The genus Anthoxanthum (sweet vernal grass, Poaceae) represents a taxonomically intricate polyploid complex with large phenotypic variation and its evolutionary relationships still poorly resolved. In order to get insight into the geographic distribution of ploidy levels and assess the taxonomic value of genome size data, we determined C- and Cx-values in 628 plants representing all currently recognized European species collected from 197 populations in 29 European countries. The flow cytometric estimates were supplemented by conventional chromosome counts. In addition to diploids, we found two low (rare 3x and common 4x) and one high (~16x-18x) polyploid levels. Mean holoploid genome sizes ranged from 5.52 pg in diploid A. alpinum to 44.75 pg in highly polyploid A. amarum, while the size of monoploid genomes ranged from 2.75 pg in tetraploid A. alpinum to 9.19 pg in diploid A. gracile. In contrast to Central and Northern Europe, which harboured only limited cytological variation, a much more complex pattern of genome sizes was revealed in the Mediterranean, particularly in Corsica. Eight taxonomic groups that partly corresponded to traditionally recognized species were delimited based on genome size values and phenotypic variation. Whereas our data supported the merger of A. aristatum and A. ovatum, eastern Mediterranean populations traditionally referred to as diploid A. odoratum were shown to be cytologically distinct, and may represent a new taxon. Autopolyploid origin was suggested for 4x A. alpinum. In contrast, 4x A. odoratum seems to be an allopolyploid, based on the amounts of nuclear DNA. Intraspecific variation in genome size was observed in all recognized species, the most striking example being the A. aristatum/ovatum complex. Altogether, our study showed that genome size can be a useful taxonomic marker in Anthoxathum to not only guide taxonomic decisions but also help resolve evolutionary relationships in this challenging grass genus.

Central European Institute of Technology Masaryk University Brno Czech Republic

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

Department of Botany Faculty of Science Charles University Prague Prague Czech Republic; Institute of Botany The Czech Academy of Sciences Průhonice Czech Republic

Department of Botany Faculty of Science Charles University Prague Prague Czech Republic; Institute of Botany The Czech Academy of Sciences Průhonice Czech Republic; Biotechnological Centre Faculty of Agriculture University of South Bohemia České Budějovice Czech Republic

Zobrazit více v PubMed

Soltis DE, Burleigh JG. Surviving the K-T mass extinction: New perspectives of polyploidization in angiosperms. Proc Natl Acad Sci USA. 2009;106: 5455–5456. 10.1073/pnas.0901994106 PubMed DOI PMC

Symonds W, Soltis PS, Soltis DE. Dynamic polyploid formation in Tragopogon (Asteraceae): Recurrent formation, gene flow, and population structure. Evolution. 2010;64: 1984–2003. 10.1111/j.1558-5646.2010.00978.x PubMed DOI

Jiao YN, Wickett NJ, Ayyampalayam S, Chanderbali AS, Landherr L, Ralph PE, et al. Ancestral polyploidy in seed plants and angiosperms. Nature. 2011;473: 97–100. 10.1038/nature09916 PubMed DOI

Favarger C. Cytogeography and biosystematics In: Grant WF, editor. Plant biosystematics. Toronto, New York: Academic Press; 1984.

Suda J, Weiss-Schneeweiss H, Tribsch A, Schneeweiss GM, Trávníček P, Schönswetter P. Complex distribution patterns of di-, tetra- and hexaploid cytotypes in the European high mountain plant Senecio carniolicus (Asteraceae). Amer J Bot. 2007;94: 1391–1401. PubMed

Perný M, Kolarčik V, Majeský L, Mártonfi P. Cytogeography of the Phleum pratense group (Poaceae) in the Carpathians and Pannonia. Bot J Linn Soc. 2008;157: 475–485.

Jersáková J, Castro S, Sonk N, Milchreit K, Schödelbauerová I, Tolasch T, et al. Absence of pollinator-mediated premating barriers in mixed-ploidy populations of Gymnadenia conopsea s.l. (Orchidaceae). Evol Ecol. 2010;24: 1199–1218.

Baack EJ. Cytotype segregation on regional and microgeographic scales in snow buttercups (Ranunculus adoneus, Ranunculaceae). Am J Bot. 2004;91: 1783–1788. 10.3732/ajb.91.11.1783 PubMed DOI

Baack EJ, Stanton ML. Ecological factors influencing tetraploid speciation in snow buttercups (Ranunculus adoneus, Ranunculaceae): Niche differentiation and tetraploid establishment. Evolution. 2005;59: 1936–1944. PubMed

Kron P, Suda J, Husband BC. Applications of flow cytometry to evolutionary and population biology. Annu Rev Ecol Evol Syst. 2007;38: 847–876.

Suda J, Pyšek P. Flow cytometry in botanical research: introduction. Preslia. 2010; 82: 1–2.

Loureiro J, Trávníček P, Rauchová J, Urfus T, Vít P, Štech M, et al. The use of flow cytometry in the biosystematics, ecology and population biology of homoploid plants. Preslia. 2010;82: 3–21.

Dixon CJ, Schönswetter P, Suda J, Wiedermann MM, Schneeweiss GM. Reciprocal Pleistocene origin and postglacial range formation of an allopolyploid and its sympatric ancestors (Androsace adfinis group, Primulaceae). Molec Phylogen Evol. 2009;50: 74–83. PubMed

Suda J, Trávníček P, Mandák B, Berchová-Bímová K. Genome size as a marker for the recognition of invasive alien taxa in Fallopia section Reynoutria . Preslia. 2010;82: 97–106.

Trávníček P, Kubátová B, Čurn V, Rauchová J, Krajníková E, Jersáková J, et al. Remarkable coexistence of multiple cytotypes of the Gymnadenia conopsea aggregate (the fragrant orchid): evidence from flow cytometry. Ann Bot. 2011;107: 77–87. 10.1093/aob/mcq217 PubMed DOI PMC

Kúr P, Štech M, Koutecký P, Trávníček P. Morphological and cytological variation in Spergularia echinosperma and S. rubra, and notes on potential hybridization of these two species. Preslia. 2012;84: 905–924.

Mandák B, Trávníček P, Paštová L, Kořínková D. Is hybridization involved in the evolution of the Chenopodium album aggregate? An analysis based on chromosome counts and genome size estimation. Flora. 2012;207: 530–540.

Prančl J, Kaplan Z, Trávníček P, Jarolímová V. Genome Size as a Key to Evolutionary Complex Aquatic Plants: Polyploidy and Hybridization in Callitriche (Plantaginaceae). PLoS ONE 2014;9(9): e105997 10.1371/journal.pone.0105997 PubMed DOI PMC

Soltis DE, Bell CD, Kim S, Soltis PS. The origin and early evolution of the angiosperms. Ann NY Acad Sci. 2008;1133: 3–25. 10.1196/annals.1438.005 PubMed DOI

Matsuoka Y. Evolution of Polyploid Triticum Wheats under Cultivation: The Role of Domestication, Natural Hybridization and Allopolyploid Speciation in their Diversification. Plant Cell Physiol. 2011;52: 750–764. 10.1093/pcp/pcr018 PubMed DOI

Petersen G, Seberg O. On the Origin of the Tetraploid Species Hordeum capense and H. secalinum (Poaceae). Syst Bot. 2004;29: 862–873.

Chen JF, Huang QF, Gao DY, Wang JY, Lang YS, Liu TY, et al. Whole-genome sequencing of Oryza brachyantha reveals mechanisms underlying Oryza genome evolution. Nat Commun. 2013;4:1595 10.1038/ncomms2596 PubMed DOI PMC

Betekthin A, Jenkins G, Hasterok R. Reconstructing the evolution of Brachypodium genomes using comparative chromosome painting. Plos ONE 2014;9(12): e115108 10.1371/journal.pone.0115108 PubMed DOI PMC

Ainouche ML, Fortune PM, Salmon A, Parisod C, Grandbastien M- A, Fukunaga K, et al. Hybridization, polyploidy and invasion: lessons from Spartina (Poaceae). Biol Invasions. 2009;11: 1159–1173.

Clayton WD, Renvoize S. Genera Graminum: Grasses of the world. Kew Bull, Addit Ser. 1986;13: 1–389.

Watson L, Dallwitz MJ. The grass genera of the world Wallingford: CAB; 1992.

Allred KW, Barkworth ME. Anthoxanthum L In: Flora of North America 24, Barkworth ME, Capels KM & Long S, editors. Oxford University Press; 2007.

Muyt A. Bush invaders of Southeast Australia RG & Richardson FJ, editors. Meredith, Victoria, Australia; 2001.

Pimentel M, Sahuquillo E. Relationships between the close congeners Anthoxanthum odoratum and A. alpinum (Poaceae, Pooideae) assessed by morphological and molecular methods. Bot J Linn Soc. 2008;156: 237–252.

Valdés B. Revisión de las especies anuales del género Anthoxanthum (Graminae) [Revision of the annual species of the genus Anthoxanthum (Graminae)]. Lagascalia. 1973;3: 99–141.

Tutin TG. Anthoxanthum L In.: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA, editors. Europaea Flora, Vol. 5 Cambridge: Cambridge University Press; 1980.

Teppner H. Anthoxanthum maderense spec. nova und A. odoratum (Poaceae-Aveneae) von Madeira und deren Chromosomen-Morphologie. Phyton. 1998;38: 307–321.

Pignatti S. Anthoxanthum In.: Pignatti S, editor. Flora d'Italia, Vol. 3 Bolonia Edagricole, Italy; 1982.

Pimentel M, Estévez G, Sahuquillo E. European sweet vernal grasses (Anthoxanthum, Poaceae; Pooideae; Aveneae): a morphometric taxonomical approach. Syst Bot. 2007;32: 43–59.

Pimentel M, Sahuquillo E, Catalán P. Genetic diversity and spatial correlation patterns unravel the biogeographic history of the European sweet vernal grasses (Anthoxanthum L., Poaceae). Molec Phylogen Evol. 2007;44: 667–684. PubMed

Pimentel M, Catalán P, Sahuquillo E. Morphological and molecular taxonomy of the annual diploids Anthoxanthum aristatum and A. ovatum (Poaceae) in the Iberian Peninsula. Evidence of introgression in natural populations. Bot J Linn Soc. 2010;164: 53–71.

Felber-Girard M, Felber F, Buttler A. Habitat differentiation in a narrow hybrid zone between diploid and tetraploid Anthoxanthum alpinum . New Phytol. 1996;133(3): 531–540.

Fernándes A, Queirós M. Contribution à la connaissance cytotaxinomique des spermatophyta du Portugal I. Gramineae [Contribution to the understanding of cytotaxonomy of angiosperms from Portugal I. Gramineae]. Bol Soc Brot. 1969;43: 20–140.

Jones K. Chromosomes and the origin of Anthoxanthum odoratum L. Chromosoma. 1964;15: 248–274.

Teppner H. Karyotypen europaischer, perennierender Sippen der Gramineen-Gattung Anthoxanthum . Oesterr Bot Z. 1970;118: 280–292.

Felber F. Contribution à l'étude phytogéographique, biosystématique et experimentale du complexe polyploïd Anthoxanthum odoratum L. s. lat. [Contribution to the study of phytogeography, biosystematics and experiments in the polyploid complex of Anthoxanthum odoratum L. s. lat.]. Thèse à l'UNINe [PhD thesis], Neuchâtel; 1987.

Hedberg I. Cytotaxonomic studies on Anthoxanthum odoratum L. s. lat. IV. Karyotypes, meiosis and the origin of tetraploid A. odoratum . Hereditas. 1970;48: 471–502.

Zeroual-Humbert-Droz C, Felber F. Evidence from isozymes analysis of autopolyploidy in Anthoxanthum alpinum A. & D. Löve. Bot Helv. 1999;109: 217–227.

Felber F. Sensitivity of the four cytodemes of Anthoxanthum odoratum l. s. Lat. (Poaceae) to Puccinia sardonensis Gäumann (Uredinales). Taxon. 1987;36(3): 573–577.

Hedberg I. The genesis of tetraploid Anthoxanthum odoratum . Symb Bot Upsal. 1986;27: 147–154.

Hedberg I. Morphological, cytotaxonomic and evolutionary studies in Anthoxanthum odoratum L. s. lat.–a critical review. Sommerfeltia. 1990;11: 97–107.

Pimentel M, Sahuquillo E. Infraspecific variation and phylogeography of the high-polyploid Iberian endemic Anthoxanthum amarum Brot. (Poaceae; Pooideae) assessed by random amplified polymorphic DNA markers (RAPDs) and morphology. Bot J Linn Soc. 2007;155: 179–192.

Pimentel M, Sahuquillo E, Torrecilla Z, Popp M, Catalán P, Brochmann C. Hybridization and long-distance colonization at different time scales: towards resolution of long-term controversies in the sweet vernal grasses (Anthoxanthum). Ann Bot. 2013;112: 1015–1030. 10.1093/aob/mct170 PubMed DOI PMC

Conert HJ. Gramineae In.: Hegi G, editor. Illustrierte Flora von Mitteleuropa, I/3 Verlag Paul Parey, Berlin, Hamburg; 1988.

Filipová L, Krahulec F. The transition zone of Anthoxanthum alpinum and A. odoratum in the Krkonoše Mts. Preslia. 2006;78: 317–330.

Greilhuber J, Doležel J, Lysák MA, Bennett MD. The origin, evolution and proposed stabilization of the terms ‘Genome size’ and ‘C-value’ to describe nuclear DNA contents. Ann Bot. 2005;95: 255–260. PubMed PMC

Otto F. DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA In: Crissman HA, Darzynkiewicz Z (eds). Methods in cell biology, Vol. 33 New York, NY, Academic Press; 1990. PubMed

Doležel J, Greillhuber J, Suda J. Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc. 2007;2: 2233–2244. PubMed

Suda J, Krahulcová A, Trávníček P, Krahulec F. Ploidy level versus DNA ploidy level: an appeal for consistent terminology. Taxon. 2006;55: 447–450.

Clayton WD, Vorontsova MS, Harman KT, Williamson H. GrassBase–The online world grass flora. 2006 onwards. Available: http://www.kew.org/data/grasses-db.html.

López-González G. Nota sobre el género Anthoxanthum L. (Gramineae) [Some notes on genus Anthoxanthum L. (Gramineae)]. Anales Jard. Bot. Madrid. 1994;51: 309–312.

Pimentel M, Sahuquillo E. An approach to the study of morphological relationships among the sweet vernal grass (Anthoxanthum L. Poaceae, Pooideae) in the Iberian Peninsula. Bocconea. 2003;16: 731–737.

Kattermann G. Über die Bildung polyvalenter Chromosomenverbande bei einigen Gramineen [About creation of polyvalent chromosome connections by some grasses]. Planta. 1931;12: 732–774.

Parthasarathy N. Cytogenetical studies in Oryzeae and Phalarideae. III. Cytological studies in Phalarideae. Ann Bot. 1939;3: 43–76.

Östergren G. Chromosome numbers in Anthoxanthum . Hereditas. 1942;33: 242–243.

Hedberg I. Cytotaxonomic studies on Anthoxanthum odoratum L. s. lat. II. Investigations of some Swedish and of a few Swiss population samples. Symb Bot Upsal. 1967;18: 5–88.

Eilam T, Anikster Y, Millet E, Manisterski J, Feldman M. Genome size in natural and synthetic autopolyploids and in a natural segmental allopolyploid of several Triticeae species. Genome. 2009;52: 275–285. 10.1139/G09-004 PubMed DOI

Borrill M. Experimental studies of evolution in Anthoxanthum (Gramineae). Genetica. 1963;34: 183–210.

Greilhuber J. Intraspecific variation in genome size in angiosperms: identifying its existence. Ann Bot. 2005;95: 91–98. PubMed PMC

Šmarda P, Bureš P. Understanding intraspecific variation in genome size in plants. Preslia. 2010;82: 41–61.

Pečinka A, Suchánková P, Lysák MA, Trávníček B, Doležel J. Nuclear DNA content variation among central European Koeleria taxa. Ann Bot. 2006;98: 117–122. PubMed PMC

Šmarda P & Bureš P. Intraspecific DNA content variability in Festuca pallens on different geographical scales and ploidy levels. Ann Bot. 2006;98: 665–78. PubMed PMC

Slovák M, Urfus T, Vít T, Marhold K. Balkan endemic Picris hispidissima (Compositae): morphology, DNA content and relationship to polymorphic P. hieracioides . Plant Syst Evol. 2009;278: 187–201.

Trávníček P, Jersáková J, Kubátová B, Krejčíková J, Bateman RM, Lučanová M, et al. Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity. Ann Bot. 2012;110: 977–986. 10.1093/aob/mcs171 PubMed DOI PMC

Teppner H. Poaceae in the greenhouses of the Botanic Garden of the Institute of Botany in Graz (Austria, Europe). Fritschiana. 2002;31: 1–42.

Bennet MD. Nuclear DNA content and minimum generation time in herbaceous plants. Proc Roy Soc London, Ser B Biol Sci. 1972;181: 109–135. PubMed

Creber HMC, Davies MS, Francis D, Walker HD. Variation in DNA C-value in natural-populations of Dactylis glomerata L. New Phytol. 1994;128: 555–561. PubMed

Reeves G, Francis D, Davies MS, Rogers HJ, Hodkinson TR. Genome size is negatively correlated with altitude in natural populations of Dactylis glomerata . Ann Bot. 1998;82 (Suppl A): 99–105.

Laurie DA, Bennett MD. Nuclear DNA content in the genera Zea and Sorghum. Intergeneric, interspecific, and intraspecific variation. Heredity. 1985;55: 307–313.

Duchoslav M, Šafářová L, Jandová M. Role of adaptive and non-adaptive mechanisms forming complex patterns of genome size variation in six cytotypes of polyploid Allium oleraceum (Amaryllidaceae) on a continental scale. Ann Bot. 2013;111: 419–431. 10.1093/aob/mcs297 PubMed DOI PMC

Trávníček P, Kirschner J, Chudáčková H, Rooks F, Štěpánek J. Substantial Genome Size Variation in Taraxacum stenocephalum (Asteraceae, Lactuceae). Folia Geobot. 2013;48(2): 271–284.

Diploid and tetraploid cytotypes of the flagship Cape species Dicerothamnus rhinocerotis (Asteraceae): variation in distribution, ecological niche, morphology and genetics

Morphological and environmental differentiation as prezygotic reproductive barriers between parapatric and allopatric Campanula rotundifolia agg. cytotypes

Integrative Study of Genotypic and Phenotypic Diversity in the Eurasian Orchid Genus Neotinea

Tetraploids expanded beyond the mountain niche of their diploid ancestors in the mixed-ploidy grass Festuca amethystina L

On the Origin of Tetraploid Vernal Grasses (Anthoxanthum) in Europe

Polyploid evolution: The ultimate way to grasp the nettle

Shoot apical meristem and plant body organization: a cross-species comparative study

The Mediterranean: the cradle of Anthoxanthum (Poaceae) diploid diversity