Hybridization, polyploidization, and apomixis are evolutionary forces that obscure genetic differentiation and boost morphological variability. These processes have shaped the family Rosaceae, particularly the genus Crataegus, which includes both diploid and polyploid species reproducing sexually or via apomixis. In Central Europe, C. monogyna and C. laevigata are predominantly diploid sexuals, while C. rhipidophylla is mainly a polyploid apomict. These species hybridize to form C. × media, C. × macrocarpa, and C. × subsphaerica. Our aim was to assess how hybridization, apomixis, and polyploidy shape Crataegus diversity by integrating genetic, morphological, and cytological data. Leaves and fruits were collected from ten natural populations where all three species coexist and hybridize. Species identification was performed with novel nuclear microsatellites, marking the first genetic-based Crataegus taxonomy in Central Europe. Ploidy levels were estimated by flow cytometry (FCM), including seed screening to infer reproductive modes. A combined morphological analysis of leaves and fruits was used to distinguish parental species and evaluate hybrid variability. Genotyping identified distinct genetic clusters for parental species and their hybrids, with geographic structuring within C. laevigata and C. rhipidophylla. Morphological data clearly separated genetically defined parental species, although hybrids can be difficult to distinguish from parents due to a big overlap in morphology. FCM indicated that C. × media is predominantly a diploid sexual hybrid like its diploid parents, while other tri- or tetraploid hybrids with polyploid C. rhipidophylla as a parent are apomictic. Ploidy rather than hybridization dictates the mode of reproduction.

Department of Biomedical Imaging and Image Guided Therapy Medical University of Vienna Währinger Gürtel 18 20 1090 Vienna Austria

Department of Botany Faculty of Science Charles University Benátská 2 128 00 Prague Czech Republic

Department of Conservation Biology University of Göttingen Bürgerstrasse 50 37073 Göttingen Germany

Department of Forest Biodiversity and Nature Conservation Austrian Research Centre for Forests Seckendorff Gudent Weg 8 1131 Vienna Austria

Department of Forest Growth Silviculture and Genetics Austrian Research Centre for Forests Seckendorff Gudent Weg 8 1131 Vienna Austria

Department of Silviculture BOKU University Peter Jordan Straße 82 2 1190 Vienna Austria

Faculty of Biology University of Barcelona Diagonal 643 08028 Barcelona Spain

Faculty of Life Science Leipzig University Talstraße 33 04103 Leipzig Germany

Faculty of Life Sciences University of Vienna Rennweg 14 1030 Vienna Austria

Institute of Botany Czech Academy of Sciences Zámek 1 252 43 Průhonice Czech Republic

Russian Academy of Sciences Komarov Botanical Institute Ulitsa Professora Popova 2 197022 St Petersburg Russian Federation

Zobrazit více v PubMed

Adams  DC, Otárola-Castillo  E. Geomorph: an r package for the collection and analysis of geometric morphometric shape data. Methods Ecol Evol  2013;4:393–9. 10.1111/2041-210X.12035 DOI

Albarouki  E, Peterson  A. Molecular and morphological characterization of DOI

Bankevich  A, Nurk  S, Antipov  D  et al.  SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol  2012;19:455–77. 10.1089/cmb.2012.0021 PubMed DOI PMC

Baranec  T. Karyotaxonomical notes of some taxa of genus

Baranec  T. Biosystematické štúdium rodu

Beck  JB, Alexander  PJ, Allphin  L  et al.  Does hybridization drive the transition to asexuality in diploid PubMed DOI

Böcher  TW. Cytogenetical and embryological studies in the amphiapomictic

Browicz  K.

Byatt  JI. Hybridization between

Byatt  JI. The structure of some

Camacho  C, Coulouris  G, Avagyan  V  et al.  BLAST+: architecture and applications. BMC Bioinformatics  2009;10:421. 10.1186/1471-2105-10-421 PubMed DOI PMC

Campbell  CS, Dickinson  TA. Apomixis, patterns of morphological variation, and species concepts in subfam. Maloideae (Rosaceae). Syst Bot  1990;15:124–35. 10.2307/2419022 DOI

Canché-Delgado  A, García-Jain  SE, Vaca-Sánchez  MS  et al.  Cambios en la morfología floral y foliar en

Christensen  KI. A biometric study of some hybridizing DOI

Christensen  KI. Revision of DOI

Clark  LV, Jasieniuk  M. Polysat: an R package for polyploid microsatellite analysis. Mol Ecol Resour  2011;11:562–6. 10.1111/j.1755-0998.2011.02985.x PubMed DOI

Dar  AH, Kumar  S, Mukesh  M  et al.  Genetic characterization and population structure of different coat colour variants of Badri cattle. Mol Biol Rep  2020;47:8485–97. 10.1007/s11033-020-05890-w PubMed DOI

Depypere  L, Mijnsbrugge  KV, Cock  KD  et al.  Indigenous species of DOI

Dickinson  TA. Sex and Rosaceae apomicts. Taxon  2018;67:1093–107. 10.12705/676.7 DOI

Dickinson  TA, Lo  E, Talent  N. Polyploidy, reproductive biology, and Rosaceae: understanding evolution and making classifications. Plant Syst Evol  2007;266:59–78. 10.1007/s00606-007-0541-2 DOI

Dickinson  TA, Phipps  JB. Studies in DOI

Dickinson  TA, Phipps  JB. Studies in PubMed DOI

Dickinson  TA, Yan  BX, Han  S  et al.  Niche shifts, hybridization, polyploidy and geographic parthenogenesis in Western North American hawthorns ( DOI

Dobeš  C, Lückl  A, Hülber  K  et al.  Prospects and limits of the flow cytometric seed screen—insights from PubMed DOI PMC

Doležel  J, Greilhuber  J, Lucretti  S  et al.  Plant genome size estimation by flow cytometry: inter-laboratory comparison. Ann Bot  1998;82:17–26. 10.1093/oxfordjournals.aob.a010312 DOI

Du  X, Zhang  X, Bu  H  et al.  Molecular analysis of evolution and origins of cultivated hawthorn ( PubMed DOI PMC

Earl  DA, Vonholdt  BM. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour  2012;4:359–61. 10.1007/s12686-011-9548-7 DOI

Emami  A, Shabanian  N, Rahmani  M-S  et al.  Genetic characterization of the DOI

Evanno  G, Regnaut  S, Goudet  J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol  2005;14:2611–20. 10.1111/j.1365-294X.2005.02553.x PubMed DOI

Ferrazzini  D, Monteleone  I, Belletti  P. Small-scale genetic diversity in oneseed hawthorn ( DOI

Fichtner  A. DOI

Fineschi  S, Salvini  D, Turchini  D  et al. DOI

Franco  J.

Gladkova  VN. Caryological studies on the genera

Gosler  G. Introgressive hybridization between

Güney  M, Kafkas  S, Keles  H  et al.  Characterization of hawthorn ( PubMed DOI PMC

Hojsgaard  D, Hörandl  E. The rise of apomixis in natural plant populations. Front Plant Sci  2019;10:358. 10.3389/fpls.2019.00358 PubMed DOI PMC

Hörandl  E, Hojsgaard  D, Caperta  A  et al.  Apomixis in systematics, evolution and phylogenetics of angiosperms: current developments and prospects. CRC Crit Rev Plant Sci  2024;44:1–43. 10.1080/07352689.2024.2396259 DOI

Jakobsson  M, Rosenberg  NA. CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinformatics  2007;23:1801–6. 10.1093/bioinformatics/btm233 PubMed DOI

Jensen  RJ, Ciofani  KM, Miramontes  LC. Lines, outlines, and landmarks: morphometric analyses of leaves of DOI

Jensen  RJ, Hokanson  SC, Isebrands  JG  et al.  Morphometric variation in oaks of the Apostle Islands in Wisconsin: evidence of hybridization between DOI

Kalkman  C. Rosaceae. In: Kubitzki  K (ed.) Flowering Plants, Dicotyledons: Celastrales, Oxalidales, Rosales, Cornales, Ericales. Berlin, Heidelberg: Springer, 2004, 343–86.

Karbstein  K, Kösters  L, Hodač  L  et al.  Species delimitation 4.0: integrative taxonomy meets artificial intelligence. Trends Ecol Evol  2024;39:771–84. 10.1016/j.tree.2023.11.002 PubMed DOI

Kerényi-Nagy  V. Nomenclature, taxonomy and distribution of DOI

Khan  MMH, Rafii  MY, Ramlee  SI  et al.  DNA fingerprinting, fixation-index (Fst), and admixture mapping of selected Bambara groundnut ( PubMed DOI PMC

Khiari  S, Boussaid  M, Messaoud  C. Genetic diversity and population structure in natural populations of Tunisian Azarole ( DOI

Klatt  S, Schinkel  CCF, Kirchheimer  B  et al.  Effects of cold treatments on fitness and mode of reproduction in the diploid and polyploid alpine plant PubMed DOI PMC

Klingenberg  CP. Morphoj: an integrated software package for geometric morphometrics. Mol Ecol Resour  2011;11:353–7. 10.1111/j.1755-0998.2010.02924.x PubMed DOI

Kolarčik  V, Kocová  V, Mikoláš  V  et al.  Variability of reproduction pathways in the Central-European populations of hawthorns with emphasis on triploids. Plants  2022;11:1–25. 10.3390/plants11243497 DOI

Kuhn  T, Jancso  B, Ruprecht  E. Hawthorn ( DOI

Kuhn  T, Ruprecht  E. Flowering phenology may shape hybridization patterns of hawthorn ( DOI

Lê  S, Josse  J, Husson  F. FactoMineR: an DOI

Lipnerová  I, Bureš  P, Horová  L  et al.  Evolution of genome size in PubMed DOI PMC

Lippert  W. Chromosomenzahlen von Pflanzen aus Bayern und anderen Gebieten. Ber Bayer Bot Ges  2006;76:85–110.

Lo  EYY, Stefanović  S, Dickinson  TA. Population genetic structure of diploid sexual and polyploid apomictic hawthorns ( PubMed DOI

Lo  EYY, Stefanović  S, Ritland  K  et al.  Fine-scale comparisons of genetic variability in seed families of asexually and sexually reproducing PubMed DOI

Matzk  F. Reproduction mode screening. In: Doležel  J, Greilhuber  J, Suda  J (eds.) Flow Cytometry with Plant Cells. Weinheim: Wiley-VCH, 2007, 131–52.

Matzk  F, Meister  A, Schubert  I. An efficient screen for reproductive pathways using mature seeds of monocots and dicots. Plant J  2000;21:97–108. 10.1046/j.1365-313x.2000.00647.x PubMed DOI

Meglécz  E, Costedoat  C, Dubut  V  et al.  QDD: a user-friendly program to select microsatellite markers and design primers from large sequencing projects. Bioinformatics  2010;26:403–4. 10.1093/bioinformatics/btp670 PubMed DOI

Meirmans  PG. Genodive version 3.0: easy-to-use software for the analysis of genetic data of diploids and polyploids. Mol Ecol Resour  2020;20:1126–31. 10.1111/1755-0998.13145 PubMed DOI PMC

Muniyamma  M, Phipps  JB. Cytological proof of apomixis in DOI

Muniyamma  M, Phipps  JB. Studies in DOI

Nicolè  F, Tellier  F, Vivat  A  et al.  Conservation unit status inferred for plants by combining interspecific crosses and AFLP. Conserv Genet  2007;8:1273–85. 10.1007/s10592-006-9277-8 DOI

Nordborg  M, Hu  TT, Ishino  Y  et al.  The pattern of polymorphism in PubMed DOI PMC

Oklejewicz  K, Chwastek  E, Szewczyk  M  et al.  Distribution of

Ortiz  JPA, Quarin  CL, Pessino  SC  et al.  Harnessing apomictic reproduction in grasses: what we have learned from PubMed DOI PMC

Paun  O, Stuessy  TF, Hörandl  E. The role of hybridization, polyploidization and glaciation in the origin and evolution of the apomictic PubMed DOI

Pedregosa  F, Varoquaux  G, Gramfort  A  et al.  Scikit-learn: machine learning in python. J Mach Learn Res  2011;12:2825–30. 10.5555/1953048.2078195 DOI

Phipps  JB.

Piedra-Malagón  EM, Albarrán-lara  AL, Rull  J  et al.  Using multiple sources of characters to delimit species in the genus DOI

Píšová  S, Fér  T. Intraspecific differentiation of DOI

Píšová  S, Hroudová  Z, Chumová  Z  et al.  Ecological hybrid speciation in central-European species of DOI

Pritchard  JK, Stephens  M, Donnelly  P. Inference of population structure using multilocus genotype data. Genetics  2000;155:945–59. 10.1093/genetics/155.2.945 PubMed DOI PMC

Ptak  K. Cyto-embryological investigations on the Polish representatives of the genus

Ptak  K. Cyto-embryological investigations on the Polish representatives of the genus

QGIS Development Team .

R Core Team . R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing, 2021.

Rahmani  M-S, Shabanian  N, Khadivi-Khub  A  et al.  Population structure and genotypic variation of PubMed DOI

Rohlf  FJ, Marcus  LF. A revolution in morphometrics. Trends Ecol Evol  1993;8:129–32. 10.1016/0169-5347(93)90024-J PubMed DOI

Rosenberg  NA. Distruct: a program for the graphical display of population structure: PROGRAM NOTE. Mol Ecol Notes  2004;4:137–8. 10.1046/j.1471-8286.2003.00566.x DOI

Rozen  S, Skaletsky  H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol  2000;132:365–86. 10.1385/1-59259-192-2:365 PubMed DOI

Sagbas  HI, Ercisli  S, Ozkan  G  et al.  Inter- and intraspecific genetic variation of native hawthorn ( DOI

Šarhanová  P, Sharbel  TF, Sochor  M  et al.  Hybridization drives evolution of apomicts in PubMed DOI PMC

Schlager  S. Morpho and Rvcg—shape analysis in R: R-packages for geometric morphometrics, shape analysis and surface manipulations. In: Zheng  G, Li  S, Székely  G (eds.) Statistical Shape and Deformation Analysis. London: Academic Press, 2017, 217–56. 10.1016/B978-0-12-810493-4.00011-0 DOI

Schneider  CA, Rasband  WS, Eliceiri  KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods  2012;9:671–5. 10.1038/nmeth.2089 PubMed DOI PMC

Schuelke  M. An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol  2000;18:233–4. 10.1038/72708 PubMed DOI

Šlenker  M, Koutecký  P, Marhold  K. MorphoTools2: an R package for multivariate morphometric analysis. Bioinformatics  2022;38:2954–5. 10.1093/bioinformatics/btac173 PubMed DOI

Smith  PG, Phipps  JB. Studies in DOI

Sołtys-Lelek  A, Barabasz-Krasny  B, Turis  P  et al.  Chorológia niektorých taxónov kritických rodov

Sołtys-Lelek  A, Barabasz-Krasny  B, Turis  P  et al.  The genus DOI

Sołtys-Lelek  A, Barabasz-Krasny  B, Turis  P  et al.  Chorology of some taxa from the critical genera

Steffens  DL, Sutter  SL, Roemer  SC. An alternate universal forward primer for improved automated DNA sequencing of M13. BioTechniques  1993;15:580–82. PubMed

Suda  J, Kron  P, Husband  BC  et al.  Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology. In: Doležel  J, Greilhuber  J, Suda  J (eds.) Flow Cytometry with Plant Cells. Weinheim: Wiley-VCH, 2007, 103–30. 10.1002/9783527610921 DOI

Talent  N. Evolution of gametophytic apomixis in flowering plants: an alternative model from Maloid Rosaceae. Theory Biosci  2009;128:121–38. 10.1007/s12064-009-0061-4 PubMed DOI

Talent  N, Dickinson  TA. Polyploidy in DOI

Talent  N, Dickinson  TA. Endosperm formation in aposporous PubMed DOI

Talent  N, Dickinson  TA. The potential for ploidy level increases and decreases in DOI

Ufimov  R, Zeisek  V, Píšová  S  et al.  Relative performance of customized and universal probe sets in target enrichment: a case study in subtribe Malinae. Appl Plant Sci  2021;9:e11442. 10.1002/aps3.11442 PubMed DOI PMC

van Buuren  S, Groothuis-Oudshoorn  K. Mice: multivariate imputation by chained equations in DOI

Vašková  D, Kolarčik  V. Breeding systems in diploid and polyploid Hawthorns ( DOI

Venables  WN, Ripley  BD. Modern Applied Statistics with S. New York, NY: Springer New York, 2002.

Viscosi  V, Fortini  P, Slice  DE  et al.  Geometric morphometric analyses of leaf variation in four oak species of the subgenus DOI

Wickham  H. ggplot2: Elegant Graphics for Data Analysis. New York: Springer International Publishing, 2016.

Zarrei  M, Stefanović  S, Dickinson  TA. Reticulate evolution in North American black-fruited hawthorns ( PubMed DOI PMC

Zelditch  ML. Geometric Morphometrics for Biologists: a Primer. Amsterdam Boston: Elsevier Academic Press, 2004.

Zhang  Y, Dai  H, Zhang  Q. Assessment of genetic relationship in DOI

Zhang  L, Hu  J, Han  X  et al.  A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour. Nat Commun  2019;10:1494. 10.1038/s41467-019-09518-x PubMed DOI PMC

Zhang  S-D, Jin  J-J, Chen  S-Y  et al.  Diversification of Rosaceae since the Late Cretaceous based on plastid phylogenomics. New Phytol  2017;214:1355–67. 10.1111/nph.14461 PubMed DOI