BACKGROUND: Transposable elements (TEs) are considered to be an important source of genome size variation and genetic and phenotypic plasticity in eukaryotes. Most of our knowledge about TEs comes from large genomic projects and studies focused on model organisms. However, TE dynamics among related taxa from natural populations and the role of TEs at the species or supra-species level, where genome size and karyotype evolution are modulated in concert with polyploidy and chromosomal rearrangements, remain poorly understood. We focused on the holokinetic genus Eleocharis (Cyperaceae), which displays large variation in genome size and the occurrence of polyploidy and agmatoploidy/symploidy. We analyzed and quantified the long terminal repeat (LTR) retrotransposons Ty1-copia and Ty3-gypsy in relation to changes in both genome size and karyotype in Eleocharis. We also examined how this relationship is reflected in the phylogeny of Eleocharis. RESULTS: Using flow cytometry, we measured the genome sizes of members of the genus Eleocharis (Cyperaceae). We found positive correlation between the independent phylogenetic contrasts of genome size and chromosome number in Eleocharis. We analyzed PCR-amplified sequences of various reverse transcriptases of the LTR retrotransposons Ty1-copia and Ty3-gypsy (762 sequences in total). Using real-time PCR and dot blot approaches, we quantified the densities of Ty1-copia and Ty3-gypsy within the genomes of the analyzed species. We detected an increasing density of Ty1-copia elements in evolutionarily younger Eleocharis species and found a positive correlation between Ty1-copia densities and C/n-values (an alternative measure of monoploid genome size) in the genus phylogeny. In addition, our analysis of Ty1-copia sequences identified a novel retrotransposon family named Helos1, which is responsible for the increasing density of Ty1-copia. The transition:transversion ratio of Helos1 sequences suggests that Helos1 recently transposed in later-diverging Eleocharis species. CONCLUSIONS: Using several different approaches, we were able to distinguish between the roles of LTR retrotransposons, polyploidy and agmatoploidy/symploidy in shaping Eleocharis genomes and karyotypes. Our results confirm the occurrence of both polyploidy and agmatoploidy/symploidy in Eleocharis. Additionally, we introduce a new player in the process of genome evolution in holokinetic plants: LTR retrotransposons.

Department of Botany and Zoology Masaryk University Kotlářská 2 61137 Brno Czech Republic

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Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O. et al.A unified classification system for eukaryotic transposable elements. Nature Reviews Genetics. 2007;8(12):973–982. doi: 10.1038/nrg2165. PubMed DOI

Kumar A, Bennetzen JL. Plant retrotransposons. Annual Review of Genetics. 1999;33:479–532. doi: 10.1146/annurev.genet.33.1.479. PubMed DOI

Kumar A, Bennetzen J. Retrotransposons: central players in the structure, evolution and function of plant genomes. Trends in plant science. 2000;5(12):509–510. doi: 10.1016/S1360-1385(00)01760-X. PubMed DOI

Boeke JD, Corces VG. Transcription and reverse transcription of retrotransposons. Annual Review of Microbiology. 1989;43:403–434. doi: 10.1146/annurev.mi.43.100189.002155. PubMed DOI

Wilhelm M, Wilhelm FX. Reverse transcription of retroviruses and LTR retrotransposons. Cellular and Molecular Life Sciences. 2001;58(9):1246–1262. doi: 10.1007/PL00000937. PubMed DOI PMC

Kashkush K, Feldman M, Levy AA. Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat. Nature Genetics. 2003;33(1):102–106. doi: 10.1038/ng1063. PubMed DOI

Hollister JD, Gaut BS. Epigenetic silencing of transposable elements: A trade-off between reduced transposition and deleterious effects on neighboring gene expression. Genome Research. 2009;19(8):1419–1428. doi: 10.1101/gr.091678.109. PubMed DOI PMC

Lockton S, Gaut BS. The Contribution of Transposable Elements to Expressed Coding Sequence in Arabidopsis thaliana. Journal of Molecular Evolution. 2009;68(1):80–89. doi: 10.1007/s00239-008-9190-5. PubMed DOI

Lonnig WE, Saedler H. Chromosome rearrangements and transposable elements. Annual Review of Genetics. 2002;36:389–410. doi: 10.1146/annurev.genet.36.040202.092802. PubMed DOI

Vitte C, Panaud O. LTR retrotransposons and flowering plant genome size: emergence of the increase/decrease model. Cytogenetic and Genome Research. 2005;110(1-4):91–107. doi: 10.1159/000084941. PubMed DOI

Grandbastien M, Audeon C, Bonnivard E, Casacuberta JM, Chalhoub B, Costa APP, Le QH, Melayah D, Petit M, Poncet C. et al.Stress activation and genomic impact of Tnt1 retrotransposons in Solanaceae. Cytogenetic and Genome Research. 2005;110(1-4):229–241. doi: 10.1159/000084957. PubMed DOI

Mansour A. Epigenetic activation of genomic retrotransposons. Journal of Cell and Molecular Biology. 2007;6(2):99–107.

Okamoto H, Hirochika H. Silencing of transposable elements in plants. Trends in Plant Science. 2001;6(11):527–534. doi: 10.1016/S1360-1385(01)02105-7. PubMed DOI

Matsuoka Y, Tsunewaki K. Evolutionary dynamics of Ty1-copia group retrotransposons in grass shown by reverse transcriptase domain analysis. Molecular Biology and Evolution. 1999;16(2):208–217. PubMed

Navarro-Quezada A, Schoen DJ. Sequence evolution and copy number of Ty1-copia retrotransposons in diverse plant genomes. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(1):268–273. doi: 10.1073/pnas.012422299. PubMed DOI PMC

Baucom RS, Estill JC, Leebens-Mack J, Bennetzen JL. Natural selection on gene function drives the evolution of LTR retrotransposon families in the rice genome. Genome Research. 2009;19(2):243–254. doi: 10.1101/gr.083360.108. PubMed DOI PMC

Capy P, Gasperi G, Biemont C, Bazin C. Stress and transposable elements: co-evolution or useful parasites? Heredity. 2000;85(2):101–106. doi: 10.1046/j.1365-2540.2000.00751.x. PubMed DOI

Comai L, Tyagi AP, Winter K, Holmes-Davis R, Reynolds SH, Stevens Y, Byers B. Phenotypic instability and rapid gene silencing in newly formed arabidopsis allotetraploids. Plant Cell. 2000;12(9):1551–1567. doi: 10.1105/tpc.12.9.1551. PubMed DOI PMC

Baumel A, Ainouche M, Kalendar R, Schulman AH. Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica CE Hubbard (Poaceae) Molecular Biology and Evolution. 2002;19(8):1218–1227. PubMed

Parisod C, Alix K, Just J, Petit M, Sarilar V, Mhiri C, Ainouche M, Chalhoub B, Grandbastien MA. Impact of transposable elements on the organization and function of allopolyploid genomes. New Phytologist. 2010;186(1):37–45. doi: 10.1111/j.1469-8137.2009.03096.x. PubMed DOI

Piegu B, Guyot R, Picault N, Roulin A, Saniyal A, Kim H, Collura K, Brar DS, Jackson S, Wing RA. et al.Doubling genome size without polyploidization: Dynamics of retrotransposition-driven genomic expansions in Oryza australiensis, a wild relative of rice. Genome Research. 2006;16(10):1262–1269. doi: 10.1101/gr.5290206. PubMed DOI PMC

Soleimani VD, Baum BR, Johnson DA. Quantification of the retrotransposon BARE-1 reveals the dynamic nature of the barley genome. Genome. 2006;49(4):389–396. doi: 10.1139/G05-119. PubMed DOI

Kalendar R, Tanskanen J, Immonen S, Nevo E, Schulman AH. Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(12):6603–6607. doi: 10.1073/pnas.110587497. PubMed DOI PMC

Wendel JF, Wessler SR. Retrotransposon-mediated genome evolution on a local ecological scale. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(12):6250–6252. doi: 10.1073/pnas.97.12.6250. PubMed DOI PMC

Bennett MD, Leitch LI. Plant DNA C-values database (release 4.0, Oct. 2005) 2004. http://www.kew.org/cvalues/

Roalson EH, Hinchliff CE, Trevisan R, da Silva CRM. Phylogenetic Relationships in Eleocharis (Cyperaceae): C-4 Photosynthesis Origins and Patterns of Diversification in the Spikerushes. Systematic Botany. 2010;35(2):257–271. doi: 10.1600/036364410791638270. DOI

Blomberg SP, Garland T, Ives AR. Testing for phylogenetic signal in comparative data: Behavioral traits are more labile. Evolution. 2003;57(4):717–745. PubMed

Roalson EH, Friar EA. Infrageneric classification of Eleocharis (Cyperaceae) revisited: Evidence from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Systematic Botany. 2000;25(2):323–336. doi: 10.2307/2666645. DOI

Yano O, Katsuyama T, Tsubota H, Hoshino T. Molecular phylogeny of Japanese Eleocharis (Cyperaceae) based on ITS sequence data, and chromosomal evolution. Journal of Plant Research. 2004;117(5):409–419. doi: 10.1007/s10265-004-0173-3. PubMed DOI

Preston ML, Pearman DA, Dines TD. New atlas of the British & Irish Flora. New York: Oxford Univ. Press; 2002.

Nishikawa K, Furuta Y, Ishitobi K. Chromosomal evolution in genus Carex as viewed from nuclear-dna content, with special reference to its aneuploidy. Japanese Journal of Genetics. 1984;59(5):465–472. doi: 10.1266/jjg.59.465. DOI

Hipp AL, Rothrock PE, Roalson EH. The Evolution of Chromosome Arrangements in Carex (Cyperaceae) Botanical Review. 2009;75(1):96–109. doi: 10.1007/s12229-008-9022-8. DOI

Roalson EH, McCubbin GA, Whitkus R. Chromosome evolution in Cyperales. Vol. 23. Claremont, CA, ETATS-UNIS: Rancho Santa Ana Botanic Garden; 2007.

Roalson EH. A synopsis of chromosome number variation in the Cyperaceae. Botanical Review. 2008;74(2):209–393. doi: 10.1007/s12229-008-9011-y. DOI

Da Silva CRM, Trevisan R, González-Elizondo MS, Ferreira JM, Vanzela ALL. Karyotypic diversification and its contribution to the taxonomy of Eleocharis (Cyperaceae) from Brazil. Australian Journal of Botany. 2010;58(1):49–60. doi: 10.1071/BT09185. DOI

Smarda P, Bures P, Horova L, Foggi B, Rossi G. Genome size and GC content evolution of Festuca: Ancestral expansion and subsequent reduction. Annals of Botany. 2008;101(3):421–433. doi: 10.1093/aob/mcm307. PubMed DOI PMC

Flavell AJ, Dunbar E, Anderson R, Pearce SR, Hartley R, Kumar A. Ty1-copia group retrotransposons are ubiquitous and heterogeneous in higher-plants. Nucleic Acids Research. 1992;20(14):3639–3644. doi: 10.1093/nar/20.14.3639. PubMed DOI PMC

Flavell AJ, Smith DB, Kumar A. Extreme heterogeneity of Ty1-copia group retrotransposons in plants. Molecular & General Genetics. 1992;231(2):233–242. PubMed

Kumekawa N, Ohtsubo E, Ohtsubo H. Identification and phylogenetic analysis of gypsy-type retrotransposons in the plant kingdom. Genes & Genetic Systems. 1999;74(6):299–307. PubMed

Park JM, Schneeweiss GM, Weiss-Schneeweiss H. Diversity and evolution of Ty1-copia and Ty3-gypsy retroelements in the non-photo synthetic flowering plants Orobanche and Phelipanche (Orobanchaceae) Gene. 2007;387(1-2):75–86. doi: 10.1016/j.gene.2006.08.012. PubMed DOI

Ungerer MC, Strakosh SC, Stimpson KM. Proliferation of Ty3/gypsy-like retrotransposons in hybrid sunflower taxa inferred from phylogenetic data. Bmc Biology. 2009;7 doi: 10.1186/1741-7007-7-40. PubMed DOI PMC

Whitford R, Baumann U, Sutton T, Gumaelius L, Wolters P, Tingey S, Able JA, Langridge P. Identification of transposons, retroelements, and a gene family predominantly expressed in floral tissues in chromosome 3DS of the hexaploid wheat progenitor Aegilops tauschii. Functional & Integrative Genomics. 2007;7(1):37–52. PubMed

Felsenstein J. Phylogenies and the comparative method. American Naturalist. 1985;125(1):1–15. doi: 10.1086/284325. PubMed DOI

Garland T, Harvey PH, Ives AR. Procedures for the analysis of comparative data using phylogenetically independent contrasts. Systematic Biology. 1992;41(1):18–32.

Webb CO, Ackerly DD, Kembel SW. Phylocom: software for the analysis of phylogenetic community structure and trait evolution. Bioinformatics. 2008;24(18):2098–2100. doi: 10.1093/bioinformatics/btn358. PubMed DOI

Svenson HK. Monographic studies in the genus Eleocharis V. Rhodora. 1939;41:1–19. 43-77, 90-110.

Oliver KR, Greene WK. Transposable elements: powerful facilitators of evolution. Bioessays. 2009;31(7):703–714. doi: 10.1002/bies.200800219. PubMed DOI

Zeh DW, Zeh JA, Ishida Y. Transposable elements and an epigenetic basis for punctuated equilibria. Bioessays. 2009;31(7):715–726. doi: 10.1002/bies.200900026. PubMed DOI

Bures P. A high polyploid Eleocharis uniglumis sl (Cyperaceae) from Central and Southeastern Europe. Folia Geobotanica. 1998;33(4):429–439. doi: 10.1007/BF02803644. DOI

Bureš P, Zedek F, Šmarda P, Rotreklová O, Hrálová I. The Comparative Biology of the Monocotyledons: 2008; Copenhagen, Denmark. University of Copenhagen; 2008. Genome size in Cyperaceae; pp. 13–14.

da Silva CRM, Gonzalez-Elizondo MS, Rego L, Torezan JMD, Vanzela ALL. Cytogenetical and cytotaxonomical analysis of some Brazilian species of Eleocharis (Cyperaceae) Australian Journal of Botany. 2008;56(1):82–90. doi: 10.1071/BT07017. DOI

Matzke MA, Matzke AJM. Polyploidy and transposons. Trends in Ecology & Evolution. 1998;13(6):241–241. PubMed

Matzke MA, Scheid OM, Matzke AJM. Rapid structural and epigenetic changes in polyploid and aneuploid genomes. Bioessays. 1999;21(9):761–767. doi: 10.1002/(SICI)1521-1878(199909)21:9<761::AID-BIES7>3.0.CO;2-C. PubMed DOI

Picot S, Wallau GL, Loreto ELS, Heredia FO, Hua-Van A, Capy P. The mariner transposable element in natural populations of Drosophila simulans. Heredity. 2008;101(1):53–59. doi: 10.1038/hdy.2008.27. PubMed DOI

Löve A, Löve D. Arctic polyploidy. Proc Genet Soc Can. 1957;2:23–27.

Stebbins GL. Polyploidy and the distribution of the arctic-alpine flora-new evidence and a new approach. Botanica Helvetica. 1984;94(1):1–13.

Brochmann C, Brysting AK, Alsos IG, Borgen L, Grundt HH, Scheen AC, Elven R. Polyploidy in arctic plants. Biological Journal of the Linnean Society. 2004;82(4):521–536. doi: 10.1111/j.1095-8312.2004.00337.x. DOI

Guggisberg A, Mansion G, Conti E. Disentangling Reticulate Evolution in an Arctic-Alpine Polyploid Complex. Systematic Biology. 2009;58(1):55–73. doi: 10.1093/sysbio/syp010. PubMed DOI

Haizel T, Lim YK, Leitch AR, Moore G. Molecular analysis of holocentric centromeres of Luzula species. Cytogenetic and Genome Research. 2005;109(1-3):134–143. doi: 10.1159/000082392. PubMed DOI

Bures P, Wang YF, Horova L, Suda J. Genome size variation in Central European species of Cirsium (Compositae) and their natural hybrids. Annals of Botany. 2004;94(3):353–363. doi: 10.1093/aob/mch151. PubMed DOI PMC

Dolezel J, Greilhuber J, Lucretti S, Meister A, Lysak MA, Nardi L, Obermayer R. Plant genome size estimation by flow cytometry: Inter-laboratory comparison. Annals of Botany. 1998;82:17–26. doi: 10.1006/anbo.1998.0730. DOI

Dolezel J, Sgorbati S, Lucretti S. Comparison of 3 DNA fluorochromes for flow cytometric estimation of nuclear-DNA content in plants. Physiologia Plantarum. 1992;85(4):625–631. doi: 10.1111/j.1399-3054.1992.tb04764.x. DOI

Park JM, Schneeweiss GM, Weiss-Schneeweiss H. Diversity and evolution of Ty1-copia and Ty3-gypsy retroelements in the non-photo synthetic flowering plants Orobanche and Phelipanche (Orobanchaceae) Gene. 2007;387(1-2):75–86. doi: 10.1016/j.gene.2006.08.012. PubMed DOI

Ruas CF, Weiss-Schneeweiss H, Stuessy TF, Samuel MR, Pedrosa-Harand A, Tremetsberger K, Ruas PM, Schluter PM, Herrera MAO, Konig C. et al.Characterization, genomic organization and chromosomal distribution of Ty1-copia retrotransposons in species of Hypochaeris (Asteraceae) Gene. 2008;412(1-2):39–49. doi: 10.1016/j.gene.2008.01.009. PubMed DOI

Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution. 2007;24(8):1596–1599. doi: 10.1093/molbev/msm092. PubMed DOI

Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser. 1999;41:95–98.

Gonzalez-Elizondo MS, Peterson PM. A classification of and key to the supraspecific taxa in Eleocharis (Cyperaceae) Taxon. 1997;46(3):433–449. doi: 10.2307/1224386. DOI

Bures P, Rotreklova O, Stoneberg Holt SD, Pikner R. Cytogeographical survey of Eleocharis subser. Eleocharis in Europe-1: Eleocharis palustris. Folia Geobotanica. 2004;39(3):235–257. doi: 10.1007/BF02804780. DOI

Rotreklova O, Bures P, Grulich V. Chromosome numbers for some species of vascular plants from Europe. Biologia. 2004;59(4):425–433.

Complex patterns of ploidy in a holocentric plant clade (Schoenus, Cyperaceae) in the Cape biodiversity hotspot

Chromosome size matters: genome evolution in the cyperid clade

Horizontally Acquired nrDNAs Persist in Low Amounts in Host Hordeum Genomes and Evolve Independently of Native nrDNA

Evolution of genome size and genomic GC content in carnivorous holokinetics (Droseraceae)

Flow cytometry may allow microscope-independent detection of holocentric chromosomes in plants

Holokinetic centromeres and efficient telomere healing enable rapid karyotype evolution

Evolution of genome size in Carex (Cyperaceae) in relation to chromosome number and genomic base composition

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GU976788, GU976789, GU976790, GU976791, GU976792, GU976793, GU976794, GU976795, GU976796, GU976797, GU976798, GU976799, GU976800, GU976801, GU976802, GU976803, GU976804, GU976805, GU976806, GU976807, GU976808, GU976809, GU976810, GU976811, GU976812, GU976813, GU976814, GU976815, GU976816, GU976817, GU976818, GU976819, GU976820, GU976821, GU976822, GU976823, GU976824, GU976825, GU976826, GU976827, GU976828, GU976829, GU976830, GU976831, GU976832, GU976833, GU976834, GU976835, GU976836, GU976837, GU976838, GU976839, GU976840, GU976841, GU976842, GU976843, GU976844, GU976845, GU976846, GU976847, GU976848, GU976849, GU976850, GU976851, GU976852, GU976853, GU976854, GU976855, GU976856, GU976857, GU976858, GU976859, GU976860, GU976861, GU976862, GU976863, GU976864, GU976865, GU976866, GU976867, GU976868, GU976869, GU976870, GU976871, GU976872, GU976873, GU976874, GU976875, GU976876, GU976877, GU976878, GU976879, GU976880, GU976881, GU976882, GU976883, GU976884, GU976885, GU976886, GU976887, GU976888, GU976889, GU976890, GU976891, GU976892, GU976893, GU976894, GU976895, GU976896, GU976897, GU976898, GU976899, GU976900, GU976901, GU976902, GU976903, GU976904, GU976905, GU976906, GU976907, GU976908, GU976909, GU976910, GU976911, GU976912, GU976913, GU976914, GU976915, GU976916, GU976917, GU976918, GU976919, GU976920, GU976921, GU976922, GU976923, GU976924, GU976925, GU976926, GU976927, GU976928, GU976929, GU976930, GU976931, GU976932, GU976933, GU976934, GU976935, GU976936, GU976937, GU976938, GU976939, GU976940, GU976941, GU976942, GU976943, GU976944, GU976945, GU976946, GU976947, GU976948, GU976949, GU976950, GU976951, GU976952, GU976953, GU976954, GU976955, GU976956, GU976957, GU976958, GU976959, GU976960, GU976961, GU976962, GU976963, GU976964, GU976965, GU976966, GU976967, GU976968, GU976969, GU976970, GU976971, GU976972, GU976973, GU976974, GU976975, GU976976, GU976977, GU976978, GU976979, GU976980, GU976981, GU976982, GU976983, GU976984, GU976985, GU976986, GU976987, GU976988, GU976989, GU976990, GU976991, GU976992, GU976993, GU976994, GU976995, GU976996, GU976997, GU976998, GU976999, GU977000, GU977001, GU977002, GU977003, GU977004, GU977005, GU977006, GU977007, GU977008, GU977009, GU977010, GU977011, GU977012, GU977013, GU977014, GU977015, GU977016, GU977017, GU977018, GU977019, GU977020, GU977021, GU977022, GU977023, GU977024, GU977025, GU977026, GU977027, GU977028, GU977029, GU977030, GU977031, GU977032, GU977033, GU977034, GU977035, GU977036, GU977037, GU977038, GU977039, GU977040, GU977041, GU977042, GU977043, GU977044, GU977045, GU977046, GU977047, GU977048, GU977049, GU977050, GU977051, GU977052, GU977053, GU977054, GU977055, GU977056, GU977057, GU977058, GU977059, GU977060, GU977061, GU977062, GU977063, GU977064, GU977065, GU977066, GU977067, GU977068, GU977069, GU977070, GU977071, GU977072, GU977073, GU977074, GU977075, GU977076, GU977077, GU977078, GU977079, GU977080, GU977081, GU977082, GU977083, GU977084, GU977085, GU977086, GU977087, GU977088, GU977089, GU977090, GU977091, GU977092, GU977093, GU977094, GU977095, GU977096, GU977097, GU977098, GU977099, GU977100, GU977101, GU977102, GU977103, GU977104, GU977105, GU977106, GU977107, GU977108, GU977109, GU977110, GU977111, GU977112, GU977113, GU977114, GU977115, GU977116, GU977117, GU977118, GU977119, GU977120, GU977121, GU977122, GU977123, GU977124, GU977125, GU977126, GU977127, GU977128, GU977129, GU977130, GU977131, GU977132, GU977133, GU977134, GU977135, GU977136, GU977137, GU977138, GU977139, GU977140, GU977141, GU977142, GU977143, GU977144, GU977145, GU977146, GU977147, GU977148, GU977149, GU977150, GU977151, GU977152, GU977153, GU977154, GU977155, GU977156, GU977157, GU977158, GU977159, GU977160, GU977161, GU977162, GU977163, GU977164, GU977165, GU977166, GU977167, GU977168, GU977169, GU977170, GU977171, GU977172, GU977173, GU977174, GU977175, GU977176, GU977177, GU977178, GU977179, GU977180, GU977181, GU977182, GU977183, GU977184, GU977185, GU977186, GU977187