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Seasonal cold hardiness in maritime pine assessed by different methods


Three screening methods—visual scoring (V), relative conductivity (C) and fluorometry (F)—were used to study the genetic variation in cold hardiness among six populations of maritime pine (Pinus pinaster Ait.) comprising both Atlantic and Mediterranean origins. Freezing damage assessments were carried out in three organs—needles, stems and buds—in two seasons, spring and autumn. We found high levels of genetic differentiation among populations for cold hardiness in autumn, but not in spring. Within populations, differences were always significant (p < 0.05) no matter which organ or screening method was used. Measuring F was the fastest and most easily replicated method to estimate cold hardiness and was as reliable as V and C for predicting the species performance. In autumn, there was a positive correlation between the damage measured in all three types of organs assessed, whereas in spring, correlation among organs was weak. We conclude that sampling date in spring has a crucial impact to detect genetic differences in maritime pine populations, whereas autumn sampling allows more stable comparisons. We also conclude that the fluorometry method provides a more efficient and stable comparison of cold hardiness in maritime pine.

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  1. Aitken SN, Adams WT (1996) Genetics of fall and winter cold hardiness of coastal Douglas-fir in Oregon. Can J For Res 26:1828–1837. doi:10.1139/x26-208

  2. Aitken SN, Adams WT (1997) Spring cold hardiness under strong genetic control in Oregon populations of Pseudotsuga menziesii var. menziesii. Can J For Res 27:1773–1780

  3. Alía R, Gil L, Pardos JA (1995) Performance of 43 Pinus pinaster Ait. provenances on 5 locations in Central Spain. Silvae Genet 44:75–80

  4. Anekonda TS, Adams WT (2000) Cold hardiness testing for Douglas-Fir tree improvement programs: guidelines for a simple, robust, and inexpensive screening method. West J Appl For 15:129–136

  5. Anekonda TS, Adams WT, Aitken SN et al (2000) Genetics of cold hardiness in a cloned full-sib family of coastal Douglas-fir 1. Can J For Res 30:837–840

  6. Aranda I, Alía R, Ortega U et al (2010) Intra-specific variability in biomass partitioning and carbon isotopic discrimination under moderate drought stress in seedlings from four Pinus pinaster populations. Tree Genet Genomes 6:169–178. doi:10.1007/s11295-009-0238-5

  7. Berrang PC, Steiner KC (1986) Seasonal changes in the cold tolerance of pitch pine. Can J For Res 16:408–410

  8. Binder WD, Fielder P (1996a) Chlorophyll fluorescence as an indicator of frost hardiness in white spruce seedlings from different latitudes. New For 11:233–253

  9. Binder WD, Fielder P (1996b) Seasonal changes in chlorophyll fluorescence of white spruce seedlings from different latitudes in relation to gas exchange and winter storability. New For 11:207–232

  10. Binder WD, Fielder P, Mohammed GH, L’Hirondelle SJ (1996) Applications of chlorophyll fluorescence for stock quality assessment with different types of fluorometers. New For 13:63–89

  11. Bouvarel P (1960) Note sur la resistance au froid de quelques provenances de pin maritime. Rev For Fr 12:495–508

  12. Bower AD, Aitken SN (2006) Geographic and seasonal variation in cold hardiness of whitebark pine. Can J For Res 36:1842–1850. doi:10.1139/x06-067

  13. Burr KE, Tinus RW, Wallner SJ, King RM (1990) Comparison of three cold hardiness tests for conifer seedlings. Tree Physiol 6:351–369

  14. Burr KE, Hawkings CDB, L’Hirondelle S et al (2001) Methods for measuring cold hardiness of conifers. In: Bigras FJ, Colombo SJ (eds) Conifer cold hardiness. Kluwer Academic, Dordrecht, pp 369–401

  15. Calkins JB, Swanson BT (1990) The distinction between living and dead plant tissue—viability tests in cold hardiness research. Cryobiol 27:194–211

  16. Climent J, Costa e Silva F, Chambel MR et al (2009) Freezing injury in primary and secondary needles of Mediterranean pine species of contrasting ecological niches. Ann For Sci 66:407–407. doi:10.1051/forest/2009016

  17. Colombo SJ (1997) Frost hardening spruce container stock for overwintering in Ontario. New For 13:449–467

  18. Corcuera L, Gil-Pelegrin E, Notivol E (2010) Phenotypic plasticity in Pinus pinaster δ13C: environment modulates genetic variation. Ann For Sci, p67

  19. Corcuera L, Gil-Pelegrin E, Notivol E (2011) Intraspecific variation in Pinus pinaster PSII photochemical efficiency in response to winter stress and freezing temperatures. PloS One 6(12):e28772. doi:10.1371/journal.pone.0028772

  20. Correia I, Almeida MH, Aguiar A et al (2008) Variations in growth, survival and carbon isotope composition (delta(13)C) among Pinus pinaster populations of different geographic origins. Tree Physiol 28:1545–1552

  21. Darychuk N, Hawkins BJ, Stoehr M (2012) Trade-offs between growth and cold and drought hardiness in submaritime Douglas-fir. Can J For Res 42:1530–1541. doi:10.1139/x2012-092

  22. Deans JD, Billington HL, Harvey FJ (1995) Assessment of frost damage to leafless stem tissues of Quercus petraea: a reappraisal of the method of relative conductivity. Forestry 68:25–34

  23. Díaz R, Johnsen O, Fernández-López J (2009) Variation in spring and autumn freezing resistance among and within Spanish wild populations of Castanea sativa. Ann For Sci 66:708–720

  24. Dutkowski GW, Silva JC, Gilmour AR, Lopez GA (2002) Spatial analysis methods for forest genetic trials. Can J For Res 32:2201–2214. doi:10.1139/x02-111

  25. EUFORGEN (2009) Distribution map of maritime pine (Pinus pinaster). Accessed 1 Oct 2013

  26. European Commission (2005) 2005/853/EC: Commission decision of 30 November 2005 authorising France to prohibit the marketing to the end user, with a view to seeding or planting in certain regions of France, of reproductive material of Pinus pinaster Ait. of Iberian Peninsula origin. 14–16

  27. Gaspar MJ, Alves A, Louzada JL, Morais J, Santos A, Fernandes C, Almeida MH et al (2011) Genetic variation of chemical and mechanical traits of maritime pine (Pinus pinaster Aiton). Correlations with wood density components. Ann For Sci 68(2):255–265. doi:10.1007/s13595-011-0034-x

  28. Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92

  29. Grivet D, Sebastiani F, Alía R et al (2011) Molecular footprints of local adaptation in two Mediterranean conifers. Mol Biol Evol 28:101–116. doi:10.1093/molbev/msq190

  30. Guardia M, Díaz R, Savé R, Aleta N (2013) Autumn Frost resistance on several walnut species: methods comparison and impact of leaf fall. For Sci 1–7

  31. Hannerz M, Aitken SN, King JN, Budge S (1999) Effects of genetic selection for growth on frost hardiness in western hemlock. Can J For Res 29:509–516. doi:10.1139/x99-019

  32. Illy G (1966) Recherches sur l’amélioration génétique du Pin maritime. Ann Sci For 23:765–948. doi:10.1051/forest/19660401

  33. Institute Inc SAS (2004) SAS® 9.1 SQL Procedure user’s guide. SAS Institute Inc, Cary

  34. IPCC (2007) Summary for policymakers. In: Climate change 2007: The physical science basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. (Solomon, S., D. Qin, M. Manning, Z. Chen, M. Marquis, K.Bk Av

  35. Jensen JS, Deans JD (2004) Late autumn frost resistance of twelve north European provenances of Quercus species. Scand J For Res 19:390–399

  36. Jonsson A, Eriksson G, Franzen A (1986) Within-population variation in frost damage in Pinus contorta Dougl. seedlings after simulated autumn or late-winter conditions. Silvae Genet 35:96–102

  37. Kalberer SR, Wisniewski M, Arora R (2006) Deacclimation and reacclimation of cold-hardy plants: current understanding and emerging concepts. Plant Sci 171:3–16. doi:10.1016/j.plantsci.2006.02.013

  38. Kremer A, Vincenti B, Alia R et al (2011) Forest ecosystem genomics and adaptation: EVOLTREE conference report. Tree Genet Genom 7:869–875

  39. L’Hirondelle SJ, Simpson DG, Binder WD (2006) Overwinter storability of conifer planting stock: operational testing of fall frost hardiness. New For 32:307–321

  40. Lamy J-B, Bouffier L, Burlett R, Plomion C, Cochard H, Delzon S (2011) Uniform selection as a primary force reducing population genetic differentiation of cavitation resistance across a species range. PloS One 6(8):e23476. doi:10.1371/journal.pone.0023476

  41. Lamy J-B, Delzon S, Bouche PS et al (2013) Limited genetic variability and phenotypic plasticity detected for cavitation resistance in a Mediterranean pine. New Phytol. doi:10.1111/nph.12556

  42. Le Tacon F, Bonneau M, Gelpe J et al (1994) Le dépérissement du pin maritime dans les landes de gascogne à la suite des introductions de graines d’origine ibérique et des grands froids des années 1962–1963 et 1985. Rev For Fr 46:474–484

  43. Lindgren K, Hällgren JE (1993) Cold acclimation of Pinus contorta and Pinus sylvestris assessed by chlorophyll fluorescence. Tree Physiol 13:97–106

  44. Lindner M, Maroschek M, Netherer S et al (2010) Climate change impacts, adaptive capacity, and vulnerability of European forest ecosystems. For Ecol Manag 259:698–709. doi:10.1016/j.foreco.2009.09.023

  45. Littell RC, Milliken GA, Stroup WW, Wolfinger RD, Schabenberger O (2006) SAS for Mixed models, Secondth edn. SAS Institute Inc, Cary, p 840

  46. Luoranen J, Repo T, Lappi J (2004) Assessment of the frost hardiness of shoots of silver birch (Betula pendula) seedlings with and without controlled exposure to freezing. Can J For Res 34:1108–1118

  47. McKay HM (1994) Frost hardiness and cold-storage tolerance of the root system of Picea sitchensis, Pseudotsuga menziesii, Larix kaempferi and Pinus sylvestris bare-root seedlings. Scand J For Res 9:203–213

  48. Morgenstern E (1996) Geographic variation in forest trees: genetic basis and application of knowledge in silviculture. UBC, Vancouver, 209 p

  49. Neale DB, Kremer A (2011) Forest tree genomics: growing resources and applications. Nat Rev Genet 12(2):111–122. doi:10.1038/nrg2931

  50. Nilsson JE (2001) Seasonal changes in phenological traits and cold hardiness of F1-populations from plus-trees of Pinus sylvestris and Pinus contorta of various geographical origins. Scand J For Res 16:7–20. doi:10.1080/028275801300004361

  51. O’Neill GA, Adams WT, Aitken SN (2001) Quantitative genetics of spring and fall cold hardiness in seedlings from two Oregon populations of coastal Douglas-fir. For Ecol Manag 149:305–318. doi:10.1016/S0378-1127(00)00564-8

  52. Peguero-Pina JJ, Morales F, Gil-Pelegrín E (2008) Frost damage in Pinus sylvestris L. stems assessed by chlorophyll fluorescence in cortical bark chlorenchyma. Ann For Sci 65:813, p1–p6

  53. Perks MP, Monaghan S, O’Reilly C et al (2001) Chlorophyll fluorescence characteristics, performance and survival of freshly lifted and cold stored Douglas fir seedlings. Ann For Sci 58:225–236

  54. Perks MP, Osborne BA, Mitchell DT (2004) Rapid predictions of cold tolerance in Douglas-fir seedlings using chlorophyll fluorescence after freezing. New For 28:49–62

  55. Persson T, Andersson B, Ericsson T (2010) Relationship between autumn cold hardiness and field performance in northern Pinus sylvestris. Silva Fenn 44(2):255–266

  56. Repo T, Mononen K, Alvila L, Pakkanen TT, Hänninen H (2008) Cold acclimation of pedunculate oak (Quercus robur L.) at its northernmost distribution range. Environ Exp Bot 63:59–70

  57. Royo A, Fernandez M, Gil L, Pardos JA (2003) Assessing the hardiness of Aleppo pine, maritime pine, and holm oak seedlings by electrolyte leakage and water potential methods. Tree Planters’ Notes 50:38–43

  58. Ryyppö A, Repo T, Vapaavuori E (1998) Development of freezing tolerance in roots and shoots of Scots pine seedlings at nonfreezing temperatures. Can J For Res 28:557–565

  59. Sakai A, Larcher R (1987) Frost survival of plants—responses and adaptation to freezing stress. Springer, Berlin, 321 pp

  60. Sakai A, Weiser CJ (1973) Freezing resistance of trees in North America with reference to tree regions. Ecol 54:118–126

  61. Salmela MJ, Cavers S, Cottrell JE et al (2011) Seasonal patterns of photochemical capacity and spring phenology reveal genetic differentiation among native Scots pine (Pinus sylvestris L.) populations in Scotland. For Ecol Manag 262:1020–1029. doi:10.1016/j.foreco.2011.05.037

  62. Shortt RL, Hawkings BJ, Woods JH (1996) Inbreeding effects on the spring frost hardiness of coastal Douglas-fir. Can J For Res 26:1049–1054

  63. Sutinen ML, Palta JP, Reich PB (1992) Seasonal differences in freezing stress resistance of needles of Pinus nigra and Pinus resinosa: evaluation of the electrolyte leakage method. Tree Physiol 11:241–254

  64. Visscher PM (1998) On the sampling variance of intraclass correlations and genetic correlations. Genetics 149:1605–1614

  65. Weng YH, Parker WH (2008) Adaptive variation in fall cold hardiness of aspen from northwestern Ontario. Tree Physiol 28:143–150

  66. Wilson AJ (2008) Why h2 does not always equal V A/V P? J Evol Biol 21(3):647–650. doi:10.1111/j.1420-9101.2008.01500.x

  67. Wright S (1951) The genetical structure of populations. Ann Eugenics 15(4):323–354

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This research was developed as part of the project 09MDS019502PR from the Galician government. The field trial was installed as part of project TREESNIPS (QLK3-CT2002-01973) and currently is part of the Spanish Network of Genetic Trials (GENFORED). We thank all persons and institutions linked to the establishment of the field trials and to the maintenance of the network. We thank the C.I.F. Lourizán staff, and also Antonio Soliño and the students Rebeca Rodas, Marta Paraños and Carmen Muiña for field and laboratory assistance.

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Correspondence to Eva Prada.

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Communicated by S. González-Martínez

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Prada, E., Alía, R., Climent, J. et al. Seasonal cold hardiness in maritime pine assessed by different methods. Tree Genetics & Genomes 10, 689–701 (2014).

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  • Cold hardiness
  • Maritime pine
  • Visual scoring
  • Relative conductivity
  • Fluorometry