DNA ploidy variation and distribution in the Lepidosperma costale complex (Cyperaceae): implications for conservation and restoration in a biodiversity hotspot
Mark J. Wallace A B E F , Lydia K. Guja A C E , Marie A. Jouault A , Kathy A. Fuller D , Russell L. Barrett A B E , Siegfried L. Krauss A B and Matthew D. Barrett A B
+ Author Affiliations
- Author Affiliations
A Botanic Gardens and Parks Authority, Kings Park and Botanic Garden, West Perth, WA 6005, Australia.
B School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia.
C Curtin Institute for Biodiversity and Climate, Curtin University, Bentley, WA 6102, Australia.
D Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Crawley, WA 6009, Australia.
E Present address: Centre for Australian National Biodiversity Research, Canberra, ACT 2601, Australia.
F Corresponding author. Email: m.wallace@csiro.au
Australian Journal of Botany 65(2) 120-127 https://doi.org/10.1071/BT16197
Submitted: 30 September 2016 Accepted: 24 January 2017 Published: 21 February 2017
Abstract
Intraspecific ploidy variation is an important component of angiosperm biodiversity; however, this variation is rarely considered in conservation programs. This is of particular concern when conservation activities include augmentation, reintroduction or ecological restoration because there are potentially negative consequences when ploidy variants are unintentionally mixed within populations. We surveyed regional ploidy variation in the Lepidosperma costale Nees species complex (Schoeneae: Cyperaceae) in the South West Australian Floristic Region, an international biodiversity hotspot. Several L. costale sensu lato populations are threatened by iron-ore extraction, including the rare L. gibsonii R.L.Barrett, and these populations are the subject of ecological restoration programs. The DNA ploidy of 2384 individuals from 28 populations across the range of the species complex was determined and four DNA ploidy levels were discovered, namely, diploid, triploid, tetraploid and pentaploid. Diploids and tetraploids were the most common cytotypes and were largely geographically segregated, even at an exhaustively studied contact zone. Triploids were found at a low frequency in two populations. The rarity of triploids suggests substantial interploidy sterility, and that mixing of ploidy variants should, therefore, be avoided when restoring L. costale s.l. populations. These data provide a guide for L. costale s.l. germplasm collection and suggest that polyploidy may be an important driver of diversification in these sedges.
Additional keywords: ironstone, contact zone, cytotype, flow cytometry, genome size, granite, polyploid.
References
Baack EJ (2004) Cytotype segregation on regional and microgeographic scales in snow buttercups (Ranunculus adoneus: Ranunculaceae). American Journal of Botany 91, 1783–1788.| Cytotype segregation on regional and microgeographic scales in snow buttercups (Ranunculus adoneus: Ranunculaceae).Crossref | GoogleScholarGoogle Scholar |
Barker MS, Arrigo N, Baniaga AE, Li Z, Levin DA (2016) On the relative abundance of autopolyploids and allopolyploids. New Phytologist 210, 391–398.
| On the relative abundance of autopolyploids and allopolyploids.Crossref | GoogleScholarGoogle Scholar |
Barrett RL (2007) New species of Lepidosperma (Cyperaceae) associated with banded ironstone formations in southern Western Australia. Nuytsia 17, 37–60.
Barrett RL (2012) Description of six Lepidosperma species (Cyperaceae) based on type specimens. Nuytsia 22, 295–322.
Barrett RL (2013) Ecological importance of sedges: a survey of the Australasian Cyperaceae genus Lepidosperma. Annals of Botany 111, 499–529.
| Ecological importance of sedges: a survey of the Australasian Cyperaceae genus Lepidosperma.Crossref | GoogleScholarGoogle Scholar |
Barrett RL, Wilson KL (2012) A review of the genus Lepidosperma Labill. (Cyperaceae: Schoeneae). Australian Systematic Botany 25, 225–294.
| A review of the genus Lepidosperma Labill. (Cyperaceae: Schoeneae).Crossref | GoogleScholarGoogle Scholar |
Barrett MD, Wallace MJ, Anthony JM (2012) Characterization and cross application of novel microsatellite markers for a rare sedge, Lepidosperma gibsonii (Cyperaceae). American Journal of Botany 99, e14–e16.
| Characterization and cross application of novel microsatellite markers for a rare sedge, Lepidosperma gibsonii (Cyperaceae).Crossref | GoogleScholarGoogle Scholar |
Bennett MD, Leitch IJ (2012) ‘Plant DNA C-values database (release 6.0, December 2012).’ Available at: http://data.kew.org/cvalues/ [Verified 1 September 2016].
Bennett MD, Leitch IJ, Hanson L (1998) DNA amounts in two samples of angiosperm weeds. Annals of Botany 82, 121–134.
| DNA amounts in two samples of angiosperm weeds.Crossref | GoogleScholarGoogle Scholar |
Bennett MD, Leitch IJ, Price HJ, Johnston JS (2003) Comparisons with Caenorhabditis (~100 Mb) and Drosophila (~175 Mb) using flow cytometry show genome size in Arabidopsis to be ~157 Mb and thus ~25% larger than the Arabidopsis Genome Initiative estimate of ~125 Mb. Annals of Botany 91, 547–557.
| Comparisons with Caenorhabditis (~100 Mb) and Drosophila (~175 Mb) using flow cytometry show genome size in Arabidopsis to be ~157 Mb and thus ~25% larger than the Arabidopsis Genome Initiative estimate of ~125 Mb.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXjsVyitr8%3D&md5=68927f3e9f79cee206c0fd64436837c5CAS |
Bowers JE, Chapman BA, Rong J, Paterson AH (2003) Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. Nature 422, 433–438.
| Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXitlGgtbk%3D&md5=f39fd18134603cc00c894212509efaf7CAS |
Brown AHD, Young AG (2000) Genetic diversity in tetraploid populations of the endangered daisy Rutidosis leptorrhynchoides and implications for its conservation. Heredity 85, 122–129.
| Genetic diversity in tetraploid populations of the endangered daisy Rutidosis leptorrhynchoides and implications for its conservation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXnsFChtr8%3D&md5=604ef453644eb2e0019af902aec9e5e8CAS |
Burton TL, Husband BC (1999) Population cytotype structure in the polyploid Galax urceolata (Diapensiaceae). Heredity 82, 381–390.
| Population cytotype structure in the polyploid Galax urceolata (Diapensiaceae).Crossref | GoogleScholarGoogle Scholar |
Byrne M, Hopper SD (2008) Granite outcrops as ancient islands in old landscapes: evidence from the phylogeography and population genetics of Eucalyptus caesia (Myrtaceae) in Western Australia. Biological Journal of the Linnean Society. Linnean Society of London 93, 177–188.
| Granite outcrops as ancient islands in old landscapes: evidence from the phylogeography and population genetics of Eucalyptus caesia (Myrtaceae) in Western Australia.Crossref | GoogleScholarGoogle Scholar |
Chung K-S, Weber JA, Hipp AL (2011) Dynamics of chromosome number and genome size variation in a cytogenetically variable sedge (Carex scoparia var. scoparia, Cyperaceae). American Journal of Botany 98, 122–129.
| Dynamics of chromosome number and genome size variation in a cytogenetically variable sedge (Carex scoparia var. scoparia, Cyperaceae).Crossref | GoogleScholarGoogle Scholar |
Clarke CB (1908) New genera and species of Cyperaceae. Bulletin of Miscellaneous Information: Additional Series 8, 91
De Bodt S, Maere S, Van de Peer Y (2005) Genome duplication and the origin of angiosperms. Trends in Ecology & Evolution 20, 591–597.
| Genome duplication and the origin of angiosperms.Crossref | GoogleScholarGoogle Scholar |
de Lange PJ, Murray BG, Datson PM (2004) Contributions to a chromosome atlas of the New Zealand flora – 38. Counts for 50 families. New Zealand Journal of Botany 42, 873–904.
| Contributions to a chromosome atlas of the New Zealand flora – 38. Counts for 50 families.Crossref | GoogleScholarGoogle Scholar |
Delaney JT, Baack EJ (2012) Intraspecific chromosome number variation and prairie restoration—a case study in northeast Iowa, USA. Restoration Ecology 20, 576–583.
| Intraspecific chromosome number variation and prairie restoration—a case study in northeast Iowa, USA.Crossref | GoogleScholarGoogle Scholar |
Doležel J, Sgorbati S, Lucretti S (1992) Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum 85, 625–631.
| Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants.Crossref | GoogleScholarGoogle Scholar |
Fowler NL, Levin DA (1984) Ecological constraints on the establishment of a novel polyploid in competition with its diploid progenitor. American Naturalist 124, 703–711.
| Ecological constraints on the establishment of a novel polyploid in competition with its diploid progenitor.Crossref | GoogleScholarGoogle Scholar |
Gibson N, Coates DJ, Thiele KR (2007) Taxonomic research and the conservation status of flora in the Yilgarn banded iron formation ranges. Nuytsia 17, 1–12.
Gibson N, Yates CJ, Dillon R (2010) Plant communities of the ironstone ranges of South Western Australia: hotspots for plant diversity and mineral deposits. Biodiversity and Conservation 19, 3951–3962.
| Plant communities of the ironstone ranges of South Western Australia: hotspots for plant diversity and mineral deposits.Crossref | GoogleScholarGoogle Scholar |
Grime JP, Shacklock JML, Brand SR (1985) Nuclear DNA contents, shoot phenology and species co-existence in a limestone grassland community. New Phytologist 100, 435–445.
| Nuclear DNA contents, shoot phenology and species co-existence in a limestone grassland community.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL2MXlt1Sgsrc%3D&md5=812e26b71bd8138e29e56e0aa00c1b71CAS |
Halverson K, Heard SB, Nason JD, Stireman JO (2008) Origins, distribution, and local co-occurrence of polyploid cytotypes in Solidago altissima (Asteraceae). American Journal of Botany 95, 50–58.
| Origins, distribution, and local co-occurrence of polyploid cytotypes in Solidago altissima (Asteraceae).Crossref | GoogleScholarGoogle Scholar |
Henry IM, Dilkes BP, Young K, Watson B, Wu H, Comai L (2005) Aneuploidy and genetic variation in the Arabidopsis thaliana triploid response. Genetics 170, 1979–1988.
| Aneuploidy and genetic variation in the Arabidopsis thaliana triploid response.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtFeis7%2FF&md5=eff652e8f71e5f1ffb04868622e50ed6CAS |
Hipp AL (2007) Nonuniform processes of chromosome evolution in sedges (Carex: Cyperaceae). Evolution 61, 2175–2194.
| Nonuniform processes of chromosome evolution in sedges (Carex: Cyperaceae).Crossref | GoogleScholarGoogle Scholar |
Hipp AL, Rothrock PE, Roalson EH (2009) The evolution of chromosome arrangements in Carex (Cyperaceae). Botanical Review 75, 96–109.
| The evolution of chromosome arrangements in Carex (Cyperaceae).Crossref | GoogleScholarGoogle Scholar |
Hodgon J, Bruhl JJ, Wilson KL (2006) Systematic studies in Lepidosperma (Cyperaceae: Schoeneae) with particular reference to L. laterale. Australian Systematic Botany 19, 273–288.
| Systematic studies in Lepidosperma (Cyperaceae: Schoeneae) with particular reference to L. laterale.Crossref | GoogleScholarGoogle Scholar |
Husband BC (2000) Constraints on polyploid evolution: a test of the minority cytotype exclusion principle. Proceedings. Biological Sciences 267, 217–223.
| Constraints on polyploid evolution: a test of the minority cytotype exclusion principle.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c7nslemtQ%3D%3D&md5=cd0e007aef332ac3f2ba71adeca39dbcCAS |
Husband BC, Sabara HA (2004) Reproductive isolation between autotetraploids and their diploid progenitors in fireweed, Chamerion angustifolium (Onagraceae). New Phytologist 161, 703–713.
| Reproductive isolation between autotetraploids and their diploid progenitors in fireweed, Chamerion angustifolium (Onagraceae).Crossref | GoogleScholarGoogle Scholar |
Husband BC, Schemske DW (1998) Cytotype distribution at a diploid-tetraploid contact zone in Chamerion (Epilobium) angustifolium (Onagraceae). American Journal of Botany 85, 1688–1694.
| Cytotype distribution at a diploid-tetraploid contact zone in Chamerion (Epilobium) angustifolium (Onagraceae).Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3MnkvVCjtQ%3D%3D&md5=62454170a221612303bb18b25ad4576dCAS |
Kaur N, Datson PM, Murray BG (2012) Genome size and chromosome number in the New Zealand species of Schoenus (Cyperaceae). Botanical Journal of the Linnean Society 169, 555–564.
| Genome size and chromosome number in the New Zealand species of Schoenus (Cyperaceae).Crossref | GoogleScholarGoogle Scholar |
Kodym A, Temsch EM, Bunn E, Delpratt J (2012) Ploidy stability of somatic embryo-derived plants in two ecological keystone sedge species (Lepidosperma laterale and L. concavum, Cyperaceae). Australian Journal of Botany 60, 396–404.
| Ploidy stability of somatic embryo-derived plants in two ecological keystone sedge species (Lepidosperma laterale and L. concavum, Cyperaceae).Crossref | GoogleScholarGoogle Scholar |
Kolář F, Štech M, Trávníček P, Rauchová J, Urfus T, Vít P, Kubešova M, Suda J (2009) Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales. Annals of Botany 103, 963–974.
| Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales.Crossref | GoogleScholarGoogle Scholar |
Levin DA (1975) Minority cytotype exclusion in local plant populations. Taxon 24, 35–43.
| Minority cytotype exclusion in local plant populations.Crossref | GoogleScholarGoogle Scholar |
Levin DA (2002) ‘The role of chromosomal change in plant evolution.’ (Oxford University Press: Melbourne)
Luceño M, Vanzela ALL, Guerra M (1998) Cytotaxonomic studies in Brazilian Rhynchospora (Cyperaceae), a genus exhibiting holocentric chromosomes. Canadian Journal of Botany 76, 440–449.
| Cytotaxonomic studies in Brazilian Rhynchospora (Cyperaceae), a genus exhibiting holocentric chromosomes.Crossref | GoogleScholarGoogle Scholar |
Maceira NO, Jacquard P, Lumaret R (1993) Competition between diploid and derivative autotetraploid Dactylis glomerata L. from Galicia. Implications for the establishment of novel polyploid populations. New Phytologist 124, 321–328.
| Competition between diploid and derivative autotetraploid Dactylis glomerata L. from Galicia. Implications for the establishment of novel polyploid populations.Crossref | GoogleScholarGoogle Scholar |
Millar MA, Byrne M, Coates DJ (2008) Seed collection for revegetation: guidelines for Western Australian flora. Journal of the Royal Society of Western Australia 91, 293–299.
Murray BG, Young AG (2001) Widespread chromosome variation in the endangered grassland forb Rutidosis leptorrhynchoides F.Muell. (Asteraceae: Gnaphalieae). Annals of Botany 87, 83–90.
| Widespread chromosome variation in the endangered grassland forb Rutidosis leptorrhynchoides F.Muell. (Asteraceae: Gnaphalieae).Crossref | GoogleScholarGoogle Scholar |
Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403, 853–858.
| Biodiversity hotspots for conservation priorities.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXhs1Olsr4%3D&md5=714524c78ac3b71f700b2f775cd1d2ceCAS |
Nees von Esenbeck CGD (1846) Lepidosperma. In ‘Plantae Preissianae. Vol. 2’. (Ed. JGC Lehmann) pp. 89–93. (Meissner: Hamburg, Germany)
Nishikawa K, Furuta Y, Ishitobi K (1984) Chromosomal evolution in genus Carex as viewed from nuclear DNA content, with special reference to its aneuploidy. Japanese Journal of Genetics 59, 465–472.
| Chromosomal evolution in genus Carex as viewed from nuclear DNA content, with special reference to its aneuploidy.Crossref | GoogleScholarGoogle Scholar |
Parisod C, Holderegger R, Brochmann C (2010) Evolutionary consequences of autopolyploidy. New Phytologist 186, 5–17.
| Evolutionary consequences of autopolyploidy.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXkvVOksbo%3D&md5=a52a31a1d6c51cf45f124fa294444d17CAS |
Petit C, Bretagnolle F, Felber F (1999) Evolutionary consequences of diploid-polyploid hybrid zones in wild species. Trends in Ecology & Evolution 14, 306–311.
| Evolutionary consequences of diploid-polyploid hybrid zones in wild species.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC2sbgslWgsQ%3D%3D&md5=ab99b72e94af8e3d4db4ee5d1fef43fcCAS |
Ramsey J, Schemske DW (1998) Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annual Review of Ecology and Systematics 29, 467–501.
| Pathways, mechanisms, and rates of polyploid formation in flowering plants.Crossref | GoogleScholarGoogle Scholar |
Rice A, Glick L, Abadi S, Einhorn M, Kopelman NM, Salman-Minkov A, Mayzel J, Chay O, Mayrose I (2015) The Chromosome Counts Database (CCDB) – a community resource of plant chromosome numbers. New Phytologist 206, 19–26.
| The Chromosome Counts Database (CCDB) – a community resource of plant chromosome numbers.Crossref | GoogleScholarGoogle Scholar |
Roberts AV (2007) The use of bead beating to prepare suspensions of nuclei for flow cytometry from fresh leaves, herbarium leaves, petals and pollen. Cytometry. Part A 71A, 1039–1044.
| The use of bead beating to prepare suspensions of nuclei for flow cytometry from fresh leaves, herbarium leaves, petals and pollen.Crossref | GoogleScholarGoogle Scholar |
Severns PM, Liston A (2008) Intraspecific chromosome number variation: a neglected threat to the conservation of rare plants. Conservation Biology 22, 1641–1647.
| Intraspecific chromosome number variation: a neglected threat to the conservation of rare plants.Crossref | GoogleScholarGoogle Scholar |
Severns PM, Bradford E, Liston A (2013) Whole genome duplication in a threatened grassland plant and the efficacy of seed transfer zones. Diversity & Distributions 19, 455–464.
Soltis DE, Soltis PS, Tate JA (2004) Advances in the study of polyploidy since Plant speciation. New Phytologist 161, 173–191.
| Advances in the study of polyploidy since Plant speciation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXmsVWluw%3D%3D&md5=f34ca9a58de1eec0695b139f8c6c3233CAS |
Soltis DE, Soltis PS, Schemske DW, Hancock JF, Thompson JN, Husband BC, Judd WS (2007) Autopolyploidy in angiosperms: have we grossly underestimated the number of species? Taxon 56, 13–30.
Soltis DE, Mavrodiev EV, Doyle JJ, Rauscher J, Soltis PS (2008) ITS and ETS sequence data and phylogeny reconstruction in allopolyploids and hybrids. Systematic Botany 33, 7–20.
| ITS and ETS sequence data and phylogeny reconstruction in allopolyploids and hybrids.Crossref | GoogleScholarGoogle Scholar |
Soltis DE, Visger CJ, Marchant DB, Soltis PS (2016) Polyploidy: pitfalls and paths to a paradigm. American Journal of Botany 103, 1146–1166.
| Polyploidy: pitfalls and paths to a paradigm.Crossref | GoogleScholarGoogle Scholar |
Španiel S, Marhold K, Hodálová I, Lihová J (2008) Diploid and tetraploid cytotypes of Centaurea stoebe (Asteraceae) in central Europe: morphological differentiation and cytotype distribution patterns. Folia Geobotanica 43, 131–158.
| Diploid and tetraploid cytotypes of Centaurea stoebe (Asteraceae) in central Europe: morphological differentiation and cytotype distribution patterns.Crossref | GoogleScholarGoogle Scholar |
Stuessy TF, Weiss-Schneeweiss H, Keil DJ (2004) Diploid and polyploid cytotype distribution in Melampodium cinereum and M. leucanthum (Asteraceae, Heliantheae). American Journal of Botany 91, 889–898.
| Diploid and polyploid cytotype distribution in Melampodium cinereum and M. leucanthum (Asteraceae, Heliantheae).Crossref | GoogleScholarGoogle Scholar |
Suda J, Krahulcová A, Trávníček P, Krahulec F (2006) Ploidy level versus DNA ploidy level: an appeal for consistent terminology. Taxon 55, 447–450.
| Ploidy level versus DNA ploidy level: an appeal for consistent terminology.Crossref | GoogleScholarGoogle Scholar |
Suda J, Weiss-Schneeweiss H, Tribsch A, Schneeweiss GM, Trávníček P, Schönswetter P (2007) Complex distribution patterns of di-, tetra-, and hexaploid cytotypes in the European high mountain plant Sencio carniolicus (Asteraceae). American Journal of Botany 94, 1391–1401.
| Complex distribution patterns of di-, tetra-, and hexaploid cytotypes in the European high mountain plant Sencio carniolicus (Asteraceae).Crossref | GoogleScholarGoogle Scholar |
Vanzela ALL, Luceño M, Guerra M (2000) Karyotype evolution and cytotaxonomy in Brazilian species of Rhynchospora Vahl (Cyperaceae). Botanical Journal of the Linnean Society 134, 557–566.
| Karyotype evolution and cytotaxonomy in Brazilian species of Rhynchospora Vahl (Cyperaceae).Crossref | GoogleScholarGoogle Scholar |
Wallace MJ, Barrett MD, Barrett RL (2011) Novel chloroplast markers for the study of intraspecific variation and hybridisation in the Lepidosperma costale species complex (Cyperaceae). Conservation Genetics Resources 3, 355–360.
| Novel chloroplast markers for the study of intraspecific variation and hybridisation in the Lepidosperma costale species complex (Cyperaceae).Crossref | GoogleScholarGoogle Scholar |
Waters C, Murray BG, Melville G, Coates D, Young A, Virgona J (2010) Polyploidy and possible implications for the evolutionary history of some Australian Danthonieae. Australian Journal of Botany 58, 23–34.
| Polyploidy and possible implications for the evolutionary history of some Australian Danthonieae.Crossref | GoogleScholarGoogle Scholar |
Wood TE, Takebayashi N, Barker MS, Mayrose I, Greenspoon PB, Rieseberg LH (2009) The frequency of polyploid speciation in vascular plants. Proceedings of the National Academy of Sciences, USA 106, 13875–13879.
| The frequency of polyploid speciation in vascular plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtFWksLnF&md5=9723db00ce0c2f91f369f0295f5a13b4CAS |
Yano O, Hoshino T (2005) Molecular phylogeny and chromosomal evolution of Japanese Schoenoplectus (Cyperaceae), based on ITS and ETS 1f sequences. Acta Phytotaxonomica et Geobotanica 56, 183–195.
Zedek F, Šmerda J, Šmarda P, Bureš P (2010) Correlated evolution of LTR retrotransposons and genome size in the genus eleocharis. BMC Plant Biology 10, 265
| Correlated evolution of LTR retrotransposons and genome size in the genus eleocharis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhsFagtrjK&md5=ff5dacdd8c0738f1cf83a1b536441b98CAS |
