Abstract: Genetic variation in fall cold damage in coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. menziesii) was measured by exposing excised branches of seedlings from 666 source locations grown in a common garden to freezing temperatures in a programmable freezer. Considerable variation was found among populations in fall cold hardiness of stems, needles, and buds compared with bud burst, bud set, and biomass growth after 2 years. Variation in fall cold hardiness was strongly correlated (r = 0.67) with cold-season temperatures of the source environment. Large population differences corresponding with environmental gradients are evidence that natural selection has been important in determining genetic variation in fall cold hardiness, much more so than in traits of bud burst (a surrogate for spring cold hardiness), bud set, and growth. Seed movement guidelines and breeding zones may be more restrictive when considering genetic variation in fall cold hardiness compared with growth, phenology, or spring cold hardiness. A regional stratification system based on ecoregions with latitudinal and elevational divisions, and roughly corresponding with breeding zones used in Oregon and Washington, appeared to be adequate for minimizing population differences within regions for growth and phenology, but perhaps not fall cold hardiness. Although cold hardiness varied among populations, within-population and within-region variation is sufficiently large that responses to natural or artificial selection may be readily achieved.
Key words: cold hardiness, genetic variation, adaptation, Psuedotsuga menziesii.
Resume : Les auteurs ont mesure la variation genetique des dommages par le froid automnal, chez le sapin Douglas (Pseudotsuga menziesii (Mirb.) Franco var. menziesii), en exposant les rameaux excises de plantules provenant de 666 localite s sources, cultivees dans un jardin commun, a des temperatures de congelation dans un congelateur programmable. On a trouve une variation considerable au sein de la population quant a la resistance au froid automnal chez les tiges, les aiguilles et les bourgeons, comparativement a l'ouverture des bourgeons et la croissance de la biomasse apres deux ans. La variation de la resistance au froid automnal est fortement correlee (r = 0,67) avec les temperatures de la saison froide de l'environnement source. Les grandes differences observees dans les populations correspondant aux gradients environnementaux, sont des preuves que la selection naturelle a joue un role important dans la determination de la variation genetique de la resistance au froid automnal, beaucoup plus que pour les caracteres de l'ouverture des bourgeons (l'equivalant de la resistance au froid printanier), la formation des bourgeons, et la croissance. Les prescriptions pour le mouvement des graines et les zones de croisement pourraient etre plus restrictives, lorsqu'on considere la variation de la resistance au froid automnal comparativement a la croissance, la phenologie, ou la resistance au froid printanier. Un systeme de stratification regional, base sur des ecoregions avec des divisions latitudinales et altitudinales correspondant grossierement aux zones de croisement utilisees en Oregon et Washington, semble adequat pour minimiser les differences dans les regions, quant a la croissance et la phenologie mais possiblement pas pour la resistance au froid automnal. Bien que la resistance au froid varie entre les populations, dans les populations et dans les regions, la variation est suffisamment importante pour que les reactions a la selection naturelle ou artificielle se realisent rapidement.
Mots cles : resistance au froid, variation genetique, adaptation, Pseudotsuga menziesii.
[Traduit par la Redaction]
Introduction
Susceptibility to damage from cold is important to the adaptation of Douglas-fir (Psuedotsuga menziesii (Mirb.) Franco), particularly because of the highly variable environments within its range, both spatially and temporally. Cold hardiness varies among populations of Douglas-fir, and much of the variation is clinally related to gradients in temperature and moisture (Campbell and Sorensen 1973; Rehfeldt 1979, 1986; White 1987; Loopstra and Adams 1989). Large population differences and a consistent, strong association of a trait with environments provide indirect evidence that a trait may be adaptive and that natural selection was important in shaping variation (Endler 1986). Cold hardiness traits also vary considerably within populations, and this variation may be subject to natural or artificial selection (e.g., tree improvement programs). Bud burst and spring cold hardiness in coastal Douglas-fir (var. menziesii) are under strong genetic control and highly genetically correlated (Aitken and Adams 1997; O'Neill et al. 2000, 2001). Heritabilities for bud set and cold hardiness in the fall and winter are low to moderate (Aitken et al. 1996; Aitken and Adams 1997; O'Neill et al. 2000, 2001). Correlations between bud set and fall cold hardiness vary among studies, but are often weak (Aitken et al. 1996; O'Neill et al. 2000). Over a large geographic and climatic scale, tradeoffs exist between cold hardiness and growth, although at smaller scales, the correlations are weaker and less consistent (Howe et al. 2003); a strong correlation was found between fall cold injury and height (r = 0.70) in seedlings among interior Douglas-fir (var. glauca) populations in northern Idaho (Rehfeldt 1979), whereas most genetic and family mean correlations were near zero for fall cold injury and height in coastal Douglas-fir saplings in two Washington breeding zones (Aitken et al. 1996). Stevenson et al. (1999) found that genetically improved Douglas-fir seedlings were less cold hardy than unimproved seedlings grown at two sites in British Columbia.
Previous studies of genetic variation in cold hardiness in coastal Douglas-fir have included only a few populations or breeding zones, or have been of limited geographic range. Thus, conclusions about the structure and patterns of genetic variation are incomplete. In this study, I report on genetic variation in fall cold hardiness using artificial freeze testing on a large number of families distributed across much of the range of coastal Douglas-fir in western Oregon and Washington. The objective is to explore structure and patterns of genetic variation in fall cold hardiness by considering the relative magnitudes of among- and within-population variation and the relationship of population variation to environmental variation. In so doing, I hope to evaluate the importance of cold hardiness in determining adaptation of Douglas-fir populations to their local environments. I also considered the effect of geographic scale to conclusions about the magnitudes of variation and relationships to environment by dividing the study area into regions. This also allowed comparisons among regions in means, family variances, and correlations, and facilitated comparisons with other studies done at the scale of breeding zones.
Materials and methods
Common garden test
Samples for this study are part of a larger study of the genecology of coastal Douglas-fir that focuses on geographic variation in traits of emergence, bud phenology, growth, and partitioning as measured on seedlings grown in a common garden (St. Clair et al. 2005). The present study reports on genetic variation in fall cold hardiness as measured by artificial freeze tests on approximately two-thirds of the seedlings from the earlier study. To allow comparisons of fall cold hardiness to other traits, bud phenology and seedling biomass are reanalyzed using the same subset of seedlings. Spring cold damage was not measured, since only two branches per seedling could be destructively sampled, and because bud burst appears to be a surrogate for spring cold damage based on findings of high genetic correlations in previous studies (Aitken and Adams 1997; O'Neill et al. 2000). Bud set, however, does not appear to be strongly correlated to fall cold hardiness (Aitken et al. 1996; Aitken and Adams 1997; O'Neill et al. 2000, 2001).
The sampling design for parents from native stands and common garden procedures are described by St. Clair et al. (2005). In brief, wind-pollinated seeds were collected from parent trees in naturally regenerated stands throughout the range of Douglas-fir in western Oregon and Washington. Progeny from the parents were grown for 2 years in raised nursery beds in Corvallis, Oregon. To evaluate a large number of parent trees, tests were established in 3 successive years (1994-1996) using different sets of families, but with a common set of families included in all 3 years to allow for adjustment of year effects (see White and Hodges 1989). Each year families were randomly assigned to five-tree row plots (of which four trees were used for cold hardiness testing) in each of four raised beds with each bed treated as a block. A total of 792 families from 666 source locations (i.e., populations) were evaluated for cold hardiness over the 3 establishment years, with 10 families measured in all 3 years.
Cold hardiness testing
Branch samples were taken for cold hardiness testing in the fall after the second growing season, and subjected to artificial freeze tests following methods described by Anekonda et al. (2000). The method involved removing 4-6 cm long shoot tips from two lateral branches of each seedling, with each branch labeled to ensure identity. Branches from 50 seedlings were wrapped in a packet of moist cheesecloth and aluminum foil with each of the two branches of a seedling in a separate packet. Branches were frozen in a programmable freezer at two different test temperatures chosen to give between 30% and 70% damage (one temperature for each branch sample from a seedling). Packets were placed in the freezer at -2 [degrees]C for approximately 10 h to allow the samples to equilibrate and to freeze extracellular water. The temperature was then lowered 3 [degrees]C per h to the target test temperature and maintained at that temperature for 1 h. After treatment, packets were removed from the freezer and put in a 4 [degrees]C refrigerator overnight to allow them to slowly thaw. The packets …
