managing genetic diversity in a tree improvement program

Forest Ecology and Management, 35 ( 1990 ) 131-149 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

131

Managing Genetic Diversity in a Tree Improvement Program

JAY. H. KITZMILLER USDA Forest Service, Pacific Southwest Region Tree Improvement Center, 2741 Cramer Lane, Chico,

CA 95928 (U.S.A.)

ABSTRACT

Kitzmiller, J.H., 1990. Managing genetic diversity in a tree improvement program. For. Ecol. Man- age., 35: 131-149.

Maintenance of species and within-species diversity is a goal of the USDA Forest Service, Pacific Southwest Region, which is examining and monitoring the genetic effects of silvicultural activities using isozyme analysis in conjunction with traditional methods. An effective monitoring and evaluation program will help improve silvicultural practices. Regional policies on gene conservation focus mainly on artificial regeneration. Tree improvement, nursery, and reforestation activities in the PSW Region may significantly affect adaptability, genetic diversity, and economic timber traits. The Base- Level Tree Improvement Program (TIP) aims for: ( 1 ) high adaptability via use of native species and local wild seed; (2) high within-species genetic diversity via designs for sampling several local seed- stands and via long-term plans for improvement using simple recurrent selection and bulking among the seed-production-area plantations; and (3) moderate improvement in performance via mild selection for superior phenotypes. The ceiling on genetic diversity is established by the seed collected. Nursery and reforestation practices may result in some loss of original (seed collection) diversity. Nursery practices aim to maximize plantable seedlings per unit of seed and thereby preserve diversity through sound management. Reforestation practices aim to achieve high local diversity by mixing appropriate species at planting, allowing natural regeneration to established in plantations, encouraging high survival and using higher planting densities so that natural selection can operate effectively. The High-Level TIP involving selective breeding and orchards will supplant the wild collections for major species and productive sites. Orchard seed will have a base of 50-60 unrelated, interbreeding parents, each representing a separate stand. Cooperative programs will enhance the genetic base by providing a broad range of material for breeding. New genes may be infused into breeding populations from material selected in natural forest reserves to increase diversity. Production plantations will be genetically more diverse than naturally regenerated stands.

INTRODUCTION

Silvicultural practices have great potential to alter the genetic composition of natural forests. Species and genotypic mixtures are strongly controlled through removal of mature or undesirable trees, followed by renewal with similar or different genotypes. Regeneration by planting is more likely to re-

132 J.H. KITZMILLER

suit in genetic changes than is natural regeneration; therefore, it is appropriate to scrutinize and monitor the genetic effects of tree improvement, nursery, and reforestation practices on plantations relative to natural stands.

A silvicultural goal of the USDA Forest Service, Pacific Southwest Region (PSW), which includes California, Hawaii, and U.S. territories in the Pacific, is to improve economically important timber traits such as growth rate, form, wood quality, and pest resistance, while maintaining high genetic diversity and adaptability of the populations. Silviculturists are trained to address the effects of management activities on diversity, adaptability, and economic timber traits in silvicultural prescriptions. Silvicultural activities with the greatest potential effects include: tree breeding; delimitation of seed collection zones and seed transfer; selection of seed trees for natural or artificial regeneration; seed and seedling handling; and plantation establishment.

This paper reviews artificial-regeneration practices of the Pacific South- west Region and their potential effects on genetic diversity. Emphasis is on changes in diversity due to management practices. Diversity is important, since it is the foundation for genetic change (improvement) and the primary factor allowing populations to adapt to environmental change. Diversity ex- ists among and within species. Maintenance of native species on reforested sites is a regional goal for species diversity. Genetic differences among individuals (genotypic diversity) and within individuals (allelic diversity), also important for management, are the focus of this paper. The tree improvement, nursery, and reforestation programs of the PSW Region are discussed below in terms of how they may affect genetic diversity.

BASE-LEVEL TREE IMPROVEMENT PROGRAM

General description

The Pacific Southwest Region currently uses a simple but effective low- intensity program of tree selection and seed collection for reforestation. This Base-Level Program is designed to meet all needs for improved seed for the next 10 years. It will meet long-term needs for those species and areas not included in the High-Level (seed orchard) Program (Kitzmiller, 1976 ).

The Base-Level Program is a practical approach to gene conservation. It is incorporated into continuing reforestation and plantation-management activities. California seed-zones were established in 1970 (Fig. 1 ), and only native species and local sources or those from adjacent zones are used, so stock is expected to be climatically adapted to the planting site.

Key elements of the Base-Level Program are: ( 1 ) native species and local seed (zone of origin) are collected; (2) several stands and trees are sampled to provide broad genetic diversity; (3) mild selection is applied for desirable stand and tree characteristics; and (4) the resulting plantations will be con-

MANAGING GENETIC DIVERSITY IN A TREE IMPROVEMENT PROGRAM 13 3

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vetted to seed production areas (SPA's), with subsequent bulking of seed among SPA's.

Seed-collection guidelines

A special sampling design is used to collect seed for reforestation in each seed-zone. The objective is to collect a balanced, representative, and broad genetic sample from these stands and trees apparently favored by natural se-

134 J.H. KITZMILLER

lection. Environmental differences among planting sites within a zone may require different genotypic mixtures for optimum performance. Since these mixtures are not predictable for every site, planting a broad genetic sample provides latitude for nature to select the best mix. The seed-collection guidelines are:

( 1 ) Seed should come from a minimum of 20 stands in each seed-zone. This sample size is adequate to capture most, if not all, of the allelic diversity among stands in a zone. A seed stand is an area whose boundary is delineated by the location of one or more seed trees which are within 200 yards ( ~ 180 m) of each other, and which are separated from the nearest seed-trees of another seed-stand by at least 200 yards.

(2) Stands are separated by a minimum of 200 yards and, preferably, are well-distributed throughout the seed-zone to correspond with planned regenerated units. This minimum distance increases the probability that parents from different stands will be unrelated.

(3) Stands contribute nearly equally to the bulked seedlot; no stand may contribute more than 10% of the seed in a lot.

(4) The number of trees within each stand is nearly equal and varies with overall seed needs.

(5) The amount of seed per tree is similar, to achieve a balanced genetic base.

(6) Cone collection is concentrated in the mid- to upper-crown to minimize inbreeding and improv e seed quality.

(7) Cone and seed insects and seed ripeness are monitored, and only mature cones that are relatively free of insects are collected.

(8) Cone collection is restricted to moderate to heavy crop-years, when the size of the breeding population is large, genetic diversity and seed quality is high, and collection costs are relatively low.

(9) Cone collection in different years will usually sample different stands and trees.

(10) Records are maintained on seed origin and genetic base. Seed inven- tory and procurement plans are administered via the computer program SIPPLAN.

Together, these guidelines are designed to provide high-quality seed from a diverse genetic sample. Once seed is collected, the maximum potential genotypic diversity for that seedlot is fixed. Subsequent nursery and reforestation activities reduce genotypic diversity through selective mortality.

Seed bulking and the genetic base

At the Placerville Nursery, cones or processed seeds are bulked across tree and stand collections within each seed-zone and 500-ft (152-m) elevation band. The size of seedlots varies by seed needs for a zone. Given the number

MANAGING GENETIC DIVERSITY IN A TREE IMPROVEMENT PROGRAM 135

of seed-trees required for collection, preference is placed on dispersing them over many stands rather than concentrating them in a smaller area of few stands. Thus, small lots may include only one tree from each of 20 stands. Large lots may include 10 or more trees from 20-30 stands. The number of trees sampled commonly ranges from 20 to 300, sometimes more. The amount of seed collected per tree depends on species, crop size, and method of collection. The range is commonly from 0.5 to 5 lbs (0.22-2.2 kg) per tree.

Each bulked seedlot is coded to identify species, seed-zone and elevation, Forest, District, type of collection, genetic base, and collection year; e.g., respectively 122-741.65-05-57-4-2001-84. Genetic base (bold type) consists of 2 digits for number of stands and 2 digits for average number of trees within stands. This information is kept on computer to track seedlots from nursery through plantation.

When a seedlot is composed of less than 20 stands, an attempt is made to combine it with another lot from the same zone and of similar quality and age. This enhances genetic diversity and reduces the number of lots to be managed. Although it contributes a small amount of variation, the among- stand component may be important to sample. Sampling at the stand level may capture locally common, but restricted, alleles or multi-allelic combina- tions that may reflect adaptation to specific stand conditions.

In 1988, a small preliminary study compared the isozyme diversity of ponderosa pine (Pinus ponderosa Dougl.) seedlots derived from varying the numbers of stands and trees contributing to the bulked lots (Table 1 ). All five lots were collected from mid-elevation stands in the same seed-zone in comparable seed-crop-years. Allelic diversity is similar among the four lots,

TABLE 1

The genetic base and isozyme diversi ty in ponderosa-pine seedlots f rom nor thwes tern Califor- nia, mid-e levat ion Zone 312 (23 loci used)

Genet ic base Measures of allelic diversi ty

Lot No. No. of trees No. of alleles PL 2 H Z 3 G D 4

no. s tands (%) Stand Total Total Locus I

1 83 5 415 48 2.1 65 0.279 - - 2 27 2 54 49 2.2 65 0.278 0.002 3 21 1 21 52 2.3 74 0.303 0.002 4 17 1 17 51 2.2 70 0.294 0.022 5 6 2 12 44 1.9 56 0.198 0.039

IAverage numbers. 2Polymorphological loci. 3Heterozygosity (average). 4Genetic distance ( > Lot 1 ).

136 J.H. KITZMILLER

which range from 17 to 83 stands. However, Lot 5 with only six stands but nearly as many total trees as Lot 4 ( 12 vs. 17) had less allelic diversity than the other lots (fewer alleles, more fixation, and less expected heterozygosity).

Allelic frequency difference, as measured by genetic distances (from Lot 1 ), was more sensitive to stand sampling and showed a slightly different pat- tern (Table 1 ). Lots 1, 2, and 3, which all had greater than 20 stands, were similar, but these differed from Lot 4 with 17 stands, which in turn differed from Lot 5 with six stands. These data suggest that a 20-stand sample provides an adequate representation of alleles and allelic frequencies within a collection zone, and perhaps even a 17-stand sample is adequate based on allelic-diversity measures other than genetic distance. However, since only 20 seeds were analyzed per lot, the diversity of the larger lots may have been limited by sample size. Further study is underway using other sources, sample sizes, and improved techniques to confirm these results.

At present, records indicate that many seedlots in the bank do not com- pletely meet the target guidelines. The number of stands in many lots are fewer than could be sampled in good crop-years. Part of the problem is the higher cost of collecting from several different stands. Another part is due to lack of access to the total areas within a collection zone. Most zones have a limited road network. Only a fraction of the zone may actually be accessible for cone collection, and part of that will not produce cones of the needed species. On the other hand, it is likely that the genetic base is larger than the records indicate. Foresters have often confused the definition of a seed stand to mean a much larger area of forest. Thus, they have often underestimated the number of unrelated groups of trees (stands).

A new method of cone collection in California, aerial cone harvest (ACH) using the Fandrich cone rake attached to a helicopter, greatly enhances genetic diversity of seedlots. ACH was used to collect a bumper crop of white fir (Abies concolor) in 1984 ( 1089 bushels I ) and a bumper crop of red fir (A. magnifica) in 1986 (1526 bushels) in the central Sierra Nevada. A major advantage of ACH is unrestricted access to large interior stands, which greatly expands the seed-tree base. ACH enables collection from tree-tops where the pollen cloud may be more diverse, and also from ripe cones having better seed quality. Comparing ACH with harvest by climbing for red fir, ACH collected from 5 times as many trees as climbing, but fewer cones per tree (ACH=0.7 bu tree-1; c l imb= 3.6 bu tree -1 ). This broadened base is important to true firs, since they normally sustain higher mortality in the nursery and in outplanting than pines and Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) and thus they incur greater risk of losing diversity.

Selection of seed-trees for average or better phenotypic characteristics such as growth rate, crown and stem form, and freedom from pest injury will result

J Bushel ( b u ) ~ 35.2 1 ( 1.24 f t 3 ) .

MANAGING GENETIC DIVERSITY IN A TREE IMPROVEMENT PROGRAM | 37

in some low level of improvement, without narrowing the genetic base significantly for any single trait.

Plantations and future seed production

Base-Level seedlots will have an adequate proportion of adaptive genotypes for all sites in the zone, especially if initial plantation density is kept high (500-600 trees acre-l ; ~ 1250-1500 h a - l ) . Lots are expected to be highly heterozygous, heterogeneous, and buffered to withstand adversities of environmental extremes and pest attacks. Higher planting densities help ensure a uniform distribution of adapted crop trees on marginal sites (Camp- bell, 1987 ).

When plantations from Base-Level seed are silviculturally thinned, some will emerge as potential seed-production areas (SPA's). These will then be heavily thinned to promote flowering and interbreeding among the best trees. Much of the seed will come from interpollination among selected trees. How- ever, contamination from surrounding stands cannot be avoided. This will broaden the genetic base but dilute any effects of selection. Selection intensity can be kept relatively high (retaining the best 25% ) by heavy thinning. And when combined with collection from several such SPA's to form a new bulked seedlot, diversity will be maintained. A significant increment of improvement (about 5-7%) in growth due to 'forced outcrossing' may be obtained automatically, to the extent that the interbreeding trees are from unrelated parentages (Friedman, 1988). Such improvement stems from reduced inbreeding relative to natural forests where related trees may occur in proxim- ity, facilitating crosses among relatives.

If the plantation was to be replaced by natural regeneration rather than replanting, the scenario would be similar. A shelterwood or seed-tree cut would leave the best phenotypes to interbreed and reseed the area. In this case, however, seed from other seed-production areas would not be available to enhance diversity. Seed cast from the surrounding stand may introduce some new variation on the site, but, in this case, natural regeneration is likely to produce a new stand with less diversity than plantations from large, bulked seedlots from SPA's, assuming seed and seedling handling operations do not significantly reduce the genetic base and that planting density is adequate to sample genotypes and microsites.

The process of seed collection from SPA's may be repeated for an indefinite number of generations without loss of diversity, if properly managed. The amount of immigrant pollen from natural stands, the number of unrelated interbreeding trees in the SPA's, and the number of SPA's contributing to the bulked seedlot will determine its level of diversity.

Genetic variation should be monitored in each generation using the best available technology (e.g., DNA or isozyme analyses by electrophoresis).

138 J.H. K1TZMILLER

Trees in SPA's can be genotyped to determine, along with performance, which trees to thin and which to interbreed to achieve the desired diversity (Fried- man, 1988).

Adequate natural reserves (e.g., streamside management zones, travel-influence zones, and other set-asides) exist in most areas in California. These genetic reservoirs for natural variation could be sampled when needed.

NURSERY ACTIVITIES

Seed collected and bulked using Base-Level guidelines initially has a broad base. The amount of change in the initial genotypic mixture depends on the amount and nature of seed and seedling losses in the nursery. Random losses will not change the initial genotypic mixture. Some nursery stresses may actually favor heterozygotes and thereby increase individual (or genic) diversity even though the genotypic mixture is changed. However, directional selection (e.g., differential survival of large or small seedlings) reduces diversity (Campbell and Sorensen, 1984 ).

Seed and seedling handling involves several activities that are laborious and costly. Procedures are often standardized to achieve efficiency. Standard treatments may work for most genotypes, but not all, yet using a different procedure for each seedlot and activity could be unmanageable. The benefits of tailoring procedures to each seedlot must be balanced against costs.

Major nursery activities include: cone handling and storage; cone drying; seed extraction and cleaning; seed storage; seed stratification; seed sowing; seedling culture; lifting; sorting; and packing. Their expected effects on diversity and performance are considered below.

Cone handling and storage. No change is predicted if seed is mature and bags are properly filled, stacked, and protected from overheating. Improper handling and storage reduce the genetic base. Red fir and sugar pine (P. lamber- tiana Dougl. ) are the most critical species with regard to time of collection and seed ripeness.

Cone drying. No change is predicted if seed is mature. Immature seed may die, remain immature, or ripen to some degree. Immature seed germinates slowly and poorly and is susceptible to pests, providing potential for loss.

Seed extraction and cleaning. This process removes empty, defective, and weak seed and some good seed, but effects should be minimal since loss of good seed is closely monitored. Seed weight is affected by maturity of seed, year of collection, and environment and genotype of parent tree. Heavy grading may eliminate all seed from some trees and reduce the genetic base (Silen and Osterhaus, 1979), but the absence of grading does not ensure that no change


will occur; e.g., small seed produces small seedlings which may be eliminated by competition or by culling during lifting when grown in mixture with large seedlings. Scots pine (Pinus sylvestris L.) embryos from heavy seeds were found to be more heterozygous than embryos from light seeds, and even con- tained a slight heterozygote excess, whereas light seeds had a homozygote excess (Szmidt, 1987 ). Removal of homozygotes would improve performance and increase diversity.

Seed storage. Proper storage conditions at Placerville (low seed moisture, cold temperatures, and minimum oxygen) maintain viability and reduce muta- tions (Belcher, 1980), but seed with low initial viability stores poorly. Peri- odic germination tests often identify problem lots before much damage occurs in storage. Storage will not negatively affect diversity and performance when initial viability is high, as is usual in hard pines and Douglas fir.

Seed stratification (pre-chilling). The period of stratification affects speed of germination. Dormancy-breaking requirements vary widely by lot. Long (60- 90 days) stratification meets requirements of most seeds and favors early, rapid germination. The PSW Region uses standard or longer stratification periods. The germination tests that use standard stratification methods are used to identify lots needing special treatment. Short stratification promotes variability in germination time, both among and within lots. Since early germinators have a competitive edge in capturing environmental resources, late germinators will be weaker and more likely to damp-off or succumb to heat stress or be culled. Thus, short stratification selects against late germinators and reduces diversity, while long stratification generally maintains a broad base. A relatively long stratification is often best, but not always. For example, if heavier seed is more heterozygous and promotes earlier germination, then short stratification may produce higher heterozygosity levels among sur- vivors. In addition, the potential for damage from molds is higher with long stratification. Also, if seed has been mechanically damaged, a short stratification is beneficial, and some true fir lots germinate best without any prechill.

Sowing date and environment. Genotypes react differently to date of sowing and seedbed environment. Sowing date partly determines germination temperature. Cool temperature may occur with early sowing and cause large variation in date of emergence, especially for seed given a short prechill. Late sowings may risk high mortality from heat. Early sowing at some California nurseries reduces losses in sugar pine and other species due to Fusarium (Jenkinson et al., 1982), but this advantage is countered somewhat by the threat of damage from heavy rains in early spring (B. Scheuner, USDA Forest Service, Placerville Nursery, personal communication, 1989). Long stratification, combined with properly timed sowing date, produces least change in

140 J.H. KITZMILLER

the genotypic mixture of a lot. For most sources of Douglas fir, a 90-day stratification combined with an early March sowing at Humboldt Nursery pro- vided the most uniform, rapid and complete germination (Jenkinson and Nelson, 1986 ). Date of emergence also affects growth rhythm as well as size and shape of seedlings (Campbell and Sorenson, 1984 ). By shifting the sowing-date, managers can favor one lot and discriminate against another due to different climatic requirements. Managers often selectively place species and lots in the nursery on the sowing-date that provides the best germination. High germination maintains diversity.

Seedling culture. Genotypes respond differently to irrigation, fertilization, and spacing in seedbeds. Seedlings that respond best to abundant water and nu- trients are favored. In the nursery, seedlings adapted to survive harsh sites may be less competitive than those adapted to high sites. Thus, plantation performance and diversity could be reduced by nursery culture. However, since nursery stress is low and culling is moderate, the general result is that most seedlings survive to be outplanted. A notable exception is disease outbreaks, which can drastically reduce diversity. Disease resistance is often an inherited trait, so outbreaks selectively reduce the genetic base. Sugar pine and true firs are particularly susceptible to nursery root diseases in California.

Lifting. Root damage could affect outplanting performance dramatically and reduce diversity. Lifting date affects root growth capacity and survival after planting. Genotypes respond differently to lifting date and storage period (Jenkinson, 1984). Managers tailor practices to seedlot requirements when these are known. Even so, some very good trees have growing root-tips when lifting occurs, and these get damaged. Potential for genetic change is high when seedlot requirements are unknown.

Sorting. Culling the smallest 20-30% of seedlings that would not be expected to survive outplanting is a necessary reduction in genotypic diversity which mimics natural selection. Many 'runts' are inbred seedlings from related mat- ings or from small or immature seed or from late germinants. Some may have been well-adapted to the planting site if they had arrived via natural regeneration. Double-sorting - i.e. separating stock into small and large groups, usually with the small stock planted on 'easy' sites and the large for harsh sites - reduces the genotypic base of stock on a given site. Even if the small stock is transplanted as 1-1 (one year in nursery and one year in transplant bed) the result is a reduction in diversity unless the transplants are deliberately mixed with other lots to broaden the genotypic base. Hold-over stock - initially grown for 1-0 but later changed for use as 2-0 - results in a reduction in the genotypic base because the largest 1-0 stock will grow too large as 2-0 and be culled. Since stock that is too small or too large (for ease of planting) is culled


in normal operations, sorting will reduce the genetic base but may have little effect on mean size of a seedlot.

Packing and cold storage of seedlings. Lengthy storage, inadequate precondi- tioning, and above-freezing temperature can have strong effects on carbohy- drate levels and subsequent outplanting performance. Genotypes may react differently to cold-storage conditions, including the absence of light.

Any nursery practice that tends to produce seedlings that cannot survive after outplanting reduces genotypic diversity. Lifting- and sowing-dates are prime examples. Nursery environment also influences the growth rhythm of seedlings. If the nursery environment is greatly different from the outplanting environment, then the genotypic mixture will change from the nursery to the plantation, resulting in a considerable reduction in diversity. This is because selection has occurred at the nursery and at the plantation for different types of trees; trees lost in the nursery may have been adapted to the plantation site.

Nursery practices that maximize tree percent (percentage of seeds sown that develop to plantable trees) and seedling survival after outplanting will minimize changes in the genotypic mixture of a seedlot and preserve the maximum potential genetic diversity. Nurseries and species with large nursery factors (high seedling losses) undergo the greatest genetic change. In partic- ular, red fir and some small-seeded species, e.g. giant sequoia (Sequoiaden- dron giganteum (Lindl.) Buchholz) and Port Orford cedar (a.k.a. Lawson cypress, Chamaecyparis lawsoniana (Murr.) Parlatore ), suffer great losses in the nursery. Also, sugar pine and white fir require close attention. Normally, 40-50% of the viable seed genotypes are lost in the nursery for ponderosa and Jeffrey pines (Pinusjeffreyi Murr. ) and Douglas fir, 50-60% for white fir and sugar pine, while 60-75% of the viable seed of 'problem' species are lost (B. Scheuner, personal communication, 1989). Nursery activities should aim to maximize the proportion of seeds that become healthy, plantable seedlings. The better the nursery management, the less is the effect on the genetic resource (Campbell and Sorensen, 1984). When seedlot requirements are known, genetic change can usually be managed.

PLANTATION ESTABLISHMENT

Many nursery influences can continue to affect field survival and growth for a few years, but field operations also influence planting success. Shipping and storage of stock prior to planting may cause failure if stock is exposed to high temperatures or desiccating conditions. Length of storage may affect growth for several years and contribute to reduced survival. Poor handling of planting stock, such as long root-exposure time, will decrease diversity by dis-

142 J.H. KITZMILLER

criminating against small and large seedlings, which may reflect family differences.

When germination is low or losses from disease are high in the nursery, then diversity will be decreased, adaptation to the field-planting site will be reduced, and plantation survival will decrease. Plantations that fail (i.e., have less than 30% survival) are replanted. Natural selection, combined with selection due to nursery practices, can cause significant changes in the genotypic mixture and reduce variation when survival is very low.

Regional averages for third-year plantation survival from 1982-88 were: ponderosa pine, 70%; Jeffrey pine, 63%; sugar pine, 53%; Douglas fir, 57%; and white fir and red fir, each 40%. Area planted averaged about 33 000 acres (13 355 ha) annually. The proportion planted, by species, was: ponderosa pine, 40%; Jeffrey pine, 15%; sugar pine, 3%; Douglas fir, 30%; white fir, 7%; red fir, 3%; and other species, 2%. These data indicate that, on average, 15%- 35% of the viable seed sown in the nursery become established trees in plantations.

Additional effort is required to broaden the genetic base of those species, such as true firs, that have nursery and plantation survival problems. This effort begins with collection of seed, continues through the nursery, and cul- minates in plantation culture. Foresters often enhance the base by judicious mixing of species or seedlots at the planting site. For example, they favor mesic species in drainages and moist micro-sites, and more-xeric species on more exposed sites with gravelly soils. They also encourage the immigration of natural regeneration into plantations, which has a positive effect on genetic diversity and adaptation. When the optimum mix of species and genotypes for a site is not known, foresters often plant more trees (genotypes) per unit area. The higher densities provide more diversity on which natural selection can act.

HIGH-LEVEL TREE I M P R O V E M E N T P R O G R A M

General description

The High-Level, or intensive, seed-orchard program, is designed to increase the quantity and quality of timber on a sustained basis in the PSW Region (KJtzmiller, 1976). It involves four components: (1) superior tree selection in wild stands; (2) evaluation of selections in genetic test plantations (progeny tests); (3) production seed orchards; and (4) long-term breeding populations in clone banks.

Selection and propagation of superior trees in natural stands preserves the best timber trees that nature has produced. Wind-pollinated seeds from the wild-stand parent trees are used to establish genetic test plantations located


in forest environments near their origin. These plantations are well-protected and maintained for one rotation.

Meanwhile, scions from the superior trees are grafted and established in clone banks and production seed orchards. The orchards will be rogued to leave only the best clones, based on progeny performance and seed production. The clone banks will maintain all the initial selections for use in pedigree breeding. The program will eventually involve seven major species, with 4-9 breeding zones per species. Between 1000 and 2000 superior trees will be selected and propagated per species. Current work includes four species and about 4000 superior selections.

In many of the breeding zones, the USDA Forest Service has formed co- operatives with state and private sectors. Normally, the Forest Service will select 200 trees and other cooperators select 100 trees in each zone. These 300 trees are then grafted into cooperative clone-banks located in National For- ests. Seed collected for genetic tests are grown in a nursery, and the seedlings are planted out on each cooperator's land. These cooperative efforts are ex- cellent for gene conservation because they save a large sample of genes and genotypes from land bases outside the National Forests for the clone-bank breeding projects.

Grafts of superior germplasm are organized into three clone-banks and three seed-orchards, totalling about 60 and 600 acres (24 and 243 ha), respectively. About 70 genetic tests on 700 acres (283 ha) are distributed throughout Cal- ifornia. Together these ex-situ plantations preserve a broad, representative, genetic base for future use.

Breeding strategy

Long-term breeding work in clone banks is the key to successful management of the genetic resources included in the High-Level program. Clone banks are physically and functionally separate from production orchards. The great uncertainty in predicting future forest environments and products requires a highly flexible gene pool, maintained through breeding. Inbreeding must be skillfully managed. New technology must be developed to shorten the re- sponse time between new-product needs and availability of genetic stock that meets the needs.

The general approach to long-term breeding is to develop multiple varieties from each base population of select trees to serve different breeding objec- tives (e.g., short-rotation fiber production or long-rotation quality wood production). This is the 'shotgun' to future uncertainty. For each objective (va- riety), the base population is subdivided into several small breeding groups called replicate lines (or sublines). The reason for sublines is that genetic change will be accelerated by the combined effect of selection and drift in small, isolated groups, which will provide the opportunity for rapid improve-

144 J.H. KITZMILLER

ment. Trees of different sublines will be brought together in seed orchards, which avoids inbreeding because crosses (other than selfs) will be between, not within, sublines. Trees forming each replicate line were near-neighbors in nature. These 'local' breeding groups will preserve important allelic combi- nations, if they exist.

Orchard seed

Seed from production orchards will eventually supply 65-75% of the Re- gion's planting needs, and the Base-Level Program will supply the rest. The proportion of orchard seed varies by species; e.g., most of the Douglas-fir and ponderosa-pine plantings will be from orchard seed, but most of the true-fir plantings will be from Base-Level (wild-stand) seed collections. Orchards will begin producing some seed in 1990, and large quantities will be produced for ponderosa pine and Douglas fir by 2000. Production levels of sugar and Jef- frey pines, and white and red firs, will be available by 2010 or 2015.

The first-generation orchards will result in t imber stands with improved growth rate, stem and crown form, pest resistance, and wood quality. Mod- erate to intensive selection for these traits will change the frequencies for genes that affect them, and may even eliminate some undesirable genes in the High- Level Program. Since any gene may have future value, it should be accessible from natural forest reserves and the base-level plantations indefinitely.

The genetic base will be maintained or narrowed for the economic traits

TABLE 2

Comparison between base-level (wild) seed and high-level (orchard) seed for genetic diversity as affected by selection and breeding factors

Factors influencing diversity Diversity comparisons for seed collected from:

Base-level (wild) High-level (orchard)

Superior tree selection (economic traits)

No. unrelated seed trees (female parentage)

Pollen background (male parentage)

Inbreeding level

Low intensity (higher diversity)

20-100 (higher diversity )

some related, unlimited cloud from forest (higher diveristy)

high intensity (lower diversity)

50-60 (lower diversity)

unrelated, limited to 60 clones (lower diversity)

low to moderate none to very low (lower diversity) (higher diversity)


under selection, but the base should be maintained or broadened for characteristics that affect adaptability to environmental diversity and pests (Zobel and Talbert, 1984). The broadened base derives from crossing unrelated parents from different stands. The genetic independence of traits for economic value and adaptability makes this simultaneous narrowing and broadening possible. Environmental differences within a breeding zone are not suffi- ciently large to cause problems in adaptation as a result of interstand crossing. The maximum geographic distance in a zone is 160 km, and breeding groups are composed of trees within a distance of 50 km or less.

Because orchard seed will be genetically heterogeneous, it will perform well across a range of environments. Seed will come from 50-60 interbreeding parents having the best combination of seed production and progeny performance. This is more than adequate to meet the needs for production plantations. Although the diversity of a plantation from High-Level orchard seed is likely to be less than Base-Level wild seed for both economic and adaptability traits (Table 2), it has more than adequate diversity to meet the needs of production plantations and will normally exceed the level of diversity in natural stands.

GENETIC DIVERSITY NEEDS IN PLANTATIONS AND COMMERCIALLY MANAGED

STANDS

Controlling diversity

Most biologists would agree that breeding populations and natural-resource populations need high levels of diversity. The amount of diversity needed in plantations and commercially managed stands is more controver- sial. Vulnerability to pests and environmental extremes is a problem of production forestry, because narrowing the genetic base provides opportunity for great loss as well as for great gain (Ledig, 1986, 1988 ). Conservative forestry protects variation from loss.

In deploying genetically improved planting stock, foresters can control the distribution of genetic diversity within and among stands. This may be ac- complished by: ( 1 ) varying the genetic composition of the seedlot (i.e., varying the families and stands in pure or mixed lots); (2) varying the timing of planting various lots; and (3) varying the spatial patterns of genetic groups (Nance, 1986).

How much diversity is needed in a plantation of locally adapted stock? Do plantations require more or less diversity than natural stands? Should more than one species be planted? How many families or clones should be included in single- and mixed-species settings? These decisions are made with limited knowledge of site ecology, including potential damaging agents and their risk factors, levels of resistance and variation in the planting stock, and manage-

146 J.H. KITZMILLER

ment responses to mortality (i.e., policies on planting density, replanting, re- lease, pesticide use, thinning, etc. ).

Risk of loss from physical versus biotic events

When the risk of loss is within acceptable limits, genetic mixtures are considered superior to genetic monotypes for crop reliability over a long rotation. However, when the risk of loss from damaging physical events is high, then a mosaic of small monotypic plantations may require less replanting than mixtures (Libby, 1982).

Risks may be higher near the margins of species ranges in harsh environments where, unfortunately for several California species, seed crops are less reliable and may possess a lower genetic base. Environmental extremes are more frequent and contribute to secondary pest epidemics. Such low growth conditions justify less silvicultural input in thinning, salvage, and replanting. Under such high risks, a mosaic of small, single-family plantations may be a better strategy than mixed plantations.

Biotic events can be more complicated in their effects than physical events. Unlike physical events, a pest may successfully colonize a plantation, and its subsequent generations may evolve an increasingly precise adaptation to a genetic monotype (Libby, 1982). Thus, genetically diverse plantations are less vulnerable to pests because they break the spread of an infection or infestation.

A pest that successfully colonizes a seedling plantation is unlikely to adapt narrowly to a single genotype, but will remain genetically diverse. And over several of its generations it may continue to adapt to similar phenotypes until more and more trees are damaged each generation (Libby, 1982 ). Therefore, to control damage from pests the magnitude of difference both among and within genetic groups is very important, in addition to the number of groups in a plantation. Although no two genotypes are identical in seedling plantations, many are very similar. Seedling plantations form a genetic continuum because the variability within families is large (Libby, 1982 ). The number of genetic monotypes must be high enough (7-10) to assure plantation success and low enough (20-25) to allow sufficient genetic differences among them so that cross-adaption of a narrowly-adapted pest is unlikely to spread among groups (Libby, 1982 ). Mixed-species plantations provide the greatest protec- tion against cross-adaption of pests (Libby, 1982). Most commercial forest tree species reproduce naturally by seed, and many occur in pure, even-aged stands of predominantly one species. These natural stands do not appear threatened as predicted by the 'genetic continuum and cross-adaptation of pests' concept, although some infections or infestations could be associated with members of one or a few families that tend to cluster in natural stands.

The general concept of maintaining a broad difference between genetic groups and using 7-25 groups per plantation is a reasonable guide to seedling


plantations. The PSW Region plants multiple species where ecologically appropriate, and uses seedlots collected from 10-20 stands (unrelated groups) and one to a few trees per stand. Species, stands and families provide genetic discontinuity and diversity in seedling plantations.

Knowledge of the genetic structure of natural stands based on isozyme or DNA analyses may serve as a model to set minimum levels of diversity for plantations. Evaluation of wild stands before and after exposure to various hazards may help to define diversity requirements.

Plantation replacement

A major factor influencing the amount of diversity that is needed is how the plantation or managed stand will be replaced. Diversity should be maintained so that managers can exercise the option of natural regeneration. Stands replaced by planting need sufficient diversity to cope with the environmental time-frame of a single rotation. Stands to be regenerated naturally must provide for offspring that are adapted to the environment over at least a second rotation. Even if production stands will be replaced by planting, they should possess enough diversity that seeds could be collected and grown for the next generation of plantations. This would reduce reliance on seed orchards and allow evolution to proceed in managed stands.

M O N I T O R I N G G E N E T I C D I V E R S I T Y

The PSW Tree Improvement Program will monitor genetic diversity to assess the effects of tree improvement and management activities, providing an opportunity to modify practices, if necessary. Genetic diversity of forest trees has been difficult to measure directly until relatively recently. Applications of biochemical techniques developed in forestry over the past 20 years now per- mit us to measure and evaluate the genetic effects of various practices. Indi- viduals can be readily genotyped, and stocks can be characterized by assaying enzyme variants, called isozymes (Conkle, 1986). In combination with more traditional studies of morphological and physiological traits, isozyme analysis can help identify levels and patterns of genetic diversity (Libby and Critchfield, 1986).

Isozymes were used effectively in a study of genetic structure of Scots pine in Sweden after natural and artificial regeneration. An excess of homozygotes was detected in the seed, evidence of inbreeding as a result of self-fertilization. However, inbreds in the natural regeneration that originated after a shelterwood cut were apparently eliminated by natural selection in 10-20 years (Yazdani et al., 1985 ). With artificial regeneration, inbreds were eliminated much earlier, by age 3 (Muona et al., 1987 ). Additional studies revealed that the genetic changes were greater in natural stands than in nurseries, and that

148 J.H. KITZM1LLER

allelic frequency changed little in the passage from seed to bareroot seedlings, and not at all from seed to containerized seedlings (Muona et al., 1988).

In 1988, the USDA Forest Service established an enzyme-electrophoresis laboratory at Placerville, California; called the National Forest Genetic Elec- trophoresis Laboratory (NFGEL), it was created to generate genetic information rapidly and inexpensively to support timber-management programs, and to service all Regions of the Forest Service with state-of-the-art technology. Since isozyme markers are useful for monitoring genetic changes and evaluating the genetic consequences of almost any management practice, some anticipated uses of NFGEL include: ( 1 ) mapping the current level and pat- tern of genetic diversity in natural stands, which provides a natural baseline for comparing managed populations and also helps to delineate breeding zones; (2) examining the effects of harvesting methods and regeneration practices on genetic diversity and the differences between natural and planted stands; and, (3) determining breeding patterns in seed orchards and identi- fying reproductive materials suspected of being mislabelled.

Isozyme analysis will be used to assess the effects of various management activities on genetic diversity. Certainly, it will provide information that can help improve silvicuRural practices as forest-management and tree-improvement activities expand throughout the National Forests.

CONCLUSION

The PSW Region has examined various phases of artificial regeneration, recognized potential problem activities, and taken positive steps to reduce or eliminate factors that lower diversity. PSW Region silviculturists aim to maintain diversity by: ( 1 ) collecting seed from a broad base of trees and stands of local origin; (2) using practices that favor high survival in the nursery and forest; (3) planting appropriate mixtures of species and/or genotypes; (4) planting as densely as economics allow; ( 5 ) encouraging natural regeneration in plantations; and (6) developing schedules and spatial patterns for deploying planting stock.

REFERENCES

Belcher, E.W., 1980. Germ plasm storage by ex situ method. In: R. Miller (Editor), Proc. Serv- ice-Wide Workshop on Gene Resource Management, 14-16 April 1980. USDA Forest Serv- ice, Sacramento, California, pp. 300-311.

Campbell, R.K., 1987. Biogeographical distribution limits of Douglas-fir in southwest Oregon. For. Ecol. Manage. 18: 1-34.

Campbell, R.K. and Sorensen, F.C., 1984. Genetic implications of nursery practices. In: M.L.


Duryea and T.D. Landis (Editors), Forest Nursery Manual: Production of Bareroot Seed- lings. Martinus Nijhoff/Dr. W. Junk, Dordrecht, pp. 183-191.

Conkle, M.T., 1986. Decoding Tree DNA. J. For., 84: 34-37. Friedman, S., 1988. Appropriate technology in tree breeding: Gain with less pain. In: M.E. De-

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Jenkinson, J.L. and Nelson, J.A., 1986. Winter sowing for production of 1-0 Douglas-fir planting stock. In: T. Landis (Editor), Proc. Western Forest Nursery Council/Intermountain Nurseryman's Association Combined Meeting, Tumwater, Wash., 12-15 August 1986. USDA For. Serv., Rocky Mount. For. Range Exp. Stn., Fort Collins, CO, GTR-137, pp. 35-51.

Jenkinson, J.L., 1984. Seed source lifting windows improve plantation establishment of Pacific slope Douglas-fir. In: M.L. Dureay and G.N. Brown (Editor), Seedling Physiology and Re- forestation Success. Martinus Nijhoff/Dr. W. Junk, Dordrecht, pp. 115-141.

Jenkinson, J.L., Bega, R.V. and McCain, A.H., 1982. Winter sowings increase size of nursery seedlings of sugar pine in the Sierra Nevada of California. In: INIA-IUFRO Reunion Techn- ica Principios de Introduccion de Especies. 4-8 October 1982, Lourizan, Spain. Centro de Investigaciones Forestales de Lourizan, Conselleria de Agricultura, Xunta de Galicia 19 pp. (on file).

Kitzmiller, J.H., 1976. Tree improvement master plan for the California region. USDA Forest Service, San Francisco, Calif., 96 pp.

Ledig, F.T., 1986. Conservation strategies for forest gene resources. For. Ecol. Manage., 14: 7 7 - 90.

Ledig, F.T., 1988. The conservation of diversity in forest trees. BioScience, 38: 471-479. Libby, W.J., 1982. What is a safe number of clones per plantation? In: H.M. Heybroek, B.R.

Stephen, and K. von Weissenberg (Editors), Proc. 3rd Int. Workshop on Genetics of Host- Parasite Interactions in Forestry, Wageningen, The Netherlands, 14-21 September 1980. Pudoc, Wageningen, pp. 342-360.

Libby, W.J. and Critchfield, W.B., 1986. Patterns of genetic architecture. In: 18th IUFRO Con- gress Workshop, Latest Achievements and Prospects in Forest Genetics and Tree Improve- ment, Dubrovnik, Yugoslavia. 20-21 September 1986. Ann. For., 13: pp. 77-92.

Muona, O., Yazdani, R. and Rudin, D., 1987. Genetic changes between life stages in Pinus sylvestris: Allozyme variation in seeds and planted seedlings. Silvae Genet., 36: 39-42.

Muona, O., Haru, A. and Karkkainen, K., 1988. Genetic comparison of natural and nursery grown seedlings of Pinus sylvestris using allozymes. Scand. J. For. Res., 3: 37-46.

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managing genetic diversity in a tree improvement program

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