BACKGROUND AND AIMS: Reproductive isolation and local establishment are necessary for plant speciation. Polyploidy, the possession of more than two complete chromosome sets, creates a strong postzygotic reproductive barrier between diploid and tetraploid cytotypes. However, this barrier weakens between polyploids (e.g. tetraploids and hexaploids). Reproductive isolation may be enhanced by cytotype morphological and environmental differentiation. Moreover, morphological adaptations to local conditions contribute to plant establishment. However, the relative contributions of ploidy level and the environment to morphology have generally been neglected. Thus, the extent of morphological variation driven by ploidy level and the environment was modelled for diploid, tetraploid and hexaploid cytotypes of Campanula rotundifolia agg. Cytotype distribution was updated, and morphological and environmental differentiation was tested in the presence and absence of natural contact zones. METHODS: Cytotype distribution was assessed from 231 localities in Central Europe, including 48 localities with known chromosome counts, using flow cytometry. Differentiation in environmental niche and morphology was tested for cytotype pairs using discriminant analyses. A structural equation model was used to explore the synergies between cytotype, environment and morphology. KEY RESULTS: Tremendous discrepancies were revealed between the reported and detected cytotype distribution. Neither mixed-ploidy populations nor interploidy hybrids were detected in the contact zones. Diploids had the broadest environmental niche, while hexaploids had the smallest and specialized niche. Hexaploids and spatially isolated cytotype pairs differed morphologically, including allopatric tetraploids. While leaf and shoot morphology were influenced by environmental conditions and polyploidy, flower morphology depended exclusively on the cytotype. CONCLUSIONS: Reproductive isolation mechanisms vary between cytotypes. While diploids and polyploids are isolated postzygotically, the environmental niche shift is essential between higher polyploids. The impact of polyploidy and the environment on plant morphology implies the adaptive potential of polyploids, while the exclusive relationship between flower morphology and cytotype highlights the role of polyploidy in reproductive isolation.

Czech Academy of Sciences Institute of Botany Department of Evolutionary Plant Biology Zámek Průhonice Czech Republic

Faculty of Ecology and Environmental Sciences Technical University in Zvolen T G Masaryka Zvolen Slovakia

Faculty of Science Department of Botany Charles University Benátská Prague Czech Republic

Faculty of Science Department of Ecosystem Biology University of South Bohemia Branišovská České Budějovice Czech Republic

Institute of Botany Plant Science and Biodiversity Centre Slovak Academy of Sciences Dúbravská cesta Bratislava Slovakia

Erratum v

Ann Bot. 2023 Feb 7;131(1):243. doi: 10.1093/aob/mcac011. PubMed

Zobrazit více v PubMed

Anderson  MJM. 2006. Distance‐based tests for homogeneity of multivariate dispersions. Biometrics 62: 245–253. PubMed

Balao  F, Herrera J, Talavera S. 2011. Phenotypic consequences at the microevolutionary scale of polyploidy and genome size dynamics. Journal of Evolutionary Biology 192: 256–265. PubMed

Barker  M, Rayens W. 2003. Partial least squares for discrimination. Journal of Chemometrics 17: 166–173.

te Beest  M, Le Roux JJ, Richardson DM, et al.  2012. The more the better? The role of polyploidy in facilitating plant invasions. Annals of Botany 109: 19–45. PubMed PMC

Bjørnstad  ON. 2019.

Bjørnstad  ON, Falck W. 2001. Nonparametric spatial covariance functions: estimation and testing. Environmental and Ecological Statistics 8: 53–70.

Blackith  RE, Reyment RA. 1971. Multivariate morphometrics. London: Academic Press.

Böcher  TW. 1936. Cytological studies on

Brady  KU, Kruckeberg AR, Bradshaw HD. 2005. Evolutionary ecology of plant adaptation to serpentine soils. Annual Review of Ecology, Evolution, and Systematics 36: 243–266.

Brochmann  C, Brysting AK, Alsos IG, et al.  2004. Polyploidy in arctic plants. Biological Journal of the Linnean Society 82: 521–536.

Broennimann  O, Fitzpatrick MC, Pearman PB, et al.  2012. Measuring ecological niche overlap from occurrence and spatial environmental data. Global Ecology and Biogeography 21: 481–497.

Caperta  AD, Castro S, Loureiro J, et al.  2017. Biogeographical, ecological and ploidy variation in related asexual and sexual

Castro  M, Castro S, Figueiredo A, Husband BC, Loureiro J. 2018. Complex cytogeographical patterns reveal a dynamic tetraploid–octoploid contact zone. AoB Plants 10: 1–18. PubMed PMC

Castro  M, Loureiro J, Figueiredo A, Serrano M, Husband BC, Castro S. 2020. Different patterns of ecological divergence between two tetraploids and their diploid counterpart in a parapatric linear coastal distribution polyploid complex. Frontiers in Plant Science 11: 315. PubMed PMC

Castro  M, Loureiro J, Husband BC, Castro S. 2020. The role of multiple reproductive barriers: strong post-pollination interactions govern cytotype isolation in a tetraploid–octoploid contact zone. Annals of Botany 126: 991–1003. PubMed PMC

Čertner  M, Fenclová E, Kúr P, et al.  2017. Evolutionary dynamics of mixed-ploidy populations in an annual herb: dispersal, local persistence and recurrent origins of polyploids. Annals of Botany 120: 303–315. PubMed PMC

Christensen  RHB. 2019.

Chumová  Z, Krejčíková J, Mandáková T, Sud J, Trávníček P. 2015. Evolutionary and taxonomic implications of variation in nuclear genome size: lesson from the grass genus Anthoxanthum (Poaceae). Plos One 10: e0133748. doi: 10.1371/journal.pone.0133748. PubMed DOI PMC

Claude  J. 2008. Morphometrics with R. New York: Springer Science & Business Media.

De Marco  P, Nóbrega CC. 2018. Evaluating collinearity effects on species distribution models: an approach based on virtual species simulation. PLoS One 13: e0202403. PubMed PMC

Doležel  J, Greilhuber J, Suda J. 2007. Estimation of nuclear DNA content in plants using flow cytometry. Nature Protocols 2: 2233–2244. PubMed

Doyle  JJ, Sherman-Broyles S. 2017. Double trouble: taxonomy and definition of polyploidy. New Phytologist 213: 487–493. PubMed

Eriksson  L, Johansson E, Kettaneh-Wold N, Trygg J, Wikström C, Wold S. 2006. Multi- and megavariate data analysis. Part II. Advanced applications and method extensions. Umea: Umetrics Academy.

Fenaroli  F, Pistarino A, Peruzzi L, Cellinese N. 2013.

Flagel  LE, Wendel JF. 2009. Gene duplication and evolutionary novelty in plants. New Phytologist 183: 557–564. PubMed

Flatscher  R, García PE, Hülber K, et al.  2015. Underestimated diversity in one of the world’s best studied mountain ranges: the polyploid complex of PubMed PMC

Gadella  TWJ. 1964. Cytotaxonomic studies in the genus

Glennon  KL, Ritchie ME, Segraves KA. 2014. Evidence for shared broad-scale climatic niches of diploid and polyploid plants. Ecology Letters 17: 574–582. PubMed

Hanušová  K, Čertner M, Urfus T, et al.  2019. Widespread co-occurrence of multiple ploidy levels in fragile ferns ( PubMed PMC

Hopkins  R. 2013. Reinforcement in plants. New Phytologist 197: 1095–1103. PubMed

Hülber  K, Sonnleitner M, Suda J, et al.  2015. Ecological differentiation, lack of hybrids involving diploids, and asymmetric gene flow between polyploids in narrow contact zones of PubMed PMC

Husband  BC, Baldwin SJ, Sabara HA. 2016. Direct vs. indirect effects of whole-genome duplication on prezygotic isolation in chamerion angustifolium: Implications for rapid speciation. American Journal of Botany 103: 1259–1271. PubMed

Husband  BC, Schemske DW. 2000. Ecological mechanisms of reproductive isolation between diploid and tetraploid

Jiao  Y, Wickett NJ, Ayyampalayam S, et al.  2011. Ancestral polyploidy in seed plants and angiosperms. Nature 473: 97–100. PubMed

Jiménez-Valverde  A, Nakazawa YJ, Lira-Noriega A, Peterson Townsend A. 2009. Environmental correlation structure and ecological niche model projections. Biodiversity Informatics 6: 28–35.

Kaplan  Z. 2012. Flora and phytogeography of the Czech Republic. Preslia 84: 505–573.

Karunarathne  P, Schedler M, Martínez EJ, Honfi AI, Novichkova A, Hojsgaard D. 2018. Intraspecific ecological niche divergence and reproductive shifts foster cytotype displacement and provide ecological opportunity to polyploids. Annals of Botany 121: 1183–1196. PubMed PMC

Kennedy  BF, Sabara HA, Haydon D, Husband BC. 2006. Pollinator-mediated assortative mating in mixed ploidy populations of PubMed

Kirkpatrick  M. 2000. Reinforcement and divergence under assortative mating. Proceedings of the Royal Society B: Biological Sciences 267: 1649–1655. PubMed PMC

Kobrlová  L, Hroneš M, Koutecký P, Štech M, Trávníček B. 2016.

Köhler  C, Mittelsten Scheid O, Erilova A. 2010. The impact of the triploid block on the origin and evolution of polyploid plants. Trends in Genetics 26: 142–148. PubMed

Kolář  F, Čertner M, Suda J, Schönswetter P, Husband BC. 2017. Mixed-ploidy species: progress and opportunities in polyploid research. Trends in Plant Science 22: 1041–1055. PubMed

Kolář  F, Fér T, Štech M, et al.  2012. Bringing together evolution on serpentine and polyploidy: spatiotemporal history of the diploid-tetraploid complex of PubMed PMC

Kovačić  S. 2004. The genus

Kovanda  M. 1966. Some chromosome counts in the

Kovanda  M. 1967. Polyploidie a variabilita v komplexu Campanula rotundifolia L. PhD Thesis, Charles University, Czech Republic.

Kovanda  M. 1970

Kovanda  M. 1970

Kovanda  M. 1977. Polyploidy and variation in the

Kovanda  M. 1983. Chromosome numbers in selected Angiosperms. Preslia 55: 193–205.

Kovanda  M. 2002. A range extension for

Krejčíková  J, Sudová R, Lučanová M, et al.  2013. High ploidy diversity and distinct patterns of cytotype distribution in a widespread species of PubMed PMC

Laane  MM, Croff BE, Wahlstrøm R. 1983. Cytotype distribution in the PubMed

Lafon-Placette  C, Johannessen IM, Hornslien KS, et al.  2017. Endosperm-based hybridization barriers explain the pattern of gene flow between PubMed PMC

Landis  JB, Soltis DE, Li Z, et al.  2018. Impact of whole-genome duplication events on diversification rates in angiosperms. American Journal of Botany 105: 348–363. PubMed

Laport  RG, Hatem L, Minckley RL, Ramsey J. 2013. Ecological niche modeling implicates climatic adaptation, competitive exclusion, and niche conservatism among

Laport  RG, Minckley RL, Ramsey J. 2016. Ecological distributions, phenological isolation, and genetic structure in sympatric and parapatric populations of the PubMed

Laport  RG, Muhia P, Kennell C, Hasan H. 2017. Assessing water use variation among the cytotypes of the autopolyploid southwestern desert Creosotebush (

Laport  RG, Ramsey J. 2015. Morphometric analysis of the North American creosote bush (

Levin  DA. 1975. Minority cytotype exclusion in local plant populations. Taxon 24: 35–43.

Levin  DA. 1983. Polyploidy and novelty in flowering plants. The American Naturalist 122: 1–25.

Levin  DA. 2019. Why polyploid exceptionalism is not accompanied by reduced extinction rates. Plant Systematics and Evolution 305: 1–11.

López-Jurado  J, Mateos-Naranjo E, Balao F. 2019. Niche divergence and limits to expansion in the high polyploid PubMed

Lüdecke  D, Makowski D, Waggoner P. 2020.

Maad  J, Armbruster WS, Fenster CB. 2013. Floral size variation in

Macková  L, Nosková J, Ďurišová Ľ, Urfus T. 2020. Insights into the cytotype and reproductive puzzle of

Macková  L, Vít P, Urfus T. 2018. Crop-to-wild hybridization in cherries—empirical evidence from PubMed PMC

Mandáková  T, Kovařík A, Zozomová-Lihová J, et al.  2013. The more the merrier: recent hybridization and polyploidy in Cardamine. The Plant Cell 25: 3280–3295. PubMed PMC

Mandáková  T, Pouch M, Harmanová K, Zhan SH, Mayrose I, Lysak MA. 2017. Multispeed genome diploidization and diversification after an ancient allopolyploidization. Molecular Ecology 26: 6445–6462. PubMed

Manly  BFJ. 2006. Randomization, bootstrap and Monte Carlo methods in biology. Boca Raton: CRC Press.

Mansion  G, Parolly G, Crowl AA, et al.  2012. How to handle speciose clades? Mass taxon-sampling as a strategy towards illuminating the natural history of PubMed DOI PMC

Mao  Y, Gabel A, Nakel T, et al.  2020. Selective egg cell polyspermy bypasses the triploid block. eLife 9: 1–15. PubMed PMC

Marhold  K, Mártonfi P, Mereďa P, Mráz P,

Mayrose  I, Zhan SH, Rothfels CJ, et al.  2015. Methods for studying polyploid diversification and the dead end hypothesis: a reply to Soltis PubMed

Meeus  S, Šemberová K, De Storme N, Geelen D, Vallejo-Marín M. 2020. Effect of whole-genome duplication on the evolutionary rescue of sterile hybrid monkeyflowers. Plant Communication PubMed PMC

Mevik  B-H, Wehrens R, Liland KH. 2019.

Molina-Henao  YF, Hopkins R. 2019. Autopolyploid lineage shows climatic niche expansion but not divergence in PubMed

Moore  RC, Purugganan MD. 2005. The evolutionary dynamics of plant duplicate genes. Current Opinion in Plant Biology 8: 122–128. PubMed

Mráz  P. 2005. Chromosome number and DNA ploidy level reports from Central Europe - 1. Biologia, Bratislava 60: 99–103.

Mráz  P, Ronikier M. 2016. Biogeography of the Carpathians: evolutionary and spatial facets of biodiversity. Biological Journal of the Linnean Society 119: 528–559.

Mráz  P, Šingliarová B, Urfus T, Krahulec F. 2008. Cytogeography of PubMed PMC

Muñoz-Pajares  AJ, Perfectti F, Loureiro J, et al.  2017. Niche differences may explain the geographic distribution of cytotypes in PubMed

Murdoch  D, Chow ED. 2018.

Nakagawa  S, Johnson PCD, Schielzeth H. 2017. The coefficient of determination R PubMed PMC

Nierbauer  KU, Paule J, Zizka G. 2017. Heteroploid reticulate evolution and taxonomic status of an endemic species with bicentric geographical distribution. AoB Plants 9: plx002. PubMed PMC

Oksanen  J, Blanchet FG, Friendly M, et al.  2019.

Pannell  JR, Obbard DJ, Buggs RJA. 2004. Polyploidy and the sexual system: what can we learn from

Parisod  C, Besnard G. 2007. Glacial in situ survival in the Western Alps and polytopic autopolyploidy in PubMed

Paule  J, Wagner ND, Weising K, Zizka G. 2017. Ecological range shift in the polyploid members of the South American genus PubMed PMC

Peskoller  A, Silbernagl L, Hülber K, Sonnleitner M, Schönswetter P. 2021. Do pentaploid hybrids mediate gene flow between tetraploid DOI

Pinheiro  J, Bates D. 2000. Mixed-effects models in S and S-PLUS. New York: Springer Science & Business Media.

Pinheiro  J, Bates D, DebRoy S, Sarkar D, Team RC. 2019.

Podlech  D. 1965. Revision der europäischen und nordafrikanischen Vertreter der Subsect. Heterophylla (Wit.) Fed. der Gattung

Porturas  LD, Anneberg TJ, Curé AE, Wang S, Althoff DM, Segraves KA. 2019. A meta-analysis of whole genome duplication and the effects on flowering traits in plants. American Journal of Botany 106: 469–476. PubMed

Preite  V, Stöcklin J, Armbruster GFJ, Scheepens JF. 2015. Adaptation of flowering phenology and fitness-related traits across environmental gradients in the widespread

R Core Team. 2019. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing.

Ramsey  J, Schemske DW. 1998. Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annual Review of Ecology and Systematics 29: 467–501.

Ramsey  J, Schemske DW. 2002. Neopolyploidy in flowering plants. Annual Review of Ecology and Systematics 33: 589–639.

Rauchová  J. 2007. Karyologická, fenetická a genetická diferenciace českého subendemického taxonu Campanula gentilis Kovanda. MSc Thesis, Charles University, Czech Republic.

Ray‐Mukherjee  J, Nimon K, Mukherjee S, et al.  2014. Using commonality analysis in multiple regressions: a tool to decompose regression effects in the face of multicollinearity. Methods in Ecology and Evolution 5: 320–328.

Rejlová  L, Chrtek J, Trávníček P, Lučanová M, Vít P, Urfus T. 2019. Polyploid evolution: the ultimate way to grasp the nettle. PLoS One 14: 1–24. PubMed PMC

Rice  A, Glick L, Abadi S, et al.  2015. The Chromosome Counts Database (CCDB) - a community resource of plant chromosome numbers. New Phytologist 206: 19–26. PubMed

Rice  A, Šmarda P, Novosolov M, et al.  2019. The global biogeography of polyploid plants. Nature Ecology and Evolution 3: 265–273. PubMed

Roquet  C, Sanmartín I, Garcia-Jacas N, et al.  2009. Reconstructing the history of Campanulaceae with a Bayesian approach to molecular dating and dispersal–vicariance analyses. Molecular Phylogenetics and Evolution 52: 575–587. PubMed

Schneider  U, Becker A, Finger P, Meyer-Christoffer A, Ziese M, Rudolf B. 2014. GPCC’s new land surface precipitation climatology based on quality-controlled in situ data and its role in quantifying the global water cycle. Theoretical and Applied Climatology 115: 15–40.

Schönswetter  P, Lachmayer M, Lettner C, et al.  2007. PubMed

Schönswetter  P, Suda J, Popp M, Weiss-Schneeweiss H, Brochmann C. 2007 PubMed

Scott  RJ, Spielman M, Bailey H, Dickinson HG. 1998. Parent-of-origin effects on seed development in PubMed

Segraves  KA, Thompson JN, Soltis PS, Soltis DE. 1999. Multiple origins of polyploidy and the geographic structure of

Šemberová  K. 2013. Population cytotype structure and phenotypic variation in Campanula moravica. MSc Thesis, Charles University, Czech Republic.

Shipley  B. 2009. Confirmatory path analysis in a generalized multilevel context. Ecology 90: 363–368. PubMed

Sliwinska  E, Loureiro J, Leitch IJ, Šmarda P, Bainard J, Bureš P, et al.  2021. Application-based guidelines for best practices in plant flow cytometry. Cytometry A. doi: 10.1002/cyto.a.24499. PubMed DOI

Simón-Porcar  VI, Silva JL, Meeus S, Higgins JD, Vallejo-Marín M. 2017. Recent autopolyploidization in a naturalized population of

Soltis  DE, Albert VA, Leebens-Mack J, et al.  2009. Polyploidy and angiosperm diversification. American Journal of Botany 96: 336–348. PubMed

Soltis  DE, Burleigh GJ. 2009. Surviving the K-T mass extinction: new perspectives of polyploidization in angiosperms. Proceedings of the National Academy of Sciences of the United States of America 106: 5455–5456. PubMed PMC

Sonnleitner  M, Flatscher R, Escobar García P, et al.  2010. Distribution and habitat segregation on different spatial scales among diploid, tetraploid and hexaploid cytotypes of PubMed PMC

Sonnleitner  M, Hülber K, Flatscher R, et al.  2016. Ecological differentiation of diploid and polyploid cytotypes of PubMed PMC

Stevens  CJ, Wilson J, McAllister HA. 2012. Biological Flora of the British Isles:

Suda  J, Herben T. 2013. Ploidy frequencies in plants with ploidy heterogeneity: fitting a general gametic model to empirical population data. Proceedings of the Royal Society B: Biological Sciences 280: 20122387. PubMed PMC

Sutherland  BL, Galloway LF. 2016. Postzygotic isolation varies by ploidy level within a polyploid complex. New Phytologist 213: 404–412. PubMed

Sutherland  BL, Galloway LF. 2018. Effects of glaciation and whole genome duplication on the distribution of the PubMed

Sutherland  BL, Galloway LF. 2021. Variation in heteroploid reproduction and gene flow across a polyploid complex: one size does not fit all. Ecology and Evolution 11: 9676–9688. PubMed PMC

Sutherland  BL, Miranda-Katz T, Galloway LF. 2020. Strength in numbers? Cytotype frequency mediates effect of reproductive barriers in mixed-ploidy arrays. Evolution 74: 2281–2292. PubMed

Sutherland  BL, Quarles BM, Galloway LF. 2018. Intercontinental dispersal and whole-genome duplication contribute to loss of self-incompatibility in a polyploid complex. American Journal of Botany 105: 1–8. PubMed

Trávníček  P, Eliášová A, Suda J. 2010. The distribution of cytotypes of

Trávníček  P, Jersáková J, Kubátová B, et al.  2012. Minority cytotypes in European populations of the PubMed PMC

Trygg  J, Wold S. 2002. Orthogonal projections to latent structures (O-PLS). Journal of Chemometrics 16: 119–128.

Tutz  G, Hennevogl W. 1996. Random effects in ordinal regression models. Computational Statistics & Data Analysis 22: 537–557.

Vít  P, Lepší M, Lepší P. 2012. There is no diploid apomict among Czech

Wehrens  R. 2011. Chemometrics with R. Berlin: Springer-Verlag.

Wei  N, Tennessen JA, Liston A, Ashman TL. 2017. Present-day sympatry belies the evolutionary origin of a high-order polyploid. New Phytologist 216: 279–290. PubMed PMC

Weiss  H, Dobeš C, Schneeweiss GM, Greimler J. 2002. Occurrence of tetraploid and hexaploid cytotypes between and within populations in

Weiss-Schneeweiss  H, Emadzade K, Jang TS, Schneeweiss GM. 2013. Evolutionary consequences, constraints and potential of polyploidy in plants. Cytogenetic and Genome Research 140: 137–150. PubMed PMC

Wendel  JF. 2015. The wondrous cycles of polyploidy in plants. American Journal of Botany 102: 1753–1756. PubMed

Westerhuis  JA, Hoefsloot HC, Smit S, et al.  2008. Assessment of PLSDA cross validation. Metabolomics 4: 81–89.

Wickham  H. 2016. ggplot2: elegant graphics for data analysis. New York: Springer.

Wilson  J, Perry A, Shepherd JR, Durán-Castillo M, Jeffree CE, Cavers S. 2020. Invasion, isolation and evolution shape population genetic structure in PubMed PMC

Wold  S, Sjöström M, Eriksson L. 2001. PLS-regression: a basic tool of chemometrics. Chemometrics and Intelligent Laboratory Systems 58: 109–130.

Wos  G, Mořkovská J, Bohutínská M, et al.  2019. Role of ploidy in colonization of alpine habitats in natural populations of PubMed PMC

Overcoming Ploidy Barriers: The Role of Triploid Bridges in the Genetic Introgression of Cardamine amara