect’; Stebbins, 1971) and hence produce bigger organs (te Beest et al., 2012). In addition, changes in cell volume associated with WGD may promote phenotypic novelty (Soltis etal., 2014; Wendel, 2015). The most common phenotypic changes in neopolyploid plants include larger stomatal size, thicker leaf morphology, and more chloroplasts within each guard cell, which can modify physiological traits such as transpiration, and photosynthetic and growth rates (Masterson, 1994; Levin, 2002; Maherali et al., 2009; Liu etal., 2011). Following WGD, polyploids start an adaptive process that can be divided into two evolutionary time-frames (Dodsworth etal., 2016). In the short term, neopolyploids need to survive and to establish by overcoming several intrinsic disadvantages (such as ‘genomic shock’ and aberrant meiosis; Comai, 2005), as well as extrinsic ones (i.e. minority cytotype exclusion, a frequency-dependent mating disadvantage; Levin, 1975). In this initial stage, polyploids need to outcompete their parents or ecologically differentiate from them. Hence, the adaptive shifts required for the establishment of neopolyploids (Soltis etal., 2014; Glick etal., 2016) usually bring them to inhabit new niches, especially under unstable environmental conditions (te Beest etal., 2012; López-Jurado etal., 2020). Polyploidymediated changes in physiological and ecological tolerances can enhance selective advantages and lead to the initial short-term survival and establishment of polyploids (Pandit etal., 2011; Soltis etal., 2014; Münzbergová and Haisel, 2019; Rice etal., 2019). In spite of the importance of ecophysiological aspects of niche divergence in autopolyploids, actual supporting evidence is scarce and is usually focused on drought tolerance (Maherali etal., 2009; Soltis etal., 2010; Liu etal., 2011; Spoelhof etal., 2017). Furthermore, the role of photosynthetic-related shifts in the establishment of polyploids has largely been overlooked (but see Vyas etal., 2007 and López-Jurado etal., 2020). Once a polyploid has been effectively established, a longer-term process of evolution begins as it becomes a quasidiploid, through the diploidization process. The most common phenomenon during diploidization is genome downsizing caused by the loss of duplicate genes (fractionation) and small genomic fragments (Cheng etal., 2018), which might be noticeable when comparing the monoploid genome size (1Cx) among different ploidy levels (Balao etal., 2009). In addition, there are other evolutionary processes that act in parallel and shape the genomic, transcriptomic, and phenotypic structure of the mesopolyploids to allow their functional diploidization, such as gene subfunctionalization and neofunctionalization, gene-silencing, activation of transposable elements, or genome rearrangements (Dodsworth etal., 2016). Notwithstanding all these well-known effects of WGD at the genomic, morphological, anatomical, physiological, and ecological scales (Ramsey and Schemske, 2002; Madlung etal., 2005; Soltis etal., 2014; Spoelhof etal., 2017), discerning the immediate impact of WGD from the short-term and long-term adaptive effects is challenging. Most previous studies have been conducted in naturally occurring polyploid complexes (Thompson and Merg, 2008; Balao et al., 2011; Manzaneda etal., 2012; Pegoraro etal., 2019) whose lineages have already experienced selection and diploidization across many generations. In contrast, synthetic neopolyploids provide an opportunity to study the immediate consequences of WGD on plant morphology and physiology (Maherali etal., 2009; Madlung, 2013; Spoelhof etal., 2017; Van Drunen and Husband, 2018). Artificial induction of autopolyploids using antimitotic chemicals (e.g. colchicine) has been successfully carried out in both plant breeding and research (Stupar et al., 2007; Vyas et al., 2007; Liu etal., 2011) and has provided valuable insights into the mechanisms by which polyploidization per se affects plant phenotypes. Discerning immediate WGD versus post-WGD phenotypic effects is possible by comparing the synthetic polyploids with parental diploids and natural polyploids (Martin and Husband, 2012; Husband etal., 2016; Pavlíková et al., 2017; Van Drunen and Husband, 2018). On the one hand, differences between diploids and synthetic neopolyploids in functional traits will reveal the instantaneous effects of WGD. On the other hand, comparisons between synthetic neopolyploids and naturally established polyploids might indicate which polyploidy-mediated shifts are adaptive (because they are retained in natural polyploids) and which ones are not advantageous in the longterm. Dianthus broteri (Caryophyllaceae) provides an excellent system for discerning the immediate effects of WGD from the subsequent evolutionary processes. This Iberian taxon encompasses the largest known autopolyploid complex within the genus (Balao et al., 2009), including diploid (2n=2x=30), tetraploid (2n=4x=60), hexaploid (2n=6x=90), and dodecaploid (2n=12x=180) monocytotypic populations. This complex seems to have evolved recently (0.9–2.1 Mya) following a rapid cytotypic adaptive divergence in concert with an extensive geographical disjunction and an ecological niche differentiation within an aridity gradient, with the higher cytotypes (6x and 12x) inhabiting more restricted and extreme habitats (Balao etal., 2010; López-Jurado etal., 2019). Diploids occupy the most benign Mediterranean subclimate, whilst tetraploids have enhanced ecological tolerances, encompassing the most diverse environmental conditions. This niche divergence is accompanied by numerous phenotypic differences (vegetative and reproductive) between the cytotypes (Balao etal., 2011), as well as different strategies regarding light-harvesting and photoprotection under temperature stress, therefore suggesting divergent adaptations (López-Jurado etal., 2020). All these phenotypic differences seem to be due to a mixture of both immediate and postWGD effects. For example, stomatal sizes vary with ploidy 5524 | Domínguez-Delgado etal. according to the nucleotypic effect, and some floral parts show within-cytotype differences in phenotypic optima, such as in tetraploids due to their two unrelated lineages. In addition, genome-downsizing that is linage-dependent is evident from the 1Cx values (Balao etal., 2009) and higher cytotypes show increased levels of DNA methylation and epigenetic variability (Alonso etal., 2016). Within this framework, the artificial induction of D.broteri neopolyploids provides a potential opportunity to distinguish between the influence of WGD per se and subsequent adaptive changes that vary across natural populations due to reproductive isolation and divergence by selective pressures (Maherali etal., 2009; Spoelhof etal., 2017; Van Drunen and Husband, 2018). In this study, we established a procedure for the induction of autotetrapolyploidy by treating diploid seeds with colchicine, and we then compared several ecophysiological traits of diploids and synthetic and natural tetraploids to address the following three questions. Is genome-downsizing a direct effect of polyploidization? Are there physiological shifts directly linked to polyploidization in D.broteri? And to what extent can post-polyploidization mechanisms explain the functional basis of the niche differentiation observed in naturally occurring populations of D.broteri? Materials andmethods Plant material and induction of synthetic neopolyploidy Seeds were collected in 2018 from a diploid population (15 plants) of Dianthus broteri located in south-eastern Spain (37º00´N, 3º01´W). The ploidy level and DNA content of this population have previously been characterized by flow cytometry together with chromosome counts (Balao etal., 2009), and its morphology and ecophysiology are known to be representative of D.broteri diploids (Balao etal., 2011). Treatments with colchicine (Sigma-Aldrich) consisted of two different incubation times (24h or 48h) in combination with four different concentrations in water: 0% (w/v, control), 0.2%, 0.4%, and 0.6%. Seeds were incubated in 2-ml Eppendorf tubes in darkness on a shaker, after which they were rinsed with 25ml of distilled water and sown on water-saturated filter paper in Petri dishes that were kept under a 12/12h photoperiod at 25.5/17ºC (130µmol m–2 s–1). Germination was recorded every 2 d for 21 d, and the seedlings obtained were sown in individual 2.5-l pots filled with a mixture of a commercial organic substrate (Gramoflor) and perlite (3:1) and placed in a greenhouse with controlled temperature of 21–25 ºC, 40–60 % relative humidity (RH), and natural daylight ranging from 200–1200 μmol m−2 s−1 photosynthetic photon flux density (PPFD) incident on leaves during the day. The pots were adequately irrigated with tap water and seedling survival was checked weekly for 90 d after sowing (at which point all plants displayed sufficient vigour to be considered as definitely established). In addition, diploid and tetraploid plants from natural populations spanning the whole range of distribution of this species (Table 1) were also grown under the same greenhouse conditions. The aim of including such among-population variation was to make our results conservative (i.e. based exclusively on intrinsic characteristics of each group beyond the possible dissimilarities caused by local adaptations and genetic drift) and our conclusionsrobust. For the ecophysiological characterization, 10 plants per group (i.e. 2x, 4xNeo, and 4xNat) were randomly selected 15 d prior to analysis and placed in a controlled-environment chamber (Aralab/Fitoclima 18.000EH, Lisbon, Portugal) with a 14/10h photoperiod (300μmol m−2 s−1) at 25/18°C and 40–60 % RH. This period under controlled conditions was to avoid interference by possible border effects within the greenhouse. Determination of DNA content and ploidylevels The DNA content and ploidy level of the seedlings were estimated by flow cytometry (FC) following a specific protocol for D.broteri (Balao et al., 2009). The FC measurements were conducted using a Coulter CYTOMICS FC500-MPL (Beckman Coulter, Fullerton, CA, USA) equipped with a 20 mW argon-ion laser at 488nm. Fresh leaf material (100mg) from glasshouse-grown D.broteri seedlings was used for nuclear suspensions together with Pisum sativum L.cv. ‘Ctirad’ as the genome size control (2C nuclear DNA=9.09 pg). All the FC measurements showed high quality (>5000 nuclei per peak and CV <5 %) and peak means were determined through manual gating using the Kaluza Analysis 2.1 software (Beckman Coulter). DNA content (2C) was estimated for the main FC peak (phase G0/G1; Supplementary Fig. S1), which would correspond to ~1.80 pg for diploids and ~3.60 pg for tetraploids (Balao etal., 2009). Mixoploidy (mixture of diploid and polyploid cells within the same tissue) was found, with some FC samples showing a second large peak representing more than 40% of the sample events, plus a third peak corresponding to nuclei in mitosis (phase G2) of the higher-ploidy-level cells. According to these results, each plant was classified as diploid (2n=2x), neotetraploid (2n=4xNeo), diploid-tetraploid mixoploid (2n=2x+4x), or tetraploid-octoploid mixoploid (2n=4x+8x). The mixoploids were discarded for further analysis. Monoploid genome size (1Cx) was estimated as the amount of 2C nuclear DNA divided by the ploidy level. Table 1. Population localities and numbers of plants of D.broteri used for the ecophysiological characterization Cytotype Population Coordinates No. of plants 2xLanjarón (Spain) 36º54´N 3º29´W 2 2xLaroles (Spain)* 37º00´N 3º01´W 7 2xMecina Alfahar (Spain) 36º59´N 3º04´W 2 2xÓrgiva (Spain) 36º53´N 3º24´W 1 2xSão Brás de Alportel (Portugal) 37º09´N 7º50´W 2 4xNat Archidona (Spain) 37º11´N 4º31´W 2 4xNat La Barrosa (Spain) 36º23´N 6º07´W 3 4xNat Pinar de la Breña (Spain) 36º11´N 5º58´W 2 4xNat Doñana, Peladillo (Spain) 37º05´N 6º35´W 2 4xNat Puerto del Boyar (Spain) 36º45´N 5º23´W 2 All 4xNeo (n=10) were induced from seeds collected in the diploid population of Laroles (marked with an asterisk). Phenotypic diploidization in plant functional traits | 5525 In addition, 1Cx data for the naturally occurring tetraploids (4xNat) were obtained from Balao etal. (2009). Stomatal measurements Stomatal measurements were performed 80–90 d after the colchicine treatment of seeds on 11 plants for each cytotype (all the confirmed 4xNeo plants, and randomly chosen plants for 2x and 4xNat). Epidermal impressions of the region near the leaf tip were taken on the adaxial surface of the youngest fully expanded leaf using Germolene New Skin Liquid Plaster (PharmaPac, UK) and examined under a Carl-Zeiss Axiophot photomicroscope equipped with a Sony DXC-390P Exwave HAD camera. The resulting images were analysed using ImageJ v.1.51j8, and the mean stomatal density together with mean lengths and widths were determined on the basis of 10 images per plant, at 10× magnification for density and 40× for the dimensions (Balao etal., 2011). Measurement of leaf gas exchange A LI-COR LI-6400 infrared gas analyser in an open system equipped with a Li-6400-02B LED light source was used for instantaneous gasexchange measurements and for constructing A/Ci curves. Five fully developed leaves were randomly selected for measurement on each cytotype. Net photosynthetic rate (AN), stomatal conductance (gs), and intercellular CO2 concentration (Ci) were recorded at a PPFD of 1000µmol m−2 s−1 (with 15% blue light to maximize stomatal aperture), vapour pressure deficit of 2.0–3.0 kPa, ambient CO2 concentration (Ca) of 400 µmol CO2 mol−1, leaf temperature of 25±2°C, and 50±5% RH. In addition, leaves were dark-adapted for 10min for measurement of dark respiration (Rd). We estimated mesophyll conductance (gm) and maximum carboxylation activity of Rubisco (Vc,max) by the curve-fitting method (Ethier and Livingston, 2004) and using the software package developed by Sharkey etal. (2007). A/Ci responses were measured once the leaves had reached a steady-state under the conditions described above (~20min), at which point Ca was decreased in the following steps: 400, 350, 300, 250, 200, 150, 100, and 50µmol mol−1. The chamber conditions were then restored to their initial levels and Ca was increased in the following steps to complete the curve: 500, 750, 1000, 1250, 1500, 1750, and 2000µmol mol−1. At each step, gas exchange was allowed to equilibrate to avoid significant variations in Rubisco activity (generally <180s; Long and Bernacchi, 2003). CO2 leakages into and out of the leaf chamber were determined using photosynthetically inactive leaves and the correction was applied to all the curves, as described previously by Flexas etal. (2013). Analysis of leaf chlorophyll fluorescence Modulated Chlorophyll a fluorescence was measured in dark-adapted leaves of each cytotype using a FluorPen FP100 PAM (Photon Systems Instruments, Czech Republic). The maximum quantum efficiency of PSII (Fv/Fm) was determined by using a 0.8s saturating light beam with an intensity of 8000μmol m−1 s−1 (Schreiber etal., 1986), with 10 replicates per cytotype. In addition, Kautsky curves (which indicate the fast kinetics of Chlorophyll a) were obtained via OJIP tests implemented in the pre-programmed protocols of the FluorPen FP100 (n=10). Fv/Fm, absorbed energy flux (ABS/CS), trapped energy flux (TR/CS), electron transport energy flux (ET/CS), and dissipated energy flux (DI/CS) per leaf cross-section, together with performance index (PI) derived from OJIP were calculated according to Strasser etal. (2004, 2010). These energy transduction fluxes on a leaf cross-section basis were employed due to differences in the density of reaction centres (RC/CS) among the cytotypes, according to López-Jurado etal. (2020). Maximum electron transport rate (ETRmax) was also obtained using the pre-programmed rapid light curve (RLC) protocol (n=6), which consisted of the exposure of dark-adapted leaves (30min) to increasing light levels (100, 200, 300, 500, and 1000µmol m−2 s−1) and recording the quantum yield at each step. Statistical analyses Data analyses were carried out using R v.3.6.3 (http://www.R-project. org/). The effects of the colchicine treatments (concentration and duration as main factors) on seed germination and seedling survival (dependent variables) were tested by fitting generalized linear models (GLMs) using a binomial error structure and the ‘logit’ link function for each dependent variable. The effectiveness of the colchicine treatments in producing viable polyploids was examined using the Fisher’s exact test. One-way ANOVA and post hoc Tukey’s tests were used for detecting differences between the cytotypes in the 1Cx DNA contents and the ecophysiological parameters derived from the OJIP tests and RLC protocol. Results Effects of colchicine on germination, seedling survival, and neopolyploidy inductionrate Germination was significantly affected by colchicine concentration and incubation time, and there was no interaction between them (Table 2). Higher concentrations of colchicine resulted in lower germination success, from 61% in the control to 39% in seeds treated with 0.6 % colchicine. Seeds incubated for 48h showed 29.3% higher germination overall than those incubated for 24h (Fig. 1A). Seedling survival was also significantly affected by the colchicine concentration and by the incubation time, and again there was no interaction. An increase Table 2. Summary of generalized linear model (GLM) results for seed germination and seedling survival in response to colchicine concentration and incubation time Response variable Factor L-R χ 2d.f. P-value Seed germination Colchicine concentration 21.3072 3 <0.001 Incubation time 14.2144 1 <0.001 Colchicine concentration × Incubation time 7.1943 3 >0.05 Seedling survival Colchicine concentration 82.779 3 <0.001 Incubation time 3.935 1 <0.05 Colchicine concentration × Incubation time 1.746 3 >0.05 L-R χ 2, likelihood-ratio chi-square; d.f., degrees of freedom. 5526 | Domínguez-Delgado etal. in colchicine concentration resulted in a decrease in survival rate, from 77.5% in the control to 13.74% in seeds treated with 0.6 % colchicine; however, in contrast to germination, survival was 20.8% lower overall in seedlings from the 48h treatment compared to 24h (Fig. 1B). A total of 94 plants were subjected to flow cytometry analysis (21 controls and 73 that survived the colchicine treatments) of which 74 were diploids (the 21 controls and 53 treated plants), 11 were tetraploids, and nine were mixoploids (eight 2x+4x and one 4x+8x). The frequency of polyploidy induction was significantly higher as the dose (i.e. concentration and incubation time) of colchicine increased (Fisher’s exact test, P<0.05; Fig. 1C). The control treatment invariably failed to induce polyploidization. The highest induction of neopolyploidy (tetraploids 37.5% of the established plants) was obtained at 0.6 % colchicine concentration and Fig. 1. Effects of colchicine treatments on seeds of Dianthus broteri. Seeds were treated with colchicine at concentrations of 0, 0.2, 0.4, or 0.6% for either 24h or 48h. (A) Germination rate (n=100) and (B) survival rate of seedlings. The boxplots show the median, the interquartile range, and the non-outlier range. (C) Frequency of cytotypes obtained under the different incubation times and concentrations. (D) Monoploid genome sizes of the 2x diploid, the 4xNat natural tetraploid, and the 4xNeo neotetraploid. In the violin plots, white circles represent the median, thick black bars correspond to the interquartile range, and the thin lines show the 95% confidence interval. Different letters indicate significant differences among the cytotypes as determined using ANOVA followed by Tukey’s test (P<0.05). Phenotypic diploidization in plant functional traits | 5527 24h incubation. The highest frequency of mixoploids (17.6 % of the established plants) was obtained after 48h. Genome size variation The nuclear DNA amount of the synthetic 4xNeo plants (3.60±0.07 pg; mean±s.d.) was about twice that of the diploids (1.82±0.03 pg), whilst the natural tetraploids showed a mean genome size of 3.47±0.13 pg. Accordingly, the monoploid DNA amount (1Cx) was significantly dependent on cytotype (F2,170=59.49, P<0.001; Fig. 1D). Whilst no significant differences were found when 2x (0.910±0.014 pg) and 4xNeo (0.900±0.017 pg) were compared (P>0.05), 4xNat showed a significantly lower 1Cx DNA value than either of them (P<0.05), varying from 0.77–0.94pg. Polyploidy-mediated differences in functionaltraits Stomatal traits were affected differently by polyploidization (Fig. 2). Comparisons among the cytotypes revealed significant Fig. 2. Stomatal sizes of diploids (2x), natural tetraploids (4xNat), and colchicine-induced neotetraploids (4xNeo) of Dianthus broteri. (A) Epidermal impressions of leaves of 2x and 4xNeo plants (4xNat was similar 2x and is therefore not shown). Scale bars are 100µm. (B) Stomatal length and (C) density of the three cytotypes. The boxplots show the median, the interquartile range, and the non-outlier range. Different letters indicate significant differences among cytotypes as determined using ANOVA followed by Tukey’s test (P<0.05). 5528 | Domínguez-Delgado etal. differences in stomatal length (F2,30=25.79, P<0.001) and width (F2,30=9.61, P<0.001), as well as density (F2,30=28.49, P<0.001). Whilst length, width, and density were indistinguishable between 2x and 4xNat (P>0.05), varying between 29–39μm, 19–28μm, and 66–154mm−2, respectively, the synthetic neotetraploids showed significantly longer (40.58±3.77 μm; P<0.05) and wider (28.24±2.97 μm; P<0.05) stomata than either of them, but had a lower density (38.91±6.91mm−2; P<0.05). Notably, the synthetic tetraploids showed an increased variation in stomatal length (CV 6.1%, 6.4%, and 9.3% in 2x, 4xNat, and 4xNeo, respectively), but a more consistent density (CV 26.3%, 27.4%, and 17.8% in 2x, 4xNat, and 4xNeo, respectively). Photosynthetic performance was also affected by polyploidization, but 4xNat did not always display the same trends than 4xNeo. Analysis of leaf gas exchange showed that the synthetic neopolyploids had a significant increase in net photosynthetic rate (AN; F2,13=14.72, P<0.001), stomatal conductance (gs; F2,13=17.71, P<0.001), and mesophyll conductance (gm; F2,11=4.48, P<0.05) in comparison to the diploids and natural tetraploids, which showed similar values to each other (Fig. 3). AN and gs were almost doubled in 4xNeo compared to 2x and 4xNat (P<0.05), which had similar values. The 4xNeo plants also showed ~2-fold greater gm than 2x (P<0.05), whilst 4xNat showed intermediate values between those of 2x and 4xNeo. Although the results showed broadly similar patterns, no significant differences were found among the cytotypes for the other photosynthetic parameters that were measured, namely intercellular CO2 concentration (Ci), maximum rate of Rubisco activity (Vc,max), and the rate of dark respiration (Rd) (Supplementary Fig. S2; ANOVA, P>0.05). Analysis of leaf chlorophyll fluorescence also showed several distinct patterns of variation between the cytotypes (Fig. 4). Significant differences were found for maximum electron transport rate (ETRmax; F2,20=5.64, P<0.05) and maximum quantum efficiency of PSII (Fv/Fm; F2,48=4.17, P<0.05). In both cases, higher values were found in 4xNeo compared to 2x, whilst vales in 4xNat were intermediate (Fig. 4A, B), indicating a reduction in the polyploidy-mediated direct effect on these traits. Such an after-WGD reduction was not found in the performance index (PI), with 4xNeo and 4xNat showing higher values (71.5% on average) than 2x (F2,47=8.70, P<0.001; Fig. 4C). A different pattern was observed for the energy transduction fluxes. We did not observe a direct effect of WGD but a postpolyploidization divergence was found for the absorbed energy flux per cross-section (ABS/CS; F2,47=4.87, P<0.05; Fig. 4D). The values for 2x and 4xNeo were similar (P>0.05), whilst 4xNat showed a 30.8% increase in ABS/CS compared to 2x (P<0.01). No significant difference was found between 4xNat and 4xNeo (P>0.05). The same post-WGD divergence was also observed for the trapped energy flux per cross-section (TR/ CS; F2,47=5.31, P<0.01; Fig. 4E), with 4xNat showing a 33.0% increase compared to the diploids. Asimilar trend was found in the electron transport energy flux per cross-section (ET/CS), but there were no significant differences among the cytotypes (F2,47=1.75, P>0.05), with mean values of 1717, 1960, and 2065 for 2x, 4xNeo, and 4xNat, respectively (Supplementary Fig. S2). Finally, the dissipated energy flux per cross-section (DI/ CS) was only marginally affected by chromosome doubling (F2,45=2.57, P=0.09),with 4xNeo showing ~0.3-fold lower dissipation values than 2x and 4xNat (Fig. 4F). Discussion Exposure to colchicine through seed imbibition was an efficient method for obtaining synthetic neopolyploids of Dianthus broteri, and our results could have implications for the optimization of autopolyploidy induction in general (Fig. 1). Fig. 3. Effects of polyploidization in Dianthus broteri on leaf gas exchange. Measurements were taken on diploids (2x), natural tetraploids (4xNat), and colchicine-induced neotetraploids (4xNeo). (A) Net photosynthetic rate (AN), (B) stomatal conductance (gs), and (C) and mesophyll conductance (gm). Data are means (±SD), n=5. Different letters indicate significant differences among cytotypes as determined using ANOVA followed by Tukey’s test (P<0.05). Phenotypic diploidization in plant functional traits | 5529 Despite polyploidization increasing in parallel with colchicine concentration, large reductions in germination and seedling survival at high dosages made treatments with intermediate doses more suitable for inducing neopolyploidy. This was consistent with results of previous studies (Thiebaut and Kasha, 1978; Chen etal., 2006; Lehrer etal., 2008; Omidbaigi etal., 2010; Xing et al., 2011; Pavlíková et al., 2017; Sadat Noori etal., 2017), although the different methodologies (different antimitotic agents and/or different target tissues such as shoot apical meristem or callus) and the species-specific responses to colchicine do not make it possible to establish a general protocol for neopolyploidy induction. Quantification of nuclear DNA through flow cytometry provides a practical method for quickly determining ploidy levels (Balao etal., 2009) and it was also effective for discerning mixoploidy (Fig. 1C). Analysis of the monoploid genome size (1Cx) revealed remarkable genome size stability in nascent neopolyploids but a moderate genomic diploidization in the natural tetraploid populations (Fig. 1D), as previously shown by Balao etal. (2009). This is a common phenomenon in mesopolyploids (Dodsworth etal., 2016; Mandákova etal., 2010)and could be mainly due to gene loss and chromosomal rearrangements contributing to restore successful cyto–nuclear interactions (Sharbrough etal., 2017). Although genetic change seems to be subtle (but spread locally) in this D.broteri polyploid complex due to rapid diversification (Balao etal., 2010), genomic adjustments could have played a crucial role in adaptation after WGD, not only providing genomic stability but also accounting for transcriptomic distinctness, which frequently results in phenotypic novelty and ecophysiological differentiation (Chen etal., 2007; Chelaifa etal., 2010; Soltis etal., 2014; Dodsworth etal., 2016). In this regard, genomic diploidization could have boosted local adaptations following the post-WGD niche expansion, and fostered the remarkable ecological features of D.broteri tetraploids, which show greater niche breadth compared with the rest of the cytotypes (López-Jurado etal., Fig. 4. Effects of polyploidization in Dianthus broteri on leaf chlorophyll fluorescence. Measurements were taken on diploids (2x), natural tetraploids (4xNat), and colchicine-induced neotetraploids (4xNeo). (A) Maximum electron transport rate (ETRmax), (B) maximum quantum efficiency of PSII (Fv/Fm), (C) performance index (PI), (D) absorbed energy flux (ABS/CS), (E) trapped energy flux (TR/CS), and (F) dissipated energy flux (DI/CS) per leaf cross-section. Data are means (±SD), n=10, except (A) where n=6. Different letters indicate significant differences among cytotypes as determined using ANOVA followed by Tukey’s test (P<0.05). 5530 | Domínguez-Delgado etal. 2019) and also distinct light-harvesting and photoprotection strategies (López-Jurado etal., 2020). The few previous studies that have examined the effects of autopolyploidy on plant physiology have focused on synthetic neopolyploids (Stupar etal., 2007; Liu etal., 2011; Dong etal., 2017; Wei etal., 2020) or on divergence in natural populations (Balao etal., 2011; Manzaneda etal., 2012; Thompson etal., 2014; Pacey etal., 2020). In contrast, our approach was able to distinguish not just the post-WGD effects on several physiological and development traits, but also the immediate effects that help to explain the divergent evolution of the ecological niche in the polyploids. For example, it is well established that stomatal characteristics have a great impact on the physiology of land plants, and climatic conditions usually operate as selective forces for these traits (Veselý et al., 2020). Our data indicated that autopolyploidization in D.broteri directly modified the size of guard cells (i.e. the ‘gigas effect’), resulting in larger stomata with greater aperture area available for gas exchange (Fig. 2). Conversely, the density of stomata immediately after WGD decreased, but interestingly tetraploid populations in the wild did not show these changes in stomatal size and density. A reduction in stomatal size and an increase in density after WGD has also been shown in other polyploid systems (e.g. Maherali et al., 2009; Münzbergová, 2017). Thus, a ‘phenotypic diploidization’ could occur after WGD on these stomatal traits, which become diploid-like either due to directional selection after establishment (Vyas etal., 2007) or because the establishment of polyploid individuals with unfit stomatal characteristics is prevented (Comai, 2005; Soltis etal., 2014). In addition, stomatal downsizing in 4xNat D.broteri, together with an increased number of stomata per unit area, would be related to the genome downsizing (i.e. genomic diploidization) in the natural tetraploids. Variation in cell size (related to the loss of DNA content) will play a crucial role in optimizing gas exchange, potential carbon gain, and water use after WGD (Simonin and Roddy, 2018; Lawson and Vialet-Chabrand, 2019; Roddy et al., 2020). Given the fact that higher stomatal densities have been associated with more arid climates (Carlson etal., 2016), the phenotypic and genomic diploidization in the higher-order neopolyploids (6x, 12x) of D.broteri could have also triggered adaptation to harsh environments (Balao etal., 2011). In addition, the remarkable within-cytotype variation of stomatal size in naturally occurring 2x and 4x populations (Fig. 2) suggests that local adaptations in D.broteri might reside in adjusting stomatal density rather than stomatal size (Wei etal., 2019). Physiological traits also showed a mixture of immediate and post-WGD effects. However, we did not find any differences between cytotypes (either synthetic or natural polyploids) in maximum carboxylation rate of Rubisco (Vc,max), dark respiration (Rd), CO2 concentration in the substomatal cavities (Ci), or the electron transport energy flux per leaf cross-section (ET/CS) (Supplementary Fig. S2), so the few (if any) changes following WGD would be deleterious (e.g. under purifying selection). Interestingly, a previous study showed significant down-regulation in linear electron transport (i.e. ET/CS) only in the higher-ploidy levels (6x and 12x) of the D.broteri complex, suggesting a photoprotective adaptation to their stressful ecological niches (López-Jurado etal., 2020). Phenotypic diploidization was evident in the stomatal and mesophyll conductances (gs and gm; Fig. 3) and hence the increases gained through WGD were not maintained in 4xNat. In agreement with our results (i.e. 4xNeo having higher conductances than 2x), neopolyploids frequently display increased gs and gm. (Vyas etal., 2007; Wang etal., 2019; Monda etal., 2016). This might be partially explained by structural differences between the ploidy levels, with greater stomatal area and larger intercellular spaces in the neopolyploids. Nevertheless, other non-structural factors directly affected by WGD could help to explain the differences in conductances, such as osmotic adjustment and metabolic regulation of stomatal opening (Mouhaya etal., 2010; Monda etal., 2016). It is worth noting that our physiological measurements were taken under optimal conditions and that a different picture might be seen under stressful ones (López-Jurado etal., 2020). The tendency of a return to diploid-like gs and gm values after WGD has not been previously reported to our knowledge, and a single study comparing diploids, neopolyploids, and established polyploids found that increases in gs and gm observed immediately after WGD tended to then increase even further (Vyas etal., 2007). However, this diploidization of conductances could accompany the structural diploidization of the stomata. Higher densities of smaller stomata are advantageous for gas exchange because they result in shorter diffusion path lengths (Franks and Beerling, 2009), and they can protect plants from xylem embolisms as well as facilitate the reduction of water loss (Li etal., 1996; te Beest etal., 2012). The net photosynthetic rate (AN), which correlates with gs and gm (among other parameters), showed higher values for 4xNeo whilst it did not differ between 2x and 4xNat (Fig. 3). A greater photosynthetic capacity in combination with faster growth rate is common in neopolyploids (Ramsey and Schemske, 2002; Saleh etal., 2008; Münzbergová, 2017) and seem to be the rule in established mesopolyploids (Hull-Sanders etal., 2009; Ježilová etal., 2015; Liao etal., 2016). Accordingly, the noticeably lower net photosynthetic rate that we observed in natural tetraploids (similar to that in the diploids) would also be related to the genomic diploidization and the consequent stomatal adaptations to optimize the balance between CO2 acquisition and water losses under a wide range of environmental conditions (Simonin and Roddy, 2018; Roddy etal., 2020). The influence of phenotypic diploidization was also observed for the efficiency of the PSII apparatus, as indicated by the differences between the cytotypes in several chlorophyll fluorescence parameters (Fig. 4) (Maxwell and Johnson, 2000). The 4xNeo plants showed higher values for maximum