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Physiological Genetics of Contrasting Loblolly and Slash Pine Families and Clones

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PAGE 1

PHYSIOLOGICAL GENETICS OF CONTRA STING LOBLOLLY AND SLASH PINE FAMILIES AND CLONES By VERONICA INGRID EMHART A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Veronica I. Emhart

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To my parents and brothers, who love d and supported me along my journey. To my friends, whose company filled my heart with happiness all five years.

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iv ACKNOWLEDGMENTS During my study, Drs. Timothy White a nd Timothy Martin provided guidance, support, direction, and encouragement. I owe a great deal to them. My immense appreciation also goes to Dr. Dudley Huber for his excellent suggestions, disposition, and help in understanding difficult statistical i ssues and enlightening discussion of results. Thanks also go to Dr. Eric Jokela and Dr. Kenneth Boote for their participation as supervisory committee members and their i nvaluable guidance throughout my study. My study was made possible by the fina ncial support of the United States Department of Agriculture (USDA) Forest Service and the Forest Biology Research Cooperative. Rayonier Inc. provided the study site and access to valuable information. Thanks go to Dave Nolletti, Greg Powell, and Greg Starr for their availability and help in field activities and for making my life easier in th e lab. I want to express my appreciation to my fellow graduate stude nts (Salvador Gezan, Maheteme Gebremedhin, Alex Medina, and Xiabo Li); and field technicians (Sean Gallagher, Tim Walton, Jason Martin, Paul Proctor, Chris Cabrera, a nd Kate Kritcher) who collaborated measuring trees, downloading data, collecting shoot and foliage samples, grinding foliage, and processing data. Thanks also go to my friends (Gabriela Luciani, Belkys Bracho, Gladys Vergara, Rosana Higa, and Tatiana Verissimo) for being such great company in afterwork activities, and for their support in difficult times.

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v I thank God for giving me the opportunity to start a new advent ure far from home, and to celebrate victory at the end of this road. Finally, and most important, I thanks my parents and brothers for their love, hel p, support, and belief in this journey.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES.............................................................................................................x ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION........................................................................................................1 2 CLONAL VARIATION IN CRO WN STRUCTURE, ABSORBED PHOTOSYNTHETICALLY ACTIVE RA DIATION, AND GROWTH OF LOBLOLLY AND SLASH PINE................................................................................5 Introduction................................................................................................................... 5 Materials and Methods.................................................................................................7 Site Description and Plant Material.......................................................................7 Growth and Crown Architectural Traits................................................................8 Estimating Absorbed Photosyntheti cally Active Radiation (APAR)....................9 Statistical Analysis................................................................................................9 Genetic Parameter Estimation.............................................................................10 Results........................................................................................................................ .11 Genetic Variation in St em and Crown Traits......................................................11 Within-Family Individual-Tree Broad-Sense Heritabilities................................13 Within-Family Genetic and Environmental Correlations....................................15 Discussion...................................................................................................................16 3 GENETIC VARIATION IN BASALAREA INCREMENT PHENOLOGY AND ITS CORRELATION WITH GROWTH RATE IN LOBLOLLY AND SLASH PINE FAMILIES AND CLONES..............................................................................25 Introduction.................................................................................................................25 Material and Methods.................................................................................................28 Study Site and Plant Material..............................................................................28 Basal-area Incremen t Measurements...................................................................29 Phenological Traits..............................................................................................30

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vii Meteorological Data and Water Balance.............................................................30 Statistical Analyses and Genetic Parameters.......................................................31 Results and Discussion...............................................................................................33 Genetic Variation among Species and Families..................................................33 Clonal Variation and Within-Family Inhe ritance of Phenol ogical Traits and Stem Growth....................................................................................................41 Genetic Correlations among Phenol ogical Traits and Stem Growth...................43 Analysis across Years 2002-2003.......................................................................47 4 CARBON ISOTOPE DISCRIMINATION, CROWN CONDUCTANCE, GROWTH AND THEIR GENETIC PARAMETERS IN LOBLOLLY AND SLASH PINE FAMILIES AND CLONES................................................................50 Introduction.................................................................................................................50 Material and Methods.................................................................................................54 Study Site and Plant Material..............................................................................54 Tree Growth and Carbon Isotope Discrimination...............................................55 Meteorological Data............................................................................................56 Individual Tree Transpiration..............................................................................57 Crown Conductance and Stomatal Sensitivity Calculations...............................58 Genetic Parameters and Statistical Analyses.......................................................60 Results........................................................................................................................ .62 Carbon Isotope Discrimination............................................................................62 Stem Growth........................................................................................................65 Whole-Tree Crown Conductance and Stomatal Sensitivity................................66 Genetic Correlations between Carbon Isotope Discrimination and Growth and Whole-Tree Crown Conductance..............................................................68 Discussion...................................................................................................................69 5 SUMMARY AND CONCLUSIONS.........................................................................76 Genetic Variation among Species and Families.........................................................77 Clonal Variation and Within-Family Inheritance.......................................................78 Correlations.................................................................................................................79 APPENDIX A DESIGN AND LAYOUT STUDY SITE...................................................................82 B SILVICULTURAL TREATM ENTS AT STUDY SITE...........................................83 REFERENCES..................................................................................................................84 BIOGRAPHICAL SKETCH.............................................................................................97

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viii LIST OF TABLES Table page 2-1 Significance levels (p-values), sp ecies means and pooled within-family heritabilities (H2 WF) for individual-tree growth and cr own structural variables for 5 and 6 year-old loblolly and slash pine families in north central Florida..................14 2-2 Age 5 y and 6 y within-family indi vidual-tree broad-sense heritability (H2 WF) for growth and crown structural traits in f our slash pine families in north central Florida......................................................................................................................18 2-3 Within-family genetic correlations among individual-tree volume increment between age 5-6 and crown structural variables at age 5, for slash (S1, S2, S3 and S10) and loblolly (L4) pine families in north central Florida..................................19 3-1 Significance levels (p-values), and sp ecies and family least square means for individual tree stem growth and phenologi cal traits for two growing seasons for loblolly and slash pine familie s in north central Florida..........................................35 3-2 Significance levels (p-val ues) for clone within-family for tree stem growth and phenological traits for two growing seasons in loblolly and slash pine families in north central Florida.................................................................................................42 3-3 Within-family individual-tree broad-sens e heritabilities for growth phenology traits and basal-area growth increment by year in loblolly and slash pine families growing in north central Florida...............................................................................43 3-4 Within-family genetic correlations betw een growth phenology tr aits in 2002 (above the diagonal) and 2003 (below the diagonal) in loblolly and slash pine families growing in north central Florida...............................................................................46 3-5 Within-family genetic correlations between growth and phenology traits by year in loblolly and slash pine families gr owing in north central Florida............................46 4-1 Significance levels (p-values), species and within family least square means for volume, carbon isotope discrimination ( 13C) for three growing periods, and crown conductance variables (Gsref and Gssensitivity ) in slash and loblolly pine families in north central Florida.................................................................................................64

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ix 4-2 Significance levels (p-values) for clone within family in carbon isotope discrimination for two growing periods and crown conductance variables for loblolly and slash pine familie s in north central Florida..........................................64 4-3 Within-family individual-tree broad-sens e heritabilities for stable carbon isotope discrimination ( 13C) by year, and crown conductan ce variables in loblolly and slash pine families growing in north-central Florida...............................................65 4-4 Genetic correlations between year s 2001 and 2003 by family for carbon isotope discrimination ( 13C) and between 4-5 yr and 6-7 yr stem volume increment (VI) for loblolly and slash pine families in north central Florida....................................65 4-5 Within-family correlatio ns between volume increment of the growing season and stable carbon isotope discrimination ( 13C) by year in loblo lly and slash pine families growing in north-central Florida................................................................68 4-6 Genetic and environmental correl ations between mean carbon isotope discrimination (mean 13C) and GSref and GSsensitivity for loblolly and slash pine families in north central Florida...............................................................................69 B-1 Treatment regimes applied in the research location at Rayonier, Inc......................83

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x LIST OF FIGURES Figure page 2-1 Family means and standard error bars for individual-tree growth and crown structural traits for 5 and 6 year-old loblo lly and slash pine families in north central Florida......................................................................................................................12 3-1 Family mean cumulative basal-area growth curves for years 2002 and 2003 in loblolly and slash pine in north central Florida........................................................35 3-2 Species mean daily basal-area growth incr ement for loblolly and slash pine in north central Florida and environmental variables............................................................36 3-3 Relationship between individual tree daily basal-area increment and simulated daily plot-level soil water balance in loblolly and slash pine in 2002 (A) and 2003 (B)..40 4-1 Family means and standard errors in carbon isotope discrimination in year 2001 and 2003...................................................................................................................63 4-2 Accumulated monthly precipitation in years 2001, 2003 and mean normal year from Gainesville Regional Airpor t, Gainesville, Florida (NOAA 2003).................63 4-3 Representative relationship between canopy average stomatal conductance ( GS) and vapor pressure deficit (D) on a half hourly basis for a typical slash pine ramet......67 A-1 Design and layout of full-sib family block plot study at Rayonier, Inc...................82

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xi Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PHYSIOLOGICAL GENETICS OF CONTRA STING LOBLOLLY AND SLASH PINE FAMILIES AND CLONES By Veronica Ingrid Emhart December 2005 Chair: Timothy L. White Cochair: Timothy A. Martin Major Department: Forest Resources and Conservation My study focused on the biology and geneti c structure of 300 clones from five different full-sib loblolly and slash pine fa milies. The study was divided into three main areas of research: (1) detailed quantification of crown structure and estimation of annual absorbed photosynthetically active radi ation (APAR); (2) seasonal dynamics and phenology of basal area growth a nd its association with soil water balance; and (3) leaf carbon isotope discrimination and whole-tree sap flow. Genetic variation in crown structural tr aits, APAR, stem volume growth, basal area growth phenology, basal area growth rates, leaf carbon isotope discrimination ( 13C), and crown conductance were more apparent at the clonal level than at the species and family levels. The one loblolly pine family we studied tended to grow fast er, developed larger crowns with more acute branch angles, had mo re leaf area and intercepted more radiation than the four slash pine families average d. Loblolly and slash pine within-family

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xii individual-tree broad-se nse heritabilities (H2 WF) ranged from 0.00 to 0.41 for growth and crown structural traits, and most were between 0.10 and 0.25 when estimated from a combined analysis across families. Genetic correlations of crown size, leaf area, and APAR with volume increment were generally positive. Basal-area growth spanned March through Octo ber for both species. In both years, peaks in basal-area increment occurred in short (2-3 week) periods in the early spring for all families, followed by relatively constant rates of basal-area growth until cessation. The H2 WF ranged from 0.01 to 0.37 for basal ar ea growth phenology. Both the strength and direction of correlation estimates of phenologi cal traits with growth rate varied across families and years. Clonal mean values for 13C ranged from 19‰ to 25.45‰. The H2 WF for 13C and crown conductance parameters ranged from 0.01 to 0.32. Genetic and environmental correlations of stem growth with 13C or with crown conductance were low. There was no evidence of clone-by-year interaction in stem growth, basal-ar ea growth phenology, and 13C for any family. Understanding the biology of physiological processes and their genetic parameters gives us insight into the ke y functional and structural tr aits that determine genotype performance differences in southern pines.

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1 CHAPTER 1 INTRODUCTION Loblolly pine ( Pinus taeda L.) and slash pine ( Pinus elliottii Engelm. var elliottii ) are widely planted as commercial timber species in the southeastern Un ited States (Smith et al. 2004). From the early 1950s, large-scale tree-breeding programs in both species improved forest productivity by selecting trees fo r superior growth rate, form, and disease resistance (McKeand et al. 2003). The genetically improved material currently being established in commercial plantations is de ployed from bulked or chard seed, half-sib families, and full-sib families (with growing interest in the deployment of outstanding clones). Tree breeding has proven to be a very effective tool, and breeding will continue to be the most important mechanism for de veloping recombinant genotypes to achieve increasing genetic gains (White and Carson 2004). Management of southern pine plantations in the United States is being transformed from a relatively extensive system of planting coupled with isolated individual treatment, to a much more intensive system in which ge netic and site resources are manipulated in concert, to optimize stand productivity (Fox et al. 2004). Increased productivity in southern pines create more value for the fo rest industry, and decrea ses pressure on native pine forests, considering that the demand for forest products will continue to increase and intensive management will be needed to meet this demand. Increased forest productivity also provides great po tential for sequestering atmospheric carbon (Johnsen et al. 2004). Clonal forestry may be an excellent way to increase the produc tivity of southern pine plantations in the near term (Fox et al. 2004). Many potential benefits of clonal

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2 forestry have been previously describe d (Libby 1982; Libby and Rauter 1984; Carson 1986) and include: Gains arising from testing and selection of clones; Clone/site matching to increase genetic gains by capturing favorable genotype by environment effects (G x E), and by targeti ng expression to existi ng site properties; Greater uniformity (little impact on growth and yield traits, but extremely valuable for log and wood quality and disease resi stance traits, and for harvesting and processing); Greater repeatability (better yield predicti on and planning). Specific clones can be identified that are most adapted to differe nt site qualities. Identifying these clones help us take advantage of positive genot ype X environment interaction and to optimize silvicultural practi ces, including spacing, weed control, and fertilizer regimes. To identify superior clone s within-family we must understand the biological basis for growth differences a nd identify key struct ural and functional attributes at the organ, tree, and stand level. Growth involves many integrated physiol ogical processes influenced by genetic and environmental factors (Kozlowski a nd Pallardy 1997). Processes like radiation absorption, carbon gain capacity, crown conduc tance responses to changes in the environment, growth phenology, nutrient assim ilation, and growth regulation may control stem volume growth in contrasting southern pine families and clones. Better understanding the physiologial proce sses underlying genetic differences in growth performance may allow geneticists to be more deliberate in their selections, focusing specific objectives and allowing for more predictable gains. Martin et al. (2005) described several potential obstacles to ecophysiological cont ributions to tree improvement programs: Selecting wrong physiological parameters for screening; Making physiological measurements at inap propriate spatial and temporal scales; Attempting to use seedlings to predic t field performance of adult trees.

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3 My overall goal was to inves tigate biological traits and their genetic structure in 300 clones from five different full-sib loblolly and slash pine families. My study used measurements that integrated biological info rmation over space and time, with the intent of studying biological traits th at correspond more closely to the spatial and temporal scales at which growth was observed (e .g., whole trees over seasons to years). The study was divided into three main areas of investigation: The first phase used detailed crown struct ural information for each ramet withinclone to parameterize the process model MAESTRA, which was then used to estimate the total amount of radiation intercepted by each ramet over a year; Second, repeated basal-area measurements of each ramet was used to estimate seasonal dynamics and phenology of basalarea growth, associated with a soilwater balance calculation to examine rela tionships between basal area growth and integrated environmental variables; Third, leaf carbon isotope discrimination an alysis (integrates leaf physiology over the time of leaf formation) and wholetree sap flow analysis (integrates leaf physiology over the tree crown over long peri ods of time) was used to analyze integrated gas exchange properties and its relationship with growth. My study focused on the following specific objectives, organized by main areas of investigation: Specific aim 1a: quantify growth and crown structural variation among species, families and clones representing a range of growth performance in loblolly and slash pines; Specific aim 1b: integrate crown structural variable s into a radiative transfer model to estimate variation in intercepted radi ation for different genotypes for a given period of time and their rela tionship with growth rate; Specific aim 1c: estimate within-family genetic control and environmental influence on crown structural attributes and growth; Specific aim 2a: compare two years basal area growth phenology among species, families and clones; Specific aim 2b: estimate genetic parameters fo r basal area growth phenology, its correlation with growth rates, and th e genotype interaction with seasonal environmental changes;

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4 Specific aim 3a: determine whether genetic va riation for leaf carbon isotope discrimination occurs among slash and loblo lly pine genotypes (species, families, or clones); Specific aim 3b: examine genetic variation in cr own-level stomatal conductance (crown conductance) sensitivity to vapor pressure deficit be tween species, among slash pine families, and among clones within slash and loblolly pine families; Specific aim 3c: determine broad-sense heritabili ties and genetic correlations for leaf carbon isotope discrimination, growth and crown conductance. Results from my study should positively im pact future tree growth modeling and will help in decisions that involve genotype deployment and silvicultural treatments. Results will also aid in examining genetic and environmental control of several key structural and functional pro cesses that determine producti vity in different full-sib families and clones within-family in southern forest plantations.

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5 CHAPTER 2 CLONAL VARIATION IN CROWN STRUCTURE, ABSORBED PHOTOSYNTHETICALLY ACTIVE RADIAT ION, AND GROWTH OF LOBLOLLY AND SLASH PINE Introduction Crown structural characteris tics (such as crown size, br anching frequency, branch diameter, branch angle, and leaf area quant ity and spatial distri bution) influence the efficiency and magnitude of ra diation interception and competitive interactions with other trees (Wang and Jarvis 1990; Stenberg et al. 1994; Vose et al. 1994; McCrady and Jokela 1996, 1998). As a result, crown architecture is an important determinan t of both tree-level and stand-level productivity (Da lla-Tea and Jokela 1991; Stenberg et al. 1994; McCrady and Jokela 1996). This linkage is often reflect ed in ideotypes or conceptual models of desirable tree phenotypes intended to guide plant genetic research and breeding programs (Donald 1968; Dickmann et al. 1994). For example, the published ideotypes for Populus (Dickmann 1985; Dickmann and K eathley 1996) and Scandinavi an conifers (Karki and Tigerstedt 1985) incorporate numerous crown st ructural variables. Genetic variation in growth has been the subject of much research (White 1996), and forms the basis of most commercial tree improvement programs (White et al. 1993; McKeand and Bridwater 1998; Li et al. 2000). In contrast, the genetic archite cture of crown structure has been much less intensively studied, and is seldom used in tree improvement programs (Martin et al. 2001). Xiao et al. (2003) showed that signi ficant differences in growth between loblolly pine ( Pinus taeda L.) and slash pine ( Pinus elliottii Engelm. var elliottii ) were associated

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6 with variation in crown structure and bioma ss allocation. At age 3 and 4 y, loblolly pine had more branches per tree and allocated more biomass to branches than slash pine. Greater branch/leaf biomass as a growth strategy might deve lop spacious crowns facilitating faster growth by increasing th e leaf-area carrying cap acity in the crown. Knowledge of heritabilities and genetic correlations is need ed to understand the genetic structure of breeding populations, and to determine deployment strategies in tree improvement programs (White 1987). Broadsense heritabilities for a number of structural and growth propert ies have been estimated for Populus and Eucalyptus (Wilcox and Farmer 1967; Weber et al. 1984; Borralho et al. 1992; Lambeth et al. 1994; Osorio 1999). Genetic correlations between grow th traits and crown st ructural attributes are scarce, but studies in Populus Eucalyptus loblolly and slash pine have identified positive genetic correlations between growth performance and branching patterns, and growth performance and crown vigor (Wilcox and Farmer 1967; Lambeth et al. 1994; Lambeth and Huber 1997; Xiao et al. 2003). Incorporating new information on crown structural attributes (such as crown si ze, crown shape ratio, and arrangement and diameter of branches) would improve our understanding of how canopy structure affects absorbed photosynthetic activ e radiation and stand devel opment. Crown structural attributes also may prove useful in selection of families or clones for silvicultural programs, and development of new crop tree ideotypes. Our objectives were as follows: Quantify growth and crown structural variation among species, families, and clones, representing a range of growth pe rformance in loblolly and slash pines; Integrate crown structural variables into a radiative transfer model to estimate variation in intercepted radiation for di fferent genotypes for a given period of time and to estimate their relati onship with growth rate;

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7 Estimate within-family genetic control and environmental influence on crown structural attri butes and growth. According to Martin et al. (2005), one reason ecophysiolo gical research has failed to contribute to southern pine tree improve ment programs is that researchers have focused on small spatial and short temporal sc ales that are too fa r removed in space and time from growth processes. Accordingly, we hypothesized that tree growth would be genetically correlated with crown structural traits, and that trai ts which integrated information over space and/or time would be more highly correlated with growth than would less-integrated traits. Materials and Methods Site Description and Plant Material The study area was located on lands manage d by Rayonier Inc. in Bradford County, Florida. The climate is humid and subtropi cal, with a mean annual temperature of 21 C, mean annual rainfall of 1316 mm, and over 50% of the rainfall occurr ing in June through September. Periods of drought are normal in the spring and fall. Mean annual rainfall during 1999-2001 was 967 mm, in contrast to 1405 mm in year 2002 (NOAA 2002). The soils are classified as Pomona and consis t of very deep, somewhat poorly to poorly drained soils that are formed in sandy a nd loamy marine sediments (sandy, siliceous, hyperthermic Ultic Alaquods). Slopes are 0 to 2 %. In a typical profile, the spodic horizon occurs at 30-60 cm, with an argi llic horizon at 90-120 cm. Water table is typically at a depth of 15 to 45 cm for one to three months and a depth of 25 to 100 cm for six months or more, during most years (Soil Survey Staff 1998). The study took place in an area containing 16 full-sib and half-sib loblolly and slash pine families planted in 337 m2 family plots in January 1997. The experiment was

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8 designed as a randomized complete block with four replicates (Appendix A). We used one full-sib loblolly pine family and four full-sib slash pine families. Each family plot contained 60 clones propagated as rooted cut tings from a single family, planted at 1.7 m x 3.4 m spacing (1730 trees ha-1). Cuttings were taken from donor hedges in the spring, and were rooted and grown in a greenhouse for six months before planting. Each of the four plots of the same family contained the same 60 genotypes, but with the ramets planted into different, randomly-determined pl anting locations in the plot. In total we studied approximately 1,200 trees: 60 trees per family plot x 5 families x 4 replications. Fertilization and weed control were app lied periodically to reduce interspecific competition and prevent nutrient deficiency (Appendix B). Growth and Crown Architectural Traits Stem volume growth in the 2000, 2001, and 2002 growing seasons (ages 4, 5, and 6 y, respectively) was determined from dormant -season measurements of tree diameter at 1.37 m height (DBH) and total tree height (H T). Outside-bark individual-tree stem volume was calculated with a general equation (Hodge et al. 1996) as shown in Equation 2-1, where DBH and HT were entered in m: VOL (dm3) = (0.25 3.14 (DBH)2 (1.37 + 0.33 (HT 1.37)))*1000 (2-1) Crown architecture was assessed by meas uring length and width of the living crown and basal diameter of all living br anches at the end of the 2001 and 2002 growing seasons. Also, branch angle was measured in four branches in the 2000 cohort of each tree, with a protractor. Other traits derived from these r ecords included total number of branches per tree, crown shape ratio (CSR= crown height/ crown width), and branch-free stem height. Individual-tree leaf area at age 5 y was calculated by summing individual branch leaf area estimated from regiona l allometric equations (McGarvey 2000).

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9 Regression equations were developed betw een crown size at age 5 y (independent variable) and leaf area at age 5 y (dependent variable) by family; and then leaf area at age 6 y was predicted using crown volume at age 6 y by family. Estimating Absorbed Photosynthet ically Active Radiation (APAR) Total APAR was simulated for each tree in the study from January 1, 2002 to December 31, 2002, using hourly radiation data fr om a weather station at the site, input into the process model MAES TRA, a modification of th e MAESTRO model (Wang and Jarvis 1990; Medlyn 2004). MAESTRA uses Norman and Welles (1983) method to calculate PAR at grid points within the crown, taking into account the spatial distribution of foliage in the target crow n and in adjacent tree crowns. Crown shapes were assumed to be ellipsoidal. Vertical foliage distributi on was specified by a Beta function developed for loblolly pine in North Carolina (Luo et al 2001), while horizontal foliage distribution was assumed to be uniform. Simulations were run for each tree, in each of the 20 study plots. For each tree, the locati on, crown radius in two directio ns, total tree height, height to the base of the live crown, and leaf area were specified. Tree locations, crown dimensions, and leaf area for a two-tree border surrounding each study plot were also specified. When study plots were near non-study plots, crown dimensions and leaf area of border trees were predicted from measured he ight and diameter. Crown dimensions were assumed to increase linearly from March 1st to December 1st. Statistical Analysis Analysis of variance (ANOVA) was used to analyze growth, crown structural traits, and APAR data. PROC GLM in the SAS Syst em were used to test for significance of random effects (clone), while PROC MIXED was used to test the fixed effects (species and families). Equation 2-2 shows the ANOVA model for the analyses, where Yijkl is the

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10 performance of the ramet of the lth clone within the kth family nested in the jth species in the ith replication; i = 1, 2, 3, and 4 for replica tions; j = slash, loblolly; k = 1, 2, 3, 4, and 10 for families; l = 60 identification numbers for 60 clones within each of the five families: Yijkl = + bi + Sj + Fk(j) + cl(jk) + bSij + bFik(j) + ijkl (2-2) = population mean, bi = random variable of replication ~ NID (0, 2 b), Sj = fixed effect of species (slash or loblolly), Fk(j) = fixed effect of family nested within species, cl(jk) = random variable of clone nested within-family and species ~ NID (0, 2 c), bSij = random variable for replicatio n x species interaction ~ NID (0, 2 bS), bFik(j) = random variable for replication x family(species) interaction ~ NID (0, 2 bF), and ijkl = error term ~ NID (0, 2 ). Genetic Parameter Estimation For each species and family, two types of parameters were estimated: within-family heritability for each trait, and genetic a nd environmental correlations among traits. Within-family variance and covariance com ponents were obtained using Multiple Trait Derivate-free Restricted Maximum Like lihood (MTDFREML) software (Boldman et al. 1995). Within-family individual-tree broad-sense heritability was calculated as 2 2 2 2 c c WFH (2-3) Theoretically, broad-sense within-family heritability for full-sib families contains the additive genetic variance, of the dominance genetic varian ce, and most of the epistatic genetic variance (F alconer and Mackay 1996). The st andard error for heritability estimates was calculated using a method de scribed by Dickerson (1962). The residual likelihood ratio test (Wolfinger 1996) was used to test heterogeneity of variances among

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11 slash pine families, and heritabilities were estimated separately for each family (X2 (6, 0.05)= 12.6), or pooled, as appropriate.We estimated all genetic parameters from data collected from only one experimental site; ther efore, the clonal genetic variance contains the clone-environment interac tion variance in the above resu lts, and the estimated genetic parameters are biased upward if the in teraction is non-zero (Hodge and White 1992). Within-family genetic and environmenta l correlations among growth traits and crown structural variables (Falconer and Mackay 1996) we re calculated as shown in Equation 2-4, where xy is the covariance (clonal or re sidual) between two traits, and x y corresponds to the square root of the pr oduct of the clonal or re sidual variance withinfamily of each trait: y x xy xyr (2-4) Results Genetic Variation in Stem and Crown Traits We examined variation in cumulative stem volume, annual stem volume growth, and crown architectural and f unctional traits. Comparisons were made between species (loblolly vs. slash pine), among families within species (four full-sib slash pine families), and among clones within species (60 clones with in each of the four slash pine families and one loblolly pine family). By age 6 y, loblolly pine stem volume was almost 25% larger than mean slash pine stem volume (p=0.0727, 31.42 dm3 vs. 25.47 dm3, respectively), reflecting fairly consistent species-level differe nces in annual stem volume increment (Table 2-1). Within slash pine, there were consistent differences among families (p<0.10) in stem volume and stem volume increment, with the exception of age 5-6 yr increment (Table 2-1, Figure 2-

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12 1). Within-family clonal variation in stem volume and stem volume increment was highly significant for all years for sl ash pine (p<0.0001, Table 2-1). Stem volume (dm 3 tree -1 ) 1213141516171819 20 22 24 26 28 30 32 34 S1 S2 S3 S10 L4 Stem volume increment (dm 3 tree -1 ) 6.06.57.07.58.08.59.09.510.0 9 10 11 12 13 14 Crown length (m tree -1 ) 3.63.84.04.24.44.64.8 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 Crown radius (m tree -1 ) 0.850.900.951.001.051.101.151.201.25 Age 6 0.9 1.0 1.1 1.2 1.3 1.4 Crown shape ratio 1.81.92.02.12.22.3 2.05 2.10 2.15 2.20 2.25 2.30 2.35 2.40 Crown volume (m 3 tree -1 ) 456789101112 6 8 10 12 14 16 18 Number of branches per tree 22242628303234 26 27 28 29 30 31 32 33 34 Branch diameter (cm)Age 5 1.301.351.401.451.501.551.601.651.70 1.42 1.44 1.46 1.48 1.50 1.52 1.54 1.56 1.58 1.60 Branches per unit of crown length (# m -1 ) 5.56.06.57.07.58.08.59.09.5 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 Figure 2-1. Family means and standard error bars for individual-t ree growth and crown structural traits for 5 and 6 year-old lo blolly and slash pine families in north central Florida. S1=family slash 1; S2= family slash 2; S3=family slash 3; S10=family slash 10; L4=family loblolly 4. There were species-level differences in a number of crown structural traits. Loblolly pine had longer and wider crowns at age 5 and 6 y, resulting in species differences in crown volume on the order of 85% (Table 2-1). Slash pine crowns of a given length were slightly narrower than l oblolly pine crowns of a similar length, as

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13 quantified by the crown shape ratio: 2.23 vs. 2.10 at age 6 y fo r slash and loblolly pine, respectively (Table 2-1). Loblolly pine bran ches were displayed at a more acute angle than were slash pine branches: 51.1 vs. 56.9 , respectively (Table 2-1). Age 5-6 y radiation interception, simulated with the MA ESTRA radiative transfer model, was about 20% greater in loblolly pi ne (11,901 MJ/tree) than the m ean slash pine annual APAR (9,901 MJ/tree). Numbers of branches per cr own, branch diameter, and number of branches per unit crown length were not different between species (Table 2-1). Crown structure also varied at the family level, with crown size and shape traits (length, radius, volume, and crown shape ra tio) all varying signifi cantly among the four slash pine families (p<0.10). Slash pine families also differed in numbers of branches, branch diameter (at age 5 y), numbers of branches per unit crown length, and branch angle (Table 2-1, Figure 2-1). Th ere was no significant family-lev el variation in tree leaf area or annual APAR. Within families, there was significant clonal variation for all traits measured (p<0.0001, Table 2-1). Within-Family Individual-Tree Broad-Sense Heritabilities Within-family individual-tree broad-sense heritabilities (H2 WF) were low to moderate for stem volume and crown structural traits. In loblolly pi ne, a number of crown structural traits were moderately heritable, with crown radius at age 5 yr, crown volume at age 6 yr, leaf area at age 6 yr, number of br anches at age 5 yr, and branch angle at age 5 yr having H2 WF between 0.20 and 0.27. Stem volume and stem volume growth traits had lower H2 WF, ranging between 0.05 and 0.18 (Table 2-1). For volume in slash pine at different ages, H2 WF was between 0.17 and 0.19, and crown structural traits sh owed similar ranges of variation (Table 2-1). When H2 WF values were estimated separately by family due to heterogeneous variance components among

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14Table 2-1. Significance levels (p-values), species means and pooled within-f amily heritabilities (H2 WF) for individual-tree growth and crown structural variables for 5 and 6 year-old loblolly and slash pine families in north central Florida. Significance level by effect Species mean H2 WF Trait Species Family Clone Slash Loblolly Slasha Loblolly Inventory Volume age 4 (dm3 tree-1) 0.2240 0.0797 <0.0001 7.13 8.72 -0.05 (0.06) Volume age 5 (dm3 tree-1) 0.1007 0.0451 <0.0001 14.46 18.13 0.17 (0.04) 0.08 (0.07) Volume age 6 (dm3 tree-1) 0.0727 0.0802 <0.0001 25.47 31.42 0.17 (0.04) 0.18 (0.08) Volume increment age 4-5 (dm3 tree-1) 0.0455 0.0567 <0.0001 7.32 9.41 0. 19 (0.04) 0.12 (0.07) Volume increment age 5-6 (dm3 tree-1) 0.0754 0.3324 <0.0001 11.02 13.29 -0.18 (0.08) Crown structure Live crown length age 5 (m) 0.0055 0.2358 <0.0001 3.85 4.55 0.16 (0.04) 0.09 (0.07) Live crown length age 6 (m) 0. 0028 0.0812 <0.0001 4.61 5.46 -0.11 (0.07) Crown radius age 5 (m) 0.0067 0. 0106 <0.0001 0.94 1.20 -0.20 (0.08) Crown radius age 6 (m) 0.0041 0. 0233 <0.0001 1.05 1.33 -0.18 (0.08) Crown shape ratio age 5 0.1001 0. 0012 <0.0001 2.07 1.92 -0.13 (0.07) Crown shape ratio age 6 0.0519 0. 0095 <0.0001 2.23 2.10 -0.00 (0.00) Crown volume age 5 (m3) 0.0039 0.0913 <0.0001 5.80 10.88 -0.19 (0.08) Crown volume age 6 (m3) 0.0020 0.0723 <0.0001 8.91 16.42 -0.25 (0.09) Leaf area age 5 (m2) 0.0450 0.4940 <0.0001 33.11 44.07 0.12 (0.04) 0.08 (0.06) Leaf area age 6 (m2) 0.1197 0.5562 <0.0001 47.14 54.58 -0.25 (0.09) Number branches age 5 0.1214 0.0021 <0.0001 30 33 -0.27 (0.09) Number branches age 6 0.1610 0.0225 <0.0001 30 33 -0.16 (0.07) Branch diameter age 5 (cm) 0.5230 0.0793 <0.0001 1.49 1.54 0.14 (0.04) 0.10 (0.07) Branch diameter age 6 (cm) 0.3292 0.1584 0.0003 1.49 1.54 -0.19 (0.08) Number branches/crown length age 5 0.2087 0.0018 <0.0001 7.90 7.30 -0.11 (0.07) Number branches/crown length age 6 0.1047 0.0136 <0.0001 6.63 6.02 -0.14 (0.07) Branch angle age 5 ( ) 0.0099 <0.0001 <0.0001 56.9 51.1 0.18 (0.04) 0.26 (0.08) Light interception age 5-6 (MJ tree-1) 0.0293 0.1041 <0.0001 9,901 11,901 0. 17 (0.01) 0.17 (0.08) Note: Values in parentheses are standard errors a-Values of H2 WF in slash pine were estimated separa tely by family and are in Table 2-2.

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15 slash pine families, there was a tendency for higher heritabilities in family S2 (Table 2-2). For example, crown radius at ages 5 and 6 showed H2 WF of 0.41, and crown volume at ages 5 yr and 6 yr had moderate he ritability values between 0.34 and 0.36. Within-Family Genetic and Environmental Correlations Within slash pine families, the genetic correlations between individual-tree stem volume increment (ages 5 y and 6 y) were positive and moderate to high with: APAR between age 5 y and 6 y, crown size traits at age 5 y, and tree leaf area at age 5 y (rg=0.35 to 0.74). Individual-tree stem volume incremen t had low positive or low negative genetic correlations with crown shape ratio and branch angle at age 5 y (rg=-0.33 to 0.39, Table 2-3). For loblolly pine family L4, indivi dual-tree volume increment between age 5 y and 6 y was moderately genetica lly correlated with APAR between age 5 y and 6 y (rg=0.64), and crown size traits at age 5 y such as crown volume (rg=0.51), crown radius (rg=0.47), and crown length (rg=0.53). Stem volume increment was positively, but less strongly correlated with leaf area at age 5 y (rg=0.31). As in slash pine, traits such as crown shape ratio and branch angle at ag e 5 yr had much weaker genetic correlations with stem volume growth (rg=-0.20 and 0.20, respectively Table 2-3). Environmental correlations are measures of microsite enviro nmental fluctuation between two traits measured on the same ramets. In slash pine families, moderately to highly positive environmental correlations were found between stem volume increment age 5 and 6 y and light interception age 5 and 6 y (re=0.43 to 0.83), implying that microsites that enhanced APAR also enhanced stem growth. At the same time, positive environmental correlations were found betw een stem volume increment age 5 and 6 y and crown size at age 5 (crown volume, crown radius, and crown length, re=0.52 to 0.76), and between stem volume increment age 5 and 6 y and leaf area age 5 y, number of

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16 branches age 5 y, and branch diameter age 5 y (re=0.36 to 0.72, Table 2-3). Finally, crown shape ratio age 5 y and branch angle age 5 had low positive or negative environmental correlations with stem vol ume increment age 5 and 6 y, implying that microsites that favored growth did not affect crown shape ratio age 5 y and branch angle age 5 y. For loblolly pine, environmental corre lations had similar tendencies as in slash pine, with moderate positive environmental correlations between stem volume increment age 5 and 6 y and crown size at age 5 (crown volume, crown radius, and crown length, re=0.49 to 0.0.58), between stem volume incremen t age 5 and 6 y and leaf area age 5 y (re=0.46), and also between stem volume increm ent age 5 and 6 y and branch diameter age 5 y (re=0.43, Table 2-3). Weakly positive or negative environmental correlations were found between stem volume increment age 5 and 6 y and number of branches age 5 y, branch angle age 5 y and crown shape ration age 5 y (re=-0.12 to 0.29). Both APAR and crown volume at age 5 y proved to be good integrators of crown characteristics for individual trees. In gene ral, APAR and crown volume at age 5 y had stronger genetic correlations with stem volume growth than did any other crown traits. Discussion At the species level, the one loblolly pine family we st udied tended to grow faster than the average of our four slash pine families at ages 5 y and 6 y. At the same time, loblolly pine developed larger crowns with more acute bran ch angles and had more leaf area per individual-tree at age 5 y and 6 y than did the slash pine families (Table 2-1). Xiao et al. (2003) found similar species-level contrasts in juvenile loblol ly and slash pine in north central Florida, where loblolly pi ne accumulated more crown volume per tree, allocated more biomass to branches, and had greater amount of leaf area than slash pine at ages 3 and 4 y. Stand-level st udies have similarly confirmed the ability of loblolly pine

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17 to develop and retain higher levels of leaf area than slash pine (Dalla-Tea and Jokela 1991; Martin and Jokela 2004). Growth differences among slash pine families were subtle, probably because the families selected for my study were all chosen for superior growth potential. In spite of the apparent similarities in stem volume growth rate, the four slash pine families differed in a number of crown architectural traits. Contrasting families had different arrangements and sizes of branches within the crown, a nd varied in crown shape ratio (Table 2-1, Figure 2-1). This suggests that any of a numbe r of crown traits may be associated with high growth rate in southern pi ne families (see also McGarvey et al. 2004). In contrast, McCrady and Jokela (1996) concluded that, am ong the five loblolly pine families they studied, there were significant differences in height growth but none for most branching attributes. Within-family clonal variation was highly significant for all growth and crown structural traits, reflecting a wide spectrum of clonal performance in growth and crown development at these ages. There are few reports in the literature on clonal variation in loblolly or slash pine growth. Paul et al. (1997) reported that hei ght of loblolly pine clones varied significantly at different ages, but that DBH and volume did not. To our knowledge, no published studies have quantified clonal variation in crown characteristics in loblolly or slash pine, but th ese traits have been studied in other forest tree species. For example, Lambeth et al. (1994) found large differences among Eucalyptus grandis clones in growth, branching, and crown density. In Populus, clonal differences in branch characteristics and branching pa tterns were found that resulted in striking differences in crown form and arch itecture (Ceulemans et al. 1990). Sylleptic branches and the

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18 considerable leaf area that th ey carry have important impli cations for whole tree light interception, and thus, play a critical role in the superior growth and productivity of certain hybrid poplar clones. The considerable variation in branch ch aracteristics implies a strong justification for in cluding them in selecti on and breeding programs for Populus Table 2-2. Age 5 y and 6 y within-family i ndividual-tree broad-sense heritability (H2 WF) for growth and crown structural traits in four slash pine families in north central Florida. H2 WF Trait Family S1 Family S2 Family S3 Family S10 Stem volume (age 4 y) 0.16 (0.08) 0.22 (0.09) 0.08 (0.08) 0.00 (0.00) Stem volume increment (age 5-6 y) 0.21 (0.08) 0.24 (0.09) 0.02 (0.07) 0.10 (0.07) Crown length (age 6 y) 0.21 (0.08) 0.31 (0.10) 0.13 (0.08) 0.18 (0.08) Crown radius (age 5 y) 0.11 (0.07) 0.41 (0.11) 0.12 (0.09) 0.09 (0.07) Crown radius (age 6 y) 0.17 (0.07) 0.41 (0.11) 0.12 (0.08) 0.09 (0.08) Crown shape ratio (age 5 y) 0.27 (0.09) 0.33 (0.10) 0.22 (0.10) 0.25 (0.09) Crown shape ratio (age 6 y) 0.32 (0.09) 0.05 (0.07) 0.26 (0.10) 0.17 (0.08) Crown volume (age 5 y) 0.13 (0.07) 0.34 (0.11) 0.10 (0.08) 0.10 (0.07) Crown volume (age 6 y) 0.17 (0.08) 0.36 (0.10) 0.12 (0.08) 0.16 (0.08) Leaf area (age 6 y) 0.13 (0.07) 0.34 (0.10) 0.15 (0.09) 0.16 (0.08) Number of branches (age 5 y) 0.10 (0.07) 0.26 (0.09) 0.22 (0.10) 0.14 (0.08) Number of branches (age 6 y) 0.26 (0.09) 0.15 (0.08) 0.14 (0.09) 0.10 (0.07) Branch diameter (age 6 y) 0.09 (0.06) 0.08 (0.07) 0.03 (0.07) 0.02 (0.06) Number of branches per unit of crown length (age 5 y) 0.00 (0.00) 0.12 (0.07) 0.27 (0.10) 0.03 (0.06) Number of branches per unit of crown length (age 6 y) 0.04 (0.06) 0.01 (0.07) 0.24 (0.09) 0.05 (0.06) Note: Values in parentheses are standard errors

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19 Table 2-3. Within-family genetic correlati ons among individual-tree volume increment between age 5-6 and crown structural variables at age 5, for slash (S1, S2, S3 and S10) and loblolly (L4) pine families in north central Florida. Trait Family S1 Family S2 Fam ily S3 Family S10 Family L4 Stem Volume Increment (age 5-6 yr) Genetic correlations Light interception (age 5-6 y) 0.70 (0.08) 0.74 (0.05) 0.62 (0.47) 0.67 (1.00) 0.64 (0.10) Crown volume (age 5 y) 0.71 (0.11) 0.61 (0.10) 0.69 (0.78) 0.41 (0.33) 0.51 (0.14) Leaf area (age 5 y) 0.64 (0.19) 0.64 (0.10) 0.35 (0.93) 0.61 (0.45) 0.31 (0.34) Crown shape ratio (age 5 y) 0.39 (0.23) -0.33 (0.23) 0.02 (0.72) 0.01 (0.37) -0.20 (0.32) Branch diameter (age 5 y) 0.51 (0.21) 0.75 (0.10) 0.40 (0.75) 0.02 (0.71) 0.16 (0.36) Branch angle (age 5 y) -0.05 (0.29) 0.01 (0.26) 0.01 (0.70) -0.22 (0.45) 0.26 (0.25) Number of branches (age 5 y) 0.43 (0.26) 0.41 (0.18) -0.06 (0.75) 0.03 (0.41) 0.20 (0.24) Crown radius (age 5 y) 0.55 (0.17) 0.66 (0.09) 0.42 (0.56) 0.20 (0.43) 0.47 (0.16) Crown length (age 5 y) 0.77 (0.09) 0.55 (0.13) 0.37 (0.76) 0.31 (0.36) 0.53 (0.23) Environmental correlations Light interception (age 5-6 y) 0.71 (0.03) 0.78 (0.03) 0.83 (0.03) 0.43 (0.07) 0.68 (0.04) Crown volume (age 5 y) 0.69 (0.04) 0.70 (0.04) 0.76 (0.04) 0.60 (0.05) 0.58 (0.05) Leaf area (age 5 y) 0.44 (0.06) 0.60 (0.05) 0.72 (0.07) 0.58 (0.05) 0.46 (0.06) Crown shape ratio (age 5 y) -0.10 (0.08) 0.09 (0.08) 0.03 (0.09) 0.01 (0.08) -0.12 (0.08) Branch diameter (age 5 y) 0.36 (0.07) 0.52 (0.06) 0.70 (0.05) 0.50 (0.06) 0.43 (0.06) Branch angle (age 5 y) -0.16 (0.08) -0.06 (0.08) -0.19 (0.09) -0.24 (0.08) 0.01 (0.08) Number of branches (age 5 y) 0.39 (0.06) 0.41 (0.07) 0.44 (0.07) 0.39 (0.07) 0.29 (0.07) Crown radius (age 5 y) 0.62 (0.04) 0.62 (0.05) 0.74 (0.04) 0.58 (0.05) 0.56 (0.05) Crown length (age 5 y) 0.52 (0.05) 0.64 (0.05) 0.76 (0.04) 0.57 (0.05) 0.49 (0.06) Note: Values in parentheses are standard errors

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20 (Ceulemans et al. 1990). Wu (1994a) also reported significant clonal variation in Populus hybrids in crown structural traits at the leaf, branch, and whole-tree levels. Traditionally, most complex traits, such as growth rate and crown architecture, are thought to be polygenic, determined by the expression of many genes (Falconer and Mackay 1996). This seems intuitive, given that growth rate and crown architecture are affected by many physiological parameters, phe nological patterns, organ growth rates, and also by environmental factors like competition interactions, seasonal variation in water availability, nutrient status, light intens ity and duration, air and soil temperature, pest and pathogen pressure. Our results agreed with the polygenic model in that crown architectural and growth traits had low to moderate within-family br oad-sense heritabilitie s, and are therefore likely determined by the expression of many ge nes. It is possible that the low genetic variation may be due to the nature of the trai ts we measured and thei r role in determining fitness. Traits connected with fitness often show low heritability, since natural selection for these traits reduces genetic variation, wh ile traits which are less intimately tied to fitness may have higher genetic variability and so higher heritability (Falconer and Mackay 1996). Tree growth rate and crown si ze are potentially impor tant components of fitness. Broad-sense heritabilities estimated from my study were expected to be smaller than broad-sense heritabilities values usually re ported in the literature, because they were estimated within full-sib families and half the additive genetic variation and one fourth of the dominance variation as well as a portion of the epistatic variance occurs among fullsib families (Falconer and Mackay 1996). Cons idering this, our results were comparable

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21 with other clonal studies. With respect to stem growth traits, Paul et al. (1997) reported a H2 of 0.14 for loblolly pine stem volume, while Borralho et al. (1992) estimated H2 between 0.08 and 0.18 for height and sapwood area in E. globulus. In crown structural traits, reported H2 values ranged from 0.27 to 0.78 in E. grandis and hybrid poplars (Lambeth et al. 1994; Wu 1994a). Narrow-sense heritability which includes only the additive genetic variation is necessarily smaller than broad-sense heritability for the same trait. For stem growth and crown structural traits, low to moderate narrow-sense heri tabilities (0.0 to 0.62) have been reported in loblolly and slash pine at young ages (Lambeth and Huber 1997; Xiao et al. 2003), as well as in other pine species as Pinus brutia, P. radiata and P. sylvestris (0.02 to 0.53; Espinel and Aragons 1997; Haapanen et al. 1997; Arregui et al. 1999; Isik and Isik 1999). One interesting finding was the heteroge neity of the variance components among families, which resulted in significantly differe nt within-family broadsense heritabilities for many traits. Slash pine family S2 showed higher H2 WF values compared to the other three slash pine families (Table 2-2). Higher within-family broad-sense heritability can reflect either a larger cl onal variance component ( 2 c in the numerator of H2 WF) or a smaller residual variance ( 2 in the denominator of H2 WF), or both. In family S2, a larger proportion of clonal variance and smaller re sidual variance component, were found with respect to the rest of the slash pine families, and resulted in larger H2 WF. Smaller residual variances in S2 corresponded also to a sma ller interaction between clone and microsite for that particular family than the other slash pine families. It is possible that the two parents of family S2 had great er proportion of heterozygosit y at gene loci determining

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22 crown size, producing more segregation among their progeny than in other slash pine families. If this is true, then even for polyge nic traits, it is possible to find specific pairs of parents producing more variable offspring for growth or crown traits. These families might be useful for quantitative trait loci (QTL) mapping and gene discovery (Bradshaw and Stettler 1995; Wu and St ettler 1996; Wu 1998). An understanding of the relationship betw een crown architecture and tree growth might provide a basis for predicting tree gr owth, and could aid in the search for discovering genes involved in growth and for developing new crop ideotypes (Kuuluvainen 1988; Dickmann and Keathley 1996; Martin et al. 2001). Evidence of positive phenotypic association between crown architecture and tree growth is common in many species, including l oblolly and slash pines, w ith many authors reporting the importance of the amount of light intercepted by the ca nopy and its correlation with growth rate (Linder 1987; Cannell 1989; Dalla-Tea and Jokela 1991; McCrady and Jokela 1998; Will et al. 2001). In my study, a number of crown architectural traits were consistently genetically correlated with growth (Table 2-3), which is co nsistent with previous quantitative genetic analysis of crown architectural traits in other species (Wu 1994b; Espinel and Aragons 1997; Haapanen et al. 1997; Arregui et al. 1999; Isik and Isik 1999), and production ecology work in loblolly and slash pine (e.g. McCrady and Jokela 1998; Martin and Jokela 2004). As we hypothesized, the more in tegrated measures of crown structure and function in my study, specifically APAR and crown volume, were consistently more strongly correlated with stem volume growth rate than we re less integrative measures such as crown radius or length, number of br anches, branch angle, or average branch

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23 diameter. APAR was a particularly comprehe nsive trait, providing a timeand spaceintegrated index of crown dimensional traits, leaf area, tree size, a nd crown dimension of surrounding competitor trees. It is interesting, ho wever, that the relatively simple trait of crown volume was as strongly or almost as st rongly correlated with stem volume growth as was APAR (Table 2-3). The quantity of APAR by tree crowns is one of the major factors determining aboveground biomass accumulation throughout stand development (Wang and Jarvis 1990). The amount of light intercepted by an indi vidual-tree crown is influenced by its leaf area quantity and disp lay, the incident radiat ion, and the distribution and size of surrounding trees (Wang and Jarvis 1990). Two crown traits consistently showed w eak or non-existent genetic relationships with growth: crown shape ratio and branch angle. Similar results were obtained by Lambeth and Huber (1997), where branch angle (zero being the closes t to horizontal) was weakly but negatively genetic co rrelated with growth rate (-0.24) (bigger trees tending to have flatter branch angle). In absolute term s, bigger trees tended to have wider crowns (rg=0.75), and large br anch diameter (rg=0.31), but when adjustments were made for size, they tended to have smaller branches a nd narrower crowns for their size and fewer branches per meter of height than smaller families. Xiao et al. (2003) reported for loblolly and slash pines families that crown shape ratio combined two important variables (crown height, crown width) that were sta tistically significant among taxa, but in ratio form as crown shape ratio appeared to have little ecological signifi cance in developing stands with respect to growth performa nce. Similarly, McCrady and Jokela (1996) observed significant intraspecific variation in crown shape ratio in young loblolly pine

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24 plantations, but they did not find an advantage of narrower crowns over wider crowns in height growth increment. In other species, such as P. radiata, P. sylvestris, Populus and E. grandis, significant positive genetic correlations were found among height, stem diameter, volume, crown diameter, and crown density and vigor. On the other hand, genetic correlations between growth and branch diam eter, and growth and branch angle were species specific and variable showing favorable or unfavor able correlations (Arregui et al. 1999; Espinel and Aragons 1997; Haapanen et al. 1997; Lambeth et al. 1994; Wu 1994b). With respect to environm ental correlations, microsites that favored the development of the crown, leaf area, and light interception also enhanced growth rate in all families. Branch angle and crown shape ratio showed non-significant environmental correlation with volume increment. Thus, micr osites with higher levels of nutrients or water availability appear to favor tree volum e growth, crown size and light interception at the same time, but do not seem to aff ect branch angle and crown shape ratio. Here we reported important linkage between crown struct ural and functional traits with stem volume growth in loblolly and slash pine families and clones. However, what is finally translated into stem volume incr ement depends on complex relations with other processes and their genetic patterns. Additiona l studies with respect to carbon gain, water relations and hydraulic conductiv ity at the individual-tree le vel will help improve our understanding of what control stem volume gr owth in contrasting families and clones.

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25 CHAPTER 3 GENETIC VARIATION IN BASAL-AREA INCREMENT PHENOLOGY AND ITS CORRELATION WITH GROWTH RATE IN LOBLOLLY AND SLASH PINE FAMILIES AND CLONES Introduction Loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm. var elliottii) are widely planted as commercial timber species in the southeastern Un ited States (Smith et al. 2004). From the early 1950s, large-scale tr ee breeding programs in the southeastern United States have worked to improve forest productivity by selecting trees for superior growth rate, form, and disease resistance (McKeand et al. 2003), and the improved material currently being established in comme rcial plantations is deployed from bulked orchard seed, half-sib families, and full-sib families with growing interest in the deployment of outstanding clones. The extensive natural range of loblolly and slash pines, spanning different environmental conditions, has resulted in accu mulation of adaptative genetic variation across time and differences in growth pot ential among sources (Burns and Honkala 1990). To develop tree breeding programs it is necessary to understand the genetic variation of selected traits their correlations and the effect of the environment on genotypic expression (White 1987) In Florida winter temper atures are rarely low enough to prohibit positive photosynthetic rates and co nsiderable transpiration (McGarvey 2000; Martin 2000). These mild winter conditions, plus an abundant rainfall through the summer, may have translated into genotypes adapted to a lo nger growing seasons and/or faster growth.

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26 Most pines experience a cycle of bud set and growth cessati on in the latter part of the growing season, followed by deepening dor mancy, cold hardening, dormancy release in the winter, and bud break in the spring (Dougherty et al. 1994). In the case of loblolly pine, the wide natural distribution, spa nning different ecotypes and environments, contains a diverse range of chilling requireme nts to promote dormancy release, length of the growing period and rate of growth. For instance, while it has been established that chilling is required for loblolly pine northern ecotypes, it is unclear that there is a true dormancy and chilling requirement for sout hern latitude sources (Carlson 1985). Increase in the diameter of tree stems o ccurs primarily from meristematic activity in the vascular cambium, a cylindrical late ral meristem located between the xylem and phloem of the stem, branches, and woody root s. The time of the year during which the cambium is active varies with climate, species crown class, seasonal development of leaf area in trees, and different pa rts of stems and branches (Kozlowski and Pallardy 1997). Fluctuations in environmental stresses affect cambial growth to a large extent by altering the supply of photosynthate to the branch es and stem (Kozlowski 1971; Sevanto et al. 2003). For example, cambial growth is sensitiv e to available water, with several aspects being responsive to the amount and seasonal di stribution of rainfall, including number of xylem cells produced and ring width, seasonal duration of cambial growth, proportion of xylem to phloem increment, time of latew ood initiation, duration of latewood production, and wood density (Kozlowski 1971; Cregg et al. 1988; Downes et al. 1999; Mkinen et al. 2000, 2001; Bouriaud et al. 2005). The amount of growth in a particular seas on is determined by the date of growth initiation, the date of growth cessation (whi ch together determine growth duration), and

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27 the average daily growth rate for the grow th period. The cessation of shoot and cambium activity is one determining factor, and the mo re fully the plant can utilize the growing season, without suffering from spring and fall frost, the greater potential annual growth, final harvest, and return on the investment in planting stock. Much of the interest in forest tree phenology is related with thes e practical questions (Lieth 1974). The total growth period fr om initiation to cessation, bot h for height and cambial activity, has been studied on an individual tree basis in many North American tree species, but little information on genetic variat ion is available. S easonal periodicity of tree growth has been studied in evergreen and deciduous trees (Jackson 1952; Harkin 1962; Langdon 1963; Emminham 1977; Li and Adams 1994; McCrady and Jokela 1996; Zhang et al. 1997; Jayawickrama et al. 1998; Yu et al. 2001). Wide variation among species in duration of the period of growth was recorded by Jackson (1952). Cambial growth of some species lasted only about 80 days and others grew for up to 200 days. Several of the species which initiated growth ear ly in the season had long periods of growth, while some of the late starti ng species exhibited shorter periods. Langdon (1963) studied growth patterns of slash pine (Pinus elliottii Engelm. var. densa Little and Dorman) in south Florida (F ort Myers) for four years and found that diameter growth occurred about ten months per year (from March through December). Initiation of diameter growth was believed to be promoted by apically produced hormones (Savidge and Wareing 1984). Diameter growth has been re ported to initiate before or almost simultaneously with height growth for loblolly pine (Zahner 1962) and for slash pine (Kaufmann 1977). Conifers usually continue diameter growth into the fall

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28 after height growth has stopped, as reported for Pseudotsuga menziesii and loblolly pine (Emmingham 1977; Jayawickrama et al. 1998). Previous research in loblolly and sl ash pine diameter growth phenology has provided important knowledge about the du ration of cambial activity (Jackson 1952; Harkin 1962; Langdon 1963; McCrady and Jokela 1996; Zhang et al. 1997; Jayawickrama et al. 1998). However, there is a lack of information about how the duration of cambial growth might influence the differences in growth rate between species planted in the same area, and also the growth differences among families within species and clones within families. My study examines the following hypotheses: There is significant genetic variation in basal-area growth phenology among slash and loblolly pine genotypes (species, families and clones); Where it exists, genetic varia tion in basal-area growth phenology is correlated with variation in annual ba sal-area increment. The specific objectives are to: Compare two years basal-area growth phenology among species, families and clones; Estimate genetic parameters for basal-ar ea growth phenology, its correlation with growth rates, and the genotype interac tion with seasonal environmental changes. Material and Methods Study Site and Plant Material The study area was located on lands manage d by Rayonier Inc. in Bradford County, Florida. The climate is humid and subtropi cal, with a mean annual temperature of 21C, mean annual rainfall of 1316 mm, and over 50% of the rainfall occurr ing in June through September. Periods of drought are normal in the spring and fall. Mean annual rainfall during 1999-2001 was 967 mm, in contrast to 1405 mm in year 2002 (NOAA 2002). The

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29 soils are classified as Pomona and consis t of very deep, somewhat poorly to poorly drained soils that are formed in sandy a nd loamy marine sediments (sandy, siliceous, hyperthermic Ultic Alaquods). Slopes are 0 to 2 %. In a typical profile, the spodic horizon occurs at 30-60 cm, with an argi llic horizon at 90-120 cm. Water table is typically at a depth of 15 to 45 cm for one to three months and a depth of 25 to 100 cm for six months or more, during most years (Soil Survey Staff 1998). The study took place in an area containing 16 full-sib and half-sib loblolly and slash pine families planted in 337 m2 family plots in January 1997. The experiment was designed as a randomized complete block with four replicates (Appendix A). We used one full-sib loblolly pine family and four full-sib slash pine families. Each family plot contained 60 clones propagated as rooted cut tings from a single family, planted at 1.7 m x 3.4 m spacing (1730 trees ha-1). Cuttings were taken from donor hedges in the spring, and were rooted and grown in a greenhouse for six months before planting. Each of the four plots of the same family contained the same 60 genotypes, but with the ramets planted into different, randomly-determined pl anting locations in the plot. In total we studied approximately 1,200 trees: 60 trees per family plot x 5 families x 4 replications. Fertilization and weed control were app lied periodically to reduce interspecific competition and prevent nutrient deficiency (Appendix B). Basal-area Increment Measurements Phenology was evaluated as periodic basalarea growth increment as determined from repeated DBH measurements th roughout growing seasons in 2002 and 2003. Families S1, S2, S3, L4 and S10 were monitored for diameter increment once a month in the summer time and every ten to fifteen days during the period of growth initiation and cessation in the spring and fall, respectively. Diameter increment was measured with a

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30 digital caliper (model 18 ES, Mahr, Germa ny, resolution 0.01 mm) over 4 plexiglass plates attached to the tree stem in north -south and east-west orientations. Diameter measurements were done such that two repl ications were measured on day 1 and two replications on day 2 each time period. Phenological Traits From the periodic diameter measurements, a cumulative basal-area growth curve for two growing seasons was plotted for each tree, and dates of basal-area growth initiation and cessation were estimated by interp olation as the dates when 5% and 95% of annual growth were completed (Hanover 1963). Duration of basal-area growth (in days) was calculated as the difference between da tes of cessation and initiation. Basal-area increment per year (in mm2) was calculated as the difference in individual tree basal-area between the 5% and the 95% dates of initiati on and cessation. Basal-area growth rate (in mm2/day) was calculated as the ratio of annua l basal-area increment and duration of basal-area growth. Meteorological Data and Water Balance Climatic data were collected at the Gainesville Regional Airport (about 20 km distant from the study site, NOAA 2003) and a research weather station 8 km from the study site. Meteorological variab les included hourly radiation, mean air temperatures, and daily rainfall. A simple water balance model was computed to estimate soil water reserves at daily time steps, and to quantify soil water deficit. The model was given by Equation 3-1, where Rn is soil water reserve at day n, Rn-1 is soil water reserve of the day before, Pn is precipitation and Tn is transpiration, both at day n: Rn = Rn-1 + Pn – Tn (3-1)

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31 The water holding capacity in 1 m depth for this site was estimated at 260 mm according to soil texture and flatwoods Spodosol s moisture release curves (H.L. Gholz, personal communication). Plot -level transpiration (Tn) was estimated as follows: maximum hourly potential evapotranspira tion (PET, mm) was cal culated by dividing measured hourly radiation by the latent heat of vaporization of water; maximum plotlevel transpiration was then calculated as 60% of PET, and was assumed to occur when all-sided leaf area index (LA I) was greater than 6.0. At LA I less than 6.0, transpiration was estimated to decline linearly with declin ing LAI (Martin and J okela 2004). Plot-level leaf area index was calculated from litterfall da ta as in Martin and Jokela (2004). Because understory vegetation was sparse, only pine LAI was considered. The resulting model incorporated variation in envi ronmental conditions (daily pr ecipitation, hourly radiation), as well as plot-level leaf area index to produce a plot-level index of soil water availability. Statistical Analyses and Genetic Parameters Analysis of variance (ANOVA) was us ed for phenological and growth data separately for each year. PR OC GLM in the SAS System was used to test for significance of random effects (c lone), while PROC MIXED was utilized to test the fixed effects (species and families). Equation 3-2 shows the linear model considered for the analyses, where Yijkl is the performance of the ramet of the lth clone within the kth family nested in the jth species in the ith replication; i = 1, 2, 3, and 4 for replications; j = slash, loblolly; k = 1, 2, 3, 4, and 10 for families; l = 60 identification numbers for 60 clones within each of the five families: Yijkl = + bi + Sj + Fk(j) + cl(jk) + bSij + bFik(j) + ijkl (3-2)

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32 = population mean, bi = random variable of replication ~ NID (0, 2b), Sj = fixed effect of speci es (slash or loblolly), Fk(j) = fixed effect of family nested within species, cl(jk) = random variable of clone nest ed within-family and species ~ NID (0, 2c), bSij = random variable for replicatio n x species interaction ~ NID (0, 2bS), bFik(j) = random variable for replication x family(species) interaction ~ NID (0, 2bF), and ijkl = error term ~ NID (0, 2 ). With so few families, estimates of genetic parameters were restricted to withinfamily estimates obtained from clonal variat ion expressed within each of the four slash families and one loblolly pine family. For each family two types of genetic parameters were estimated: within-family broad sense he ritability for each trait, and within-family genetic correlations among traits. Within-f amily variance and covariance components were obtained using ASREML, a statistical p ackage that fits linea r mixed models using Restricted Maximum Likelihood (Gilmour 1997). Within-family individual tree broad sense heritability was calculated using Equation 3-3, where 2 c is the variance among clones within-family and 2 is the residual variance as defined in Equation 3-2: 2 2 2 2 c c WFH (3-3) Theoretically, broad sense within-family her itability contains the additive genetic variance, of the dominance ge netic variance, and most of the epistatic genetic variance (Falconer and Mackay 1996). Th e standard error for heritabi lity estimates was calculated from Dickerson (1962). The residual likelihood ratio test (Wolfinger 1996) was used to test heterogeneity of variances among slas h pine families, and heritabilities were estimated separately ( 2 (6, 0.05)= 12.6), or pooled, as appropriate.

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33 Within-family genetic correlations among ba sal-area phenological traits and growth rate were calculated using Equation 3-4 (Falconer and Mackay 1996), where xy is the clonal covariance betw een two traits, and x and y are the square root of the product of the clonal variance within-family for traits x and y, respectively: y x xy xyr (3-4) Standard error for genetic correlations was estimated using ASREML (Gilmour 1997). The significance of the clone by year va riance component was tested using the likelihood ratio test (Wolfinger 1996). The clone by year variance component was declared different from 0 when 2 (1,0.05) was equal to or greater than 3.8. For traits with a significant clone by year vari ance component, within-family genetic correlations between years were estimated considering the two year s as two different traits using Equation 3-4. Results and Discussion Genetic Variation among Species and Families In 2002, species and families within species were not significantly different at 5% for any phenological or growth trait, while in year 2003, date of growth cessation and daily basal-area growth rate we re significant at the species level with loblolly pine ceasing growth earlier and growing more than slash pine (Table 3-1 and Figure 3-1). In 2002, the mean date of basal-area growth in itiation was March 10 (69 days after January 1) for the single loblolly pine family and one day earlier for the slash pine families. Basal-area growth cessation for the loblo lly pine family, on average occurred on November 1, resulting in a mean duration of ba sal-area growth of 236 days. In the case of

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34 slash pine families, mean cessation was on October 28, and the duration of basal-area growth was 234 days (Table 3-1). In 2003, basal-area growth started and fini shed one to two weeks sooner than in 2002 for both loblolly and slash pine families (T able 3-1). For loblolly pine, basal-area growth began in February 23 and finished by October 4, resulting in a mean duration of basal-area growth of 223 days (13 days fewe r than in 2002). For slash pine, basal-area growth began, on average, on February 25 and ended on October 17, for duration of 235 days (only one day shorter than in 2002, Table 3-1). In 2003, more growing days for slash pine compared to loblo lly pine might explain why the difference between species in total basal-area growth was less than in the previous year (26.98-23.81=3.17 cm2 in 2002 versus 24.26-21.98= 2.28 cm2 in 2003). This is supported by the fact that the slash pine families with longest (S1) and shortest (S3) duration had greater differences in basal-area growth in 2003 than in 2002 (24.94-22.12=2.82 cm2 in 2002 versus 22.8-19.69=3.11 cm2 in 2003). Annual basal-area increment and daily ba sal-area growth rate were larger for all families in 2002 than in year 2003, despite a shorter growing season for some families in 2002. Early cessation in year 2003 in comparis on with year 2002, and the difference in total annual increment between years could pos sibly be due to the differences in amount and seasonal distribution of rainfall be tween 2002 and 2003 (1405 mm and wet soil conditions by the end of the year in 2002; and 1184 mm and dry c onditions by the end of the year in 2003, Figure 3-2). In general, loblolly pine tended to accu mulate more stem volume through ages 6 and 7 y than slash pine. This was manifested by larger yearly and daily basis basal-area increments, but these differences among speci es were only significant (p<0.05) for daily

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35 basal-area growth in 2003 (Table 3-1). From my study we can conclude that the differences between loblolly and slash pine accumulated slowly over time through ages 6 and 7 y. Table 3-1. Significance levels (p-values), and species and family least square means for individual tree stem growth and phenol ogical traits for two growing seasons for loblolly and slash pine families in north central Florida. Trait Significance level by effect Species mean Slash family means Family Family Family Family Species Family Clone Slas h Loblolly S1 S2 S3 S10 Year 2002 Initiationa 0.8160 0.1120 0.0534 68.43 69.00 70.31 63.57 70.83 68.99 Cessationa 0.3542 0.1870 0.2141 302.16 304.82 306.43 301.66 301.44 299.12 Duration (days) 0.5456 0.2966 0.0131 233.71 235.82 236.13 238.03 230.59 230.10 Volume 6 y (dm3) 0.0797 0.1062 <0.0001 25.37 31.58 26.32 26.10 23.55 27.52 BA increment (cm2) 0.0784 0.0586 <0.0001 23.81 26.98 24.94 25.04 22.12 23.14 BA growth rate (mm2/day) 0.0945 0.2783 <0.0001 10.18 11.47 10.57 10.50 9.59 10.05 Year 2003 Initiationa 0.0815 0.3384 0.0062 55.85 53.86 56.21 54.94 56.79 55.45 Cessationa 0.0459 0.0853 0.0007 291.01 276.51 297.03 291.00 280.87 295.12 Duration (days) 0.0711 0.0774 0.0050 235.18 222.64 240.82 236.12 224.09 239.69 Volume 7 y (dm3) 0.0835 0.0687 <0.0001 38.31 44.30 38.67 38.87 34.67 41.03 BA increment (cm2) 0.1312 0.0894 <0.0001 21.98 24.26 22.80 22.83 19.69 22.60 BA growth rate (mm2/day) 0.0385 0.4796 <0.0001 9.35 10.81 9.45 9.63 8.87 9.45 a Initiation and cessation are days after Ja nuary 1 to complete 5 and 95% of seasonal diameter growth Day of the year 2002 050100150200250300350 Individual tree cumulative basal area (cm 2 ) 40 60 80 100 120 S1 S2 S3 L4 S10 Day of the year 2003 050100150200250300350 40 60 80 100 120 Figure 3-1. Family mean cumulative basal-ar ea growth curves for years 2002 and 2003 in loblolly and slash pine in north central Florida.

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36 050100150200250300350 -5 0 5 10 15 20 25 30 050100150200250300350 0 50 100 150 200 250 300 050100150200250300350 Individual tree daily basal area growth rate (mm2/day) -5 0 5 10 15 20 25 30 Slash Loblolly 050100150200250300350 Monthly Precipitation (mm) 0 50 100 150 200 250 300 Current Year 20 yr mean 050100150200250300350 Cumulative Precipitation (mm) 0 200 400 600 800 1000 1200 1400 1600 050100150200250300350 0 200 400 600 800 1000 1200 1400 1600 Day of Year 2002 050100150200250300350 Soil Water Balance (mm) 0 100 200 300 400 500 Day of Year 2003 050100150200250300350 0 100 200 300 400 500 A B C D EF GH* * *+ + + + + Figure 3-2. Species mean daily basal-area growth increment for loblolly and slash pine in north central Florida and environmental variables. Species mean daily basalarea growth increment in 2002 (A) and 2003 (B), where indicates significant differences between species (p<0.05) a nd + indicates significant differences among slash pine families (p<0.05); curre nt-year and 20-year mean monthly precipitation in 2002 (C) and 2003 (D); cumulative precipitation in 2002 (E) and 2003 (F); and mean plot-level soil water balance (error bars indicating standard errors) in 2002 (G) and 2003 (H). Precipitation data from Gainesville Regional Airport, NOAA (2003).

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37 Because phenology is closely related to latitude (Campbell 1986; Jayawickrama et al. 1998; Nielsen and Jrgensen 2003), growth initiation, cessation, and duration should depend on the study location and geographic orig in of the sampled seed or vegetative propagule. Our results are in agreement with what Langdon (1963) reported on growth patterns of slash pine in s outh Florida (Fort Myers). He found that diameter growth occurred about ten months during the year (from March through December), and the total amount of diameter growth and its seasonal di stribution responded to climatic variation. With respect to diameter growth cessa tion date, similar results were found by Jayawickrama et al. (1998), for example, a loblolly pine provenance from Gulf Hammock (Florida) grew until day 299 and 313 in two different years. Comparing studies done in north ern regions with slash and loblolly pine, our results showed earlier initiation date, later cessation date and longer season length. For example, in a site close to Athens, Georgia, Jacks on (1952) found that loblolly and slash pine started diameter growth betw een the end of March and the beginning of April, with a duration of five to six months. In South Ca rolina, McCrady and Jokela (1996) found that in loblolly pine diameter growth initiate d by the end of March and finished by AugustSeptember, giving mean diameter growth duration of 5 months. No significant differences were found among families in ini tiation or cessation of diameter growth. All families showed similar patterns of basal-area increment across the growing season in years 2002 and 2003, i.e. shapes of the cumulative basal-area curves were quite similar (Figure 3-1). In general, basal-area growth peaked in ear ly spring, and then remained relatively constant throughout the remainder of the growing season (Figure 3-1, 3-2). The differences at the species le vel and among families within slash pine

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38 accumulated across time. Daily average basalarea growth rate was only significantly different at the species level in 2003 (Table 31). Similar trends in diameter growth were found by others authors. Linear radial growth was observe d over the entire growing season in slash and loblolly pine trees by Jackson (1952), except for a period of slow growth in the late summer which was probabl y associated with soil moisture depletion. Similar linear trends were reported by Mc Crady and Jokela (1996) in loblolly pine families. Cregg et al. (1988) reported that unlike height growth, rapid diameter growth can be maintained over the entire growing s eason and the rate of diameter growth of loblolly pine, observed during a year when mo isture deficits did not develop was almost constant over the period from day 50 to day 290. Despite the apparent lack of variation in basal-area growth rate indicated by the cumulative growth data (Figure 3-1), peaks in basal-area increment occurred in the early spring both years (Figure 3-2). Significant sp ecies and family differences were found for critical spring periods when growth rates were highest: in year 2002 measurement period 3 (March), and in 2003 measurement periods 1 and 2 (end of February and middle March, respectively). Thes e results suggest that at least so me of the genetic differences in cumulative growth (as shown in Figure 31) are manifested not through constant expression of consistent growth rate differences, but rather through elevated growth rate during very discrete periods of time (as show n in Figure 3-2). In other words, the basalarea growth rates of taxa are remarkably sim ilar for most of the y ear, but in the spring some environmental variables or genetic di fferences in phenology trigger more rapid growth in some taxa, which essentially raises the intercept of the linear cumulative basalarea functions for the rest of the year (Figure 3-1). In other studies in loblolly and slash

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39 pine, peaks in basal-area increment in early spring also were reported by Zhang et al. (1997) and Langdon (1963); accelerating growth in spring was also reported in Norway spruce (Bouriaud et al. 2005). But these studies in c onifers did not identify genetic differences in tree growth rate at this tempor al scale. In the case of hardwoods, growth and phenology studies in hybrid aspen clones (Populus tremula x Populus tremuloides) compared growth patterns in temper ate climates throughout the year (Yu et al. 2001). Peaks in diameter growth occurred in the e nd of the spring and beginning of the summer. Hybrid clones had higher gr owth rates than the pure P. tremula and also accumulated larger annual diameter increment. In 2002, daily basal-area growth was weakly negatively correlated with soil water balance (Daily BA growth = 13.7929 – 0.0187 x soil water balance, R2=0.11, p<0.0001, Figure 3-3). In contrast, in 2003, daily basalarea growth was positively associated with calculated soil water balance (Daily BA growth = -1.9424 + 0.0478 x soil water balance, R2=0.49, p<0.0001, Figure 3-3). The total amount of rainfall in 2002 was 1405 mm, 177 mm above average. Wet conditions were pres ent especially between June and December and presumably had a negative effect on growth (Figure 3-2). On the other hand, in a year with average rainfall, like in 2003, where ra infall totaled 1184 mm (44 mm less than a normal year), a strong correlation was observed with more growth associated with higher levels of soil water availability. In year 2003, we found that daily basal-area growth followed same pattern as that of soil water balance (Figure 3-2B). For both years and both species, the highest growth rates in basa l-area were reached in conditions where the soil water balance was around 300 mm. This analys is implies that basa l-area growth rate increased as water soil ava ilability increased, when water was limiting, but excess water

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40 available in the soil had a ne gative effect on growth, perhap s caused by plant stress due to prolonged root inundation. Figure 3-3. Relationship between individua l tree daily basal-area increment and simulated daily plot-level soil water bala nce in loblolly and slash pine in 2002 (A) and 2003 (B). The line shows a li near regression thr ough data. A: Daily BA growth = 13.7929 0.0187 x soil water balance, R2=0.11, p<0.0001. B: Daily BA growth = -1.9424 + 0.0478 x soil water balance, R2=0.49, p<0.0001. Studies in flatwoods soils in north-centr al Florida have show n reduced radiation use efficiency when soil water balance was hi gh/wet, and this effect can have a direct impact on tree growth rates (Martin and J okela 2004). Langdon (1963) also reported that excess soil water appeared to depress growt h. From the four years of their study with slash pine, for the year with high rainfa ll (1929 mm) and high groundwater levels during summer and early fall, both diameter and he ight growth were considerably below the other 3 years. Water table depth was found to be associated with growth in the flatwoods in Florida (White and Pritchet t 1970). Larger height and diameter growth was reported in slash and loblolly pine with controlled wate r table depth conditions at 46 and 92 cm from the surface, in comparison with natural fl uctuating water table conditions. Bouriaud et al. (2005) studied the influence of climatic variables on annual radi al growth and wood density on Picea abies They found numerous decreases in radial growth rate closely related to the calculate d soil water deficit. Also, wood de nsity increased with decreasing Soil water balance year 2002 (mm) 0100200300400500600 Individual tree daily basal area increment (mm 2 /day) -10 0 10 20 30 Soil water balance year 2003 (mm) 0100200300400500600 -10 0 10 20 30 Slash Loblolly A B

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41 radial growth rate in the second half of the growing s eason affected by drought. Similar results were reported by Cregg et al. (1988), where early season di ameter growth rate for loblolly pine was a function of availa ble soil moisture and temperature. Clonal Variation and Within-Family Inheri tance of Phenological Traits and Stem Growth At the clone within-family level (pooled across families), differences in initiation, cessation and duration of basal-area increment in the growing season were more apparent than at the family and species level differe nces in both 2002 and 2003 (Table 3-1). Traits related to individual tree stem growth, such as volume, and yearly and daily basal-area increment were also different among clones within families in both years (Table 3-1). Analyses of the data separately by family showed that phenologica l traits differed among clones for some families and not for others in both years. Family S2 was the only one that showed significant clonal vari ation in all phenolo gy traits in 2003. Volume, yearly basalarea increment, and daily basa l-area growth had significant clonal variation within-family in 2002 and 2003 for all families (Table 3-2). For phenology traits, indivi dual tree broad sense heri tabilities were low to moderate, ranging from 0.01 to 0.24 (Table 3-3). In contrast, within-family heritabilities for stem growth traits were moderate to high in both years ranging from 0.10 to 0.37 (Table 3-3). Family S2 tended to have higher within-family broad sense heritabilities than the other slash pine families, in most cases due to higher clonal va riation within that family as opposed to lower residual envir onmental variance. These heritabilities are expected to be smaller than broad sense heri tabilities values usually reported in the literature, because they are estimated within full-sib families and half the additive genetic variation and one fourth of the dominance vari ation as well as a portion of the epistatic

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42 variance occurs among full-sib families (Falconer and Mackay 1996). Still, phenotypic expressions of phenological trai ts associated with basal-ar ea growth were under weak genetic control. Table 3-2. Significance levels (p-values) for clone within-family for tree stem growth and phenological traits for two growing seasons in loblolly and slash pine families in north central Florida. Trait Significant level within-family (clonal variation) Family L4 Family S1 Family S2 Family S3 Family S10 Year 2002 Initiation 0.0003 0.0311 0.1964 0.1577 0.1880 Cessation 0.3200 0.3489 0.1145 0.1541 0.3753 Duration 0.0695 0.1608 0.0498 0.2409 0.1593 Volume 6 y 0.0015 <0.0001 <0.0001 0.0720 0.0390 BA increment <0.0001 0.0042 <0.0001 0.0191 0.0277 BA growth rate <0.0001 0.0028 <0.0001 0.0279 0.0286 Year 2003 Initiation 0.0253 0.4211 0.0011 0.0706 0.4932 Cessation 0.2165 0.3028 0.0028 0.1302 0.1550 Duration 0.3215 0.3770 0.0068 0.1146 0.3893 Volume 7 y 0.0001 0.0003 <0.0001 0.0206 0.0318 BA increment <0.0001 0.0043 <0.0001 0.0524 0.0004 BA growth rate <0.0001 0.0016 <0.0001 0.0536 <0.0001 Heritability estimates for phenological trai ts are available for a few species and are usually presented for leaf phenology rather than basal-area or shoot phenology. For example, narrow-sense h2 estimates ranged between 0.67 to 0.96 in Juglans nigra, and between 0.28 and 0.71 in Picea glauca for initiation and cessation of leaf development, depending on experimental conditions, age, a nd method of computations (Leith 1974). In pole-size P. menziesii, individual tree heri tabilities were higher for bud burst and bud set (h2=0.73 and 0.81, respectively) than fo r duration of shoot growth (h2=0.17, Li and Adams 1993). With respect to diameter growth, Li and Adams (1994) estimated individual heritabilities for diameter growth initiation (h2=0.23), and cessation (h2=0.11) in 15 y-old P. menziesii, values that are comparable with my study. At the same time, Li and Adams (1994) did not detect significant fa mily differences in duration of diameter

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43 increment, suggesting that the small variation in date of diameter growth cessation among families may have been related to summer dr y conditions. Other studies have shown that summer drought has little effect on variation in cambial growth initiation, but reduces variation in cambial growth cessation among coastal P. menziesii provenances (Emmingham 1977). In my study, we did not detect an associa tion between clonal variation within-family for initiation or cessa tion and soil water balance and the presence of relatively dry spring or late summer. Table 3-3. Within-family individual-tree broa d-sense heritabilities for growth phenology traits and basal-area growth increment by year in loblolly and slash pine families growing in north central Florida. Trait H2 WF Family L4 Family S1 Family S2 Family S3 Family S10 Year 2002 Initiation 0.20 (0.07) 0.10 (0.06) 0.07 (0.07) 0.09 (0.08) 0.00 (0.00) Cessation 0.00 (0.00) 0.00 (0.00) 0.08 (0.07) 0.07 (0.08) 0.00 (0.00) Duration 0.08 (0.06) 0.07 (0.06) 0.11 (0.07) 0.05 (0.08) 0.00 (0.00) Volume 6 y 0.18 (0.07) 0.25 (0.07) 0.26 (0.08) 0.10 (0.08) 0.10 (0.07) BA increment 0.24 (0.07) 0.15 (0.07) 0.37 (0.08) 0.15 (0.09) 0.13 (0.07) BA growth rate 0.23 (0.07) 0.19 (0.04)a Year 2003 Initiation 0.12 (0.07) 0.03 (0.06) 0.24 (0.08) 0.10 (0.09) 0.03 (0.06) Cessation 0.05 (0.06) 0.05 (0.06) 0.19 (0.08) 0.08 (0.08) 0.09 (0.07) Duration 0.04 (0.06) 0.05 (0.06) 0.16 (0.08) 0.09 (0.08) 0.03 (0.06) Volume 7 y 0.20 (0.07) 0.20 (0.07) 0.29 (0.08) 0.14 (0.09) 0.11 (0.07) BA increment 0.34 (0.07) 0.21 (0.05)a BA growth rate 0.32 (0.07) 0.23 (0.05)a Note: Values in parentheses are standard errors a Values of H2 WF in slash pine were pooled by fam ily since variance components were homogeneous. Genetic Correlations among Phenological Traits and Stem Growth When phenological traits did not differ significantly among cl ones within-family, genetic correlations were not estimated. Am ong the estimates, many genetic correlations relating phenology traits to growth were not si gnificantly different from zero (Table 3-4). In 2002, genetic correlations between initiati on and duration were st rong and negative in family L4, S1 and S2, which indicated that cl ones with early growth initiation also had a

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44 tendency to grow longer, and that clones that initiated later also tended to have a shorter growing season. On the other hand, genetic correlations between cessation and duration were positive and strong in family S2 and S 3, meaning that clones th at had a tendency to cease growth late in the year also grew for a longer period of time. In 2003, genetic correlations between init iation and cessation were significant and moderately positive only for family S2. The ge netic correlations were positive and strong between cessation and duration for all families, meaning that clones that stopped growth later also grew for a longer period of time. In general, these results suggest that variation in duration of the growing season among individuals in these families was more a function of cessation date than initiation date, bu t all of these traits were weakly inherited (Table 3-3). With respect to genetic correlations between stem growth variables and phenological variables, signifi cant correlations were found primarily in family S2, varying from moderate to strongly po sitive (r = 0.31 to 0.85, Table 3-5). In 2002, duration had a positive strong genetic correla tion with basal-area increment in S2. In 2003, initiation, cessation, and duration had mode rate positive genetic correlations with basal-area increment in S2. At the same time initiation in 2003 for L4 showed a strong positive genetic correlation with basal-area increment (rg=0.86). Among the variables we investigated, daily basal-area growth rate in both years showed the strongest genetic correlation with yearly basal-area increment across all families. Correlations of phenology variables with total volume afte r the 2002 and 2003 growing seasons were similar to the patterns of correlation with yearly basal-area increment, reflecting consistency between phenology and increm ent during the year and phenology and

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45 cumulative stem growth. These results suggest that clones that gr ew faster between initiation and cessation were al so the ones with more yearly basal-area increment and total volume. The high genetic correlation be tween daily basal-area growth rate and yearly basal-area increment in a year was expl ained in part because of the autocorrelation between these two variables. Genetic correlations among clonal values for basal-area growth and phenological traits are scarce in the literature; most of the reported results are phenotypic correlations at the family level. One of the few studies on genetic control of cambial phenology found that P. menziesii genotypes with early growth initiati on also tended to cease growth early (rg=0.60, Li and Adams 1994). They also suggest ed that variation in growth duration among individuals is primarily a function of variation in date of growth cessation (rg=0.77). Height phenology studies in P. abies in northern Europe s howed that early start of shoot growth was genetically correlated with early shoo t growth cessation. Also, there was a consistently low or no correlation betw een the shoot elongation period and either total height or leader length (Ekberg et al. 1994). Reported phenotypic correlati ons in southern pines for both diameter and height growth are more closely relate d to growth rate, than with phenological traits such as cessation (McCrady and Jokela 1996; Jayawickrama et al. 1998). Jackson (1952) found that there was no consistent relationship betw een the starting date a nd faster growth in slash and loblolly pine trees. For P. menziesii saplings, most of the differences among populations in one season’s growth were rela ted to growth rate rather than growth duration (Emmingham 1977). In Picea mariana, cambial growth cessation and total

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46Table 3-4. Within-family genetic correlations between growth phenology tr aits in 2002 (above the di agonal) and 2003 (below the diagonal) in loblolly and slash pine familie s growing in north central Fl orida. I: initiation; C: cessation; and D: duration of basal-area growth. Trait Family L4 Family S1 Family S2 Family S3 Family S10 I C D I C D I C D I C D I C D I a -0.98 (0.33) a -0.85 (0.41) -0.47 (0.91) -0.82 (0.35) 0.46 (0.72) 0.02 (0.86) a a C 0.96 (0.61) a 0.60 (2.06) a 0.67 (0.28) 0.91 (0.21) -0.25 (0.72) 0.89 (0.17) 1.00 (2.6) a D 0.93 (1.04) 0.99 (0.06) 0.49 (2.59) 1.00 (0.09) 0.56 (0.34) 0.99 (0.01) -0.42 (0.67) 0.98 (0.02) 1.00 (2.6) 1.00 (0.31) Note: Values in parentheses are standard errors a Was not estimated because with in-family clonal variance was 0 Table 3-5. Within-family genetic correlations between growth and phenology traits by year in loblolly and slash pine families g rowing in north central Florida Traits Basal-area increment 2002 Basal-area increment 2003 L4 S1 S2 S3 S10 L4 S1 S2 S3 S10 Initiation -0.05 (0.26) -0.12 (0.45) a a a 0.86 (0.27) a 0.59 (0.22) 0.22 (0.84) a Cessation a a a a a a a 0.59 (0.23) a a Duration 0.32 (0.35) a 0.85 (0.28) a a a a 0.53 (0.25) a a BA growth rate 0.99 (0.01) 1.00 (0.01) 0. 99 (0.00) 0.99 (0.01) 0.99 (0.01) 1.00 (0.01) 0.99 (0.01) 0.99 (0.02) 0.96 (0.09) 0.99 (0.02) Volume age 6 Volume age 7 Initiation 0.03 (0.29) 0.06 (0.37) a a a 0.94 (0.32) a 0.31 (0.25) 0.76 (0.50) a Cessation a a a a a a a 0.66 (0.23) a a Duration 0.29 (0.40) a 0.83 (0.36) a a a a 0.65 (0.25) a a BA growth rate 0.99 (0.04) 1.00 (0.00) 0. 99 (0.02) 0.91 (0.11) 1.00 (0.11) 1.00 (0.03) 0.89 (0.06) 0.93 (0.04) 0.89 (0.13) 1.00 (0.13) Note: Values in parentheses are standard errors a Was not estimated because with in-family clonal variance was 0

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47 height had a positive phenotypic corr elation. Although in continental P. abies populations, this correlation was zero and sometimes negative (Dietrichson 1967, 1969). On the other hand, studies in aspen hybrids in temperate regions suggested that the fast overall growth is largely e xplained by longer vegetative period (rp between growth period and diameter was 0.67-0.91 and highly significant, Yu et al. 2001). Because cambial phenology traits appear to be weakly inherited and have small and inconsistent genetic correlati ons with growth, indirect re sponses in cambial phenology from selection of bole basal-area or volume are expected to be small. The practical implications of these findings are that select ion programs aimed at increasing growth rate are very unlikely to impact dates of initiati on or cessation; thus th ere are few concerns about increasing the likelihood of frost damage. Also, another important point to consider in indirect responses, as suggested by Langdon (1963), is the effect of length of growing season on wood properties. Trees that are capab le of growing longer into the season may produce a higher proportion of summerwood to spring wood and have higher wood density than genotypes that cease growth earl y. Future research could help to understand whether families or clones which cease gr owth earlier do, in fact, have lower wood density. If so, this could then be in corporated into selection programs. Analysis across Years 2002-2003 There were no significant cl one by year interactions fo r any basal-area phenology traits, and only basal-area increment for L4 and basal-area growth rate for S2 showed significant clone by year interact ions (data not show n). Still, genetic correlations between years were high for these two traits with sign ificant clone x year interactions (0.91 for L4 basal-area increment, and 0.93 for S2 basalarea growth rate), indicating that the interactions were not biologica lly important. From this anal ysis we can conclude that

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48 each of the basal-area growth phenology traits and each of the basal-area growth rate traits were genetically controlled by a sim ilar set of genes in years 2002 and 2003. So, the clones were consistent across years in bot h phenology and growth traits that we measured. Nevertheless, stability needs to be tested for a longer period of time, since environment played an important role in th e control of the inheri tance of basal-area phenology traits (i.e. severe environmental conditions may change the results). Clonal studies in Betula pendula revealed significant clone -by-year interactions for bud burst and also large inte rannual variation among clones in the date of bud burst and, especially, in the termination of growth (Rousi and Pusenius 2005) These interactions indicated that in addition to genetic effects, environmental factors have a strong influence on both bud burst and growth termination. In P. menziesii, provenance-by-year interaction existed for bud burst da te in a three-ye ar study (White et al. 1979). In a twoyear study with loblolly pine, Jayawickrama et al. (1998) found no significant provenance-by-year interaction; and signi ficant year-by-family within provenance interactions were found for height growth and height growth cessation. We conclude that the significant genetic variation among clones within-family in basal-area growth and the stab ility of ranks across years found in my study, contribute to understanding the potential imp act that clonal selection can have on future forest plantation productivity. Poor cons istency in direction and stre ngth of genetic correlations between basal-area increment a nd phenological traits indicate d that in these slash and loblolly pine families, initiation, cessation or duration of growth were traits that did not have biological importance in determining how much a genotype will grow during the season. Basal-area growth in loblolly and slash pine families and clones was sensitive to

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49 soil water availability, with stem growth declining both above a nd below an “optimum” soil water balance level. Finally, while ther e were significant size differences among taxa (species and families) at age 6 y and 7 y, gene tic differences in basal-area growth rate were only expressed during short, discrete time periods in the spring and fall. This finding may have important implications for the timing of investigations attempting to determine the mechanisms underlying genetic gr owth differences, since growth rate, and possibly the physiological or gene expression traits controlling growth rate, may be similar throughout most of the growing seas on among taxa with contrasting long-term cumulative growth.

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50 CHAPTER 4 CARBON ISOTOPE DISCRIMINATION, CROWN CONDUCTANCE, GROWTH AND THEIR GENETIC PARAMETERS IN LOBLOLLY AND SLASH PINE FAMILIES AND CLONES Introduction Plants fractionate carbon isotopes duri ng photosynthesis. The magnitude of the fractionation varies with photos ynthetic type, environment, genotype, and other factors, and this variation in magnitude can be used to study a variety of issues in plant physiology (O’Leary 1981, 1988, 1993; Farquhar et al. 1989). During photosynthesis, the stable isotope ratio (13C/12C) of carbon dioxide assimilated differs from that of the source CO2 and is about 2 % lower in plants than air (Farquhar et al. 1989). There are two primary processes that cause carbon isotope ratios to change during photosynthesis: diffusional fractionation and enzymatic fractionation. Carbon dioxide molecules containing 12C are lighter, and therefore, diffuse into the leaf at a faster rate (by a factor of 1.0044, or 4.4 ‰) than molecules containing 13C (Craig 1954; Farquhar and Lloyd 1993). The primary carboxylating enzyme in C3 plants, ribulose-1,5-biphosphate carboxylase, preferentially uses 12CO2 (by a factor of 1.029 or 29‰) and so discriminates against 13CO2 (Roeske and O’Leary 1984; Guy et al. 1993). The carbon isotope ratio of leaf organic material depends on the relativ e influence of diffusional and enzymatic fractionation, which in turn is determined by the ratio of intercellular CO2 (pi) and atmospheric CO2 (pa) partial pressures (Farquhar et al. 1982, 1989). Changes in the ratio pi/pa and the leaf carbon isotope ratio are a function of changes in either, or both, photosynthetic rate and stom atal conductance (Farquhar et al. 1989).

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51 Since the carbon isotope ratio in the l eaf provides information about processes integrated over the whole life of a leaf, it is particularly useful for examining subtle genetic differences in photosynthetic and water use characteristics. There has been considerable interest in usi ng carbon isotope discrimination ( 13C, determined from carbon isotope ratio of the sample with respec t to a standard; lower discrimination against 13C, means 13C value closer to 0, than high discrimination against 13C) to estimate integrated water use efficiency (WUE) in both agronomic plants and trees. WUE measures the ratio between photosynthetic rate (A) and transpiration rate (E), or in other words the ratio between carbon fixation and water losses. Genetic variation in 13C has been reported for several tree species. Family differences in 13C were reported for Pseudotsuga menziesii (Zhang et al. 1993), Larix occidentalis (Zhang et al. 1994), Picea mariana (Flanagan and Johnsen 1995; Johnsen et al. 1999), Picea glauca (Sun et al. 1996), Araucaria cunninghamii (Prasolova 2000), Pinus pinaster (Brendel et al. 2002), Castanea sativa (Lauteri et al. 2004). Significant clonal variation has also been demonstrated in foliar carbon isotope composition in F1 hybrids clones between slash pine (Pinus elliottii) and Caribbean pine (Pinus caribaea) (Xu et al. 2000; Prasolova et al. 2003, 2005), in Eucalyptus globulus (Osorio and Pereira 1994, Osorio et al. 1998), loblolly pine (Pinus taeda) (Gebremedhin 2003) and poplar hybrid clones (Marron et al. 2005). Therefore, understandi ng of the genetic basis of variation in 13C could be very useful for ranking genotypes and may serve as a guide for tree breeding programs. Most studies reported in the literature on 13C and WUE in tree species have been associated with a small number of species or genetic entries. Only a few recent

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52 publications (Johnsen et al. 1999; Prasolova et al. 2003) have reported the results of 13C with relatively large sets of genetic material s in tree breeding trials and these have been used to obtain reasonable estimates of genetic parameters such as heritability for foliar 13C and genetic correlations be tween physiological traits. Fr om an operational point of view, such information is crucial when intr oducing new genetic entries, such as untested clones, into plantation schemes. The growth of individual plants may be either positively or negatively correlated with leaf carbon isotope discrimination values depending on whether variation in discrimination is associated with change s in photosynthetic capacity or stomatal conductance (Farquhar et al. 1989). During photosynthetic ga s exchange, discrimination will be reduced in a plant when photosynthetic rate is increased, if stomatal conductance remains constant. The higher photosynthetic rate may also translate into faster growth, if other factors remain constant. Therefore, ca rbon isotope discrimination values should be negatively correlated with plant growth when variation in discrimination results from changes in photosynthetic rates (Farquhar et al. 1989). In contrast, if variation in discrimination is caused by changes in st omatal conductance, then carbon isotope discrimination values should be positively associated with growth. This results because an increase in stomatal conductance will resu lt in higher assimilation of carbon, thereby increasing growth, and will also enhance discrimination against 13C during gas exchange (Farquhar et al. 1989). Previous results have suggest ed that carbon isotope ratio in the leaf can be used in early selection in tree improvement programs (Farquhar et al.1989; Bond and Stock 1990; Zhang et al.1993; Sun et al. 1996; Johnsen et al. 1999; Xu et al. 2000; Pita et al.

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53 2001; Prasolova et al. 2003). Due to the advantages of early selection in tree improvement programs of loblolly and slas h pine, the use of the carbon isotope ratio technique might help to increase forest pr oductivity in future pl antations by selecting families or clones that show greater water us e efficiency or photosynthetic rate or a combination of both. Stomata respond to environmental varia tion and regulate water loss and carbon dioxide gain, and thus biosphere–atmosphere exchange of mass and energy. Ideally, stomatal conductance should remain in bala nce with variations in soil-leaf hydraulic conductance. This coordination would contribut e to maintenance of leaf water potential above minimum values associated with l eaf desiccation, nonstomatal inhibition of photosynthetic carbon acquisition, and xylem cavitation (Wullschleger et al. 1998). In the last decade, development and calibration of techniques that allow measurement of water movement through the sapwood and crown as sap flow, make it pos sible to calculate crown conductance parameters such as stomatal sensitivity to changes in environmental conditions, like radiation and va por pressure deficit in longer spatial and temporal scales (Granier 1987; Martin et al. 1997; Ewers et al. 1999; Martin et al. 2001; Lu et al. 2004; Martin et al. 2005). Stomatal sensitiv ity to environmental changes will affect gas exchange levels, photosynthesis, carbon fixa tion and growth (Sperry 2000; Tyree 2003). Because one of the processes that define ca rbon isotope discrimination in the leaf is stomatal conductance, it is important to know the correlation between discrimination and conductance in loblolly and slash pine clones. In this paper we examine how changes in carbon isotope discri mination are related to both differences in stem growth increm ent and differences in tree-level crown

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54 conductance observed within full-sib families and clones of loblolly and slash pine. The following hypotheses were considered: Carbon isotope discrimination varied among genotypes; Fast-growing genotypes tend to have lo wer carbon isotope discrimination and higher water use efficiency, so stem growth will be negatively correlated with leaf stable carbon isotope discrimination; and Genotypes that tend to have higher stomatal sensitivity to change s in water pressure deficits tend to have lower va lues of discrimination against 13C. Material and Methods Study Site and Plant Material The study area was located on lands manage d by Rayonier Inc. in Bradford County, Florida. The climate is humid and subtropi cal, with a mean annual temperature of 21C, mean annual rainfall of 1316 mm, and over 50% of the rainfall occurr ing in June through September. Periods of drought are normal in the spring and fall. Mean annual rainfall during 1999-2001 was 967 mm, in contrast to 1405 mm in year 2002 (NOAA 2002). The soils are classified as Pomona and consis t of very deep, somewhat poorly to poorly drained soils that are formed in sandy a nd loamy marine sediments (sandy, siliceous, hyperthermic Ultic Alaquods). Slopes are 0 to 2 %. In a typical profile, the spodic horizon occurs at 30-60 cm, with an argi llic horizon at 90-120 cm. Water table is typically at a depth of 15 to 45 cm for one to three months and a depth of 25 to 100 cm for six months or more, during most years (Soil Survey Staff 1998). The study took place in an area containing 16 full-sib and half-sib loblolly and slash pine families planted in 337 m2 family plots in January 1997. The experiment was designed as a randomized complete block with four replicates (Appendix A). We used one full-sib loblolly pine family and four full-sib slash pine families. Each family plot

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55 contained 60 clones propagated as rooted cut tings from a single family, planted at 1.7 m x 3.4 m spacing (1730 trees ha-1). Cuttings were taken from donor hedges in the spring, and were rooted and grown in a greenhouse for six months before planting. Each of the four plots of the same family contained the same 60 genotypes, but with the ramets planted into different, randomly-determined pl anting locations in the plot. For growth and carbon isotope discrimination we studied a pproximately 1,200 trees: 60 trees per family plot x 5 families x 4 ramets per clone distri buted as one ramet in each of the 4 complete blocks. For sap flow and crown conductance analysis we measured approximately 300 trees: 30 trees per family x 5 families x 2 ramets per clone distributed across the 4 complete blocks as described later. Fert ilization and weed control were applied periodically to reduce interspecific competition and prevent nutrient deficiency (Appendix B). Tree Growth and Carbon Isotope Discrimination Stem volume growth in the 2001, 2002, and 2003 growing seasons (ages 4-5 yr, 5-6 yr, and 6-7 yr, respectively) was determined from dormant season measurements of tree diameter at 1.37 m height (DBH) and total tree height (HT). Outside-ba rk individual tree stem volume was calculated using Equation 4-1 (Hodge et al. 1996), where DBH and HT were entered in m: VOL (dm3) = (0.25 3.14 (DBH)2 (1.37 + 0.33 (HT 1.37)))*1000 (4-1) Also, periodic diameter increment was m easured in March-April 2002 to calculate sapwood cross-sectional area for tree-level transpiration analysis as explained later. Foliar samples for stable carbon isotope di scrimination analysis were collected from the five families from the first flush fo rmed in the spring. Samples were taken in middle summer 2001 and 2003 from a branch on the south side of the upper canopy

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56 (exposed to full sun, to help avoid extraneous differences in isotope values). The tissue was dried at 65 C for several days, a nd finely ground. The relative abundance of 13C and 12C was determined in 3 mg subsamples with a Delta Plus isotope ratio mass spectrometer (Cornell University Stable Isot ope Laboratory). Stable carbon isotope ratio ( 13C) was expressed as 13C/12 C ratio relative to internat ional PDB (Pee Dee Belemnite, Craig 1954). Carbon isotope discrimination values ( 13C) were calculated from 13 C values using Equation 4-2 (Farquhar et al. 1989), where p is the isotope composition of the plant material and a is that of the air (assumed to be -8‰): p p a 1 (4-2) The accuracy and precision of this analysis for foliar 13C were ascertained by making repeated measurements of 13C in each batch of the samples and using an internal foliar standard in each of the sample ba tches. We concluded that carbon isotope measurements are repeatable and accura te with a standard error of 0.14‰. Use of 13C to compare A/E among genotypes requi res several assumptions: first, that leaf temperatures, and th erefore leaf-to-air vapor pre ssure differences, are similar among the plants being compared; second, that 13C of source of CO2 is identical among genotypes being compared; and third, that bi osynthetic fractionati on is similar among the genotypes. These assumptions are well met by the needle shape of conifer leaves, because they are narrow and their boundary layer conductances are therefore high (Marshall and Zhang 1993, Ewers and Oren 2000). Meteorological Data Air temperature, photosynthetically active radiation, and relative humidity were measured in year 2002 by a weather station installed in the study area. Variables were

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57 read every minute and averages recorded every 15 minutes by a data logger. Vapor pressure deficit (D) was calculated from relative humidity (RH) and air temperature (TA) measurements based on equations adap ted from Goff and Gratch (1946). Individual Tree Transpiration A subsample of 300 trees from 5 families (30 clones per family and 2 ramets per clone) was used to monitor sap flow on a daily basis. Selection of clones within families was based on three criteria: genotypes we re selected across th e range of growth performance (good, medium and poor grower s); genotype performance for growth was consistent across replications in the test; and finally genotypes we re free of disease. Selection of ramets within clone was done by selecting the two ramets more representative of the growth perfor mance category and free of disease. Water flux in the xylem was estimated usi ng the constant heat method of Granier (1987). Heat dissipation gauges we re installed in each of th e 300 trees, a constant 0.2 W of power was applied to the probe, and the degr ee to which heat is dissipated from the probe was measured. A heat probe of 20 mm l ong was inserted into the tree stem, at 40 cm from the base, and was paired with an unheated probe located 10 cm below the heated probe. To avoid thermal gradients from direct radiation, all sensors were installed in the north side of the stem and covered with aluminum shelters. Measurements of sap flux were taken every minute and stored as a 15-min average for two months (March-April in 2002) with dataloggers (Campbell Scientific, Logan, UT). Equation 4-3 shows how sap flux (JS, kg H2O m-2 s-1) was calculated using an empirical relationship (Granier 1987): JS = 0.119 (( Tm – T)/ T) 1.231 (4-3)

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58 which measured the daily maximum temp erature difference between heated and unheated probes during times of zero flux ( Tm) as a baseline. Temperature difference ( T) was also measured duri ng the day as water carried heat away from the probe. Deviation from the baseline was used to estimate water flux. Whole-tree transpiration per unit of projected crown area (EL, kg H2O m-2 AC s-1) was calculated by multiplying tree sapwood cross-sectional area (AS, m2) by the sap flux density measurement and standardized by projected crown area (AC, m2, calculated as projected crown area on the ground): EL = JS (AS/AC) (4-4) To estimate EL we assumed that water uptake, as estimated from sap-flow measurements, does not significantly lag actual canopy transpiration. Calculations of individua l tree sapwood cross-secti onal area contained two assumptions: 1) For young southern pine, the en tire cross-sectional area of the tree was composed of active sapwood; and 2) Sap flux density for sapwood further than 20 mm from the cambium (where sap flow probes ar e located) was the same as outer cambium. The first assumption was probably met in these young pine trees, while the second was almost certainly not. Several studies have show n that sap flux density tends to be higher near the cambium, and dec lines with radial depth into the sapwood (Phillips et al. 1996; Wullschleger and King 2000; James et al. 2002). However, for the purposes of my study, the estimates of whole-tree water use were acknowledged to be biased upward, but should still be useful for rela tive comparison of the genotypes. Crown Conductance and Stomatal Sensitivity Calculations We calculated whole-tree crown conductance of water vapor (GS, mm s-1) by substituting the transpiration data and meteorological meas urements into the inverted

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59 Penman-Monteith equation using the formul a suggested by Monteith and Unsworth (1990), where is the latent heat of evaporation of water (J kg-1), is the psychrometer constant (kPa K-1), a is the density of dry air (kg m-3), Cp represents the specific heat capacity of the air (J kg-1 K-1), and D is the vapor pressure deficit (kPa): GS = 1000 (EL ) / ( a Cp D) (4-5) GS values were converted from mm s1 to mmol m2 s1 using Pearcy et al. (1989). Equation 4-5 requires the following c onditions (Ewers and Oren 2000): (1) D is close to the leaf-to-air vapor pressure deficit, na mely boundary layer c onductance is high; (2) There is no vertical gradient in D through the canopy; and (3) There is negligible water stored above the JS measurement position. We assumed that these conditions were met at my study. As the vapor pressure deficit between l eaf and air increases, stomata generally respond by partial closure (Lange et al. 1971). Responses of stomatal conductance to increasing D generally follow an exponen tial decrease described by the empirical Equation 4-6 (Oren et al. 1999), where -m is the sensitivity of GS response to lnD or the slope of GS vs lnD (-dGS/dlnD) and Gsref = GS at D = 1 kPa: GS = -m lnD + Gsref (4-6) Given the uncertainties under low levels of incoming radiation (or limited light throughout the canopy) and low D situations, we filtered the data to conditions where D 0.6 kPa and incoming radiation > 500 Wm-2. This screening allowed us to keep errors in the estimation of GS below 10% (Ewers and Oren 2000).

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60 Genetic Parameters and Statistical Analyses Analysis of variance (ANOVA) was used to analyze 13C, crown conductance parameters in response to vapor pressure de ficit, and growth data by year. SAS PROC GLM was used to test for significance of random effects (clone), while PROC MIXED were utilized to test the fixed effects (sp ecies and families). Equation 4-7 shows the linear model considered for the analyses, where Yijkl is the performance of the ramet of the lth clone within the kth family nested in the jth species in the ith replication; i = 1, 2, 3, and 4 for replications; j = slash, loblolly; k = 1, 2, 3, 4, and 10 for families; l = 60 identification numbers for 60 clones within each of the five families: Yijkl = + bi + Sj + Fk(j) + cl(jk) + bSij + bFik(j) + ijkl (4-7) = population mean, bi = random variable of replication ~ NID (0, 2 b), Sj = fixed effect of species (slash or loblolly), Fk(j) = fixed effect of family nested within species, cl(jk) = random variable of clone nested within-family and species ~ NID (0, 2 c), bSij = random variable for replicatio n x species interaction ~ NID (0, 2 bS), bFik(j) = random variable for replication x family(species) interaction ~ NID (0, 2 bF), and ijkl = error term ~ NID (0, 2 ). With so few families, estimates of genetic parameters were restricted to withinfamily estimates obtained from clonal variat ion expressed within each of the four slash families and one loblolly pine family. For each family two types of genetic parameters were estimated: within-family heritability for each trait, and within-family genetic and environmental correlations among traits. Within family variance and covariance components were obtained using ASREML, a st atistical package that fits linear mixed models using Restricted Maximum Likelihood (Gilmour 1997).

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61 Within-family individual-tree broad-sense heritability was calculated as in Equation 4-8, where 2 c is the variance among clones within family and 2 is the residual variance as defined in Equation 4-7: 2 2 2 2 c c WFH (4-8) The standard error for heritability estim ates was calculated from Dickerson (1962). The residual likelihood ratio te st (Wolfinger 1996) was used to test heterogeneity of variances among slash pine families, and he ritabilities were es timated separately ( 2 (0.05,6)= 12.6), or pooled, as appropriate. Within-family genetic and envi ronmental correlations between 13C and growth rate and between 13C and crown conductance parameters were calculated with the Equation 4-9 (Falconer and Mackay 1996), where xy is the clonal or residual covariance between two traits, while x and y are the square root of the product of the clonal or residual variance within family fo r traits x and y, respectively: y x xy xyr (4-9) Standard error for genetic and environm ental correlations was estimated using ASREML (Gilmour 1997, asymptotic propertie s and the Taylor series approximation of the variance of a ratio). The significance of the clone-by-year variance component wa s tested using a likelihood ratio test (Wolfinger 1996). The clone-by-year variance component was declared different from 0 when 2 (1,0.05) was equal to or greater than 3.8. For traits with a significant clone-by-year va riance component, w ithin-family genetic correlations

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62 between years were estimated considering th e two years as two different traits, using Equation 4-9. Results Carbon Isotope Discrimination Family within-species and clone within-fam ily were significant sources of variation for foliar carbon isotope discrimination in 2001 and 2003 (Table 4-1), but at the species level no significant differences were detected (p<0.05). Mean family values for 13C ranged between 21.39‰ to 22.98‰ (Table 4-1), and at the clonal level mean values for 13C ranged from 19‰ to 25.41‰ (data not shown). Among slash pine families, family S2 tended to have the lowest values for 13C in both years 2001 and 2003 (Figure 4-1). In contrast, family S3 showed the highest value for both years. Lower values of 13C in year 2001 than in year 2003 samples for all families might be associated with lower rainfall between February and June 2001 in comparis on with same period in 2003 (near 50 % less rainfall in year 2001 than in year 2003, Figure 4-2). Clonal within-family genetic variation was significant in all 5 families for 13C in 2001, and for all families except S3 in 2003 (Table 4-2). Within-family heritabilities ranged from 0.01 to 0.32 for discrimination in years 2001 and 2003 (Table 4-3). There was no eviden ce for genotype-by-year interaction for any family. A strong within-family genetic correlation between 13C in year 2001 and year 2003 occurred for all families (Table 44), indicating that the ranking of clones remained constant across years fo r carbon isotope discrimination.

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63 Family S1S2S3S10L4 CID (‰) 21.2 21.4 21.6 21.8 22.0 22.2 22.4 22.6 22.8 23.0 23.2 2001 2003 Figure 4-1. Family means and standard erro rs in carbon isotope di scrimination in year 2001 and 2003. Months 0123456789101112 Rainfall (mm) 0 200 400 600 800 1000 1200 1400 2001 2003 Normal Figure 4-2. Accumulated m onthly precipitation in year s 2001, 2003 and mean normal year from Gainesville Regional Airpor t, Gainesville, Florida (NOAA 2003).

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64Table 4-1. Significance levels (p-values), species and within family least square means for volume, carbon isotope discriminati on ( 13C) for three growing periods, an d crown conductance variables (Gsref and Gssensitivity ) in slash and loblolly pine families in north central Florida. Significance level by effect Speci es mean Slash family means Variable Code Species Family Clone Slash Loblolly S1 S2 S3 S10 13C Year 2001 (‰) CID2001 0.9005 <0.0001 <0.0001 22.14 22.16 22.30 21.39 22.65 22.24 13C Year 2003 (‰) CID2003 0.2852 0.0175 <0.0001 22.53 22.76 22.65 22.08 22.98 22.39 Gsref (mmol m-2 s-1) Gsref 0.0840 0.4340 0.2195 754.36 553.86 798.98 702.17 735.94 780.33 Gssensitivity (mmol m-2s1 ln(kPa)-1) Gssensitivity 0.0910 0.1640 0.0801 465.91 330.55 511.17 410.11 457.54 484.84 Volume increment age 4-5 (dm3 tree-1) VI45 0.0472 0.0767 <0.0001 7.32 9.41 7.40 7.42 6.55 7.90 Volume increment age 6-7 (dm3 tree-1) VI67 0.8318 0.0786 <0.0001 12.29 12.51 12.35 12.35 10.97 13.47 Table 4-2. Significance levels (p-values) for clone within fam ily in carbon isotope discriminati on for two growing periods and crown conductance variables for loblolly and slash pine families in north central Florida. Significance level within family (clonal variation) Trait L4 S1 S2 S3 S10 13C 2001 <0.0001 0.0006 0.0009 0.0208 <0.0001 13C 2003 0.0025 <0.0001 0.0070 0.5361 <0.0001 Gsref 0.0384 0.2386 0.4424 0.3361 0.6081 Gssensitivity 0.0195 0.1801 0.3011 0.2645 0.6019 VI45 0.0112 0.0003 <0.0001 0.2153 0.0043 VI67 0.0032 0.0289 0.0001 0.0401 0.0234

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65 Table 4-3. Within-family individual-tree br oad-sense heritabilities for stable carbon isotope discrimination ( 13C) by year, and crown c onductance variables in loblolly and slash pine families growing in north-central Florida Trait Family L4 Family S1 Fam ily S2 Family S3 Family S10 13C 2001 0.23 (0.08) 0.20 (0.04)a 13C 2003 0.17 (0.07) 0.32 (0.09) 0.14 (0.07) 0.01 (0.00) 0.25 (0.08) Gsref 0.30 (0.23) 0.08 (0.09)a Gssensitivity 0.38 (0.23) 0.22 (0.19) 0.11 (0.19) 0.21 (0.19) 0.00 (0.00) VI45 0.12 (0.07) 0.19 (0.04)a VI67 0.16 (0.07) 0.15 (0.04)a Note: Values in parentheses are standard errors a Variance components were pooled across slash pine families Table 4-4. Genetic correlations betw een years 2001 and 2003 by family for carbon isotope discrimination ( 13C) and between 4-5 yr and 6-7 yr stem volume increment (VI) for loblolly and slash pine families in north central Florida. Trait Family L4 Family S1 Fam ily S2 Family S3 Family S10 13C 0.70 (0.23) 0.82 (0.17) 1.00 (0.25) --a 1.00 (0.14) VI 0.90 (0.15) 0.78 (0.13) 0.96 (0.05) 0.62 (0.42) 0.81 (0.17) Note: Values in parentheses are standard errors a -Genetic variation was not significant in carbon isot ope discrimination year 2003 Stem Growth Clonal differences in stem growth incremen ts (age 4-5 y and age 6-7 y) were more apparent than differences at the family and species levels (Table 4-1). Loblolly pine tended to growth faster than slash pine. W ithin slash pine, family S10 was the fastest grower and S3 was the slowest in both years (Table 4-1). We found greater rates of stem volume increment in year 2003 than in 2001 for all families most likely to higher rainfall during the growing season in 2003 (Figure 4-2) Clone within-family variation changed from one family to another in terms of signi ficance, with family S2 having the highest variation among clones and family S3 the sm allest clonal variation in stem volume increment (Table 4-2). This difference in clonal variation was directly related with levels of inheritance. However, when testing fo r significance of clona l variance components among slash pine families, differences were not detectable at p=0.05, so families were pooled to increase precision of heritability estimates for both years of measurements. In

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66 general, within-family heritabilities for stem growth increment were low to moderate in both loblolly and slash pine (0.12 to 0.19, Table 4-3). On the other hand, the clone-byyear interaction component wa s not significant and year-to-y ear genetic correlations were high for all families, except for family S3 in which the correlation was moderate with a wide confidence interval (Table 4-4). The lack of year-by-clone interaction indicates that clones that had a high value for stem volume in crement at age 4-5 y also had a high value for the same trait at age 6-7 y. Whole-Tree Crown Conductance and Stomatal Sensitivity Whole-tree level crown conductance was calculated at 15 minute intervals for approximately 300 trees (half of the ramets per clone, and half of the clones per family in 5 families). Tree-level stomatal conductance wa s negatively associated with D, with an exponential decrease in GS as D increased, as shown for a representative slash pine ramet (Figure 4-3). From this re lationship we estimated GSref, defined as the value of GS when D=1 kPa, and GSsensitivity which quantified the sensitivity of GS to changes in D, solving the parameters in Equation 4-6 for each ramet. Across all 5 families, there was a significant linear relationship between GSref and GSsensitivity with no intercept (averaged across family R2=0.77, data not shown). Genetic variation in GSref and GSsensitivity was difficult to detect at the species, family and clonal levels (p<0.05). This may be cau sed by microsite variation and sample size (Table 4-1). GSref and GSsensitivity were 36 and 40% higher in sl ash pine than in loblolly pine (p=0.08 and 0.09, respectively). Conducta nce in slash pine genotypes tended to be more sensitive to changes in D than loblolly pine genotypes. At the same time, slash pine families had on average higher GSref than loblolly pine, m eaning that slash pine conductance to CO2 was higher at a reference D of 1 kPa.

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67 D (kPa) 0.51.01.52.02.53.03.5 Crown conductance (mmol m-2 s-1) 0 5 10 15 20 25 Tree 7 Figure 4-3. Representative relationship between canopy average stomatal conductance (GS) and vapor pressure defi cit (D) on a half hourly ba sis for a typical slash pine ramet. When we analyzed clonal variation family by family, only family L4 showed significant differences for GSref and GSsensitivity (Table 4-2), and variab le levels of genetic control were found among slash pine families. Within-family individual-tree broad-sense heritabilities were high for GSsensitivity in family L4 (H2 WF=0.38), lower in slash pine families S1, and S3, and very low in fa milies S2 and S10 (Table 4-3). For GSref a moderate level of heri tability was found in family L4, but low heritability was found for the pooled slash pine families This result was likely influenced by lack of genetic variation in family S10. Interestingly, with in-family broad-sense heritabilities for crown conductance parameters and 13C were generally higher than for stem growth increment, meaning that these physiological traits were under stronger genetic control than growth traits. The same time, however, heritability estimates for crown conductance parameters were associated with large standard errors (Table 4-3).

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68 Genetic Correlations between Carbon Isotope Discrimination and Growth and Whole-Tree Crown Conductance Genetic correlations among families were not significantly different from zero between 13C and stem volume growth (Table 4-5). Only the genetic correlation between 13C year 2001 and stem volume increment age 4-5 yr in family S10 was significantly negative (-0.54), meaning that faster growing clones showed less discrimination against 13C during gas exchange. In 2003, family S3 had no variation for is otope composition so its correlation with growth increment could not be estimated. Of the nine estimable correlations for the 5 families across 2 years, the average genetic correlation was -0.26 which may indicate a slightly negative ge neral relationship between carbon isotope discrimination and growth in these slash and loblolly pine clones. Table 4-5. Within-family corr elations between volume incr ement of the growing season and stable carbon isotope discrimination ( 13C) by year in loblolly and slash pine families growing in north-central Florida Trait Volume increment Family L4 Family S1 Family S2 Family S3 Family S10 Genetic 13C 2001 0.09 (0.31) -0.29 (0.27) 0.05 (0.26) -1.00 (0.80) -0.54 (0.24) 13C 2003 0.01 (0.32) -0.38 (0.26) -0.33 (0.29) -a 0.01 (0.31) Environmental 13C 2001 -0.05 (0.08) -0.08 (0.08) -0.21 (0.08) -0.10 (0.09) -0.08 (0.08) 13C 2003 -0.05 (0.08) -0.40 (0.06) -0.32 (0.08) -0.33 (0.08) -0.28 (0.07) Note: Values in parentheses are standard errors a -Not estimated because genetic variati on was not significant in carbon isotope discrimination year 2003. We found non-significant envir onmental correla tions between 13C and stem volume increment in year 2001, and low nega tive environmental correlations for all families in year 2003 but family L4. We estimated genetic and environm ental correlations between mean 13C and crown conductance parameters using averages since not all measurements were made in the same year. For 13C we took the average between sa mples collected in years 2001

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69 and 2003 per individual tree (leaves formed in the springs of both years), and for crown conductance we estimated the parameters from data collected from March through April 2002. We assumed that the environmental condi tions in the springs of all years were similar. Despite the considerable effort of measuring sap flow in 300 trees, and estimating integrated crown conductance parame ters, we could not ac cept or reject our hypothesis that related low 13C with high stomatal conductan ce sensitivity to changes in D. We found that genetic and environmenta l correlations were unstable across families and had wide confidence inte rvals, so they were not significantly different from zero (Table 4-6). This lack of precision in the estimation can be related to two sources: (1) small sample size; and (2) sensitivity of the physiological measurements to subtle changes in microsite. The lack of correlations between 13C and crown conductance parameters might be due to the separation in years of sap flow measurement and leaf collection, so the assumption of similar environment was not valid. Table 4-6. Genetic and environmental co rrelations between mean carbon isotope discrimination (mean 13C) and GSref and GSsensitivity for loblolly and slash pine families in north central Florida Trait Mean 13C Family L4 Family S1 Family S2 Family S3 Family S10 All slash Genetic Gs ref -0.22 (0.40) -0.20 (0.43) 0.97 (1.07) -0.39 (0.79) --a 0.24 (0.29) Gs sensitivity -0.16 (0.35) 0.06 (0 .40) 0.84 (0.51) -0.41 (0.70) --a 0.28 (0.24) Environmental Gs ref 0.12 (0.17) -0.04 (0.17) 0.01 (0.17) 0.12 (0.19) -0.17 (0.15) 0.00 (0.08) Gs sensitivity 0.10 (0.18) -0.03 (0.17) -0.05 (0 .17) 0.17 (0.19) -0.13 (0.16) 0.00 (0.09) Note: Values in parentheses are standard errors a -Not estimated because genetic variati on was not significant in carbon isotope discrimination year 2003 Discussion Information on clonal variation in southern pines has become more common in the last two decades (Foster 1988; Paul et al. 1997; Isik et al. 2003; Schmidtling et al. 2004; Baltunis et al. 2005). At the same time, clonal forest ry appears to offer an excellent

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70 opportunity for the early capture of the be nefits generated by tree improvement and biotechnology programs (Ahuja and Libby 1993; Libby and Ahuja 1993; Schmidtling et al. 2004). The novelty of my study was its anal ysis of clonal genetic variation among families, the number of clones involved per family, and the possibility of analyzing growth and physiological tr aits under field conditions. We found significant within-family clonal genetic variation in 13C and stem volume increment in both years of measurem ents, reflecting a wide spectrum of clonal performance for growth and gas exchange. At the same time, greater rates of stem volume increment were detected in year 2003 compared to 2001 for all families. These differences may have been caused by varia tion in seasonal rainfall pattern and total amount of annual precipitation, or simply due to tree size. There ar e few reports in the literature of clonal variation in lobl olly or slash pine growth. Paul et al. (1997) reported that height of loblolly pine clones varied si gnificantly at different ages, but that DBH and volume did not. To our knowledge, no other published studies have quantified clonal variation in 13C in loblolly or slash pine under field conditions. Clonal variation in foliar 13C fluctuated from family to family, with family S3 having the lowest range of clonal values. In general, the range of phenotypic clonal mean values we found in our selected families (fro m 19 to 25.41‰) had a wider distribution in comparison to what had been reported in sim ilar studies; for example, in F1 hybrid pine clones between slash pine and Caribbean pi ne (19.6 to 20.7‰ and 18 to 21.84‰, Xu et al. 2000, Prasolova et al. 2003, respectively), loblolly pine clones (23.3 to 22.3‰, Gebremedhin 2003). In P. menziesii, family means 13C ranged between 19.7 to 22.43‰ (Zhang et al. 1993), and in E. globulus family means for 13C ranged between 16.7 to

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71 18.1‰ (Pita et al. 2001). Genetic variation in 13C should reflect differences in Ci/Ca, a consequence of the balance between stomatal supply and mesophyll demand of CO2 (Farquhar et al. 1989). In my study, low to moderate leve ls of heritabilities for growth, 13C, and crown conductance parameters suggest ed that these are complex traits determined by the expression of many genes, each one having a small effect on the phenotypic expression of the individual (Falconer and Mackay 1996). However, the heritabilities we estimated are expected to be smaller than broad sense heritabilities values us ually reported, because they are estimated within full-sib families and half the additive genetic variation and one fourth of the non-additive vari ation occurs among full-sib fam ilies (Falconer and Mackay 1996). Levels of genetic control for 13C in a clonal study reported low heritabilities of 0.08 in loblolly pine under different watering regimes (G ebremedhin 2003), and 0.09 to 0.15 in F1 hybrid slash x Caribbean pine (Prasolova et al. 2003). In other conifers, studies were carried out in full-sib families and 13C narrow-sense herita bility range from low to moderate, 0.54 for P. mariana (Johnsen et al. 1999), 0.17 in P. pinaster (Brendel et al. 2002), between 0.4 to 1.0 in A. cunninghamii (Prasolova 2000). In the hardwood C. sativa, narrow sense heritability was moderate (h2=0.31, Lauteri et al. 2004). Genetic analysis of variation in cr own conductance parameters has not been reported in the literature, so it was difficult to make compar isons with slash, loblolly or other pine species or hardwoods. Nevertheless, the within-family, indivi dual-tree broad-sense heritabilities values we reported for w hole-tree crown conductance and stomatal sensitivity were large in several cases (T able 4-3). However, the precision of our estimates were low. Increased precision likely requires a much larger number of

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72 replications per clone than wa s possible in my study. Heterogeneous soil conditions in the study site might have lowered he ritabilities due to lack of opt imal silvicultural treatments (weed control and fertilization in several years). Consistency of genotypic ranking across ye ars is essential for breeding to be effective in modifying a particular quanti tative trait. We found significant positive correlations across years for bot h stem volume increment and 13C. Similar results in consistency in across years in 13C have been reported in P. mariana (Johnsen et al. 1999). In my study we tested the hypot hesis of correlations between 13C and stomatal sensitivity to changes in D. We found negativ e results in the sense that wide confidence intervals around the es timation in genetic correlations gave small confidence in making conclusions or possible expl anations on association between these two variables. However, future research in this ave nue is needed to understand the underlying mechanism behind the intrinsic photosynthesi s-stomatal conductance relationship. Here, we analyzed the relationship between 13C and crown conductance, but the knowledge of the relationship of 13C with photosynthetic capacity is also needed. Then, we can understand if genetic variation in 13C is due to changes in stomatal conductance, or in photosynthetic capacity, or both. We hypothesized that traits which integr ated information over space and/or time would be more highly correlated with gr owth (see Chapter 2). In this case, 13C corresponds to an integrated measurement of photosynthesis and stomatal conductance during the time of formation of the leaf. Our results did not support that hy pothesis, and genetic correlations between 13C and stem volume increment were not stable across

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73 families, across years, and not significantly different from zero. We can conclude that 13C and stem growth were controlled by largel y independent sets of genes. Similarly, environmental correlations between 13C and stem volume increment were low, meaning that microsites which increased discrimination, also increased or decreased stem growth independently. The observed independence of 13C from stem growth and the absence of year-byclone interaction in both growth and 13C still provide opportunities for selecting loblolly and slash pine clones combini ng high productivity and high water-use efficiency (low 13C). The changes from year to year in gene tic and environmental correlations between these two traits might be associated with changes in seasonal weather patterns, for example the amount of rainfall and soil mois ture conditions during the time of leaf formation in the case of 13C and the total growing season in the case of stem volume increment. As in Figure 4-2, rainfall between February and June was lower in year 2001 than in 2003, and by the end of the 2001 grow ing season, the decrease in rainfall may have affected growth increment too. Unfortunately, we did not measure soil moisture content to confirm this hypothesis. On the other hand, the presence of mild weather years in my study, where stem growth was not limited by water supply and wate r use efficiency may affect the degree of correlations between 13C and stem growth increment. Condon and Richards (1993) showed that in wheat genotypes, the rela tionship between crop biomass production and leaf carbon isotope discrimination values ch anged when crops wher e grown on differentquality sites. It was only on th e driest site that the negativ e relationship between growth and leaf carbon isotope discrimination predic ted from gas exchange characteristics was

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74 supported for wheat genotypes (Condon and Richards 1993). The same situation was described in P. mariana by Flanagan and Johnsen (1995), where the strongest correlation between height and 13C was found in the driest site. In C. sativa, the genetic correlations between 13C and growth traits were generally strong and negative (-0.5 to -1.0), especially in two high temp erature treatments (Lauteri et al. 2004). In the literature, negative, posit ive, or no correlations between 13C and growth have been reported. Low, moderate and strong negative genetic correlations were reported in some conifers, as for example P. mariana (-0.96, Johnsen et al. 1999), F1 hybrid between slash pine and Caribbean pi ne (-0.19 to -0.36 depending on site and sampling season, based on clonal means, Prasolova et al. 2003; and -0.83 to -0.96 based on clonal means, Xu et al. 2000), P. menziesii (-0.65 to -0.67, Zhang et al. 1993). Positive phenotypic correlations have been demonstrated in eucalyptus species, like E. globulus (Pita et al. 2001), and some eucalyptus hybrids (Le Roux et al. 1996), and also in loblolly pine clones (0.86, but might be a ssociated to wide standard errors because of low heritability, Gebremedhin 2003). No genetic correlations at all between 13C and ring width was found in P. pinaster (0.02), and in agreement there was the lack of colocation of QTLs between both traits (Brendel et al. 2002). Similarly, Marron et al. (2005) did not find a significant correlation between discrimination and total biomass in hybrid poplar clones. Some authors explain the lack of consistent correlation between 13C and growth in pines as follows: (1) if 13C is mainly determined by assimilation rate, and if growth is not primarily determined by assimilation rate then there might be no correlation between 13C and growth (Brendel et al. 2002); (2) if genetic control is moderate for both traits,

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75 this might lower the significance of a genetic correlation (Brendel et al. 2002); and (3) if water supply is not limiting growth, then wa ter use efficiency might not be defining growth (Prasolova et al. 2003). On the other hand, differences in a llocation of carbon be tween photosynthetic tissue and root can alter the relationship between 13C values and growth when water is not limiting. For example slash or loblolly pi ne clones that have high discrimination, and a low ratio of photosynthesis to stomatal conductance, may also have a high ratio of photosynthetic tissue to root ti ssue. A higher allocation to p hotosynthetic tissue on a site that is not limited by water availability however, may overcome any restriction on growth imposed by low assimilation rates (Flanagan and Johnsen 1995). Future research in analyz ing associations between 13C and photosynthetic capacity will be needed, and also the corres ponding measurements of amount of leaf area. Some authors supported the thesis that differe nces in photosynthetic capacity have been observed to be the primary cau se of genetic variation in 13C in several coniferous species (Flanagan and Johnsen 1995; Guehl et al. 1995; Johnsen and Major 1995; Sun et al. 1996; Johnsen et al. 1999; Xu et al. 2000; Prasolova et al. 2003, 2005). Also, the possibility to repeat the study in the same field with severe weather conditions is recommended to make comparisons with our re sults. Replicated studies in different site conditions are also a good source of inform ation to capture genetic variation across different environments.

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76 CHAPTER 5 SUMMARY AND CONCLUSIONS Loblolly and slash pines are widely plan ted as commercial timber species in the southeastern United States. Knowledge about the biology of physiological processes and their genetic parameters give us insight into what are the key func tional and structural traits that determine ge notype performance differences in southern pines. The overall goal of this disse rtation was to investigate biological traits and the genetic structure of these traits in 300 clones from five differe nt full-sib loblolly and slash pine families. One peculiarity of this study was the number of clones represented in each full-sib family and also the advantage of havi ng them in field conditions at an early stage of stand development. The study was divided in three main areas of research: Detailed quantification of crown structure and estimati on of the total amount of radiation absorbed by each tree over a year using the proc ess model MAESTRA; Seasonal dynamics and phenology of basal area growth and its association with soil water balance; Leaf carbon isotope discriminati on and whole-tree sap flow The common objectives in each main research area of the study were to: Determine species, family within-species and clone within-family genetic variation for all variables measured or estimated; Where genetic variation exists, estima te genetic control and environmental influence on structural and functional variables Based on the results of the previous chapte rs the main conclusions and implications of this study were summarized into three themes:

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77 Genetic variation among species and families; Clonal variation and within-family inheritance; Correlations Genetic Variation among Species and Families Differences in stem growth and crown st ructural traits between species and among slash pine families were subtle. In general, th e one loblolly pine family we studied tended to grow faster than the average of our four sl ash pine families at ages 5 yr and 6 yr. At the same time, loblolly pine deve loped larger crowns with more acute branch angles and had more leaf area per individual-tree at age 5 yr an d 6 yr than did the slash pine families. In spite of the apparent similarities in stem volum e growth rate, the four slash pine families differed in a number of crown architectura l traits. Contrasting families had different arrangements and sizes of branches within the crown, and varied in crown shape ratio. This suggests that any of a number of crown traits may be associat ed with high growth rate in southern pine families. When we analyzed the repeated basal area growth measurements we found that loblolly pine tended to have la rger yearly and daily basis basal area increments than slash pine at ages 6 and 7 yr. From this study, we concluded that the differences between loblolly and slash pine accumulated slowly over time through ages 6 and 7 yr. Loblolly and slash pine families considered in this study tended to grow about eight months per year, from March through October. We did not find significant differe nces at species and family level in initiation, cessation or duration of basal area growth both years 2002 and 2003. In both years, peaks in basal area incremen t occurred in short (2-3 week) periods in the early spring for all families, followed by relatively constant rates of basal area growth

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78 until cessation. While there were significan t size differences among taxa (species and families) at age 6 yr and 7 yr, genetic differences in basal area growth rate were only expressed during short, discrete tim e periods in the spring and fall. When we studied environmental effects on seasonal basal area growth, we found that basal area growth rate increased during periods when water soil availability increased (up to 300 mm), but an excess in water availa bility in the soil had a negative impact on growth. Integration of climatic data with physiological variab les and soil conditions in a water balance model allowed us to better unde rstand the interactions between basal area growth in loblolly and slash pine fa milies and soil water availability. The study of leaf and crown integrated physiological processes, such as 13C and whole-tree sap flow gave us the opportunity to explore genetic variation at the species, family and clonal level in slash and loblolly pines at larger temporal and spatial scales and in much more detail than is typical in field ecophysiolo gical investigations. Family within-species was a significant source of variation for foliage carbon isotope discrimination in 2001 and 2003, but at the sp ecies level no significant differences were detected. Genetic variation in GSref and GSsensitivity was difficult to detect at the species level and among the four slash pine families. Microsite variation and the small sample size may have been responsible. Conductance in slash pine genotypes tended to be more sensitive to changes in D than loblolly pine genotypes. At the same ti me, slash pine families on average had higher crown conductance per unit of projecte d crown area than loblolly pine. Clonal Variation and Within-Family Inheritance Within-family clonal variation was highly significant for all growth and crown structural traits, reflecting a wide spectrum of clonal performance in growth and crown

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79 development at these ages. Within-family individual-tree broad-sense heritabilities (H2 WF) were low to moderate for stem volume a nd crown structural traits for all five families (0.05 to 0.41). One interesting result we found was the heterogeneity in variance components among slash pine families; there wa s a tendency for higher heritabilities in family S2 than the rest of the slash pine families in many traits, meaning that even for polygenic traits, it is possible to find specifi c pairs of parents producing more variable offspring for growth or crown structural traits. H2 WF for basal area growth phenol ogical traits ranged from low to moderate for all traits (0.00 to 0.24). In general, heritabil ities were higher for growth traits than for phenological traits for all families. Clone within-family was a significant source of variation for foliage 13C in 2001 and 2003, but not for crown conductance parameters. H2 WF for 13C and crown conductance parameters were in general higher than that for stem growth increment (0.01 to 0.38), meaning that these physiological traits were under stronger genetic control than growth traits. But, at the same time heritability estimates for crown conductance parameters were associated with large standard errors. Correlations As we hypothesized, the more integrated m easures of crown structure and function in this study, specifically AP AR and crown volume, were consistently more strongly correlated with stem volume growth rate than were less integrative measures such as crown radius or length, number of branches, branch angle, or average branch diameter. At the same time, microsites that favored th e development of the cr own, leaf area, and light interception also enhanced growth rate in all families. Branch angle and crown shape ratio showed non-significan t environmental correlation with volume increment. An

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80 understanding of the relationship between cr own architecture and tree growth might provide a basis for predicting tree growth, a nd could aid in the search for discovering genes involved in growth and fo r developing new crop ideotypes. Both the strength and direction of correla tion between basal area phenological traits and basal area growth rate varied across families and years, and many times was not significantly different from zero. There were no significant clone-by-ye ar interactions for any basal area phenology traits. We can conc lude that each of the basal area growth phenology traits and each of the basal area growth rate traits were genetically controlled by a similar set of genes in years 2002 and 2003. Genetic correlations between 13C and stem volume increment were not stable across families, across years, and not significantly different from zero. It might be that in the years 2001 and 2003, weather and field conditions were mild enough throughout the growing season that stem growth was not limited by water supply and water use efficiency, and the genetic correlation between 13C and stem volume increment did not have any biological importance. There was no evidence of genotypeby-year interaction in any family for 13C and stem volume increment, indicating that the ranking of clones remained constant between years. We f ound non-significant environmental correlations between 13C and stem volume increment in year 2001, and low negative environmental correlations for all families in year 2003. Genetic and environmental correlations between 13C and stomatal sensitivity to changes in vapor pressure deficit were di fficult to conclude due to wide confidence intervals for all families.

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81 In conclusion, for the particular loblolly and slash pine families studied here, there was a wide spectrum of clonal within-fam ily performance in stem growth, crown development, and measured physiological tra its, making interesting the possibility of clone within-family se lection for traits that increase productivity. We found low to moderate levels of within-family heritability in many key structural and functional traits (crown structure, basal area growth phenology, 13C, and crown conductance parameters), but just crown structural traits had stable and higher genetic correlation with stem growth increment. Here we reported important linkage between crown structural and functional traits with stem volume growth in loblolly and slash pine families and clones. However, what is finally translated into stem volume incr ement depends on complex relations with other processes and their genetic patterns. Additiona l studies with respect to carbon gain, water relations and hydraulic conductiv ity at the individual-tree le vel will help improve our understanding of what controls stem volume gr owth in contrasting families and clones. The results from this study should positiv ely impact future tree growth modeling and will help in decisions that involve genot ype deployment and silvicultural treatments.

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82 APPENDIX A DESIGN AND LAYOUT STUDY SITE Figure A-1.Design and layout of full-sib fam ily block plot study at Rayonier, Inc. 2 rows of buffers 1 5 12 6 7 13 9 8 4 11 3 210 14 15 16REP 2 REP 4 REP 1 REP 312 16 8 5 15 4 3 6 1 11 14 9 13 10 2 7 8 2 10 16 11 10 7 1513 2 14 3 5 9 6 1 7 14 3 13 5 16 1 4 6 12 8 9 12 4 11 15 6 beds wide(66 ft.) 10 planting spaces (55 ft.) plot corner N PropaguleType: Spp 1 =RC-clones replicatedslash 2 =RC-clones replicatedslash 3 =RC-clones replicatedslash 4 =RC-clones replicatedloblolly 10 =RC-clones replicatedslash 5=Seedlingslash 6=RC-clones not replicatedslash 7=Seedlingslash 8=RC-clones not replicatedslash 9=Seedlingslash 11=Seedlingloblolly 12=RC-clones not replicatedloblolly 13=Seedlingloblolly 14=Seedlingloblolly 15=RC-clones not replicatedloblolly 16=Seedlingslash2 rows of buffers 1 5 12 6 7 13 9 8 4 11 3 210 14 15 16REP 2 REP 4 REP 1 REP 312 16 8 5 15 4 3 6 1 11 14 9 13 10 2 7 8 2 10 16 11 10 7 1513 2 14 3 5 9 6 1 7 14 3 13 5 16 1 4 6 12 8 9 12 4 11 15 6 beds wide(66 ft.) 10 planting spaces (55 ft.) plot corner N PropaguleType: Spp 1 =RC-clones replicatedslash 2 =RC-clones replicatedslash 3 =RC-clones replicatedslash 4 =RC-clones replicatedloblolly 10 =RC-clones replicatedslash 5=Seedlingslash 6=RC-clones not replicatedslash 7=Seedlingslash 8=RC-clones not replicatedslash 9=Seedlingslash 11=Seedlingloblolly 12=RC-clones not replicatedloblolly 13=Seedlingloblolly 14=Seedlingloblolly 15=RC-clones not replicatedloblolly 16=Seedlingslash

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83 APPENDIX B SILVICULTURAL TREATM ENTS AT STUDY SITE Table B-1. Treatment regime s applied in the research location at Rayonier, Inc. Year Treatment 1997-January Double bedding and planting 1997-March Chemical weed control (arsenal imazapyr) banded 1997-June Chemical weed control (arsenal imazapyr and sulfometuron methyl) broadcast 1997-October Fertilization 220 kg/ha diammonium phosphate 1999-August Fertilization 220 kg/ ha diammonium phosphate 2000-July Mechanical weed control 2000-November Fire line plowed 2001-May Chemical weed control (glyphosate) broadcast 2002-June Fertilization 500 kg/ha ammonium nitrate

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84 REFERENCES Ahuja, M.R. and Libby, W.J. 1993. Gene tics, biotechnology and clonal forestry. In Clonal forestry I. Edited by Ahuja, M.R. and Libby, W.J. Springer-Verlag, New York, New York. pp 1-4. Arregui, A., Espinel, S., Aragons, A., a nd Sierra, R. 1999. Estimacin de parmetros genticos en un ensayo de progenie de Pinus radiata D. Don en el pas Vasco. Vest. Agr.: Sist. Recur. For. 8: 119-128. Baltunis, B.S., Huber, D.A., White, T.L., Golfar d, B., and Stelzer, H.E. Genetic effects of rooting loblolly pine stem cuttings from a partial dial lel mating design. Can. J. Forest Res. 35: 1098-1108. Boldman, K.G., Kriese, L.A., Van Vleck, L. D., Van Tassell, C.P., and Kachman S.D. 1995. A manual for use of MTDFREML. A se t of programs to obtain estimates of variances and covariances (draft). USDAARS. Washington, DC. US Gov. Print Office. 120 p. Bond, W.J. and Stock, W.D. 1990. Pre liminary assessment of the grading of Eucalyptus clones using carbon isotope discrimination. South African Forestry Journal 154: 51-55. Borralho, N.M., Almeida, I.M., and Cotterill, P.P. 1992. Genetic control of growth of young Eucalyptus globulus clones in Portugal. Silvae Genet. 41: 100-105. Bouriaud, O., Leban, J.M., Bert, D., and De leuze, C. 2005. Intraannual variations in climate influence growth and wood density of Norway spruce. Tree Physiol. 25: 651-660. Bradshaw, H.D. and Stettler, R.F. 1995. Mol ecular genetics of gr owth and development in Populus. IV. Mapping QTLs with large e ffects on growth, form, and phenology traits in a forest tr ee. Genetics 139: 963-973. Brendel, O., Pot, D., Plomion, C., Rozenberg, P., and Guehl, J.M. 2002. Genetic parameters and QTL analysis of 13C and ring width in maritime pine. Plant Cell Environ. 25: 945-953. Burns, R. M. and Honkala, B.H. 1990. Silvics of North America. Volume 1. Conifers. USDA Agriculture Handbook 654. Wash ington, DC. pp.338-345, 497-508.

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97 BIOGRAPHICAL SKETCH Veronica I. Emhart was born in the town of Osorno, in southern Chile, a region covered by lakes, mountains, native forest a nd agricultural land. In 1996, she graduated as Forestry Engineer at the Universidad Au stral de Chile in Valdivia, Chile. Between 1997 and 2000, she worked as technical manager in Tree Improvement Programs in Chilean native forest (Nothofagus), and also in Eucalyptus, at the Universidad Austral de Chile. She enrolled in the PhD program in January 2001 at the University of Florida, specializing in forest tree improvement and tree physiology. After graduation, she will continue working in Tree Improvement Progr ams and Biotechnology at Instituto Forestal de Chile in Concepcion.


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PHYSIOLOGICAL GENETICS OF CONTRASTING LOBLOLLY AND SLASH PINE
FAMILIES AND CLONES















By

VERONICA INGRID EMHART


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Veronica I. Emhart



























To my parents and brothers, who loved and supported me along my journey.


To my friends, whose company filled my heart with happiness all five years.















ACKNOWLEDGMENTS

During my study, Drs. Timothy White and Timothy Martin provided guidance,

support, direction, and encouragement. I owe a great deal to them. My immense

appreciation also goes to Dr. Dudley Huber for his excellent suggestions, disposition, and

help in understanding difficult statistical issues and enlightening discussion of results.

Thanks also go to Dr. Eric Jokela and Dr. Kenneth Boote for their participation as

supervisory committee members and their invaluable guidance throughout my study.

My study was made possible by the financial support of the United States

Department of Agriculture (USDA) Forest Service and the Forest Biology Research

Cooperative. Rayonier Inc. provided the study site and access to valuable information.

Thanks go to Dave Nolletti, Greg Powell, and Greg Starr for their availability and

help in field activities and for making my life easier in the lab. I want to express my

appreciation to my fellow graduate students (Salvador Gezan, Maheteme Gebremedhin,

Alex Medina, and Xiabo Li); and field technicians (Sean Gallagher, Tim Walton, Jason

Martin, Paul Proctor, Chris Cabrera, and Kate Kritcher) who collaborated measuring

trees, downloading data, collecting shoot and foliage samples, grinding foliage, and

processing data.

Thanks also go to my friends (Gabriela Luciani, Belkys Bracho, Gladys Vergara,

Rosana Higa, and Tatiana Verissimo) for being such great company in afterwork

activities, and for their support in difficult times.









I thank God for giving me the opportunity to start a new adventure far from home,

and to celebrate victory at the end of this road. Finally, and most important, I thanks my

parents and brothers for their love, help, support, and belief in this journey.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ............ ............................... ......... ............. ... viii

LIST OF FIGURES ............................... ... ...... ... ................. .x

ABSTRACT ........ .............. ............. ...... ...................... xi

CHAPTER

1 IN T R O D U C T IO N ............................................................................. .............. ...

2 CLONAL VARIATION IN CROWN STRUCTURE, ABSORBED
PHOTOSYNTHETICALLY ACTIVE RADIATION, AND GROWTH OF
LOBLOLLY AN D SLA SH PIN E ......................................................... ....................5

In tro d u ctio n ..................................................................................................... .... .. 5
M materials and M methods .................................................... ................................. 7
Site D description and Plant M aterial.................................................................... 7
Growth and Crown Architectural Traits...........................................................8
Estimating Absorbed Photosynthetically Active Radiation (APAR) ....................9
Statistical A naly sis ....................................................... 9
Genetic Parameter Estimation ................. .......................................... 10
Results .............. ............ ...... .... ..........................11
Genetic Variation in Stem and Crown Traits ...................................................11
Within-Family Individual-Tree Broad-Sense Heritabilities.............................13
Within-Family Genetic and Environmental Correlations...................... ..15
D iscu ssio n .........................................................................................16

3 GENETIC VARIATION IN BASAL-AREA INCREMENT PHENOLOGY AND
ITS CORRELATION WITH GROWTH RATE IN LOBLOLLY AND SLASH
PIN E FA M ILIES AN D CLON E S .............................................................................25

Introduction..................................... .................................. .......... 25
M material and M methods ........................................................... .. ............... 28
Study Site and Plant M material ........................................ ......................... 28
Basal-area Increm ent M easurem ents................................................................ 29
Phenological Traits .................. ........................... .... .... ............... ... 30









Meteorological Data and Water Balance.........................................................30
Statistical Analyses and Genetic Parameters.....................................................31
R results and D discussion .............. ...... .... ...... ......... .. ............... 33
Genetic Variation among Species and Families................................................33
Clonal Variation and Within-Family Inheritance of Phenological Traits and
Stem G row th ..................................... .............. .......... .............. 4 1
Genetic Correlations among Phenological Traits and Stem Growth .................43
A analysis across Y ears 2002-2003 ............................................ ............... 47

4 CARBON ISOTOPE DISCRIMINATION, CROWN CONDUCTANCE,
GROWTH AND THEIR GENETIC PARAMETERS IN LOBLOLLY AND
SLASH PINE FAM ILIES AND CLONES ........................................ .....................50

Intro du action ...................................... ................................................ 50
M material and M methods ........................................................... .. ............... 54
Study Site and Plant M material ........................................... ......................... 54
Tree Growth and Carbon Isotope Discrimination ............................................55
M eteorological Data .................. ............................ .. .. .. ................. 56
Individual Tree Transpiration........................................ ..... .... .. ......... 57
Crown Conductance and Stomatal Sensitivity Calculations ............................58
Genetic Parameters and Statistical Analyses...........................................60
R results ............................ ...................................................................62
Carbon Isotope Discrimination.................... ....... .......................... 62
Stem G row th ................ ............... ...... ........................6 5
Whole-Tree Crown Conductance and Stomatal Sensitivity ...............................66
Genetic Correlations between Carbon Isotope Discrimination and Growth
and W hole-Tree Crown Conductance................................... ............... 68
D discussion ................ ........... .......................... ............................69

5 SUMMARY AND CONCLUSIONS...... ................. ...............76

Genetic Variation among Species and Families ............................... ................77
Clonal Variation and Within-Family Inheritance..............................................78
Correlations ................................................................................ 79

APPENDIX

A DESIGN AND LAYOUT STUDY SITE........................................ .....................82

B SILVICULTURAL TREATMENTS AT STUDY SITE ............... .................83

R E F E R E N C E S ........................................ ........................................................... .. 8 4

B IO G R A PH IC A L SK E TCH ..................................................................... ..................97















LIST OF TABLES


Table p

2-1 Significance levels (p-values), species means and pooled within-family
heritabilities (H2WF) for individual-tree growth and crown structural variables for 5
and 6 year-old loblolly and slash pine families in north central Florida..................14

2-2 Age 5 y and 6 y within-family individual-tree broad-sense heritability (H2WF) for
growth and crown structural traits in four slash pine families in north central
F lo rid a ........................................................... ................ 18

2-3 Within-family genetic correlations among individual-tree volume increment
between age 5-6 and crown structural variables at age 5, for slash (Sl, S2, S3 and
S10) and loblolly (L4) pine families in north central Florida. ................................19

3-1 Significance levels (p-values), and species and family least square means for
individual tree stem growth and phenological traits for two growing seasons for
loblolly and slash pine families in north central Florida .......................................35

3-2 Significance levels (p-values) for clone within-family for tree stem growth and
phenological traits for two growing seasons in loblolly and slash pine families in
north central Florida. ......................... ............................. .. ...... .... ........... 42

3-3 Within-family individual-tree broad-sense heritabilities for growth phenology traits
and basal-area growth increment by year in loblolly and slash pine families
grow ing in north central Florida.................................................................... ....43

3-4 Within-family genetic correlations between growth phenology traits in 2002 (above
the diagonal) and 2003 (below the diagonal) in loblolly and slash pine families
growing in north central Florida............................ ........ .................... 46

3-5 Within-family genetic correlations between growth and phenology traits by year in
loblolly and slash pine families growing in north central Florida..........................46

4-1 Significance levels (p-values), species and within family least square means for
volume, carbon isotope discrimination (A13C) for three growing periods, and crown
conductance variables (Gsref and Gssensitivity ) in slash and loblolly pine families in
north central Florida. .......................... .......... .. ..... .............. 64









4-2 Significance levels (p-values) for clone within family in carbon isotope
discrimination for two growing periods and crown conductance variables for
loblolly and slash pine families in north central Florida .......................................64

4-3 Within-family individual-tree broad-sense heritabilities for stable carbon isotope
discrimination (A13C) by year, and crown conductance variables in loblolly and
slash pine families growing in north-central Florida ............................................ 65

4-4 Genetic correlations between years 2001 and 2003 by family for carbon isotope
discrimination (A13C) and between 4-5 yr and 6-7 yr stem volume increment (VI)
for loblolly and slash pine families in north central Florida. ..................................65

4-5 Within-family correlations between volume increment of the growing season and
stable carbon isotope discrimination (A13C) by year in loblolly and slash pine
fam ilies grow ing in north-central Florida ..................................... ............... ..68

4-6 Genetic and environmental correlations between mean carbon isotope
discrimination (mean A13C) and Gsref and Gssensitivity for loblolly and slash pine
fam ilies in north central Florida ...................................................... ........ ...... 69

B-1 Treatment regimes applied in the research location at Rayonier, Inc ....................83















LIST OF FIGURES


Figure page

2-1 Family means and standard error bars for individual-tree growth and crown
structural traits for 5 and 6 year-old loblolly and slash pine families in north central
F lo rid a ........................................................... ................ 12

3-1 Family mean cumulative basal-area growth curves for years 2002 and 2003 in
loblolly and slash pine in north central Florida...............................35

3-2 Species mean daily basal-area growth increment for loblolly and slash pine in north
central Florida and environm ental variables.. ................................................... 36

3-3 Relationship between individual tree daily basal-area increment and simulated daily
plot-level soil water balance in loblolly and slash pine in 2002 (A) and 2003 (B)..40

4-1 Family means and standard errors in carbon isotope discrimination in year 2001
an d 2 0 0 3 ............................................................................................................. ......6 3

4-2 Accumulated monthly precipitation in years 2001, 2003 and mean normal year
from Gainesville Regional Airport, Gainesville, Florida (NOAA 2003) ...............63

4-3 Representative relationship between canopy average stomatal conductance (Gs) and
vapor pressure deficit (D) on a half hourly basis for a typical slash pine ramet.....67

A-1 Design and layout of full-sib family block plot study at Rayonier, Inc .................82















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PHYSIOLOGICAL GENETICS OF CONTRASTING LOBLOLLY AND SLASH PINE
FAMILIES AND CLONES

By

Veronica Ingrid Emhart

December 2005

Chair: Timothy L. White
Cochair: Timothy A. Martin
Major Department: Forest Resources and Conservation

My study focused on the biology and genetic structure of 300 clones from five

different full-sib loblolly and slash pine families. The study was divided into three main

areas of research: (1) detailed quantification of crown structure and estimation of annual

absorbed photosynthetically active radiation aparR); (2) seasonal dynamics and

phenology of basal area growth and its association with soil water balance; and (3) leaf

carbon isotope discrimination and whole-tree sap flow.

Genetic variation in crown structural traits, APAR, stem volume growth, basal area

growth phenology, basal area growth rates, leaf carbon isotope discrimination (A13C), and

crown conductance were more apparent at the clonal level than at the species and family

levels.

The one loblolly pine family we studied tended to grow faster, developed larger

crowns with more acute branch angles, had more leaf area and intercepted more radiation

than the four slash pine families averaged. Loblolly and slash pine within-family









individual-tree broad-sense heritabilities (H2WF) ranged from 0.00 to 0.41 for growth and

crown structural traits, and most were between 0.10 and 0.25 when estimated from a

combined analysis across families. Genetic correlations of crown size, leaf area, and

APAR with volume increment were generally positive.

Basal-area growth spanned March through October for both species. In both years,

peaks in basal-area increment occurred in short (2-3 week) periods in the early spring for

all families, followed by relatively constant rates of basal-area growth until cessation.

The H2WF ranged from 0.01 to 0.37 for basal area growth phenology. Both the strength

and direction of correlation estimates of phenological traits with growth rate varied across

families and years.

Clonal mean values for A13C ranged from 19%o to 25.45%o. The H2WF for A13C and

crown conductance parameters ranged from 0.01 to 0.32. Genetic and environmental

correlations of stem growth with A13C or with crown conductance were low. There was

no evidence of clone-by-year interaction in stem growth, basal-area growth phenology,

and A13C for any family.

Understanding the biology of physiological processes and their genetic parameters

gives us insight into the key functional and structural traits that determine genotype

performance differences in southern pines.














CHAPTER 1
INTRODUCTION

Loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm. var elliottii)

are widely planted as commercial timber species in the southeastern United States (Smith

et al. 2004). From the early 1950s, large-scale tree-breeding programs in both species

improved forest productivity by selecting trees for superior growth rate, form, and disease

resistance (McKeand et al. 2003). The genetically improved material currently being

established in commercial plantations is deployed from bulked orchard seed, half-sib

families, and full-sib families (with growing interest in the deployment of outstanding

clones). Tree breeding has proven to be a very effective tool, and breeding will continue

to be the most important mechanism for developing recombinant genotypes to achieve

increasing genetic gains (White and Carson 2004).

Management of southern pine plantations in the United States is being transformed

from a relatively extensive system of planting coupled with isolated individual treatment,

to a much more intensive system in which genetic and site resources are manipulated in

concert, to optimize stand productivity (Fox et al. 2004). Increased productivity in

southern pines create more value for the forest industry, and decreases pressure on native

pine forests, considering that the demand for forest products will continue to increase and

intensive management will be needed to meet this demand. Increased forest productivity

also provides great potential for sequestering atmospheric carbon (Johnsen et al. 2004).

Clonal forestry may be an excellent way to increase the productivity of southern

pine plantations in the near term (Fox et al. 2004). Many potential benefits of clonal









forestry have been previously described (Libby 1982; Libby and Rauter 1984; Carson

1986) and include:

* Gains arising from testing and selection of clones;

* Clone/site matching to increase genetic gains by capturing favorable genotype by
environment effects (G x E), and by targeting expression to existing site properties;

* Greater uniformity (little impact on growth and yield traits, but extremely valuable
for log and wood quality and disease resistance traits, and for harvesting and
processing);

* Greater repeatability (better yield prediction and planning). Specific clones can be
identified that are most adapted to different site qualities. Identifying these clones
help us take advantage of positive genotype X environment interaction and to
optimize silvicultural practices, including spacing, weed control, and fertilizer
regimes. To identify superior clones within-family we must understand the
biological basis for growth differences and identify key structural and functional
attributes at the organ, tree, and stand level.

Growth involves many integrated physiological processes influenced by genetic

and environmental factors (Kozlowski and Pallardy 1997). Processes like radiation

absorption, carbon gain capacity, crown conductance responses to changes in the

environment, growth phenology, nutrient assimilation, and growth regulation may control

stem volume growth in contrasting southern pine families and clones.

Better understanding the physiological processes underlying genetic differences in

growth performance may allow geneticists to be more deliberate in their selections,

focusing specific objectives and allowing for more predictable gains. Martin etal. (2005)

described several potential obstacles to ecophysiological contributions to tree

improvement programs:

* Selecting wrong physiological parameters for screening;

* Making physiological measurements at inappropriate spatial and temporal scales;

* Attempting to use seedlings to predict field performance of adult trees.









My overall goal was to investigate biological traits and their genetic structure in

300 clones from five different full-sib loblolly and slash pine families. My study used

measurements that integrated biological information over space and time, with the intent

of studying biological traits that correspond more closely to the spatial and temporal

scales at which growth was observed (e.g., whole trees over seasons to years).

The study was divided into three main areas of investigation:

* The first phase used detailed crown structural information for each ramet within-
clone to parameterize the process model MAESTRA, which was then used to
estimate the total amount of radiation intercepted by each ramet over a year;

* Second, repeated basal-area measurements of each ramet was used to estimate
seasonal dynamics and phenology of basal-area growth, associated with a soil-
water balance calculation to examine relationships between basal area growth and
integrated environmental variables;

* Third, leaf carbon isotope discrimination analysis (integrates leaf physiology over
the time of leaf formation) and whole-tree sap flow analysis (integrates leaf
physiology over the tree crown over long periods of time) was used to analyze
integrated gas exchange properties and its relationship with growth.

My study focused on the following specific objectives, organized by main areas of

investigation:

* Specific aim la: quantify growth and crown structural variation among species,
families and clones representing a range of growth performance in loblolly and
slash pines;

* Specific aim Ib: integrate crown structural variables into a radiative transfer model
to estimate variation in intercepted radiation for different genotypes for a given
period of time and their relationship with growth rate;

* Specific aim Ic: estimate within-family genetic control and environmental
influence on crown structural attributes and growth;

* Specific aim 2a: compare two years basal area growth phenology among species,
families and clones;

* Specific aim 2b: estimate genetic parameters for basal area growth phenology, its
correlation with growth rates, and the genotype interaction with seasonal
environmental changes;









* Specific aim 3a: determine whether genetic variation for leaf carbon isotope
discrimination occurs among slash and loblolly pine genotypes (species, families,
or clones);

* Specific aim 3b: examine genetic variation in crown-level stomatal conductance
(crown conductance) sensitivity to vapor pressure deficit between species, among
slash pine families, and among clones within slash and loblolly pine families;

* Specific aim 3c: determine broad-sense heritabilities and genetic correlations for
leaf carbon isotope discrimination, growth and crown conductance.

Results from my study should positively impact future tree growth modeling and

will help in decisions that involve genotype deployment and silvicultural treatments.

Results will also aid in examining genetic and environmental control of several key

structural and functional processes that determine productivity in different full-sib

families and clones within-family in southern forest plantations.














CHAPTER 2
CLONAL VARIATION IN CROWN STRUCTURE, ABSORBED
PHOTOSYNTHETICALLY ACTIVE RADIATION, AND GROWTH OF LOBLOLLY
AND SLASH PINE

Introduction

Crown structural characteristics (such as crown size, branching frequency, branch

diameter, branch angle, and leaf area quantity and spatial distribution) influence the

efficiency and magnitude of radiation interception and competitive interactions with other

trees (Wang and Jarvis 1990; Stenberg et al. 1994; Vose et al. 1994; McCrady and Jokela

1996, 1998). As a result, crown architecture is an important determinant of both tree-level

and stand-level productivity (Dalla-Tea and Jokela 1991; Stenberg et al. 1994; McCrady

and Jokela 1996). This linkage is often reflected in ideotypes or conceptual models of

desirable tree phenotypes intended to guide plant genetic research and breeding programs

(Donald 1968; Dickmann et al. 1994). For example, the published ideotypes for Populus

(Dickmann 1985; Dickmann and Keathley 1996) and Scandinavian conifers (Karki and

Tigerstedt 1985) incorporate numerous crown structural variables. Genetic variation in

growth has been the subject of much research (White 1996), and forms the basis of most

commercial tree improvement programs (White et al. 1993; McKeand and Bridwater

1998; Li et al. 2000). In contrast, the genetic architecture of crown structure has been

much less intensively studied, and is seldom used in tree improvement programs (Martin

et al. 2001).

Xiao et al. (2003) showed that significant differences in growth between loblolly

pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm. var elliottii) were associated









with variation in crown structure and biomass allocation. At age 3 and 4 y, loblolly pine

had more branches per tree and allocated more biomass to branches than slash pine.

Greater branch/leaf biomass as a growth strategy might develop spacious crowns

facilitating faster growth by increasing the leaf-area carrying capacity in the crown.

Knowledge of heritabilities and genetic correlations is needed to understand the

genetic structure of breeding populations, and to determine deployment strategies in tree

improvement programs (White 1987). Broad-sense heritabilities for a number of

structural and growth properties have been estimated for Populus and Eucalyptus

(Wilcox and Farmer 1967; Weber et al. 1984; Borralho et al. 1992; Lambeth et al. 1994;

Osorio 1999). Genetic correlations between growth traits and crown structural attributes

are scarce, but studies in Populus, Eucalyptus, loblolly and slash pine have identified

positive genetic correlations between growth performance and branching patterns, and

growth performance and crown vigor (Wilcox and Farmer 1967; Lambeth et al. 1994;

Lambeth and Huber 1997; Xiao et al. 2003). Incorporating new information on crown

structural attributes (such as crown size, crown shape ratio, and arrangement and

diameter of branches) would improve our understanding of how canopy structure affects

absorbed photosynthetic active radiation and stand development. Crown structural

attributes also may prove useful in selection of families or clones for silvicultural

programs, and development of new crop tree ideotypes.

Our objectives were as follows:

* Quantify growth and crown structural variation among species, families, and
clones, representing a range of growth performance in loblolly and slash pines;

* Integrate crown structural variables into a radiative transfer model to estimate
variation in intercepted radiation for different genotypes for a given period of time
and to estimate their relationship with growth rate;









* Estimate within-family genetic control and environmental influence on crown
structural attributes and growth.

According to Martin et al. (2005), one reason ecophysiological research has failed

to contribute to southern pine tree improvement programs is that researchers have

focused on small spatial and short temporal scales that are too far removed in space and

time from growth processes. Accordingly, we hypothesized that tree growth would be

genetically correlated with crown structural traits, and that traits which integrated

information over space and/or time would be more highly correlated with growth than

would less-integrated traits.

Materials and Methods

Site Description and Plant Material

The study area was located on lands managed by Rayonier Inc. in Bradford County,

Florida. The climate is humid and subtropical, with a mean annual temperature of 210C,

mean annual rainfall of 1316 mm, and over 50% of the rainfall occurring in June through

September. Periods of drought are normal in the spring and fall. Mean annual rainfall

during 1999-2001 was 967 mm, in contrast to 1405 mm in year 2002 (NOAA 2002). The

soils are classified as Pomona and consist of very deep, somewhat poorly to poorly

drained soils that are formed in sandy and loamy marine sediments (sandy, siliceous,

hyperthermic Ultic Alaquods). Slopes are 0 to 2 %. In a typical profile, the spodic

horizon occurs at 30-60 cm, with an argillic horizon at 90-120 cm. Water table is

typically at a depth of 15 to 45 cm for one to three months and a depth of 25 to 100 cm

for six months or more, during most years (Soil Survey Staff 1998).

The study took place in an area containing 16 full-sib and half-sib loblolly and

slash pine families planted in 337 m2 family plots in January 1997. The experiment was









designed as a randomized complete block with four replicates (Appendix A). We used

one full-sib loblolly pine family and four full-sib slash pine families. Each family plot

contained 60 clones propagated as rooted cuttings from a single family, planted at 1.7 m

x 3.4 m spacing (1730 trees ha-1). Cuttings were taken from donor hedges in the spring,

and were rooted and grown in a greenhouse for six months before planting. Each of the

four plots of the same family contained the same 60 genotypes, but with the ramets

planted into different, randomly-determined planting locations in the plot. In total we

studied approximately 1,200 trees: 60 trees per family plot x 5 families x 4 replications.

Fertilization and weed control were applied periodically to reduce interspecific

competition and prevent nutrient deficiency (Appendix B).

Growth and Crown Architectural Traits

Stem volume growth in the 2000, 2001, and 2002 growing seasons (ages 4, 5, and 6

y, respectively) was determined from dormant-season measurements of tree diameter at

1.37 m height (DBH) and total tree height (HT). Outside-bark individual-tree stem

volume was calculated with a general equation (Hodge et al. 1996) as shown in Equation

2-1, where DBH and HT were entered in m:

VOL (dm3) = (0.25 3.14 (DBH)2 (1.37 + 0.33 (HT 1.37)))*1000 (2-1)

Crown architecture was assessed by measuring length and width of the living

crown and basal diameter of all living branches at the end of the 2001 and 2002 growing

seasons. Also, branch angle was measured in four branches in the 2000 cohort of each

tree, with a protractor. Other traits derived from these records included total number of

branches per tree, crown shape ratio (CSR= crown height/ crown width), and branch-free

stem height. Individual-tree leaf area at age 5 y was calculated by summing individual

branch leaf area estimated from regional allometric equations (McGarvey 2000).









Regression equations were developed between crown size at age 5 y (independent

variable) and leaf area at age 5 y (dependent variable) by family; and then leaf area at age

6 y was predicted using crown volume at age 6 y by family.

Estimating Absorbed Photosynthetically Active Radiation (APAR)

Total APAR was simulated for each tree in the study from January 1, 2002 to

December 31, 2002, using hourly radiation data from a weather station at the site, input

into the process model MAESTRA, a modification of the MAESTRO model (Wang and

Jarvis 1990; Medlyn 2004). MAESTRA uses Norman and Welles (1983) method to

calculate PAR at grid points within the crown, taking into account the spatial distribution

of foliage in the target crown and in adjacent tree crowns. Crown shapes were assumed to

be ellipsoidal. Vertical foliage distribution was specified by a Beta function developed

for loblolly pine in North Carolina (Luo et al. 2001), while horizontal foliage distribution

was assumed to be uniform. Simulations were run for each tree, in each of the 20 study

plots. For each tree, the location, crown radius in two directions, total tree height, height

to the base of the live crown, and leaf area were specified. Tree locations, crown

dimensions, and leaf area for a two-tree border surrounding each study plot were also

specified. When study plots were near non-study plots, crown dimensions and leaf area of

border trees were predicted from measured height and diameter. Crown dimensions were

assumed to increase linearly from March 1st to December 1st.

Statistical Analysis

Analysis of variance (ANOVA) was used to analyze growth, crown structural traits,

and APAR data. PROC GLM in the SAS System were used to test for significance of

random effects (clone), while PROC MIXED was used to test the fixed effects (species

and families). Equation 2-2 shows the ANOVA model for the analyses, where Yijkl is the









performance of the ramet of the 1th clone within the kth family nested in the jth species in

the ith replication; i = 1, 2, 3, and 4 for replications; j = slash, loblolly; k = 1, 2, 3, 4, and

10 for families; 1 = 60 identification numbers for 60 clones within each of the five

families:

Yijkl= [ + bi + Sj + Fkg) + Cljk) + bSij + bFikg) + ijkl (2-2)

[t = population mean,
bi = random variable of replication NID (0, C2b),
Sj = fixed effect of species (slash or loblolly),
Fkg) = fixed effect of family nested within species,
Cl(k) = random variable of clone nested within-family and species NID (0, C02),
bSij = random variable for replication x species interaction ~ NID (0, C2bS),
bFik) = random variable for replication x family(species) interaction ~ NID (0,
G2bF), and
Sijkl = error term ~ NID (0, c25).

Genetic Parameter Estimation

For each species and family, two types of parameters were estimated: within-family

heritability for each trait, and genetic and environmental correlations among traits.

Within-family variance and covariance components were obtained using Multiple Trait

Derivate-free Restricted Maximum Likelihood (MTDFREML) software (Boldman et al.

1995).

Within-family individual-tree broad-sense heritability was calculated as

2
H2 2 (2-3)


Theoretically, broad-sense within-family heritability for full-sib families contains

12 the additive genetic variance, 34 of the dominance genetic variance, and most of the

epistatic genetic variance (Falconer and Mackay 1996). The standard error for heritability

estimates was calculated using a method described by Dickerson (1962). The residual

likelihood ratio test (Wolfinger 1996) was used to test heterogeneity of variances among









slash pine families, and heritabilities were estimated separately for each family (X2(6,

0.05)= 12.6), or pooled, as appropriate.We estimated all genetic parameters from data

collected from only one experimental site; therefore, the clonal genetic variance contains

the clone-environment interaction variance in the above results, and the estimated genetic

parameters are biased upward if the interaction is non-zero (Hodge and White 1992).

Within-family genetic and environmental correlations among growth traits and

crown structural variables (Falconer and Mackay 1996) were calculated as shown in

Equation 2-4, where xy is the covariance clonall or residual) between two traits, and Cx

oy corresponds to the square root of the product of the clonal or residual variance within-

family of each trait:


r- = (2-4)

Results

Genetic Variation in Stem and Crown Traits

We examined variation in cumulative stem volume, annual stem volume growth,

and crown architectural and functional traits. Comparisons were made between species

(loblolly vs. slash pine), among families within species (four full-sib slash pine families),

and among clones within species (60 clones within each of the four slash pine families

and one loblolly pine family).

By age 6 y, loblolly pine stem volume was almost 25% larger than mean slash pine

stem volume (p=0.0727, 31.42 dm3 vs. 25.47 dm3, respectively), reflecting fairly

consistent species-level differences in annual stem volume increment (Table 2-1). Within

slash pine, there were consistent differences among families (p<0.10) in stem volume and

stem volume increment, with the exception of age 5-6 yr increment (Table 2-1, Figure 2-









12




1). Within-family clonal variation in stem volume and stem volume increment was highly



significant for all years for slash pine (p<0.0001, Table 2-1).


Stem volume
2 (dm3 tree-1) --i


















S(m tree -1
2 A


12 13 14 15 16 17 18 19


Crown radius
3 (mtree -1)




,


09
085 090 095 100 105 110 115 120 125

34
Number of branches 1 +
per tree T
32 -

31

30

29 +

28 -
28

27 -

26 2 .
22 24 26 28 30 32 34


Stem volume increment

13 (dm3tree -1)


12





.10 +



60 65 70 75 80 85 90 95 100
240
Crown shape
2 35
35 ratio

230 +

225

220

215 +

210 +

205
18 19 20 21 22 23

1 60
1 58 Branch diameter
(cm)
156
1 54


142 1 I
130 135 140 145 150 155 160 165 170


Crown length +
54
(m tree-1)
52

50

48 +

46 +
44


42
36 38 40 42 44 46 48
18
Crown volume
16 (m3tree-1)

14

12

10

8 121

6
4 5 6 7 8 9 10 11 12

74
72 Branches per unit
of crown length T
(# m-)
68
66 +
64
62
60 +
58 +
56
54
55 60 65 70 75 80 85 90 95


Age 5

Figure 2-1. Family means and standard error bars for individual-tree growth and crown

structural traits for 5 and 6 year-old loblolly and slash pine families in north

central Florida. Sl=family slash 1; S2= family slash 2; S3=family slash 3;

S10=family slash 10; L4=family loblolly 4.



There were species-level differences in a number of crown structural traits.



Loblolly pine had longer and wider crowns at age 5 and 6 y, resulting in species



differences in crown volume on the order of 85% (Table 2-1). Slash pine crowns of a



given length were slightly narrower than loblolly pine crowns of a similar length, as









quantified by the crown shape ratio: 2.23 vs. 2.10 at age 6 y for slash and loblolly pine,

respectively (Table 2-1). Loblolly pine branches were displayed at a more acute angle

than were slash pine branches: 51.1 vs. 56.9 o, respectively (Table 2-1). Age 5-6 y

radiation interception, simulated with the MAESTRA radiative transfer model, was about

20% greater in loblolly pine (11,901 MJ/tree) than the mean slash pine annual APAR

(9,901 MJ/tree). Numbers of branches per crown, branch diameter, and number of

branches per unit crown length were not different between species (Table 2-1).

Crown structure also varied at the family level, with crown size and shape traits

(length, radius, volume, and crown shape ratio) all varying significantly among the four

slash pine families (p<0.10). Slash pine families also differed in numbers of branches,

branch diameter (at age 5 y), numbers of branches per unit crown length, and branch

angle (Table 2-1, Figure 2-1). There was no significant family-level variation in tree leaf

area or annual APAR. Within families, there was significant clonal variation for all traits

measured (p<0.0001, Table 2-1).

Within-Family Individual-Tree Broad-Sense Heritabilities

Within-family individual-tree broad-sense heritabilities (H2WF) were low to

moderate for stem volume and crown structural traits. In loblolly pine, a number of crown

structural traits were moderately heritable, with crown radius at age 5 yr, crown volume

at age 6 yr, leaf area at age 6 yr, number of branches at age 5 yr, and branch angle at age

5 yr having H2WF between 0.20 and 0.27. Stem volume and stem volume growth traits

had lower H2WF, ranging between 0.05 and 0.18 (Table 2-1).

For volume in slash pine at different ages, H2WF was between 0.17 and 0.19, and

crown structural traits showed similar ranges of variation (Table 2-1). When H2WF values

were estimated separately by family due to heterogeneous variance components among














Table 2-1. Significance levels (p-values), species means and pooled within-family heritabilities (H2WF) for individual-tree growth and
crown structural variables for 5 and 6 year-old loblolly and slash pine families in north central Florida.
Trait Significance level by effect Species mean H 2w
Species Family Clone Slash Loblolly Slasha Loblolly
Inventory
Volume age 4 (dn3 tree-1) 0.2240 0.0797 <0.0001 7.13 8.72 -- 0.05 (0.06)
Volume age 5 (dn3 tree-1) 0.1007 0.0451 <0.0001 14.46 18.13 0.17(0.04) 0.08 (0.07)
Volume age 6 (dn3 tree-1) 0.0727 0.0802 <0.0001 25.47 31.42 0.17(0.04) 0.18(0.08)
Volume increment age 4-5 0.0455 0.0567 <0.0001 7.32 9.41 0.19 (0.04) 0.12 (0.07)
(dm3 tree-1)
Volume increment age 5-6 0.0754 0.3324 <0.0001 11.02 13.29 -- 0.18 (0.08)
(dm3 tree-1)
Crown structure
Live crown length age 5 (m) 0.0055 0.2358 <0.0001 3.85 4.55 0.16(0.04) 0.09 (0.07)
Live crown length age 6 (m) 0.0028 0.0812 <0.0001 4.61 5.46 -- 0.11 (0.07)
Crown radius age 5 (m) 0.0067 0.0106 <0.0001 0.94 1.20 -- 0.20 (0.08)
Crown radius age 6 (m) 0.0041 0.0233 <0.0001 1.05 1.33 -- 0.18(0.08)
Crown shape ratio age 5 0.1001 0.0012 <0.0001 2.07 1.92 -- 0.13 (0.07)
Crown shape ratio age 6 0.0519 0.0095 <0.0001 2.23 2.10 -- 0.00 (0.00)
Crown volume age 5 (m3) 0.0039 0.0913 <0.0001 5.80 10.88 -- 0.19(0.08)
Crown volume age 6 (m3) 0.0020 0.0723 <0.0001 8.91 16.42 -- 0.25 (0.09)
Leaf area age 5 (m2) 0.0450 0.4940 <0.0001 33.11 44.07 0.12 (0.04) 0.08 (0.06)
Leaf area age 6 (m2) 0.1197 0.5562 <0.0001 47.14 54.58 -- 0.25 (0.09)
Number branches age 5 0.1214 0.0021 <0.0001 30 33 -- 0.27(0.09)
Number branches age 6 0.1610 0.0225 <0.0001 30 33 -- 0.16(0.07)
Branch diameter age 5 (cm) 0.5230 0.0793 <0.0001 1.49 1.54 0.14 (0.04) 0.10 (0.07)
Branch diameter age 6 (cm) 0.3292 0.1584 0.0003 1.49 1.54 -- 0.19(0.08)
Number branches/crown 0.2087 0.0018 <0.0001 7.90 7.30 -- 0.11 (0.07)
length age 5
Number branches/crown 0.1047 0.0136 <0.0001 6.63 6.02 -- 0.14 (0.07)
length age 6
Branch angle age 5 () 0.0099 <0.0001 <0.0001 56.9 51.1 0.18 (0.04) 0.26 (0.08)
Light interception age 5-6 (VMJ 0.0293 0.1041 <0.0001 9,901 11,901 0.17(0.01) 0.17(0.08)
tree-')
Note: Values in parentheses are standard errors
a-- Values of H2WF in slash pine were estimated separately by family and are in Table 2-2.









slash pine families, there was a tendency for higher heritabilities in family S2 (Table 2-2).

For example, crown radius at ages 5 and 6 showed H2WF of 0.41, and crown volume at

ages 5 yr and 6 yr had moderate heritability values between 0.34 and 0.36.

Within-Family Genetic and Environmental Correlations

Within slash pine families, the genetic correlations between individual-tree stem

volume increment (ages 5 y and 6 y) were positive and moderate to high with: APAR

between age 5 y and 6 y, crown size traits at age 5 y, and tree leaf area at age 5 y (rg=0.35

to 0.74). Individual-tree stem volume increment had low positive or low negative genetic

correlations with crown shape ratio and branch angle at age 5 y (rg=-0.33 to 0.39, Table

2-3). For loblolly pine family L4, individual-tree volume increment between age 5 y and

6 y was moderately genetically correlated with APAR between age 5 y and 6 y (rg=0.64),

and crown size traits at age 5 y such as crown volume (rg=0.51), crown radius (rg=0.47),

and crown length (rg=0.53). Stem volume increment was positively, but less strongly

correlated with leaf area at age 5 y (rg=0.31). As in slash pine, traits such as crown shape

ratio and branch angle at age 5 yr had much weaker genetic correlations with stem

volume growth (rg=-0.20 and 0.20, respectively Table 2-3).

Environmental correlations are measures of microsite environmental fluctuation

between two traits measured on the same ramets. In slash pine families, moderately to

highly positive environmental correlations were found between stem volume increment

age 5 and 6 y and light interception age 5 and 6 y (re=0.43 to 0.83), implying that

microsites that enhanced APAR also enhanced stem growth. At the same time, positive

environmental correlations were found between stem volume increment age 5 and 6 y

and crown size at age 5 (crown volume, crown radius, and crown length, re=0.52 to

0.76), and between stem volume increment age 5 and 6 y and leaf area age 5 y, number of









branches age 5 y, and branch diameter age 5 y (re=0.36 to 0.72, Table 2-3). Finally,

crown shape ratio age 5 y and branch angle age 5 had low positive or negative

environmental correlations with stem volume increment age 5 and 6 y, implying that

microsites that favored growth did not affect crown shape ratio age 5 y and branch angle

age 5 y. For loblolly pine, environmental correlations had similar tendencies as in slash

pine, with moderate positive environmental correlations between stem volume increment

age 5 and 6 y and crown size at age 5 (crown volume, crown radius, and crown length,

re=0.49 to 0.0.58), between stem volume increment age 5 and 6 y and leaf area age 5 y

(re=0.46), and also between stem volume increment age 5 and 6 y and branch diameter

age 5 y (re=0.43, Table 2-3). Weakly positive or negative environmental correlations

were found between stem volume increment age 5 and 6 y and number of branches age 5

y, branch angle age 5 y and crown shape ration age 5 y (re=-0.12 to 0.29).

Both APAR and crown volume at age 5 y proved to be good integrators of crown

characteristics for individual trees. In general, APAR and crown volume at age 5 y had

stronger genetic correlations with stem volume growth than did any other crown traits.

Discussion

At the species level, the one loblolly pine family we studied tended to grow faster

than the average of our four slash pine families at ages 5 y and 6 y. At the same time,

loblolly pine developed larger crowns with more acute branch angles and had more leaf

area per individual-tree at age 5 y and 6 y than did the slash pine families (Table 2-1).

Xiao et al. (2003) found similar species-level contrasts in juvenile loblolly and slash pine

in north central Florida, where loblolly pine accumulated more crown volume per tree,

allocated more biomass to branches, and had greater amount of leaf area than slash pine

at ages 3 and 4 y. Stand-level studies have similarly confirmed the ability of loblolly pine









to develop and retain higher levels of leaf area than slash pine (Dalla-Tea and Jokela

1991; Martin and Jokela 2004).

Growth differences among slash pine families were subtle, probably because the

families selected for my study were all chosen for superior growth potential. In spite of

the apparent similarities in stem volume growth rate, the four slash pine families differed

in a number of crown architectural traits. Contrasting families had different arrangements

and sizes of branches within the crown, and varied in crown shape ratio (Table 2-1,

Figure 2-1). This suggests that any of a number of crown traits may be associated with

high growth rate in southern pine families (see also McGarvey et al. 2004). In contrast,

McCrady and Jokela (1996) concluded that, among the five loblolly pine families they

studied, there were significant differences in height growth but none for most branching

attributes.

Within-family clonal variation was highly significant for all growth and crown

structural traits, reflecting a wide spectrum of clonal performance in growth and crown

development at these ages. There are few reports in the literature on clonal variation in

loblolly or slash pine growth. Paul et al. (1997) reported that height of loblolly pine

clones varied significantly at different ages, but that DBH and volume did not. To our

knowledge, no published studies have quantified clonal variation in crown characteristics

in loblolly or slash pine, but these traits have been studied in other forest tree species. For

example, Lambeth et al. (1994) found large differences among Eucalyptus grandis clones

in growth, branching, and crown density. In Populus, clonal differences in branch

characteristics and branching patterns were found that resulted in striking differences in

crown form and architecture (Ceulemans et al. 1990). Sylleptic branches and the










considerable leaf area that they carry have important implications for whole tree light

interception, and thus, play a critical role in the superior growth and productivity of

certain hybrid poplar clones. The considerable variation in branch characteristics implies

a strong justification for including them in selection and breeding programs for Populus

Table 2-2. Age 5 y and 6 y within-family individual-tree broad-sense heritability (H2WF)
for growth and crown structural traits in four slash pine families in north
central Florida.
Trait HWF__
Family S1 Family S2 Family S3 Family S10
0.16 0.22 0.08 0.00
Stem volume (age 4 y) 0.08) (0.09) (0.08) (0.00

0.21 0.24 0.02 0.10
Stem volume increment (age 5-6 y) 0.) 0.) 0.0 0.0
(0.08) (0.09) (0.07) (0.07)
0.21 0.31 0.13 0.18
Crown length (age 6 y) (0.08) (0.10) (0.08) (0.08)
0.11 0.41 0.12 0.09
Crownradius(age 5 y) (0.07) (0.11) (0.09) (0.07)
0.17 0.41 0.12 0.09
Crown radius (age 6 y) (0.07) (0.11) (0.08) (0.08)

0.27 0.33 0.22 0.25
Crown shape ratio (age 5 y) 0.09) (0.10) (0.10) (0.09)

0.32 0.05 0.26 0.17
Crown shape ratio (age 6 y) 0.09) (0.07) (0.10) (0.08)

0.13 0.34 0.10 0.10
Crown volume (age 5 (0.07) (0.11) (0.08) (0.07)

Crown volume (age 6 y) (0.08) (0.10) (0.08) (0.08)
0.13 0.34 0.15 0.16
Leaf area (age 6 y) (0.07) (0.10) (0.09) (0.08)
0.10 0.26 0.22 0.14
Number of branches (age 5 y) (0.07) (0.09) (0.10) (0.08)
Number of branches (age 5 y) 02 01 01 01

0.26 0.15 0.14 0.10
Number of branches (age 6 y) (0.09) (0.08) (0.09) (0.07)

Branch diameter (age 6 y) (0.06) (0.07) (0.07) (0.06)

Number of branches per unit of crown 0.00 0.12 0.27 0.03
length (age 5 y) (0.00) (0.07) (0.10) (0.06)
Number of branches per unit of crown 0.04 0.01 0.24 0.05
length (age 6 y) (0.06) (0.07) (0.09) (0.06)
Note: Values in parentheses are standard errors










Table 2-3. Within-family genetic correlations among individual-tree volume increment
between age 5-6 and crown structural variables at age 5, for slash (Si, S2, S3
and S10) and loblolly (L4) pine families in north central Florida.
Trait Family S1 Family S2 Family S3 Family S10 Family L4
Stem Volume Increment (age 5-6 yr)
Genetic correlations
0.70 0.74 0.62 0.67 0.64
Light interception (age 5-6 y) 0
(0.08) (0.05) (0.47) (1.00) (0.10)
0.71 0.61 0.69 0.41 0.51
Crown volume (age 5 y)
(0.11) (0.10) (0.78) (0.33) (0.14)
0.64 0.64 0.35 0.61 0.31
Leaf area (age 5 y)
(0.19) (0.10) (0.93) (0.45) (0.34)
0.39 -0.33 0.02 0.01 -0.20
Crown shape ratio (age 5 y)
(0.23) (0.23) (0.72) (0.37) (0.32)
0.51 0.75 0.40 0.02 0.16
Branch diameter (age 5 y)
(0.21) (0.10) (0.75) (0.71) (0.36)
-0.05 0.01 0.01 -0.22 0.26
Branch angle (age 5 y) (0.29) (0.26) (0.70) (0.45) (0.25)
0.43 0.41 -0.06 0.03 0.20
Number of branches (age 5 y) 0.) 0.1 0.) 0.) 0.2
(0.26) (0.18) (0.75) (0.41) (0.24)
0.55 0.66 0.42 0.20 0.47
Crown radius (age 5 y)
(0.17) (0.09) (0.56) (0.43) (0.16)
0.77 0.55 0.37 0.31 0.53
Crown length (age 5 y)
(0.09) (0.13) (0.76) (0.36) (0.23)

Environmental correlations
0.71 0.78 0.83 0.43 0.68
Light interception (age 5-6 y) 0.) 0.) 0.3 0.) 0.
(0.03) (0.03) (0.03) (0.07) (0.04)
0.69 0.70 0.76 0.60 0.58
Crown volume (age 5 y)
(0.04) (0.04) (0.04) (0.05) (0.05)
0.44 0.60 0.72 0.58 0.46
Leaf area (age 5 y)
(0.06) (0.05) (0.07) (0.05) (0.06)
-0.10 0.09 0.03 0.01 -0.12
Crown shape ratio (age 5 y)
(0.08) (0.08) (0.09) (0.08) (0.08)
0.36 0.52 0.70 0.50 0.43
Branch diameter (age 5 y)
(0.07) (0.06) (0.05) (0.06) (0.06)
-0.16 -0.06 -0.19 -0.24 0.01
Branch angle (age 5 y) (0.08) (0.08) (0.09) (0.08) (0.08)

0.39 0.41 0.44 0.39 0.29
Number of branches (age 5 y) 0
(0.06) (0.07) (0.07) (0.07) (0.07)
0.62 0.62 0.74 0.58 0.56
Crown radius (age 5 y)
(0.04) (0.05) (0.04) (0.05) (0.05)
0.52 0.64 0.76 0.57 0.49
Crown length (age (0.05) (0.05) (0.04) (0.05) (0.06)

Note: Values in parentheses are standard errors









(Ceulemans et al. 1990). Wu (1994a) also reported significant clonal variation in Populus

hybrids in crown structural traits at the leaf, branch, and whole-tree levels.

Traditionally, most complex traits, such as growth rate and crown architecture, are

thought to be polygenic, determined by the expression of many genes (Falconer and

Mackay 1996). This seems intuitive, given that growth rate and crown architecture are

affected by many physiological parameters, phenological patterns, organ growth rates,

and also by environmental factors like competition interactions, seasonal variation in

water availability, nutrient status, light intensity and duration, air and soil temperature,

pest and pathogen pressure.

Our results agreed with the polygenic model in that crown architectural and growth

traits had low to moderate within-family broad-sense heritabilities, and are therefore

likely determined by the expression of many genes. It is possible that the low genetic

variation may be due to the nature of the traits we measured and their role in determining

fitness. Traits connected with fitness often show low heritability, since natural selection

for these traits reduces genetic variation, while traits which are less intimately tied to

fitness may have higher genetic variability and so higher heritability (Falconer and

Mackay 1996). Tree growth rate and crown size are potentially important components of

fitness.

Broad-sense heritabilities estimated from my study were expected to be smaller

than broad-sense heritabilities values usually reported in the literature, because they were

estimated within full-sib families and half the additive genetic variation and one fourth of

the dominance variation as well as a portion of the epistatic variance occurs among full-

sib families (Falconer and Mackay 1996). Considering this, our results were comparable









with other clonal studies. With respect to stem growth traits, Paul et al. (1997) reported a

H2 of 0.14 for loblolly pine stem volume, while Borralho et al. (1992) estimated H2

between 0.08 and 0.18 for height and sapwood area in E. globulus. In crown structural

traits, reported H2 values ranged from 0.27 to 0.78 in E. grandis and hybrid poplars

(Lambeth et al. 1994; Wu 1994a).

Narrow-sense heritability which includes only the additive genetic variation is

necessarily smaller than broad-sense heritability for the same trait. For stem growth and

crown structural traits, low to moderate narrow-sense heritabilities (0.0 to 0.62) have

been reported in loblolly and slash pine at young ages (Lambeth and Huber 1997; Xiao et

al. 2003), as well as in other pine species as Pinus brutia, P. radiata and P. sylvestris

(0.02 to 0.53; Espinel and Aragones 1997; Haapanen et al. 1997; Arregui et al. 1999; Isik

and Isik 1999).

One interesting finding was the heterogeneity of the variance components among

families, which resulted in significantly different within-family broad-sense heritabilities

for many traits. Slash pine family S2 showed higher H2WF values compared to the other

three slash pine families (Table 2-2). Higher within-family broad-sense heritability can

reflect either a larger clonal variance component (a2c in the numerator of H2WF) or a

smaller residual variance (C2, in the denominator of H2WF), or both. In family S2, a larger

proportion of clonal variance and smaller residual variance component, were found with

respect to the rest of the slash pine families, and resulted in larger H2WF. Smaller residual

variances in S2 corresponded also to a smaller interaction between clone and microsite

for that particular family than the other slash pine families. It is possible that the two

parents of family S2 had greater proportion of heterozygosity at gene loci determining









crown size, producing more segregation among their progeny than in other slash pine

families. If this is true, then even for polygenic traits, it is possible to find specific pairs

of parents producing more variable offspring for growth or crown traits. These families

might be useful for quantitative trait loci (QTL) mapping and gene discovery (Bradshaw

and Stettler 1995; Wu and Stettler 1996; Wu 1998).

An understanding of the relationship between crown architecture and tree growth

might provide a basis for predicting tree growth, and could aid in the search for

discovering genes involved in growth and for developing new crop ideotypes

(Kuuluvainen 1988; Dickmann and Keathley 1996; Martin et al. 2001). Evidence of

positive phenotypic association between crown architecture and tree growth is common

in many species, including loblolly and slash pines, with many authors reporting the

importance of the amount of light intercepted by the canopy and its correlation with

growth rate (Linder 1987; Cannell 1989; Dalla-Tea and Jokela 1991; McCrady and

Jokela 1998; Will et al. 2001).

In my study, a number of crown architectural traits were consistently genetically

correlated with growth (Table 2-3), which is consistent with previous quantitative genetic

analysis of crown architectural traits in other species (Wu 1994b; Espinel and Aragones

1997; Haapanen et al. 1997; Arregui et al. 1999; Isik and Isik 1999), and production

ecology work in loblolly and slash pine (e.g. McCrady and Jokela 1998; Martin and

Jokela 2004). As we hypothesized, the more integrated measures of crown structure and

function in my study, specifically APAR and crown volume, were consistently more

strongly correlated with stem volume growth rate than were less integrative measures

such as crown radius or length, number of branches, branch angle, or average branch









diameter. APAR was a particularly comprehensive trait, providing a time- and space-

integrated index of crown dimensional traits, leaf area, tree size, and crown dimension of

surrounding competitor trees. It is interesting, however, that the relatively simple trait of

crown volume was as strongly or almost as strongly correlated with stem volume growth

as was APAR (Table 2-3). The quantity of APAR by tree crowns is one of the major

factors determining aboveground biomass accumulation throughout stand development

(Wang and Jarvis 1990). The amount of light intercepted by an individual-tree crown is

influenced by its leaf area quantity and display, the incident radiation, and the distribution

and size of surrounding trees (Wang and Jarvis 1990).

Two crown traits consistently showed weak or non-existent genetic relationships

with growth: crown shape ratio and branch angle. Similar results were obtained by

Lambeth and Huber (1997), where branch angle (zero being the closest to horizontal) was

weakly but negatively genetic correlated with growth rate (-0.24) (bigger trees tending to

have flatter branch angle). In absolute terms, bigger trees tended to have wider crowns

(rg=0.75), and large branch diameter (rg=0.31), but when adjustments were made for size,

they tended to have smaller branches and narrower crowns for their size and fewer

branches per meter of height than smaller families. Xiao et al. (2003) reported for

loblolly and slash pines families that crown shape ratio combined two important variables

(crown height, crown width) that were statistically significant among taxa, but in ratio

form as crown shape ratio appeared to have little ecological significance in developing

stands with respect to growth performance. Similarly, McCrady and Jokela (1996)

observed significant intraspecific variation in crown shape ratio in young loblolly pine









plantations, but they did not find an advantage of narrower crowns over wider crowns in

height growth increment.

In other species, such as P. radiata, P. sylvestris, Populus and E. grandis,

significant positive genetic correlations were found among height, stem diameter,

volume, crown diameter, and crown density and vigor. On the other hand, genetic

correlations between growth and branch diameter, and growth and branch angle were

species specific and variable showing favorable or unfavorable correlations (Arregui et

al. 1999; Espinel and Aragones 1997; Haapanen et al. 1997; Lambeth et al. 1994; Wu

1994b).

With respect to environmental correlations, microsites that favored the

development of the crown, leaf area, and light interception also enhanced growth rate in

all families. Branch angle and crown shape ratio showed non-significant environmental

correlation with volume increment. Thus, microsites with higher levels of nutrients or

water availability appear to favor tree volume growth, crown size and light interception at

the same time, but do not seem to affect branch angle and crown shape ratio.

Here we reported important linkage between crown structural and functional traits

with stem volume growth in loblolly and slash pine families and clones. However, what

is finally translated into stem volume increment depends on complex relations with other

processes and their genetic patterns. Additional studies with respect to carbon gain, water

relations and hydraulic conductivity at the individual-tree level will help improve our

understanding of what control stem volume growth in contrasting families and clones.














CHAPTER 3
GENETIC VARIATION IN BASAL-AREA INCREMENT PHENOLOGY AND ITS
CORRELATION WITH GROWTH RATE IN LOBLOLLY AND SLASH PINE
FAMILIES AND CLONES

Introduction

Loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm. var elliottii)

are widely planted as commercial timber species in the southeastern United States (Smith

et al. 2004). From the early 1950s, large-scale tree breeding programs in the southeastern

United States have worked to improve forest productivity by selecting trees for superior

growth rate, form, and disease resistance (McKeand et al. 2003), and the improved

material currently being established in commercial plantations is deployed from bulked

orchard seed, half-sib families, and full-sib families with growing interest in the

deployment of outstanding clones.

The extensive natural range of loblolly and slash pines, spanning different

environmental conditions, has resulted in accumulation of adaptative genetic variation

across time and differences in growth potential among sources (Burs and Honkala

1990). To develop tree breeding programs it is necessary to understand the genetic

variation of selected traits, their correlations and the effect of the environment on

genotypic expression (White 1987). In Florida winter temperatures are rarely low enough

to prohibit positive photosynthetic rates and considerable transpiration (McGarvey 2000;

Martin 2000). These mild winter conditions, plus an abundant rainfall through the

summer, may have translated into genotypes adapted to a longer growing seasons and/or

faster growth.









Most pines experience a cycle of bud set and growth cessation in the latter part of

the growing season, followed by deepening dormancy, cold hardening, dormancy release

in the winter, and bud break in the spring (Dougherty et al. 1994). In the case of loblolly

pine, the wide natural distribution, spanning different ecotypes and environments,

contains a diverse range of chilling requirements to promote dormancy release, length of

the growing period and rate of growth. For instance, while it has been established that

chilling is required for loblolly pine northern ecotypes, it is unclear that there is a true

dormancy and chilling requirement for southern latitude sources (Carlson 1985).

Increase in the diameter of tree stems occurs primarily from meristematic activity

in the vascular cambium, a cylindrical lateral meristem located between the xylem and

phloem of the stem, branches, and woody roots. The time of the year during which the

cambium is active varies with climate, species, crown class, seasonal development of leaf

area in trees, and different parts of stems and branches (Kozlowski and Pallardy 1997).

Fluctuations in environmental stresses affect cambial growth to a large extent by altering

the supply of photosynthate to the branches and stem (Kozlowski 1971; Sevanto et al.

2003). For example, cambial growth is sensitive to available water, with several aspects

being responsive to the amount and seasonal distribution of rainfall, including number of

xylem cells produced and ring width, seasonal duration of cambial growth, proportion of

xylem to phloem increment, time of latewood initiation, duration of latewood production,

and wood density (Kozlowski 1971; Cregg et al. 1988; Downes et al. 1999; Makinen et

al. 2000, 2001; Bouriaud et al. 2005).

The amount of growth in a particular season is determined by the date of growth

initiation, the date of growth cessation (which together determine growth duration), and









the average daily growth rate for the growth period. The cessation of shoot and cambium

activity is one determining factor, and the more fully the plant can utilize the growing

season, without suffering from spring and fall frost, the greater potential annual growth,

final harvest, and return on the investment in planting stock. Much of the interest in forest

tree phenology is related with these practical questions (Lieth 1974).

The total growth period from initiation to cessation, both for height and cambial

activity, has been studied on an individual tree basis in many North American tree

species, but little information on genetic variation is available. Seasonal periodicity of

tree growth has been studied in evergreen and deciduous trees (Jackson 1952; Harkin

1962; Langdon 1963; Emminham 1977; Li and Adams 1994; McCrady and Jokela 1996;

Zhang et al. 1997; Jayawickrama et al. 1998; Yu et al. 2001). Wide variation among

species in duration of the period of growth was recorded by Jackson (1952). Cambial

growth of some species lasted only about 80 days and others grew for up to 200 days.

Several of the species which initiated growth early in the season had long periods of

growth, while some of the late starting species exhibited shorter periods.

Langdon (1963) studied growth patterns of slash pine (Pinus elliottii Engelm. var.

densa Little and Dorman) in south Florida (Fort Myers) for four years and found that

diameter growth occurred about ten months per year (from March through December).

Initiation of diameter growth was believed to be promoted by apically produced

hormones (Savidge and Wareing 1984). Diameter growth has been reported to initiate

before or almost simultaneously with height growth for loblolly pine (Zahner 1962) and

for slash pine (Kaufmann 1977). Conifers usually continue diameter growth into the fall









after height growth has stopped, as reported for Pseudotsuga menziesii and loblolly pine

(Emmingham 1977; Jayawickrama et al. 1998).

Previous research in loblolly and slash pine diameter growth phenology has

provided important knowledge about the duration of cambial activity (Jackson 1952;

Harkin 1962; Langdon 1963; McCrady and Jokela 1996; Zhang et al. 1997;

Jayawickrama et al. 1998). However, there is a lack of information about how the

duration of cambial growth might influence the differences in growth rate between

species planted in the same area, and also the growth differences among families within

species and clones within families.

My study examines the following hypotheses:

* There is significant genetic variation in basal-area growth phenology among slash
and loblolly pine genotypes (species, families and clones);

* Where it exists, genetic variation in basal-area growth phenology is correlated with
variation in annual basal-area increment.

The specific objectives are to:

* Compare two years basal-area growth phenology among species, families and
clones;

* Estimate genetic parameters for basal-area growth phenology, its correlation with
growth rates, and the genotype interaction with seasonal environmental changes.

Material and Methods

Study Site and Plant Material

The study area was located on lands managed by Rayonier Inc. in Bradford County,

Florida. The climate is humid and subtropical, with a mean annual temperature of 210C,

mean annual rainfall of 1316 mm, and over 50% of the rainfall occurring in June through

September. Periods of drought are normal in the spring and fall. Mean annual rainfall

during 1999-2001 was 967 mm, in contrast to 1405 mm in year 2002 (NOAA 2002). The









soils are classified as Pomona and consist of very deep, somewhat poorly to poorly

drained soils that are formed in sandy and loamy marine sediments (sandy, siliceous,

hyperthermic Ultic Alaquods). Slopes are 0 to 2 %. In a typical profile, the spodic

horizon occurs at 30-60 cm, with an argillic horizon at 90-120 cm. Water table is

typically at a depth of 15 to 45 cm for one to three months and a depth of 25 to 100 cm

for six months or more, during most years (Soil Survey Staff 1998).

The study took place in an area containing 16 full-sib and half-sib loblolly and

slash pine families planted in 337 m2 family plots in January 1997. The experiment was

designed as a randomized complete block with four replicates (Appendix A). We used

one full-sib loblolly pine family and four full-sib slash pine families. Each family plot

contained 60 clones propagated as rooted cuttings from a single family, planted at 1.7 m

x 3.4 m spacing (1730 trees ha-1). Cuttings were taken from donor hedges in the spring,

and were rooted and grown in a greenhouse for six months before planting. Each of the

four plots of the same family contained the same 60 genotypes, but with the ramets

planted into different, randomly-determined planting locations in the plot. In total we

studied approximately 1,200 trees: 60 trees per family plot x 5 families x 4 replications.

Fertilization and weed control were applied periodically to reduce interspecific

competition and prevent nutrient deficiency (Appendix B).

Basal-area Increment Measurements

Phenology was evaluated as periodic basal-area growth increment as determined

from repeated DBH measurements throughout growing seasons in 2002 and 2003.

Families S1, S2, S3, L4 and S10 were monitored for diameter increment once a month in

the summer time and every ten to fifteen days during the period of growth initiation and

cessation in the spring and fall, respectively. Diameter increment was measured with a









digital caliper (model 18 ES, Mahr, Germany, resolution 0.01 mm) over 4 plexiglass

plates attached to the tree stem in north-south and east-west orientations. Diameter

measurements were done such that two replications were measured on day 1 and two

replications on day 2 each time period.

Phenological Traits

From the periodic diameter measurements, a cumulative basal-area growth curve

for two growing seasons was plotted for each tree, and dates of basal-area growth

initiation and cessation were estimated by interpolation as the dates when 5% and 95% of

annual growth were completed (Hanover 1963). Duration of basal-area growth (in days)

was calculated as the difference between dates of cessation and initiation. Basal-area

increment per year (in mm2) was calculated as the difference in individual tree basal-area

between the 5% and the 95% dates of initiation and cessation. Basal-area growth rate (in

mm2/day) was calculated as the ratio of annual basal-area increment and duration of

basal-area growth.

Meteorological Data and Water Balance

Climatic data were collected at the Gainesville Regional Airport (about 20 km

distant from the study site, NOAA 2003) and a research weather station 8 km from the

study site. Meteorological variables included hourly radiation, mean air temperatures, and

daily rainfall. A simple water balance model was computed to estimate soil water

reserves at daily time steps, and to quantify soil water deficit. The model was given by

Equation 3-1, where Rn is soil water reserve at day n, Rn-1 is soil water reserve of the day

before, Pn is precipitation and Tn is transpiration, both at day n:

Rn = Rn-1 + Pn Tn (3-1)









The water holding capacity in 1 m depth for this site was estimated at 260 mm

according to soil texture and flatwoods Spodosols moisture release curves (H.L. Gholz,

personal communication). Plot-level transpiration (Tn) was estimated as follows:

maximum hourly potential evapotranspiration (PET, mm) was calculated by dividing

measured hourly radiation by the latent heat of vaporization of water; maximum plot-

level transpiration was then calculated as 60% of PET, and was assumed to occur when

all-sided leaf area index (LAI) was greater than 6.0. At LAI less than 6.0, transpiration

was estimated to decline linearly with declining LAI (Martin and Jokela 2004). Plot-level

leaf area index was calculated from litterfall data as in Martin and Jokela (2004). Because

understory vegetation was sparse, only pine LAI was considered. The resulting model

incorporated variation in environmental conditions (daily precipitation, hourly radiation),

as well as plot-level leaf area index to produce a plot-level index of soil water

availability.

Statistical Analyses and Genetic Parameters

Analysis of variance (ANOVA) was used for phenological and growth data

separately for each year. PROC GLM in the SAS System was used to test for

significance of random effects (clone), while PROC MIXED was utilized to test the fixed

effects (species and families). Equation 3-2 shows the linear model considered for the

analyses, where Yijkl is the performance of the ramet of the 1th clone within the kth family

nested in the jth species in the ith replication; i = 1, 2, 3, and 4 for replications; j = slash,

loblolly; k = 1, 2, 3, 4, and 10 for families; 1 = 60 identification numbers for 60 clones

within each of the five families:

Yijkl = t + bi + Sj + Fkg) + C(jk) + bSij + bFik() + ijkl (3-2)









t = population mean,
bi = random variable of replication NID (0, o2b),
Sj = fixed effect of species (slash or loblolly),
Fk(j) = fixed effect of family nested within species,
cl(jk) = random variable of clone nested within-family and species NID (0, o2c),
bSij = random variable for replication x species interaction ~ NID (0, o2bS),
bFik(j) = random variable for replication x family(species) interaction ~ NID (0,
o2bF), and
eijkl = error term ~ NID (0, o2s).

With so few families, estimates of genetic parameters were restricted to within-

family estimates obtained from clonal variation expressed within each of the four slash

families and one loblolly pine family. For each family two types of genetic parameters

were estimated: within-family broad sense heritability for each trait, and within-family

genetic correlations among traits. Within-family variance and covariance components

were obtained using ASREML, a statistical package that fits linear mixed models using

Restricted Maximum Likelihood (Gilmour 1997).

Within-family individual tree broad sense heritability was calculated using

Equation 3-3, where C2o is the variance among clones within-family and c25 is the residual

variance as defined in Equation 3-2:


H2 2 2 (3-3)


Theoretically, broad sense within-family heritability contains 1/2 the additive genetic

variance, 34 of the dominance genetic variance, and most of the epistatic genetic variance

(Falconer and Mackay 1996). The standard error for heritability estimates was calculated

from Dickerson (1962). The residual likelihood ratio test (Wolfinger 1996) was used to

test heterogeneity of variances among slash pine families, and heritabilities were

estimated separately (X2(6, 0.05)= 12.6), or pooled, as appropriate.









Within-family genetic correlations among basal-area phenological traits and growth

rate were calculated using Equation 3-4 (Falconer and Mackay 1996), where Cxy is the

clonal covariance between two traits, and cx and o, are the square root of the product of

the clonal variance within-family for traits x and y, respectively:


r -= (3-4)


Standard error for genetic correlations was estimated using ASREML (Gilmour

1997).

The significance of the clone by year variance component was tested using the

likelihood ratio test (Wolfinger 1996). The clone by year variance component was

declared different from 0 when X2(1,0.05) was equal to or greater than 3.8. For traits with a

significant clone by year variance component, within-family genetic correlations between

years were estimated considering the two years as two different traits using Equation 3-4.

Results and Discussion

Genetic Variation among Species and Families

In 2002, species and families within species were not significantly different at 5%

for any phenological or growth trait, while in year 2003, date of growth cessation and

daily basal-area growth rate were significant at the species level with loblolly pine

ceasing growth earlier and growing more than slash pine (Table 3-1 and Figure 3-1). In

2002, the mean date of basal-area growth initiation was March 10 (69 days after January

1) for the single loblolly pine family and one day earlier for the slash pine families.

Basal-area growth cessation for the loblolly pine family, on average occurred on

November 1, resulting in a mean duration of basal-area growth of 236 days. In the case of









slash pine families, mean cessation was on October 28, and the duration of basal-area

growth was 234 days (Table 3-1).

In 2003, basal-area growth started and finished one to two weeks sooner than in

2002 for both loblolly and slash pine families (Table 3-1). For loblolly pine, basal-area

growth began in February 23 and finished by October 4, resulting in a mean duration of

basal-area growth of 223 days (13 days fewer than in 2002). For slash pine, basal-area

growth began, on average, on February 25 and ended on October 17, for duration of 235

days (only one day shorter than in 2002, Table 3-1). In 2003, more growing days for

slash pine compared to loblolly pine might explain why the difference between species in

total basal-area growth was less than in the previous year (26.98-23.81=3.17 cm2 in 2002

versus 24.26-21.98= 2.28 cm2 in 2003). This is supported by the fact that the slash pine

families with longest (S1) and shortest (S3) duration had greater differences in basal-area

growth in 2003 than in 2002 (24.94-22.12=2.82 cm2 in 2002 versus 22.8-19.69=3.11 cm2

in 2003). Annual basal-area increment and daily basal-area growth rate were larger for all

families in 2002 than in year 2003, despite a shorter growing season for some families in

2002. Early cessation in year 2003 in comparison with year 2002, and the difference in

total annual increment between years could possibly be due to the differences in amount

and seasonal distribution of rainfall between 2002 and 2003 (1405 mm and wet soil

conditions by the end of the year in 2002; and 1184 mm and dry conditions by the end of

the year in 2003, Figure 3-2).

In general, loblolly pine tended to accumulate more stem volume through ages 6

and 7 y than slash pine. This was manifested by larger yearly and daily basis basal-area

increments, but these differences among species were only significant (p<0.05) for daily











basal-area growth in 2003 (Table 3-1). From my study we can conclude that the

differences between loblolly and slash pine accumulated slowly over time through ages 6

and 7 y.

Table 3-1. Significance levels (p-values), and species and family least square means for
individual tree stem growth and phenological traits for two growing seasons
for loblolly and slash pine families in north central Florida.
Trait Significance level by effect Species mean Slash family means
Family Family Family Family
Species Family Clone Slash Loblolly S1 S2 S3 S10
Year 2002
Initiation 0.8160 0.1120 0.0534 68.43 69.00 70.31 63.57 70.83 68.99
Cessationa 0.3542 0.1870 0.2141 302.16 304.82 306.43 301.66 301.44 299.12
Duration (days) 0.5456 0.2966 0.0131 233.71 235.82 236.13 238.03 230.59 230.10
Volume 6 y 0.0797 0.1062 <0.0001 25.37 31.58 26.32 26.10 23.55 27.52
(dm3)
BA increment 0.0784 0.0586 <0.0001 23.81 26.98 24.94 25.04 22.12 23.14
(cm2)
BA growth rate 0.0945 0.2783 <0.0001 10.18 11.47 10.57 10.50 9.59 10.05
(mm2/day)

Year 2003
Initiation 0.0815 0.3384 0.0062 55.85 53.86 56.21 54.94 56.79 55.45
Cessation 0.0459 0.0853 0.0007 291.01 276.51 297.03 291.00 280.87 295.12
Duration (days) 0.0711 0.0774 0.0050 235.18 222.64 240.82 236.12 224.09 239.69
Volume 7 y 0.0835 0.0687 <0.0001 38.31 44.30 38.67 38.87 34.67 41.03
(dm3)
BA increment 0.1312 0.0894 <0.0001 21.98 24.26 22.80 22.83 19.69 22.60
(cm2)
BA growth rate 0.0385 0.4796 <0.0001 9.35 10.81 9.45 9.63 8.87 9.45
(mm2/day)


a Initiation and cessation are days after January 1
diameter growth


cq 100 i
E
0
80-
I -
n


to complete 5 and 95% of seasonal


0 50 100 150 200 250 300 3500 50 100 150 200 250 300 350
Day of the year 2002 Day of the year 2003
Figure 3-1. Family mean cumulative basal-area growth curves for years 2002 and 2003 in
loblolly and slash pine in north central Florida.


-0- S1


_~--~t~


t L4
~ S10













30

25

20

15

10
5

S0
-5
0


(m-;

-E0


150 200 250 300 3500 50 100 150 200 250 300 350


0 50 100 150 200 250 300 3500 50 100 150 200 250 300 350


Day of Year 2002


Day of Year 2003


Figure 3-2. Species mean daily basal-area growth increment for loblolly and slash pine in
north central Florida and environmental variables. Species mean daily basal-
area growth increment in 2002 (A) and 2003 (B), where indicates significant
differences between species (p<0.05) and + indicates significant differences
among slash pine families (p<0.05); current-year and 20-year mean monthly
precipitation in 2002 (C) and 2003 (D); cumulative precipitation in 2002 (E)
and 2003 (F); and mean plot-level soil water balance (error bars indicating
standard errors) in 2002 (G) and 2003 (H). Precipitation data from Gainesville
Regional Airport, NOAA (2003).


50 100 150 200 250 300 3500 50


oUU -
250
200
150
100
50
0 -
0
1600 -
1400
1200
1000
800
600
400
200
0-


-o- Slash 25
-*- Loblolly
20

15

10


+ 0
--. -5
150 200 250 300 350
300
-a- Current Year 250
-- 20 yr mean
a 200


0


)E
.5-

aE


o






=E
0

0


E
OE


E



0

C-)
UE
03 -

'0
OT


A


200 250 300 3500


50 100 150 200 250 300 350


0 50 100


150
100
50
0

1600
1400
1200
1000
800
600
400
200
0

500

400

300

200

100

0


H


50 100 150









Because phenology is closely related to latitude (Campbell 1986; Jayawickrama et

al. 1998; Nielsen and Jorgensen 2003), growth initiation, cessation, and duration should

depend on the study location and geographic origin of the sampled seed or vegetative

propagule. Our results are in agreement with what Langdon (1963) reported on growth

patterns of slash pine in south Florida (Fort Myers). He found that diameter growth

occurred about ten months during the year (from March through December), and the total

amount of diameter growth and its seasonal distribution responded to climatic variation.

With respect to diameter growth cessation date, similar results were found by

Jayawickrama et al. (1998), for example, a loblolly pine provenance from Gulf

Hammock (Florida) grew until day 299 and 313 in two different years.

Comparing studies done in northern regions with slash and loblolly pine, our results

showed earlier initiation date, later cessation date and longer season length. For example,

in a site close to Athens, Georgia, Jackson (1952) found that loblolly and slash pine

started diameter growth between the end of March and the beginning of April, with a

duration of five to six months. In South Carolina, McCrady and Jokela (1996) found that

in loblolly pine diameter growth initiated by the end of March and finished by August-

September, giving mean diameter growth duration of 5 months. No significant

differences were found among families in initiation or cessation of diameter growth.

All families showed similar patterns of basal-area increment across the growing

season in years 2002 and 2003, i.e. shapes of the cumulative basal-area curves were quite

similar (Figure 3-1). In general, basal-area growth peaked in early spring, and then

remained relatively constant throughout the remainder of the growing season (Figure 3-1,

3-2). The differences at the species level and among families within slash pine









accumulated across time. Daily average basal-area growth rate was only significantly

different at the species level in 2003 (Table 3-1). Similar trends in diameter growth were

found by others authors. Linear radial growth was observed over the entire growing

season in slash and loblolly pine trees by Jackson (1952), except for a period of slow

growth in the late summer which was probably associated with soil moisture depletion.

Similar linear trends were reported by McCrady and Jokela (1996) in loblolly pine

families. Cregg et al. (1988) reported that unlike height growth, rapid diameter growth

can be maintained over the entire growing season and the rate of diameter growth of

loblolly pine, observed during a year when moisture deficits did not develop was almost

constant over the period from day 50 to day 290.

Despite the apparent lack of variation in basal-area growth rate indicated by the

cumulative growth data (Figure 3-1), peaks in basal-area increment occurred in the early

spring both years (Figure 3-2). Significant species and family differences were found for

critical spring periods when growth rates were highest: in year 2002 measurement period

3 (March), and in 2003 measurement periods 1 and 2 (end of February and middle

March, respectively). These results suggest that at least some of the genetic differences in

cumulative growth (as shown in Figure 3-1) are manifested not through constant

expression of consistent growth rate differences, but rather through elevated growth rate

during very discrete periods of time (as shown in Figure 3-2). In other words, the basal-

area growth rates of taxa are remarkably similar for most of the year, but in the spring

some environmental variables or genetic differences in phenology trigger more rapid

growth in some taxa, which essentially raises the intercept of the linear cumulative basal-

area functions for the rest of the year (Figure 3-1). In other studies in loblolly and slash









pine, peaks in basal-area increment in early spring also were reported by Zhang et al.

(1997) and Langdon (1963); accelerating growth in spring was also reported in Norway

spruce (Bouriaud et al. 2005). But these studies in conifers did not identify genetic

differences in tree growth rate at this temporal scale. In the case of hardwoods, growth

and phenology studies in hybrid aspen clones (Populus tremula x Populus tremuloides)

compared growth patterns in temperate climates throughout the year (Yu et al. 2001).

Peaks in diameter growth occurred in the end of the spring and beginning of the summer.

Hybrid clones had higher growth rates than the pure P. tremula and also accumulated

larger annual diameter increment.

In 2002, daily basal-area growth was weakly negatively correlated with soil water

balance (Daily BA growth = 13.7929 0.0187 x soil water balance, R2=0.11, p<0.0001,

Figure 3-3). In contrast, in 2003, daily basal-area growth was positively associated with

calculated soil water balance (Daily BA growth = -1.9424 + 0.0478 x soil water balance,

R2=0.49, p<0.0001, Figure 3-3). The total amount of rainfall in 2002 was 1405 mm, 177

mm above average. Wet conditions were present especially between June and December

and presumably had a negative effect on growth (Figure 3-2). On the other hand, in a year

with average rainfall, like in 2003, where rainfall totaled 1184 mm (44 mm less than a

normal year), a strong correlation was observed with more growth associated with higher

levels of soil water availability. In year 2003, we found that daily basal-area growth

followed same pattern as that of soil water balance (Figure 3-2B). For both years and

both species, the highest growth rates in basal-area were reached in conditions where the

soil water balance was around 300 mm. This analysis implies that basal-area growth rate

increased as water soil availability increased, when water was limiting, but excess water











available in the soil had a negative effect on growth, perhaps caused by plant stress due to

prolonged root inundation.


0)

EE
E-
cmE
-E


30- 0
A B Slash
0 Loblolly
20O 00 0

0 0 o
10 i0n. C

0 00


0 100 200 300 400 500 600 0 100 200 300 400 500 600
Soil water balance year 2002 (mm) Soil water balance year 2003 (mm)

Figure 3-3. Relationship between individual tree daily basal-area increment and
simulated daily plot-level soil water balance in loblolly and slash pine in 2002
(A) and 2003 (B). The line shows a linear regression through data. A: Daily
BA growth = 13.7929 0.0187 x soil water balance, R2=0.11, p<0.0001. B:
Daily BA growth = -1.9424 + 0.0478 x soil water balance, R2=0.49,
p<0.0001.

Studies in flatwoods soils in north-central Florida have shown reduced radiation

use efficiency when soil water balance was high/wet, and this effect can have a direct

impact on tree growth rates (Martin and Jokela 2004). Langdon (1963) also reported that

excess soil water appeared to depress growth. From the four years of their study with

slash pine, for the year with high rainfall (1929 mm) and high ground-water levels during

summer and early fall, both diameter and height growth were considerably below the

other 3 years. Water table depth was found to be associated with growth in the flatwoods

in Florida (White and Pritchett 1970). Larger height and diameter growth was reported in

slash and loblolly pine with controlled water table depth conditions at 46 and 92 cm from

the surface, in comparison with natural fluctuating water table conditions. Bouriaud et al.

(2005) studied the influence of climatic variables on annual radial growth and wood

density on Picea abies. They found numerous decreases in radial growth rate closely

related to the calculated soil water deficit. Also, wood density increased with decreasing









radial growth rate in the second half of the growing season affected by drought. Similar

results were reported by Cregg et al. (1988), where early season diameter growth rate for

loblolly pine was a function of available soil moisture and temperature.

Clonal Variation and Within-Family Inheritance of Phenological Traits and Stem
Growth

At the clone within-family level (pooled across families), differences in initiation,

cessation and duration of basal-area increment in the growing season were more apparent

than at the family and species level differences in both 2002 and 2003 (Table 3-1). Traits

related to individual tree stem growth, such as volume, and yearly and daily basal-area

increment were also different among clones within families in both years (Table 3-1).

Analyses of the data separately by family showed that phenological traits differed among

clones for some families and not for others in both years. Family S2 was the only one that

showed significant clonal variation in all phenology traits in 2003. Volume, yearly basal-

area increment, and daily basal-area growth had significant clonal variation within-family

in 2002 and 2003 for all families (Table 3-2).

For phenology traits, individual tree broad sense heritabilities were low to

moderate, ranging from 0.01 to 0.24 (Table 3-3). In contrast, within-family heritabilities

for stem growth traits were moderate to high in both years ranging from 0.10 to 0.37

(Table 3-3). Family S2 tended to have higher within-family broad sense heritabilities than

the other slash pine families, in most cases due to higher clonal variation within that

family as opposed to lower residual environmental variance. These heritabilities are

expected to be smaller than broad sense heritabilities values usually reported in the

literature, because they are estimated within full-sib families and half the additive genetic

variation and one fourth of the dominance variation as well as a portion of the epistatic










variance occurs among full-sib families (Falconer and Mackay 1996). Still, phenotypic

expressions of phenological traits associated with basal-area growth were under weak

genetic control.

Table 3-2. Significance levels (p-values) for clone within-family for tree stem growth and
phenological traits for two growing seasons in loblolly and slash pine families
in north central Florida.
Trait Significant level within-family clonall variation)
Family L4 Family S1 Family S2 Family S3 Family S10
Year 2002
Initiation 0.0003 0.0311 0.1964 0.1577 0.1880
Cessation 0.3200 0.3489 0.1145 0.1541 0.3753
Duration 0.0695 0.1608 0.0498 0.2409 0.1593
Volume 6 y 0.0015 <0.0001 <0.0001 0.0720 0.0390
BA increment <0.0001 0.0042 <0.0001 0.0191 0.0277
BA growth rate <0.0001 0.0028 <0.0001 0.0279 0.0286

Year 2003
Initiation 0.0253 0.4211 0.0011 0.0706 0.4932
Cessation 0.2165 0.3028 0.0028 0.1302 0.1550
Duration 0.3215 0.3770 0.0068 0.1146 0.3893
Volume 7 y 0.0001 0.0003 <0.0001 0.0206 0.0318
BA increment <0.0001 0.0043 <0.0001 0.0524 0.0004
BA growth rate <0.0001 0.0016 <0.0001 0.0536 <0.0001

Heritability estimates for phenological traits are available for a few species and are

usually presented for leaf phenology rather than basal-area or shoot phenology. For

example, narrow-sense h2 estimates ranged between 0.67 to 0.96 in Juglans nigra, and

between 0.28 and 0.71 in Picea glauca for initiation and cessation of leaf development,

depending on experimental conditions, age, and method of computations (Leith 1974). In

pole-size P. menziesii, individual tree heritabilities were higher for bud burst and bud set

(h2=0.73 and 0.81, respectively) than for duration of shoot growth (h2=0.17, Li and

Adams 1993). With respect to diameter growth, Li and Adams (1994) estimated

individual heritabilities for diameter growth initiation (h2=0.23), and cessation (h2=0.11)

in 15 y-old P. menziesii, values that are comparable with my study. At the same time, Li

and Adams (1994) did not detect significant family differences in duration of diameter










increment, suggesting that the small variation in date of diameter growth cessation among

families may have been related to summer dry conditions. Other studies have shown that

summer drought has little effect on variation in cambial growth initiation, but reduces

variation in cambial growth cessation among coastal P. menziesii provenances

(Emmingham 1977). In my study, we did not detect an association between clonal

variation within-family for initiation or cessation and soil water balance and the presence

of relatively dry spring or late summer.

Table 3-3. Within-family individual-tree broad-sense heritabilities for growth phenology
traits and basal-area growth increment by year in loblolly and slash pine
families growing in north central Florida.
Trait HWF_
Family L4 Family S1 Family S2 Family S3 Family S10
Year 2002
Initiation 0.20 (0.07) 0.10 (0.06) 0.07 (0.07) 0.09 (0.08) 0.00 (0.00)
Cessation 0.00 (0.00) 0.00 (0.00) 0.08 (0.07) 0.07 (0.08) 0.00 (0.00)
Duration 0.08 (0.06) 0.07 (0.06) 0.11(0.07) 0.05 (0.08) 0.00 (0.00)
Volume 6 y 0.18 (0.07) 0.25 (0.07) 0.26 (0.08) 0.10 (0.08) 0.10 (0.07)
BA increment 0.24 (0.07) 0.15 (0.07) 0.37 (0.08) 0.15 (0.09) 0.13 (0.07)
BA growth rate 0.23 (0.07) 0.19 (0.04)a

Year 2003
Initiation 0.12 (0.07) 0.03 (0.06) 0.24 (0.08) 0.10 (0.09) 0.03 (0.06)
Cessation 0.05 (0.06) 0.05 (0.06) 0.19 (0.08) 0.08 (0.08) 0.09 (0.07)
Duration 0.04 (0.06) 0.05 (0.06) 0.16 (0.08) 0.09 (0.08) 0.03 (0.06)
Volume 7 y 0.20 (0.07) 0.20 (0.07) 0.29 (0.08) 0.14 (0.09) 0.11(0.07)
BA increment 0.34 (0.07) 0.21 (0.05)a
BA growth rate 0.32 (0.07) 0.23 (0.05)a
Note: Values in parentheses are standard errors
a Values of H2WF in slash pine were pooled by family since variance components were
homogeneous.

Genetic Correlations among Phenological Traits and Stem Growth

When phenological traits did not differ significantly among clones within-family,

genetic correlations were not estimated. Among the estimates, many genetic correlations

relating phenology traits to growth were not significantly different from zero (Table 3-4).

In 2002, genetic correlations between initiation and duration were strong and negative in

family L4, S1 and S2, which indicated that clones with early growth initiation also had a









tendency to grow longer, and that clones that initiated later also tended to have a shorter

growing season. On the other hand, genetic correlations between cessation and duration

were positive and strong in family S2 and S3, meaning that clones that had a tendency to

cease growth late in the year also grew for a longer period of time.

In 2003, genetic correlations between initiation and cessation were significant and

moderately positive only for family S2. The genetic correlations were positive and strong

between cessation and duration for all families, meaning that clones that stopped growth

later also grew for a longer period of time. In general, these results suggest that variation

in duration of the growing season among individuals in these families was more a

function of cessation date than initiation date, but all of these traits were weakly inherited

(Table 3-3).

With respect to genetic correlations between stem growth variables and

phenological variables, significant correlations were found primarily in family S2,

varying from moderate to strongly positive (r = 0.31 to 0.85, Table 3-5). In 2002,

duration had a positive strong genetic correlation with basal-area increment in S2. In

2003, initiation, cessation, and duration had moderate positive genetic correlations with

basal-area increment in S2. At the same time, initiation in 2003 for L4 showed a strong

positive genetic correlation with basal-area increment (rg=0.86). Among the variables we

investigated, daily basal-area growth rate in both years showed the strongest genetic

correlation with yearly basal-area increment across all families. Correlations of

phenology variables with total volume after the 2002 and 2003 growing seasons were

similar to the patterns of correlation with yearly basal-area increment, reflecting

consistency between phenology and increment during the year and phenology and









cumulative stem growth. These results suggest that clones that grew faster between

initiation and cessation were also the ones with more yearly basal-area increment and

total volume. The high genetic correlation between daily basal-area growth rate and

yearly basal-area increment in a year was explained in part because of the autocorrelation

between these two variables.

Genetic correlations among clonal values for basal-area growth and phenological

traits are scarce in the literature; most of the reported results are phenotypic correlations

at the family level. One of the few studies on genetic control of cambial phenology found

that P. menziesii genotypes with early growth initiation also tended to cease growth early

(rg=0.60, Li and Adams 1994). They also suggested that variation in growth duration

among individuals is primarily a function of variation in date of growth cessation

(rg=0.77). Height phenology studies in P. abies in northern Europe showed that early start

of shoot growth was genetically correlated with early shoot growth cessation. Also, there

was a consistently low or no correlation between the shoot elongation period and either

total height or leader length (Ekberg et al. 1994).

Reported phenotypic correlations in southern pines for both diameter and height

growth are more closely related to growth rate, than with phenological traits such as

cessation (McCrady and Jokela 1996; Jayawickrama et al. 1998). Jackson (1952) found

that there was no consistent relationship between the starting date and faster growth in

slash and loblolly pine trees. For P. menziesii saplings, most of the differences among

populations in one season's growth were related to growth rate rather than growth

duration (Emmingham 1977). In Picea mariana, cambial growth cessation and total














Table 3-4. Within-family genetic correlations between growth phenology traits in 2002 (above the diagonal) and 2003 (below the
diagonal) in loblolly and slash pine families growing in north central Florida. I: initiation; C: cessation; and D: duration of
basal-area growth.
Trait Family L4 Family S1 Family S2 Family S3 Family S10
I C D I C D I C D I C D I C D
I a -0.98 a -0.85 -0.47 -0.82 0.46 0.02 a a
(0.33) (0.41) (0.91) (0.35) (0.72) (0.86)
r n 0 a n a ) /'7 n1 2 n1 ) n 0 n 1 nn a


(0.61) (2.06) (0.2i
D 0.93 0.99 0.49 1.00 0.5(
(1.04) (0.06) (2.59) (0.09) (0.3z
Note: Values in parentheses are standard errors
a Was not estimated because within-family clonal variance was 0


/ -. 1 ~ J -.o. 1 .J\
8) (0.21) (0.72) (0.17) (2.6)
6 0.99 -0.42 0.98 1.00 1.00
4) (0.01) (0.67) (0.02) (2.6) (0.31)


Table 3-5. Within-family genetic correlations between growth and phenology traits by year in loblolly and slash pine families growing
in north central Florida
Traits Basal-area increment 2002 Basal-area increment 2003
L4 S1 S2 S3 S10 L4 S1 S2 S3 S10
Initiation -0.05 (0.26) -0.12 (0.45) a a a 0.86 (0.27) a 0.59 (0.22) 0.22 (0.84) a
Cessation a a a a a a a 0.59(0.23) a a
Duration 0.32 (0.35) a 0.85 (0.28) a a a a 0.53 (0.25) a a
BA growth rate 0.99 (0.01) 1.00 (0.01) 0.99 (0.00) 0.99 (0.01) 0.99 (0.01) 1.00 (0.01) 0.99 (0.01) 0.99 (0.02) 0.96 (0.09) 0.99 (0.02)

Volume age 6 Volume age 7
Initiation 0.03 (0.29) 0.06 (0.37) a a a 0.94 (0.32) a 0.31 (0.25) 0.76 (0.50) a
Cessation a a a a a a a 0.66(0.23) a a
Duration 0.29 (0.40) a 0.83 (0.36) a a a a 0.65 (0.25) a a
BA growth rate 0.99 (0.04) 1.00 (0.00) 0.99 (0.02) 0.91 (0.11) 1.00 (0.11) 1.00 (0.03) 0.89 (0.06) 0.93 (0.04) 0.89 (0.13) 1.00 (0.13)
Note: Values in parentheses are standard errors
a Was not estimated because within-family clonal variance was 0









height had a positive phenotypic correlation. Although in continental P. abies

populations, this correlation was zero and sometimes negative (Dietrichson 1967, 1969).

On the other hand, studies in aspen hybrids in temperate regions suggested that the

fast overall growth is largely explained by longer vegetative period (rp between growth

period and diameter was 0.67-0.91 and highly significant, Yu et al. 2001).

Because cambial phenology traits appear to be weakly inherited and have small and

inconsistent genetic correlations with growth, indirect responses in cambial phenology

from selection of bole basal-area or volume are expected to be small. The practical

implications of these findings are that selection programs aimed at increasing growth rate

are very unlikely to impact dates of initiation or cessation; thus there are few concerns

about increasing the likelihood of frost damage. Also, another important point to consider

in indirect responses, as suggested by Langdon (1963), is the effect of length of growing

season on wood properties. Trees that are capable of growing longer into the season may

produce a higher proportion of summerwood to spring wood and have higher wood

density than genotypes that cease growth early. Future research could help to understand

whether families or clones which cease growth earlier do, in fact, have lower wood

density. If so, this could then be incorporated into selection programs.

Analysis across Years 2002-2003

There were no significant clone by year interactions for any basal-area phenology

traits, and only basal-area increment for L4 and basal-area growth rate for S2 showed

significant clone by year interactions (data not shown). Still, genetic correlations between

years were high for these two traits with significant clone x year interactions (0.91 for L4

basal-area increment, and 0.93 for S2 basal-area growth rate), indicating that the

interactions were not biologically important. From this analysis we can conclude that









each of the basal-area growth phenology traits and each of the basal-area growth rate

traits were genetically controlled by a similar set of genes in years 2002 and 2003. So, the

clones were consistent across years in both phenology and growth traits that we

measured. Nevertheless, stability needs to be tested for a longer period of time, since

environment played an important role in the control of the inheritance of basal-area

phenology traits (i.e. severe environmental conditions may change the results).

Clonal studies in Betulapendula revealed significant clone-by-year interactions for

bud burst and also large interannual variation among clones in the date of bud burst and,

especially, in the termination of growth (Rousi and Pusenius 2005). These interactions

indicated that in addition to genetic effects, environmental factors have a strong influence

on both bud burst and growth termination. In P. menziesii, provenance-by-year

interaction existed for bud burst date in a three-year study (White et al. 1979). In a two-

year study with loblolly pine, Jayawickrama et al. (1998) found no significant

provenance-by-year interaction; and significant year-by-family within provenance

interactions were found for height growth and height growth cessation.

We conclude that the significant genetic variation among clones within-family in

basal-area growth and the stability of ranks across years found in my study, contribute to

understanding the potential impact that clonal selection can have on future forest

plantation productivity. Poor consistency in direction and strength of genetic correlations

between basal-area increment and phenological traits indicated that in these slash and

loblolly pine families, initiation, cessation or duration of growth were traits that did not

have biological importance in determining how much a genotype will grow during the

season. Basal-area growth in loblolly and slash pine families and clones was sensitive to









soil water availability, with stem growth declining both above and below an "optimum"

soil water balance level. Finally, while there were significant size differences among taxa

(species and families) at age 6 y and 7 y, genetic differences in basal-area growth rate

were only expressed during short, discrete time periods in the spring and fall. This

finding may have important implications for the timing of investigations attempting to

determine the mechanisms underlying genetic growth differences, since growth rate, and

possibly the physiological or gene expression traits controlling growth rate, may be

similar throughout most of the growing season among taxa with contrasting long-term

cumulative growth.














CHAPTER 4
CARBON ISOTOPE DISCRIMINATION, CROWN CONDUCTANCE, GROWTH
AND THEIR GENETIC PARAMETERS IN LOBLOLLY AND SLASH PINE
FAMILIES AND CLONES

Introduction

Plants fractionate carbon isotopes during photosynthesis. The magnitude of the

fractionation varies with photosynthetic type, environment, genotype, and other factors,

and this variation in magnitude can be used to study a variety of issues in plant

physiology (O'Leary 1981, 1988, 1993; Farquhar et al. 1989). During photosynthesis, the

stable isotope ratio (13C/12C) of carbon dioxide assimilated differs from that of the source

CO2 and is about 2 % lower in plants than air (Farquhar et al. 1989). There are two

primary processes that cause carbon isotope ratios to change during photosynthesis:

diffusional fractionation and enzymatic fractionation. Carbon dioxide molecules

containing 12C are lighter, and therefore, diffuse into the leaf at a faster rate (by a factor

of 1.0044, or 4.4 %o) than molecules containing 13C (Craig 1954; Farquhar and Lloyd

1993). The primary carboxylating enzyme in C3 plants, ribulose-1,5-biphosphate

carboxylase, preferentially uses 12CO2 (by a factor of 1.029 or 29%o) and so discriminates

against 13CO2 (Roeske and O'Leary 1984; Guy et al. 1993). The carbon isotope ratio of

leaf organic material depends on the relative influence of diffusional and enzymatic

fractionation, which in turn is determined by the ratio of intercellular CO2 (p1) and

atmospheric CO2 (pa) partial pressures (Farquhar et al. 1982, 1989). Changes in the ratio

p,/pa and the leaf carbon isotope ratio are a function of changes in either, or both,

photosynthetic rate and stomatal conductance (Farquhar et al. 1989).









Since the carbon isotope ratio in the leaf provides information about processes

integrated over the whole life of a leaf, it is particularly useful for examining subtle

genetic differences in photosynthetic and water use characteristics. There has been

considerable interest in using carbon isotope discrimination (A13C, determined from

carbon isotope ratio of the sample with respect to a standard; lower discrimination against

13C, means A13C value closer to 0, than high discrimination against 13C) to estimate

integrated water use efficiency (WUE) in both agronomic plants and trees. WUE

measures the ratio between photosynthetic rate (A) and transpiration rate (E), or in other

words the ratio between carbon fixation and water losses.

Genetic variation in A13C has been reported for several tree species. Family

differences in A13C were reported for Pseudotsuga menziesii (Zhang et al. 1993), Larix

occidentalis (Zhang et al. 1994), Picea mariana (Flanagan and Johnsen 1995; Johnsen et

al. 1999), Picea glauca (Sun et al. 1996), Araucaria cunninghamii (Prasolova 2000),

Pinuspinaster (Brendel et al. 2002), Castanea sativa (Lauteri et al. 2004). Significant

clonal variation has also been demonstrated in foliar carbon isotope composition in Fl

hybrids clones between slash pine (Pinus elliottii) and Caribbean pine (Pinus caribaea)

(Xu et al. 2000; Prasolova et al. 2003, 2005), in Eucalyptus globulus (Osorio and Pereira

1994, Osorio et al. 1998), loblolly pine (Pinus taeda) (Gebremedhin 2003) and poplar

hybrid clones (Marron et al. 2005). Therefore, understanding of the genetic basis of

variation in A13C could be very useful for ranking genotypes and may serve as a guide for

tree breeding programs.

Most studies reported in the literature on A13C and WUE in tree species have been

associated with a small number of species or genetic entries. Only a few recent









publications (Johnsen et al. 1999; Prasolova et al. 2003) have reported the results of A13C

with relatively large sets of genetic materials in tree breeding trials, and these have been

used to obtain reasonable estimates of genetic parameters such as heritability for foliar

A13C and genetic correlations between physiological traits. From an operational point of

view, such information is crucial when introducing new genetic entries, such as untested

clones, into plantation schemes.

The growth of individual plants may be either positively or negatively correlated

with leaf carbon isotope discrimination values depending on whether variation in

discrimination is associated with changes in photosynthetic capacity or stomatal

conductance (Farquhar et al. 1989). During photosynthetic gas exchange, discrimination

will be reduced in a plant when photosynthetic rate is increased, if stomatal conductance

remains constant. The higher photosynthetic rate may also translate into faster growth, if

other factors remain constant. Therefore, carbon isotope discrimination values should be

negatively correlated with plant growth when variation in discrimination results from

changes in photosynthetic rates (Farquhar et al. 1989). In contrast, if variation in

discrimination is caused by changes in stomatal conductance, then carbon isotope

discrimination values should be positively associated with growth. This results because

an increase in stomatal conductance will result in higher assimilation of carbon, thereby

increasing growth, and will also enhance discrimination against 13C during gas exchange

(Farquhar et al. 1989).

Previous results have suggested that carbon isotope ratio in the leaf can be used in

early selection in tree improvement programs (Farquhar et al. 1989; Bond and Stock

1990; Zhang et al. 1993; Sun et al. 1996; Johnsen et al. 1999; Xu et al. 2000; Pita et al.









2001; Prasolova et al. 2003). Due to the advantages of early selection in tree

improvement programs of loblolly and slash pine, the use of the carbon isotope ratio

technique might help to increase forest productivity in future plantations by selecting

families or clones that show greater water use efficiency or photosynthetic rate or a

combination of both.

Stomata respond to environmental variation and regulate water loss and carbon

dioxide gain, and thus biosphere-atmosphere exchange of mass and energy. Ideally,

stomatal conductance should remain in balance with variations in soil-leaf hydraulic

conductance. This coordination would contribute to maintenance of leaf water potential

above minimum values associated with leaf desiccation, nonstomatal inhibition of

photosynthetic carbon acquisition, and xylem cavitation (Wullschleger et al. 1998). In the

last decade, development and calibration of techniques that allow measurement of water

movement through the sapwood and crown as sap flow, make it possible to calculate

crown conductance parameters such as stomatal sensitivity to changes in environmental

conditions, like radiation and vapor pressure deficit in longer spatial and temporal scales

(Granier 1987; Martin et al. 1997; Ewers et al. 1999; Martin et al. 2001; Lu et al. 2004;

Martin et al. 2005). Stomatal sensitivity to environmental changes will affect gas

exchange levels, photosynthesis, carbon fixation and growth (Sperry 2000; Tyree 2003).

Because one of the processes that define carbon isotope discrimination in the leaf is

stomatal conductance, it is important to know the correlation between discrimination and

conductance in loblolly and slash pine clones.

In this paper we examine how changes in carbon isotope discrimination are related

to both differences in stem growth increment and differences in tree-level crown









conductance observed within full-sib families and clones of loblolly and slash pine. The

following hypotheses were considered:

* Carbon isotope discrimination varied among genotypes;

* Fast-growing genotypes tend to have lower carbon isotope discrimination and
higher water use efficiency, so stem growth will be negatively correlated with leaf
stable carbon isotope discrimination; and

* Genotypes that tend to have higher stomatal sensitivity to changes in water pressure
deficits tend to have lower values of discrimination against 1C.

Material and Methods

Study Site and Plant Material

The study area was located on lands managed by Rayonier Inc. in Bradford County,

Florida. The climate is humid and subtropical, with a mean annual temperature of 210C,

mean annual rainfall of 1316 mm, and over 50% of the rainfall occurring in June through

September. Periods of drought are normal in the spring and fall. Mean annual rainfall

during 1999-2001 was 967 mm, in contrast to 1405 mm in year 2002 (NOAA 2002). The

soils are classified as Pomona and consist of very deep, somewhat poorly to poorly

drained soils that are formed in sandy and loamy marine sediments (sandy, siliceous,

hyperthermic Ultic Alaquods). Slopes are 0 to 2 %. In a typical profile, the spodic

horizon occurs at 30-60 cm, with an argillic horizon at 90-120 cm. Water table is

typically at a depth of 15 to 45 cm for one to three months and a depth of 25 to 100 cm

for six months or more, during most years (Soil Survey Staff 1998).

The study took place in an area containing 16 full-sib and half-sib loblolly and

slash pine families planted in 337 m2 family plots in January 1997. The experiment was

designed as a randomized complete block with four replicates (Appendix A). We used

one full-sib loblolly pine family and four full-sib slash pine families. Each family plot









contained 60 clones propagated as rooted cuttings from a single family, planted at 1.7 m

x 3.4 m spacing (1730 trees ha-1). Cuttings were taken from donor hedges in the spring,

and were rooted and grown in a greenhouse for six months before planting. Each of the

four plots of the same family contained the same 60 genotypes, but with the ramets

planted into different, randomly-determined planting locations in the plot. For growth and

carbon isotope discrimination we studied approximately 1,200 trees: 60 trees per family

plot x 5 families x 4 ramets per clone distributed as one ramet in each of the 4 complete

blocks. For sap flow and crown conductance analysis we measured approximately 300

trees: 30 trees per family x 5 families x 2 ramets per clone distributed across the 4

complete blocks as described later. Fertilization and weed control were applied

periodically to reduce interspecific competition and prevent nutrient deficiency

(Appendix B).

Tree Growth and Carbon Isotope Discrimination

Stem volume growth in the 2001, 2002, and 2003 growing seasons (ages 4-5 yr, 5-6

yr, and 6-7 yr, respectively) was determined from dormant season measurements of tree

diameter at 1.37 m height (DBH) and total tree height (HT). Outside-bark individual tree

stem volume was calculated using Equation 4-1 (Hodge et al. 1996), where DBH and HT

were entered in m:

VOL (dm3) = (0.25 3.14 (DBH)2 (1.37 + 0.33 (HT 1.37)))*1000 (4-1)

Also, periodic diameter increment was measured in March-April 2002 to calculate

sapwood cross-sectional area for tree-level transpiration analysis as explained later.

Foliar samples for stable carbon isotope discrimination analysis were collected

from the five families from the first flush formed in the spring. Samples were taken in

middle summer 2001 and 2003 from a branch on the south side of the upper canopy









(exposed to full sun, to help avoid extraneous differences in isotope values). The tissue

was dried at 65 C for several days, and finely ground. The relative abundance of 13C and

12C was determined in 3 mg subsamples with a Delta Plus isotope ratio mass

spectrometer (Cornell University Stable Isotope Laboratory). Stable carbon isotope ratio

(613C) was expressed as 13C/12 C ratio relative to international PDB (Pee Dee Belemnite,

Craig 1954). Carbon isotope discrimination values (A13C) were calculated from 613 C

values using Equation 4-2 (Farquhar et al. 1989), where 6p is the isotope composition of

the plant material and 6a is that of the air (assumed to be -8%o):

A = 8p (4-2)
1 +5p

The accuracy and precision of this analysis for foliar 613C were ascertained by

making repeated measurements of 613C in each batch of the samples and using an internal

foliar standard in each of the sample batches. We concluded that carbon isotope

measurements are repeatable and accurate with a standard error of 0.14%o.

Use of A3C to compare A/E among genotypes requires several assumptions: first,

that leaf temperatures, and therefore leaf-to-air vapor pressure differences, are similar

among the plants being compared; second, that 613C of source of C02 is identical among

genotypes being compared; and third, that biosynthetic fractionation is similar among the

genotypes. These assumptions are well met by the needle shape of conifer leaves,

because they are narrow and their boundary layer conductances are therefore high

(Marshall and Zhang 1993, Ewers and Oren 2000).

Meteorological Data

Air temperature, photosynthetically active radiation, and relative humidity were

measured in year 2002 by a weather station installed in the study area. Variables were









read every minute and averages recorded every 15 minutes by a data logger. Vapor

pressure deficit (D) was calculated from relative humidity (RH) and air temperature (TA)

measurements based on equations adapted from Goff and Gratch (1946).

Individual Tree Transpiration

A subsample of 300 trees from 5 families (30 clones per family and 2 ramets per

clone) was used to monitor sap flow on a daily basis. Selection of clones within families

was based on three criteria: genotypes were selected across the range of growth

performance (good, medium and poor growers); genotype performance for growth was

consistent across replications in the test; and finally genotypes were free of disease.

Selection of ramets within clone was done by selecting the two ramets more

representative of the growth performance category and free of disease.

Water flux in the xylem was estimated using the constant heat method of Granier

(1987). Heat dissipation gauges were installed in each of the 300 trees, a constant 0.2 W

of power was applied to the probe, and the degree to which heat is dissipated from the

probe was measured. A heat probe of 20 mm long was inserted into the tree stem, at 40

cm from the base, and was paired with an unheated probe located 10 cm below the heated

probe. To avoid thermal gradients from direct radiation, all sensors were installed in the

north side of the stem and covered with aluminum shelters.

Measurements of sap flux were taken every minute and stored as a 15-min average

for two months (March-April in 2002) with dataloggers (Campbell Scientific, Logan,

UT). Equation 4-3 shows how sap flux (Js, kg H20 m-2 s-1) was calculated using an

empirical relationship (Granier 1987):

Js = 0.119 ((ATm- AT)/ AT) 1.231 (4-3)









which measured the daily maximum temperature difference between heated and

unheated probes during times of zero flux (ATm) as a baseline. Temperature difference

(AT) was also measured during the day as water carried heat away from the probe.

Deviation from the baseline was used to estimate water flux.

Whole-tree transpiration per unit of projected crown area (EL, kg H20 m-2 Ac s-1)

was calculated by multiplying tree sapwood cross-sectional area (As, m2) by the sap flux

density measurement and standardized by projected crown area (Ac, m2, calculated as

projected crown area on the ground):

EL = Js (As/Ac) (4-4)

To estimate EL we assumed that water uptake, as estimated from sap-flow

measurements, does not significantly lag actual canopy transpiration.

Calculations of individual tree sapwood cross-sectional area contained two

assumptions: 1) For young southern pine, the entire cross-sectional area of the tree was

composed of active sapwood; and 2) Sap flux density for sapwood further than 20 mm

from the cambium (where sap flow probes are located) was the same as outer cambium.

The first assumption was probably met in these young pine trees, while the second was

almost certainly not. Several studies have shown that sap flux density tends to be higher

near the cambium, and declines with radial depth into the sapwood (Phillips et al. 1996;

Wullschleger and King 2000; James et al. 2002). However, for the purposes of my study,

the estimates of whole-tree water use were acknowledged to be biased upward, but

should still be useful for relative comparison of the genotypes.

Crown Conductance and Stomatal Sensitivity Calculations

We calculated whole-tree crown conductance of water vapor (Gs, mm s-1) by

substituting the transpiration data and meteorological measurements into the inverted









Penman-Monteith equation using the formula suggested by Monteith and Unsworth

(1990), where X is the latent heat of evaporation of water (J kg-1), y is the psychrometer

constant (kPa K-1), pa is the density of dry air (kg m-3), Cp represents the specific heat

capacity of the air (J kg-1 K-1), and D is the vapor pressure deficit (kPa):

Gs = 1000 (EL 7) / (pa Cp D) (4-5)

Gs values were converted from mm s1 to mmol m-2 s-1 using Pearcy et al. (1989).

Equation 4-5 requires the following conditions (Ewers and Oren 2000): (1) D is close to

the leaf-to-air vapor pressure deficit, namely boundary layer conductance is high; (2)

There is no vertical gradient in D through the canopy; and (3) There is negligible water

stored above the Js measurement position. We assumed that these conditions were met at

my study.

As the vapor pressure deficit between leaf and air increases, stomata generally

respond by partial closure (Lange et al. 1971). Responses of stomatal conductance to

increasing D generally follow an exponential decrease described by the empirical

Equation 4-6 (Oren et al. 1999), where -m is the sensitivity of Gs response to InD or the

slope of Gs vs InD (-dGs/dlnD) and Gsref = Gs at D = 1 kPa:

Gs = -m InD + Gsref (4-6)

Given the uncertainties under low levels of incoming radiation (or limited light

throughout the canopy) and low D situations, we filtered the data to conditions where D >

0.6 kPa and incoming radiation > 500 Wm-2. This screening allowed us to keep errors in

the estimation of Gs below 10% (Ewers and Oren 2000).











Genetic Parameters and Statistical Analyses

Analysis of variance (ANOVA) was used to analyze A13C, crown conductance

parameters in response to vapor pressure deficit, and growth data by year. SAS PROC

GLM was used to test for significance of random effects (clone), while PROC MIXED

were utilized to test the fixed effects (species and families). Equation 4-7 shows the linear

model considered for the analyses, where Yijkl is the performance of the ramet of the 1th

clone within the kth family nested in the jth species in the ith replication; i = 1, 2, 3, and 4

for replications; j = slash, loblolly; k = 1, 2, 3, 4, and 10 for families; 1 = 60 identification

numbers for 60 clones within each of the five families:

Yjkl = [t + bi + Sj + Fkg) + C(jk) + bSij + bFik() + ijkl (4-7)

[t = population mean,
bi = random variable of replication NID (0, C2b),
Sj = fixed effect of species (slash or loblolly),
Fkg) = fixed effect of family nested within species,
Cl(jk) = random variable of clone nested within-family and species ~ NID (0, 02),
bSi = random variable for replication x species interaction ~ NID (0, C2bs),
bFik) = random variable for replication x family(species) interaction ~ NID (0,
C2bF), and
Sijk = error term ~ NID (0, 02).

With so few families, estimates of genetic parameters were restricted to within-

family estimates obtained from clonal variation expressed within each of the four slash

families and one loblolly pine family. For each family two types of genetic parameters

were estimated: within-family heritability for each trait, and within-family genetic and

environmental correlations among traits. Within family variance and covariance

components were obtained using ASREML, a statistical package that fits linear mixed

models using Restricted Maximum Likelihood (Gilmour 1997).









Within-family individual-tree broad-sense heritability was calculated as in

Equation 4-8, where C2c is the variance among clones within family and c2 is the residual

variance as defined in Equation 4-7:

2
H2wF 2 (4-8)


The standard error for heritability estimates was calculated from Dickerson (1962).

The residual likelihood ratio test (Wolfinger 1996) was used to test heterogeneity of

variances among slash pine families, and heritabilities were estimated separately

(X2(0.05,6)= 12.6), or pooled, as appropriate.

Within-family genetic and environmental correlations between A13C and growth

rate and between A13C and crown conductance parameters were calculated with the

Equation 4-9 (Falconer and Mackay 1996), where oxy is the clonal or residual covariance

between two traits, while cx and o, are the square root of the product of the clonal or

residual variance within family for traits x and y, respectively:


r = (4-9)


Standard error for genetic and environmental correlations was estimated using

ASREML (Gilmour 1997, asymptotic properties and the Taylor series approximation of

the variance of a ratio).

The significance of the clone-by-year variance component was tested using a

likelihood ratio test (Wolfinger 1996). The clone-by-year variance component was

declared different from 0 when X2(1,0.05 was equal to or greater than 3.8. For traits with a

significant clone-by-year variance component, within-family genetic correlations









between years were estimated considering the two years as two different traits, using

Equation 4-9.

Results

Carbon Isotope Discrimination

Family within-species and clone within-family were significant sources of variation

for foliar carbon isotope discrimination in 2001 and 2003 (Table 4-1), but at the species

level no significant differences were detected (p<0.05). Mean family values for A13C

ranged between 21.39%o to 22.98%o (Table 4-1), and at the clonal level mean values for

A13C ranged from 19%o to 25.41%o (data not shown). Among slash pine families, family

S2 tended to have the lowest values for A13C in both years 2001 and 2003 (Figure 4-1). In

contrast, family S3 showed the highest value for both years. Lower values of A13C in year

2001 than in year 2003 samples for all families might be associated with lower rainfall

between February and June 2001 in comparison with same period in 2003 (near 50 % less

rainfall in year 2001 than in year 2003, Figure 4-2). Clonal within-family genetic

variation was significant in all 5 families for A13C in 2001, and for all families except S3

in 2003 (Table 4-2).

Within-family heritabilities ranged from 0.01 to 0.32 for discrimination in years

2001 and 2003 (Table 4-3). There was no evidence for genotype-by-year interaction for

any family. A strong within-family genetic correlation between A13C in year 2001 and

year 2003 occurred for all families (Table 4-4), indicating that the ranking of clones

remained constant across years for carbon isotope discrimination.












23.2

23.0

22.8

22.6

22.4

22.2 -
-
22.0

21.8

21.6

21.4

21.2


S1 S2 S3 S10 L4


Figure 4-1. Family means and standard
2001 and 2003.



1400

1200

1000


Family
errors in carbon isotope discrimination in year


0 1 2 3 4 5 6 7 8 9 10 11 12


Months
Figure 4-2. Accumulated monthly precipitation in years 2001, 2003 and mean normal
year from Gainesville Regional Airport, Gainesville, Florida (NOAA 2003).


* 2001
0 2003













Table 4-1. Significance levels (p-values), species and within family least square means for volume, carbon isotope discrimination
(A13C) for three growing periods, and crown conductance variables (Gsref and Gsensitity ) in slash and loblolly pine families in
north central Florida.
Variable Code Significance level by effect Species mean Slash family means
Species Family Clone Slash Loblolly S1 S2 S3 S10
A1C Year 2001 (%o) CID2001 0.9005 <0.0001 <0.0001 22.14 22.16 22.30 21.39 22.65 22.24
A13C Year 2003 (%o) CID2003 0.2852 0.0175 <0.0001 22.53 22.76 22.65 22.08 22.98 22.39
Gsref (mmol ms2 s1) Gsref 0.0840 0.4340 0.2195 754.36 553.86 798.98 702.17 735.94 780.33
Gssensitivity (mmol m2s Gsensitivity 0.0910 0.1640 0.0801 465.91 330.55 511.17 410.11 457.54 484.84
1 ln(kPa) -)
Volume increment age VI45 0.0472 0.0767 <0.0001 7.32 9.41 7.40 7.42 6.55 7.90
4-5 (dm3 tree 1)
Volume increment age VI67 0.8318 0.0786 <0.0001 12.29 12.51 12.35 12.35 10.97 13.47
6-7 (dm3 tree 1)

Table 4-2. Significance levels (p-values) for clone within family in carbon isotope discrimination for two growing periods and crown
conductance variables for loblolly and slash pine families in north central Florida.
Trait Significance level within family clonall variation)
L4 S1 S2 S3 S10
A3C 2001 <0.0001 0.0006 0.0009 0.0208 <0.0001
A1C 2003 0.0025 <0.0001 0.0070 0.5361 <0.0001
Gsref 0.0384 0.2386 0.4424 0.3361 0.6081
Gssensitivity 0.0195 0.1801 0.3011 0.2645 0.6019
VI45 0.0112 0.0003 <0.0001 0.2153 0.0043
VI67 0.0032 0.0289 0.0001 0.0401 0.0234









Table 4-3. Within-family individual-tree broad-sense heritabilities for stable carbon
isotope discrimination (A13C) by year, and crown conductance variables in
loblolly and slash pine families growing in north-central Florida
Trait Family L4 Family S1 Family S2 Family S3 Family S10
A13 2001 0.23 (0.08) 0.20 (0.04)a
A13C 2003 0.17 (0.07) 0.32 (0.09) 0.14 (0.07) 0.01 (0.00) 0.25 (0.08)
Gsref 0.30 (0.23) 0.08 (0.09)a
Gssenitivity 0.38 (0.23) 0.22 (0.19) 0.11(0.19) 0.21(0.19) 0.00 (0.00)
VI45 0.12 (0.07) 0.19 (0.04)a
VI67 0.16 (0.07) 0.15 (0.04)a
Note: Values in parentheses are standard errors
a Variance components were pooled across slash pine families

Table 4-4. Genetic correlations between years 2001 and 2003 by family for carbon
isotope discrimination (A13C) and between 4-5 yr and 6-7 yr stem volume
increment (VI) for loblolly and slash pine families in north central Florida.
Trait Family L4 Family S1 Family S2 Family S3 Family S10
A13C 0.70 (0.23) 0.82 (0.17) 1.00 (0.25) --a 1.00 (0.14)
VI 0.90 (0.15) 0.78 (0.13) 0.96 (0.05) 0.62 (0.42) 0.81(0.17)
Note: Values in parentheses are standard errors
a -- Genetic variation was not significant in carbon isotope discrimination year 2003


Stem Growth

Clonal differences in stem growth increments (age 4-5 y and age 6-7 y) were more

apparent than differences at the family and species levels (Table 4-1). Loblolly pine

tended to growth faster than slash pine. Within slash pine, family S10 was the fastest

grower and S3 was the slowest in both years (Table 4-1). We found greater rates of stem

volume increment in year 2003 than in 2001 for all families most likely to higher rainfall

during the growing season in 2003 (Figure 4-2). Clone within-family variation changed

from one family to another in terms of significance, with family S2 having the highest

variation among clones and family S3 the smallest clonal variation in stem volume

increment (Table 4-2). This difference in clonal variation was directly related with levels

of inheritance. However, when testing for significance of clonal variance components

among slash pine families, differences were not detectable at p=0.05, so families were

pooled to increase precision of heritability estimates for both years of measurements. In









general, within-family heritabilities for stem growth increment were low to moderate in

both loblolly and slash pine (0.12 to 0.19, Table 4-3). On the other hand, the clone-by-

year interaction component was not significant and year-to-year genetic correlations were

high for all families, except for family S3 in which the correlation was moderate with a

wide confidence interval (Table 4-4). The lack of year-by-clone interaction indicates that

clones that had a high value for stem volume increment at age 4-5 y also had a high value

for the same trait at age 6-7 y.

Whole-Tree Crown Conductance and Stomatal Sensitivity

Whole-tree level crown conductance was calculated at 15 minute intervals for

approximately 300 trees (half of the ramets per clone, and half of the clones per family in

5 families). Tree-level stomatal conductance was negatively associated with D, with an

exponential decrease in Gs as D increased, as shown for a representative slash pine ramet

(Figure 4-3). From this relationship we estimated Gsref, defined as the value of Gs when

D=I kPa, and Gssensitivity which quantified the sensitivity of Gs to changes in D, solving

the parameters in Equation 4-6 for each ramet. Across all 5 families, there was a

significant linear relationship between Gsref and Gssensitivity with no intercept (averaged

across family R2=0.77, data not shown).

Genetic variation in Gsref and Gssensitivity was difficult to detect at the species, family

and clonal levels (p<0.05). This may be caused by microsite variation and sample size

(Table 4-1). Gsref and Gssensitivity were 36 and 40% higher in slash pine than in loblolly

pine (p=0.08 and 0.09, respectively). Conductance in slash pine genotypes tended to be

more sensitive to changes in D than loblolly pine genotypes. At the same time, slash pine

families had on average higher Gsref than loblolly pine, meaning that slash pine

conductance to CO2 was higher at a reference D of 1 kPa.







67




25
90
0 0 Tree 7
o o
0 20 0 CP 0
E
So



S10 0
0 0
00
0 0


0 05 o
0




0.5 1.0 1.5 2.0 2.5 3.0 3.5
D (kPa)
Figure 4-3. Representative relationship between canopy average stomatal conductance
(Gs) and vapor pressure deficit (D) on a half hourly basis for a typical slash
pine ramet.

When we analyzed clonal variation family by family, only family L4 showed

significant differences for Gsref and Gssensitivity (Table 4-2), and variable levels of genetic

control were found among slash pine families. Within-family individual-tree broad-sense

heritabilities were high for Gssensitivity in family L4 (H2WF=0.38), lower in slash pine

families S1, and S3, and very low in families S2 and S10 (Table 4-3). For Gsref a

moderate level of heritability was found in family L4, but low heritability was found for

the pooled slash pine families. This result was likely influenced by lack of genetic

variation in family S10. Interestingly, within-family broad-sense heritabilities for crown

conductance parameters and A13C were generally higher than for stem growth increment,

meaning that these physiological traits were under stronger genetic control than growth

traits. The same time, however, heritability estimates for crown conductance parameters

were associated with large standard errors (Table 4-3).









Genetic Correlations between Carbon Isotope Discrimination and Growth and
Whole-Tree Crown Conductance

Genetic correlations among families were not significantly different from zero

between A13C and stem volume growth (Table 4-5). Only the genetic correlation between

A13C year 2001 and stem volume increment age 4-5 yr in family S10 was significantly

negative (-0.54), meaning that faster growing clones showed less discrimination against

13C during gas exchange. In 2003, family S3 had no variation for isotope composition so

its correlation with growth increment could not be estimated. Of the nine estimable

correlations for the 5 families across 2 years, the average genetic correlation was -0.26

which may indicate a slightly negative general relationship between carbon isotope

discrimination and growth in these slash and loblolly pine clones.

Table 4-5. Within-family correlations between volume increment of the growing season
and stable carbon isotope discrimination (A13C) by year in loblolly and slash
pine families growing in north-central Florida
Trait Volume increment
Family L4 Family S1 Family S2 Family S3 Family S10
Genetic
A3C 2001 0.09 (0.31) -0.29 (0.27) 0.05 (0.26) -1.00 (0.80) -0.54 (0.24)
A3C 2003 0.01(0.32) -0.38 (0.26) -0.33 (0.29) --a 0.01(0.31)
Environmental
A3C 2001 -0.05 (0.08) -0.08 (0.08) -0.21 (0.08) -0.10 (0.09) -0.08 (0.08)
A3C 2003 -0.05 (0.08) -0.40 (0.06) -0.32 (0.08) -0.33 (0.08) -0.28 (0.07)
Note: Values in parentheses are standard errors
a -- Not estimated because genetic variation was not significant in carbon isotope
discrimination year 2003.

We found non-significant environmental correlations between A13C and stem

volume increment in year 2001, and low negative environmental correlations for all

families in year 2003 but family L4.

We estimated genetic and environmental correlations between mean A13C and

crown conductance parameters using averages since not all measurements were made in

the same year. For A13C we took the average between samples collected in years 2001









and 2003 per individual tree (leaves formed in the springs of both years), and for crown

conductance we estimated the parameters from data collected from March through April

2002. We assumed that the environmental conditions in the springs of all years were

similar. Despite the considerable effort of measuring sap flow in 300 trees, and

estimating integrated crown conductance parameters, we could not accept or reject our

hypothesis that related low A13C with high stomatal conductance sensitivity to changes in

D. We found that genetic and environmental correlations were unstable across families

and had wide confidence intervals, so they were not significantly different from zero

(Table 4-6). This lack of precision in the estimation can be related to two sources: (1)

small sample size; and (2) sensitivity of the physiological measurements to subtle

changes in microsite. The lack of correlations between A13C and crown conductance

parameters might be due to the separation in years of sap flow measurement and leaf

collection, so the assumption of similar environment was not valid.

Table 4-6. Genetic and environmental correlations between mean carbon isotope
discrimination (mean A13C) and Gsref and Gssensitivity for loblolly and slash pine
families in north central Florida
Trait Mean A3C
Family L4 Family S1 Family S2 Family S3 Family S10 All slash
Genetic
Gs ref -0.22 (0.40) -0.20 (0.43) 0.97 (1.07) -0.39 (0.79) --a 0.24 (0.29)
Gs sensitivity -0.16 (0.35) 0.06 (0.40) 0.84 (0.51) -0.41 (0.70) --a 0.28 (0.24)
Environmental
Gs ref 0.12 (0.17) -0.04 (0.17) 0.01 (0.17) 0.12 (0.19) -0.17 (0.15) 0.00 (0.08)
Gs sensitivity 0.10 (0.18) -0.03 (0.17) -0.05 (0.17) 0.17 (0.19) -0.13 (0.16) 0.00 (0.09)
Note: Values in parentheses are standard errors
a -- Not estimated because genetic variation was not significant in carbon isotope
discrimination year 2003

Discussion

Information on clonal variation in southern pines has become more common in the

last two decades (Foster 1988; Paul et al. 1997; Isik et al. 2003; Schmidtling et al. 2004;

Baltunis et al. 2005). At the same time, clonal forestry appears to offer an excellent









opportunity for the early capture of the benefits generated by tree improvement and

biotechnology programs (Ahuja and Libby 1993; Libby and Ahuja 1993; Schmidtling et

al. 2004). The novelty of my study was its analysis of clonal genetic variation among

families, the number of clones involved per family, and the possibility of analyzing

growth and physiological traits under field conditions.

We found significant within-family clonal genetic variation in A13C and stem

volume increment in both years of measurements, reflecting a wide spectrum of clonal

performance for growth and gas exchange. At the same time, greater rates of stem

volume increment were detected in year 2003 compared to 2001 for all families. These

differences may have been caused by variation in seasonal rainfall pattern and total

amount of annual precipitation, or simply due to tree size. There are few reports in the

literature of clonal variation in loblolly or slash pine growth. Paul et al. (1997) reported

that height of loblolly pine clones varied significantly at different ages, but that DBH and

volume did not. To our knowledge, no other published studies have quantified clonal

variation in A13C in loblolly or slash pine under field conditions.

Clonal variation in foliar A13C fluctuated from family to family, with family S3

having the lowest range of clonal values. In general, the range of phenotypic clonal mean

values we found in our selected families (from 19 to 25.41%o) had a wider distribution in

comparison to what had been reported in similar studies; for example, in Fl hybrid pine

clones between slash pine and Caribbean pine (19.6 to 20.7%o and 18 to 21.84%o, Xu et

al. 2000, Prasolova et al. 2003, respectively), loblolly pine clones (23.3 to 22.3%o,

Gebremedhin 2003). In P. menziesii, family means A13C ranged between 19.7 to 22.43%o

(Zhang et al. 1993), and in E. globulus family means for A13C ranged between 16.7 to









18. 1%o (Pita et al. 2001). Genetic variation in A13C should reflect differences in Ci/Ca, a

consequence of the balance between stomatal supply and mesophyll demand of CO2

(Farquhar et al. 1989).

In my study, low to moderate levels of heritabilities for growth, A13C, and crown

conductance parameters suggested that these are complex traits determined by the

expression of many genes, each one having a small effect on the phenotypic expression

of the individual (Falconer and Mackay 1996). However, the heritabilities we estimated

are expected to be smaller than broad sense heritabilities values usually reported, because

they are estimated within full-sib families and half the additive genetic variation and one

fourth of the non-additive variation occurs among full-sib families (Falconer and Mackay

1996). Levels of genetic control for A13C in a clonal study reported low heritabilities of

0.08 in loblolly pine under different watering regimes (Gebremedhin 2003), and 0.09 to

0.15 in Fl hybrid slash x Caribbean pine (Prasolova et al. 2003). In other conifers,

studies were carried out in full-sib families and A 3C narrow-sense heritability range from

low to moderate, 0.54 for P. mariana (Johnsen et al. 1999), 0.17 in P. pinaster (Brendel

et al. 2002), between 0.4 to 1.0 in A. cunninghamii (Prasolova 2000). In the hardwood C.

sativa, narrow sense heritability was moderate (h2=0.31, Lauteri et al. 2004). Genetic

analysis of variation in crown conductance parameters has not been reported in the

literature, so it was difficult to make comparisons with slash, loblolly or other pine

species or hardwoods. Nevertheless, the within-family, individual-tree broad-sense

heritabilities values we reported for whole-tree crown conductance and stomatal

sensitivity were large in several cases (Table 4-3). However, the precision of our

estimates were low. Increased precision likely requires a much larger number of









replications per clone than was possible in my study. Heterogeneous soil conditions in the

study site might have lowered heritabilities due to lack of optimal silvicultural treatments

(weed control and fertilization in several years).

Consistency of genotypic ranking across years is essential for breeding to be

effective in modifying a particular quantitative trait. We found significant positive

correlations across years for both stem volume increment and A13C. Similar results in

consistency in across years in A13C have been reported in P. mariana (Johnsen et al.

1999).

In my study we tested the hypothesis of correlations between A13C and stomatal

sensitivity to changes in D. We found negative results in the sense that wide confidence

intervals around the estimation in genetic correlations gave small confidence in making

conclusions or possible explanations on association between these two variables.

However, future research in this avenue is needed to understand the underlying

mechanism behind the intrinsic photosynthesis-stomatal conductance relationship. Here,

we analyzed the relationship between A13C and crown conductance, but the knowledge of

the relationship of Al3C with photosynthetic capacity is also needed. Then, we can

understand if genetic variation in A 3C is due to changes in stomatal conductance, or in

photosynthetic capacity, or both.

We hypothesized that traits which integrated information over space and/or time

would be more highly correlated with growth (see Chapter 2). In this case, A13C

corresponds to an integrated measurement of photosynthesis and stomatal conductance

during the time of formation of the leaf. Our results did not support that hypothesis, and

genetic correlations between A 3C and stem volume increment were not stable across









families, across years, and not significantly different from zero. We can conclude that

A3C and stem growth were controlled by largely independent sets of genes. Similarly,

environmental correlations between A13C and stem volume increment were low, meaning

that microsites which increased discrimination, also increased or decreased stem growth

independently.

The observed independence of A13C from stem growth and the absence of year-by-

clone interaction in both growth and A13C still provide opportunities for selecting loblolly

and slash pine clones combining high productivity and high water-use efficiency (low

A13C). The changes from year to year in genetic and environmental correlations between

these two traits might be associated with changes in seasonal weather patterns, for

example the amount of rainfall and soil moisture conditions during the time of leaf

formation in the case of A13C and the total growing season in the case of stem volume

increment. As in Figure 4-2, rainfall between February and June was lower in year 2001

than in 2003, and by the end of the 2001 growing season, the decrease in rainfall may

have affected growth increment too. Unfortunately, we did not measure soil moisture

content to confirm this hypothesis.

On the other hand, the presence of mild weather years in my study, where stem

growth was not limited by water supply and water use efficiency may affect the degree of

correlations between A13C and stem growth increment. Condon and Richards (1993)

showed that in wheat genotypes, the relationship between crop biomass production and

leaf carbon isotope discrimination values changed when crops where grown on different-

quality sites. It was only on the driest site that the negative relationship between growth

and leaf carbon isotope discrimination predicted from gas exchange characteristics was









supported for wheat genotypes (Condon and Richards 1993). The same situation was

described in P. mariana by Flanagan and Johnsen (1995), where the strongest correlation

between height and A13C was found in the driest site. In C. sativa, the genetic correlations

between A13C and growth traits were generally strong and negative (-0.5 to -1.0),

especially in two high temperature treatments (Lauteri et al. 2004).

In the literature, negative, positive, or no correlations between A13C and growth

have been reported. Low, moderate and strong negative genetic correlations were

reported in some conifers, as for example P. mariana (-0.96, Johnsen et al. 1999), Fl

hybrid between slash pine and Caribbean pine (-0.19 to -0.36 depending on site and

sampling season, based on clonal means, Prasolova et al. 2003; and -0.83 to -0.96 based

on clonal means, Xu et al. 2000), P. menziesii (-0.65 to -0.67, Zhang et al. 1993).

Positive phenotypic correlations have been demonstrated in eucalyptus species, like E.

globulus (Pita et al. 2001), and some eucalyptus hybrids (Le Roux et al. 1996), and also

in loblolly pine clones (0.86, but might be associated to wide standard errors because of

low heritability, Gebremedhin 2003). No genetic correlations at all between A13C and

ring width was found in P. pinaster (0.02), and in agreement there was the lack of co-

location of QTLs between both traits (Brendel et al. 2002). Similarly, Marron et al.

(2005) did not find a significant correlation between discrimination and total biomass in

hybrid poplar clones.

Some authors explain the lack of consistent correlation between A 3C and growth in

pines as follows: (1) if A13C is mainly determined by assimilation rate, and if growth is

not primarily determined by assimilation rate, then there might be no correlation between

A13C and growth (Brendel et al. 2002); (2) if genetic control is moderate for both traits,









this might lower the significance of a genetic correlation (Brendel et al. 2002); and (3) if

water supply is not limiting growth, then water use efficiency might not be defining

growth (Prasolova et al. 2003).

On the other hand, differences in allocation of carbon between photosynthetic

tissue and root can alter the relationship between A13C values and growth when water is

not limiting. For example slash or loblolly pine clones that have high discrimination, and

a low ratio of photosynthesis to stomatal conductance, may also have a high ratio of

photosynthetic tissue to root tissue. A higher allocation to photosynthetic tissue on a site

that is not limited by water availability, however, may overcome any restriction on

growth imposed by low assimilation rates (Flanagan and Johnsen 1995).

Future research in analyzing associations between A13C and photosynthetic

capacity will be needed, and also the corresponding measurements of amount of leaf area.

Some authors supported the thesis that differences in photosynthetic capacity have been

observed to be the primary cause of genetic variation in A 3C in several coniferous

species (Flanagan and Johnsen 1995; Guehl et al. 1995; Johnsen and Major 1995; Sun et

al. 1996; Johnsen et al. 1999; Xu et al. 2000; Prasolova et al. 2003, 2005). Also, the

possibility to repeat the study in the same field with severe weather conditions is

recommended to make comparisons with our results. Replicated studies in different site

conditions are also a good source of information to capture genetic variation across

different environments.














CHAPTER 5
SUMMARY AND CONCLUSIONS

Loblolly and slash pines are widely planted as commercial timber species in the

southeastern United States. Knowledge about the biology of physiological processes and

their genetic parameters give us insight into what are the key functional and structural

traits that determine genotype performance differences in southern pines.

The overall goal of this dissertation was to investigate biological traits and the

genetic structure of these traits in 300 clones from five different full-sib loblolly and slash

pine families. One peculiarity of this study was the number of clones represented in each

full-sib family and also the advantage of having them in field conditions at an early stage

of stand development.

The study was divided in three main areas of research:

* Detailed quantification of crown structure and estimation of the total amount of
radiation absorbed by each tree over a year using the process model MAESTRA;

* Seasonal dynamics and phenology of basal area growth and its association with soil
water balance;

* Leaf carbon isotope discrimination and whole-tree sap flow

The common objectives in each main research area of the study were to:

* Determine species, family within-species and clone within-family genetic variation
for all variables measured or estimated;

* Where genetic variation exists, estimate genetic control and environmental
influence on structural and functional variables

Based on the results of the previous chapters the main conclusions and implications

of this study were summarized into three themes:









* Genetic variation among species and families;

* Clonal variation and within-family inheritance;

* Correlations

Genetic Variation among Species and Families

Differences in stem growth and crown structural traits between species and among

slash pine families were subtle. In general, the one loblolly pine family we studied tended

to grow faster than the average of our four slash pine families at ages 5 yr and 6 yr. At the

same time, loblolly pine developed larger crowns with more acute branch angles and had

more leaf area per individual-tree at age 5 yr and 6 yr than did the slash pine families. In

spite of the apparent similarities in stem volume growth rate, the four slash pine families

differed in a number of crown architectural traits. Contrasting families had different

arrangements and sizes of branches within the crown, and varied in crown shape ratio.

This suggests that any of a number of crown traits may be associated with high growth

rate in southern pine families.

When we analyzed the repeated basal area growth measurements we found that

loblolly pine tended to have larger yearly and daily basis basal area increments than slash

pine at ages 6 and 7 yr. From this study, we concluded that the differences between

loblolly and slash pine accumulated slowly over time through ages 6 and 7 yr. Loblolly

and slash pine families considered in this study tended to grow about eight months per

year, from March through October. We did not find significant differences at species and

family level in initiation, cessation or duration of basal area growth both years 2002 and

2003. In both years, peaks in basal area increment occurred in short (2-3 week) periods in

the early spring for all families, followed by relatively constant rates of basal area growth









until cessation. While there were significant size differences among taxa (species and

families) at age 6 yr and 7 yr, genetic differences in basal area growth rate were only

expressed during short, discrete time periods in the spring and fall.

When we studied environmental effects on seasonal basal area growth, we found

that basal area growth rate increased during periods when water soil availability increased

(up to 300 mm), but an excess in water availability in the soil had a negative impact on

growth. Integration of climatic data with physiological variables and soil conditions in a

water balance model allowed us to better understand the interactions between basal area

growth in loblolly and slash pine families and soil water availability.

The study of leaf and crown integrated physiological processes, such as A13C and

whole-tree sap flow gave us the opportunity to explore genetic variation at the species,

family and clonal level in slash and loblolly pines at larger temporal and spatial scales

and in much more detail than is typical in field ecophysiological investigations. Family

within-species was a significant source of variation for foliage carbon isotope

discrimination in 2001 and 2003, but at the species level no significant differences were

detected. Genetic variation in Gsref and Gssensitivity was difficult to detect at the species

level and among the four slash pine families. Microsite variation and the small sample

size may have been responsible.

Conductance in slash pine genotypes tended to be more sensitive to changes in D

than loblolly pine genotypes. At the same time, slash pine families on average had higher

crown conductance per unit of projected crown area than loblolly pine.

Clonal Variation and Within-Family Inheritance

Within-family clonal variation was highly significant for all growth and crown

structural traits, reflecting a wide spectrum of clonal performance in growth and crown









development at these ages. Within-family individual-tree broad-sense heritabilities

(H2WF) were low to moderate for stem volume and crown structural traits for all five

families (0.05 to 0.41). One interesting result we found was the heterogeneity in variance

components among slash pine families; there was a tendency for higher heritabilities in

family S2 than the rest of the slash pine families in many traits, meaning that even for

polygenic traits, it is possible to find specific pairs of parents producing more variable

offspring for growth or crown structural traits.

H2WF for basal area growth phenological traits ranged from low to moderate for all

traits (0.00 to 0.24). In general, heritabilities were higher for growth traits than for

phenological traits for all families.

Clone within-family was a significant source of variation for foliage A13C in 2001

and 2003, but not for crown conductance parameters. H2WF for A13C and crown

conductance parameters were in general higher than that for stem growth increment (0.01

to 0.38), meaning that these physiological traits were under stronger genetic control than

growth traits. But, at the same time heritability estimates for crown conductance

parameters were associated with large standard errors.

Correlations

As we hypothesized, the more integrated measures of crown structure and function

in this study, specifically APAR and crown volume, were consistently more strongly

correlated with stem volume growth rate than were less integrative measures such as

crown radius or length, number of branches, branch angle, or average branch diameter.

At the same time, microsites that favored the development of the crown, leaf area, and

light interception also enhanced growth rate in all families. Branch angle and crown

shape ratio showed non-significant environmental correlation with volume increment. An









understanding of the relationship between crown architecture and tree growth might

provide a basis for predicting tree growth, and could aid in the search for discovering

genes involved in growth and for developing new crop ideotypes.

Both the strength and direction of correlation between basal area phenological traits

and basal area growth rate varied across families and years, and many times was not

significantly different from zero. There were no significant clone-by-year interactions for

any basal area phenology traits. We can conclude that each of the basal area growth

phenology traits and each of the basal area growth rate traits were genetically controlled

by a similar set of genes in years 2002 and 2003.

Genetic correlations between A13C and stem volume increment were not stable

across families, across years, and not significantly different from zero. It might be that in

the years 2001 and 2003, weather and field conditions were mild enough throughout the

growing season that stem growth was not limited by water supply and water use

efficiency, and the genetic correlation between A13C and stem volume increment did not

have any biological importance. There was no evidence of genotype-by-year interaction

in any family for A13C and stem volume increment, indicating that the ranking of clones

remained constant between years. We found non-significant environmental correlations

between A13C and stem volume increment in year 2001, and low negative environmental

correlations for all families in year 2003.

Genetic and environmental correlations between A13C and stomatal sensitivity to

changes in vapor pressure deficit were difficult to conclude due to wide confidence

intervals for all families.









In conclusion, for the particular loblolly and slash pine families studied here, there

was a wide spectrum of clonal within-family performance in stem growth, crown

development, and measured physiological traits, making interesting the possibility of

clone within-family selection for traits that increase productivity. We found low to

moderate levels of within-family heritability in many key structural and functional traits

(crown structure, basal area growth phenology, A13C, and crown conductance

parameters), but just crown structural traits had stable and higher genetic correlation with

stem growth increment.

Here we reported important linkage between crown structural and functional traits

with stem volume growth in loblolly and slash pine families and clones. However, what

is finally translated into stem volume increment depends on complex relations with other

processes and their genetic patterns. Additional studies with respect to carbon gain, water

relations and hydraulic conductivity at the individual-tree level will help improve our

understanding of what controls stem volume growth in contrasting families and clones.

The results from this study should positively impact future tree growth modeling

and will help in decisions that involve genotype deployment and silvicultural treatments.




















APPENDIX A
DESIGN AND LAYOUT STUDY SITE


REP 2



1 7 2 10
----i--1-I --A _-1_f

5 13 3 14


12 9 11
15


6 8 4 16


13
12 15 1 1


4 10
16 11


3 2
8 14


5 6 9 7


REP 1


2 rows of buffers



REP 4



8 1 7 15


2 6 14 11


10 9 3 4


16 5 13 12


3 5 9
11


10 1 16 8
14


7 2 1 12


15 13 4 6
__ ___ I __ I ___ I


REP 3


N

Propacule Type:
1=RC-clones replicated
2=RC-clones replicated
3=RC-clones replicated
4=RC-clones replicated
10=RC-clones replicated
5=Seedling
6=RC-clones not replicated
7=Seedling
8=RC-clones not replicated
9=Seedling
11=Seedling
12=RC-clones not replicated
13=Seedling
14=Seedling
15=RC-clones not replicated
16=Seedling




10 plantig spaces (55






6 beds wide(66 ft)
plot corner


Figure A-1.Design and layout of full-sib family block plot study at Rayonier, Inc.


SpP
slash
slash
slash
loblolly
slash
slash
slash
slash
slash
slash
loblolly
loblolly
loblolly
loblolly
loblolly
slash


ft)














APPENDIX B
SILVICULTURAL TREATMENTS AT STUDY SITE


Table B-1. Treatment regimes applied in the research location at Rayonier, Inc.
Year Treatment
1997-January Double bedding and planting
1997-March Chemical weed control (arsenal imazapyr) banded
1997-June Chemical weed control (arsenal imazapyr and sulfometuron methyl)
broadcast
1997-October Fertilization 220 kg/ha diammonium phosphate
1999-August Fertilization 220 kg/ha diammonium phosphate
2000-July Mechanical weed control
2000-November Fire line plowed
2001-May Chemical weed control (glyphosate) broadcast
2002-June Fertilization 500 kg/ha ammonium nitrate
















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