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Genetic diversity and population structure of native, naturalized, and cultivated Salix purpurea


Salix purpurea is a woody perennial that is bred as a high-yielding bioenergy crop in North America. To gain an understanding of the genotypic variation associated with phenotypic diversity, this study characterized the population structure and genetic diversity of S. purpurea from its native range of Europe and naturalized range of the Northeastern United States (US). A total of 273 genotypes of S. purpurea were analyzed, which included 95 naturalized accessions and 19 horticultural cultivars from the US and 159 accessions collected from the native range of four European countries. All individuals were evaluated using a filtered set of 2287 genotyping-by-sequencing (GBS) single nucleotide polymorphism (SNP) markers. Using five clustering techniques (PCA, neighbor joining, STRUCTURE, DAPC, and affinity propagation), population structure was resolved into three broadly classified groups. Further analysis revealed seven to eight subpopulations which corresponded to geographical collection sites, where performance of the DAPC and affinity propagation methods was superior to STRUCTURE analysis for the purposes of characterizing population structure and performing population assignment. The native European accessions exhibited greater diversity and subpopulation structure than the US naturalized accessions, where there was a clear geographical delineation between the alpine/subalpine collections and the lowland collections at the Baltic Sea and Oder River. We also show that a subset of the horticultural cultivars had a higher likelihood of similarity to US naturalized populations which display hybrid ancestry, where both naturalized and cultivated genotypes appear to share a common ancestor. Additionally, several accessions collected from different sites were found to be clonal. Ongoing and future conservation and association studies will benefit from these known substructures and diversity assessments.

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  1. Ally D, Ritland K, Otto SP (2008) Can clone size serve as a proxy for clone age? An exploration using microsatellite divergence in Populus tremuloides. Mol Ecol 17:4897–4911. https://doi.org/10.1111/j.1365-294X.2008.03962.x

  2. Ally D, Ritland K, Otto SP (2010) Aging in a long-lived clonal tree. PLoS Biol 8:e1000454. https://doi.org/10.1371/journal.pbio.1000454

  3. Argus GW (1974) An experimental study of hybridization and pollination in Salix (willow). Can J Bot 52:1613–1619. https://doi.org/10.1139/b74-212

  4. Argus GW (1997) Infrageneric classification of Salix (Salicaceae) in the New World. Syst Bot Monogr 52:1–121

  5. Argus GW (2007) Salix (Salicaceae) distribution maps and a synopsis of their classification in North America, north of Mexico. Harv Pap Bot 12:335–368. https://doi.org/10.3100/1043-4534(2007)12[335:SSDMAA]2.0.CO;2

  6. Argus GW, Eckenwalder JE, Kiger RW (2010) In: Flora of North America Editorial Committee (ed) Salicaceae, vol 7. Oxford University Press, New York

  7. Beerling DJ (1998) Salix herbacea L. J Ecol 86:872–895. https://doi.org/10.1046/j.1365-2745.1998.8650872.x

  8. Beismann H, Barker JHA, Karp A, Speck T (1997) AFLP analysis sheds light on distribution of two Salix species and their hybrid along a natural gradient. Mol Ecol 6:989–993. https://doi.org/10.1046/j.1365-294X.1997.00273.x

  9. Berlin S, Fogelqvist J, Lascoux M, Lagercrantz U, Rönnberg-Wästljung AC (2011) Polymorphism and divergence in two willow species, Salix viminalis L. and Salix schwerinii E. Wolf.G3: Genes, Genomes, Genetics 1:387–400. https://doi.org/10.1534/g3.111.000539

  10. Berlin S, Trybush SO, Fogelqvist J, Gyllenstrand N, Hallingbäck HR, Åhman I, Nordh NE, Shield I, Powers SJ, Weih M, Lagercrantz U, Rönnberg-Wästljung AC, Karp A, Hanley SJ (2014) Genetic diversity, population structure and phenotypic variation in European Salix viminalis L. (Salicaceae). Tree Genet Genomes 10:1595–1610. https://doi.org/10.1007/s11295-014-0782-5

  11. Bodenhofer U, Kothmeier A, Hochreiter S (2011) APCluster: an R package for affinity propagation clustering. Bioinformatics 27:2463–2464. https://doi.org/10.1093/bioinformatics/btr406

  12. Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES (2007) TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23:2633–2635. https://doi.org/10.1093/bioinformatics/btm308

  13. Brown HP (1921) Trees of New York state : native and naturalized vol XXI, no. 5. The University, Syracuse, N.Y. https://doi.org/10.5962/bhl.title.41392

  14. Bubner B, Köhler A, Zaspel I, Zander M, Förster N, Gloger J-C, Ulrichs C, Schneck V (2018) Breeding of multipurpose willows on the basis of Salix daphnoides Vill., Salix purpurea L. and Salix viminalis L. Appl Agric Forestry Res 68:53–66. https://doi.org/10.3220/LBF1538634874000

  15. Chaves AL, Vergara CE, Mayer JE (1995) Dichloromethane as an economic alternative to chloroform in the extraction of DNA from plant tissues. Plant Mol Biol Report 13:18–25. https://doi.org/10.1007/bf02668389

  16. DeFaveri J, Viitaniemi H, Leder E, Merila J (2013) Characterizing genic and nongenic molecular markers: comparison of microsatellites and SNPs. Mol Ecol Resour 13:377–392. https://doi.org/10.1111/1755-0998.12071

  17. Dickerson J (2002) Purple osier willow Salix purpurea L.: plant fact sheet. USDA Natural Resources Conservation Service, Syracuse, NY, USA

  18. Dickmann D, Kuzovkina J (2008) Poplars and willows in the world. In: Poplars and willows in the world, meeting the needs of society and the environment. International Poplar Commission. FAO, Rome, Italy, pp 8–91

  19. Earl D, vonHoldt B (2012) STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genet Resour 4:359–361. https://doi.org/10.1007/s12686-011-9548-7

  20. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One 6:e19379. https://doi.org/10.1371/journal.pone.0019379

  21. Emanuelli F, Lorenzi S, Grzeskowiak L, Catalano V, Stefanini M, Troggio M, Myles S, Martinez-Zapater JM, Zyprian E, Moreira FM, Grando MS (2013) Genetic diversity and population structure assessed by SSR and SNP markers in a large germplasm collection of grape. BMC Plant Biol 13:39. https://doi.org/10.1186/1471-2229-13-39

  22. Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14:2611–2620. https://doi.org/10.1111/j.1365-294X.2005.02553.x

  23. Falush D, Stephens M, Pritchard JK (2003) Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164:1567–1587

  24. Flint-Garcia SA, Thornsberry JM, Buckler ES (2003) Structure of linkage disequilibrium in plants. Annu Rev Plant Biol 54:357–374. https://doi.org/10.1146/annurev.arplant.54.031902.134907

  25. Frey BJ, Dueck D (2007) Clustering by passing messages between data points. Science 315:972–976. https://doi.org/10.1126/science.1136800

  26. Glaubitz JC, Casstevens TM, Lu F, Harriman J, Elshire RJ, Sun Q, Buckler ES (2014) TASSEL-GBS: a high capacity genotyping by sequencing analysis pipeline. PLoS One 9:e90346. https://doi.org/10.1371/journal.pone.0090346

  27. González E, González-Sanchis M, Cabezas Á, Comín FA, Muller E (2010) Recent changes in the riparian forest of a large regulated mediterranean river: implications for management. Environ Manag 45:669–681. https://doi.org/10.1007/s00267-010-9441-2

  28. Gramlich S, Sagmeister P, Dullinger S, Hadacek F, Horandl E (2016) Evolution in situ: hybrid origin and establishment of willows (Salix L.) on alpine glacier forefields. Heredity 116:531–541. https://doi.org/10.1038/hdy.2016.14

  29. Hallingbäck HR, Fogelqvist J, Powers SJ, Turrion-Gomez J, Rossiter R, Amey J, Martin T, Weih M, Gyllenstrand N, Karp A, Lagercrantz U, Hanley SJ, Berlin S, Rönnberg-Wästljung A-C (2015) Association mapping in Salix viminalis L. (Salicaceae) - identification of candidate genes associated with growth and phenology. GCB Bioenergy 8:670–685. https://doi.org/10.1111/gcbb.12280

  30. Hardig TM, Brunsfeld SJ, Fritz RS, Morgan M, Orians CM (2000) Morphological and molecular evidence for hybridization and introgression in a willow (Salix) hybrid zone. Mol Ecol 9:9–24. https://doi.org/10.1046/j.1365-294X.2000.00757.x

  31. Ioannidis JP, Thomas G, Daly MJ (2009) Validating, augmenting and refining genome-wide association signals. Nat Rev Genet 10:318–329. https://doi.org/10.1038/nrg2544

  32. Jombart T, Ahmed I (2011) adegenet 1.3-1: new tools for the analysis of genome-wide SNP data. Bioinformatics 27:3070–3071. https://doi.org/10.1093/bioinformatics/btr521

  33. Jombart T, Devillard S, Balloux F (2010) Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genet 11:94. https://doi.org/10.1186/1471-2156-11-94

  34. Jones P (1997) Microclimate in open-top chambers: implications for predicting climate change effects on rice production. Trans ASAE 40:739

  35. Julkunen-Tiitto R (1996) Defensive efforts of Salix myrsinifolia plantlets in photomixotrophic culture conditions: the effect of sucrose, nitrogen and pH on the phytomass and secondary phenolic accumulation. Écoscience 3:297–303

  36. Kamvar ZN, Tabima JF, Grünwald NJ (2014) Poppr: an R package for genetic analysis of populations with clonal, partially clonal, and/or sexual reproduction. PeerJ 2:e281. https://doi.org/10.7717/peerj.281

  37. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. https://doi.org/10.1093/bioinformatics/bts199

  38. Kiddle SJ, Windram OPF, McHattie S, Mead A, Beynon J, Buchanan-Wollaston V, Denby KJ, Mukherjee S (2010) Temporal clustering by affinity propagation reveals transcriptional modules in Arabidopsis thaliana. Bioinformatics 26:355–362. https://doi.org/10.1093/bioinformatics/btp673

  39. Kikuchi S, Suzuki W, Sashimura N (2011) Gene flow in an endangered willow Salix hukaoana (Salicaceae) in natural and fragmented riparian landscapes. Conserv Genet 12:79–89. https://doi.org/10.1007/s10592-009-9992-z

  40. Kopelman NM, Mayzel J, Jakobsson M, Rosenberg NA, Mayrose I (2015) CLUMPAK: a program for identifying clustering modes and packaging population structure inferences across K. Mol Ecol Resour 15:1179–1191. https://doi.org/10.1111/1755-0998.12387

  41. Kuzovkina YA, Quigley MF (2005) Willows beyond wetlands: uses of Salix L. species for environmental projects. Water Air Soil Pollut 162:183–204. https://doi.org/10.1007/s11270-005-6272-5

  42. Kuzovkina YA, Weih M, Romero MA, Charles J, Hust S, McIvor I, Karp A, Trybush S, Labrecque M, Teodorescu TI, Singh NB, Smart LB, Volk TA (2008) Salix: botany and global horticulture. In: Horticultural reviews. John Wiley & Sons, Inc., Hoboken, NJ, pp, pp 447–489. https://doi.org/10.1002/9780470380147.ch8

  43. Lascoux M, Thorsén J, Gullberg U (1996) Population structure of a riparian willow species, Salix viminalis L. Genet Res 68:45–54. https://doi.org/10.1017/S0016672300033875

  44. Lauron-Moreau A, Pitre FE, Argus GW, Labrecque M, Brouillet L (2015) Phylogenetic relationships of American willows (Salix L., Salicaceae). PLoS One 10:e0121965. https://doi.org/10.1371/journal.pone.0121965

  45. Leone M, Sumedha, Weigt M (2007) Clustering by soft-constraint affinity propagation: applications to gene-expression data. Bioinformatics 23:2708–2715. https://doi.org/10.1093/bioinformatics/btm414

  46. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. https://doi.org/10.1093/bioinformatics/btp324

  47. Liang W, Dondini L, De Franceschi P, Paris R, Sansavini S, Tartarini S (2015) Genetic diversity, population structure and construction of a core collection of apple cultivars from Italian germplasm. Plant Mol Biol Report 33:458–473. https://doi.org/10.1007/s11105-014-0754-9

  48. Lin J, Gibbs JP, Smart LB (2009) Population genetic structure of native versus naturalized sympatric shrub willows (Salix: Salicaceae). Am J Bot 96:771–785. https://doi.org/10.3732/ajb.0800321

  49. Lu F, Lipka AE, Glaubitz J, Elshire R, Cherney JH, Casler MD (2013) Switchgrass genomic diversity, ploidy, and evolution: novel insights from a network-based SNP discovery protocol. PLoS Genet 9:e1003215. https://doi.org/10.1371/journal.pgen.1003215

  50. Meikle RD (1992) British willows; some hybrids and some problems. P Roy Soc Edinb B 98:13–20. https://doi.org/10.1017/S0269727000007429

  51. Moggridge HL, Gurnell AM (2009) Controls on the sexual and asexual regeneration of Salicaceae along a highly dynamic, braided river system. Aquat Sci 71:305–317. https://doi.org/10.1007/s00027-009-9193-3

  52. Morris GP, Grabowski PP, Borevitz JO (2011) Genomic diversity in switchgrass (Panicum virgatum): from the continental scale to a dune landscape. Mol Ecol 20:4938–4952. https://doi.org/10.1111/j.1365-294X.2011.05335.x

  53. Nagamitsu T, Hoshikawa T, Kawahara T, Barkalov VY, Sabirov RN (2014) Phylogeography and genetic structure of disjunct Salix arbutifolia populations in Japan. Popul Ecol 56:539–549. https://doi.org/10.1007/s10144-014-0434-5

  54. Nicolescu V-N, Hernea C, Bakti B, Keserű Z, Antal B, Rédei K (2018) Black locust (Robinia pseudoacacia L.) as a multi-purpose tree species in Hungary and Romania: a review. J For Res 29:1449–1463. https://doi.org/10.1007/s11676-018-0626-5

  55. Paradis E (2010) pegas: an R package for population genetics with an integrated-modular approach. Bioinformatics 26:419–420. https://doi.org/10.1093/bioinformatics/btp696

  56. Peterson G, Dong Y, Horbach C, Fu Y-B (2014) Genotyping-by-sequencing for plant genetic diversity analysis: a lab guide for SNP genotyping. Diversity 6:665–680. https://doi.org/10.3390/d6040665

  57. Plomion C, Aury JM, Amselem J, Leroy T, Murat F, Duplessis S, Faye S, Francillonne N, Labadie K, le Provost G, Lesur I, Bartholomé J, Faivre-Rampant P, Kohler A, Leplé JC, Chantret N, Chen J, Diévart A, Alaeitabar T, Barbe V, Belser C, Bergès H, Bodénès C, Bogeat-Triboulot MB, Bouffaud ML, Brachi B, Chancerel E, Cohen D, Couloux A, da Silva C, Dossat C, Ehrenmann F, Gaspin C, Grima-Pettenati J, Guichoux E, Hecker A, Herrmann S, Hugueney P, Hummel I, Klopp C, Lalanne C, Lascoux M, Lasserre E, Lemainque A, Desprez-Loustau ML, Luyten I, Madoui MA, Mangenot S, Marchal C, Maumus F, Mercier J, Michotey C, Panaud O, Picault N, Rouhier N, Rué O, Rustenholz C, Salin F, Soler M, Tarkka M, Velt A, Zanne AE, Martin F, Wincker P, Quesneville H, Kremer A, Salse J (2018) Oak genome reveals facets of long lifespan. Nature Plants 4:440–452. https://doi.org/10.1038/s41477-018-0172-3

  58. Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, Reich D (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38:904–909. https://doi.org/10.1038/ng1847

  59. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959

  60. Przyborowski JA, Sulima P, Kuszewska A, Załuski D, Kilian A (2013) Phylogenetic relationships between four Salix L. species based on DArT markers. Int J Mol Sci 14:24113–24125. https://doi.org/10.3390/ijms141224113

  61. Rechinger KH (1992) Salix taxonomy in Europe – problems, interpretations, observations. P Roy Soc Edinb B 98:1–12. https://doi.org/10.1017/S0269727000007417

  62. Reisch C, Schurm S, Poschlod P (2007) Spatial genetic structure and clonal diversity in an alpine population of Salix herbacea (Salicaceae). Ann Bot 99:647–651. https://doi.org/10.1093/aob/mcl290

  63. Rosenberg NA (2004) DISTRUCT: a program for the graphical display of population structure. Mol Ecol Notes 4:137–138. https://doi.org/10.1046/j.1471-8286.2003.00566.x

  64. Salix purpurea v1.0, DOE-JGI (2015) http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Spurpurea

  65. Setsuko S, Nagamitsu T, Tomaru N (2013) Pollen flow and effects of population structure on selfing rates and female and male reproductive success in fragmented Magnolia stellata populations. BMC Ecol 13:10. https://doi.org/10.1186/1472-6785-13-10

  66. Shield I, Macalpine W, Hanley S, Karp A (2015) Breeding willow for short rotation coppice energy cropping. In: Cruz VMV, Dierig DA (eds) Industrial crops, vol 9. Handbook of plant breeding. Springer, New York, pp 67–80. https://doi.org/10.1007/978-1-4939-1447-0_4

  67. Skvortsov AK (1999) Willows of Russia and adjacent countries. Taxonomical and geographical revision. In: University of Joensuu. Joensuu, Finland

  68. Smart LB, Cameron KD (2012) Shrub willow. In: Kole C, Joshi CP, Shonnard DR (eds) Handbook of bioenergy crop plants. CRC Press, Boca Raton, FL, pp 687–708. https://doi.org/10.1201/b11711-32

  69. Sochor M, Vašut RJ, Bártová E, Majeský Ľ, Mráček J (2013) Can gene flow among populations counteract the habitat loss of extremely fragile biotopes? An example from the population genetic structure in Salix daphnoides. Tree Genet Genomes 9:1193–1205. https://doi.org/10.1007/s11295-013-0628-6

  70. Stokes KE (2008) Exotic invasive black willow (Salix nigra) in Australia: influence of hydrological regimes on population dynamics. Plant Ecol 197:91–105. https://doi.org/10.1007/s11258-007-9363-0

  71. Sulima P, Przyborowski Jerzy A (2013) Genetic diversity of Salix purpurea L. genotypes and interspecific hybrids. abcsb 55:29–36. https://doi.org/10.2478/abcsb-2013-0020

  72. Sulima P, Przyborowski JA, Załuski D (2009) RAPD markers reveal genetic diversity in Salix purpurea L. Crop Sci 49:857–863. https://doi.org/10.2135/cropsci2008.07.0397

  73. Sulima P, Prinz K, Przyborowski J (2018) Genetic diversity and genetic relationships of purple willow (Salix purpurea L.) from natural locations. Int J Mol Sci 19:105

  74. Trybush SO, Jahodová Š, Čížková L, Karp A, Hanley SJ (2012) High levels of genetic diversity in Salix viminalis of the Czech Republic as revealed by microsatellite markers. Bioenerg Res 5:969–977. https://doi.org/10.1007/s12155-012-9212-4

  75. Urrestarazu J, Miranda C, Santesteban L, Royo J (2012) Genetic diversity and structure of local apple cultivars from Northeastern Spain assessed by microsatellite markers. Tree Genet Genomes 8:1163–1180. https://doi.org/10.1007/s11295-012-0502-y

  76. Winter DJ (2012) mmod: an R library for the calculation of population differentiation statistics. Mol Ecol Resour 12:1158–1160. https://doi.org/10.1111/j.1755-0998.2012.03174.x

  77. Wu J, Nyman T, Wang D-C, Argus GW, Yang Y-P, Chen J-H (2015) Phylogeny of Salix subgenus Salix s.l. (Salicaceae): delimitation, biogeography, and reticulate evolution. BMC Evol Biol 15:1–13. https://doi.org/10.1186/s12862-015-0311-7

  78. Zhou R, Macaya-Sanz D, Rodgers-Melnick E, Carlson CH, Gouker FE, Evans LM, Schmutz J, Jenkins JW, Yan J, Tuskan GA, Smart LB, DiFazio SP (2018) Characterization of a large sex determination region in Salix purpurea L. (Salicaceae). Mol Gen Genomics 293:1437–1452. https://doi.org/10.1007/s00438-018-1473-y

  79. Zsuffa L (1990) Genetic-improvement of willows for energy plantations. Biomass 22:35–47. https://doi.org/10.1016/0144-4565(90)90005-5

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We gratefully thank all of those who provided help and technical assistance with this study, including Juan Lin who collected naturalized S. purpurea material in the US; Jan Gloger who collected native S. purpurea in Europe; Michelle Serapiglia, Jane Lam, and Petra Knauer who assisted with leaf tissue collection and DNA extraction; and Rob Elshire and Sharon Mitchell at Cornell University’s Genomic Diversity Facility for their advice on SNP genotyping and Craig Carlson for his advice on SNP analysis.

Data archiving statement

The data reported here are archived as supplemental material in TGG. SNP genotypes used in this study can be requested from the corresponding author (lbs33@cornell.edu).


This work was funded by grants from the US Department of Agriculture National Institute of Food and Agriculture for the Northeast Woody/Warm-Season Biomass Consortium (NEWBio, Grant No. 2012-68005-19703) through the Agriculture and Food Research Initiative and from the Northeast Sun Grant Center (NE 11-48).

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Correspondence to Lawrence B. Smart.

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Gouker, F.E., DiFazio, S.P., Bubner, B. et al. Genetic diversity and population structure of native, naturalized, and cultivated Salix purpurea. Tree Genetics & Genomes 15, 47 (2019). https://doi.org/10.1007/s11295-019-1359-0

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  • Bioenergy
  • Biomass
  • Clonality
  • Genetics
  • Relatedness
  • Salicaceae