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University / "L '7 577QSv/f This is to certify that the thesis entitled INTERSPECIFIC VARIATION IN ADAPTIVE TRAITS OF TRUE FIRS (ABIES SPP.) presented by Grant Edward Jones has been accepted towards fulfillment of the requirements for the MS. degree in Horticulture ‘34» “’ Coyg, Major ProfessM Signature I Date MSU is an Affirmative Action/Equal Opportunity Institution o-n-n---I-o-o-o-o-o-a--o-a-o-I-o-a-o-o---u- ..._.-.-.-.--n-‘-_-_s'-'- PLACE [N RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 cJCTFTC/DateDuejndd-pJ 5 INTERSPECIFIC VARIATION IN ADAPTIVE TRAITS 0F. TRUE FIRS (ABIES SPP.) By Grant Edward Jones A THESIS Submitted to Michigan State University In partial fulfillment of the requirements For the degree of MASTERS OF SCIENCE Department of Horticulture 2005 ABSTRACT INTERSPECIFIC VARIATION IN ADAPTIVE TRAITS OF TRUE F IRS (ABIES SPP.) By Grant Edward Jones Recent efforts to increase conifer diversity for Christmas tree and landscape use have sparked increased interested in planting true firs and their hybrids. Expanded use of firs has been limited by their perceived intolerance of many site conditions; however, recent research shows firs are more tolerant of environmental conditions than originally thought. In this project we studied adaptive traits of 17 species and interspecific hybrids of firs at four locations in the Lower Peninsula of Michigan. Project goals include characterizing species difference in: l) budbreak and cold hardiness, 2) the influences of soil pH on foliar nutrition, physiological processes, and growth, and 3) the influence of needle morphology and shoot architecture on net photosynthesis and drought tolerance. Mean date of budbreak and growing degree days differed among species and location in both years. Fir species that broke bud early were more prone to late spring frost damage than species with late budbreak. Maximum mid-winter cold hardiness was negatively correlated with date of budbreak. Soil pH influenced nutrient availability of several important nutrients necessary for physiological processes. Net photosynthesis decreased with increased soil pH and response differed among species. Needle morphology differed among species and needle thickness was correlated with increased net photosynthesis. Needle carbon isotope discrimination was related to water use efficiency and varied among species. Continued improvement of stress tolerance of firs for the upper Midwest is possible through selection for late budbreak and tolerance to soil pH. ACKNOWLEDGMENTS I would like to thank Bert Cregg for his guidance and assistance throughout this project and allowing me to develop a better understanding of the many facets of horticultural science beyond just my own research project. I would also like to thank my committee members Brad Rowe and Dave Rothstein for their guidance. I am extremely appreciative of the farm managers and their staff for their assistance installing and maintaining my plots at each farm, including: Bill Chase, Bill Klein, Randy Kleveckis, Greg Kowalewski, and Jerry Skeltis. I am appreciative of Alison Heins for collecting budbreak data at the Northwest Horticulture Research Station. I wish to thank MAES Project GREEEN, the Michigan Nursery and Landscape Association, and the Landscape Plant Development Center for providing the funding, which made this project possible. Katrina Schneller, Becky Klingerman, Anna Arend, and Sara Tanis assisted me in the field and made to work more enjoyable. I would like to thank my family and friends for their understanding, support, and friendship throughout this endeavor. iii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES ......................................................................................................... xiii LITERATURE REVIEW Introduction ................................................................................................................. 2 Temperature ................................................................................................................ 3 Cold Hardiness ........................................................................................................ 3 High Temperature ................................................................................................... 7 Water Relations ........................................................................................................... 9 Drought tolerance .................................................................................................... 9 Water use efficiency and Carbon isotope discrimination ..................................... 10 Soil Conditions .......................................................................................................... 12 Chlorophyll and Nitrogen ..................................................................................... 14 Lime ...................................................................................................................... 1 5 Light .......................................................................................................................... 16 Biomass Allocation ............................................................................................... 16 Photoinhibition ...................................................................................................... 17 Light Response ...................................................................................................... 17 Needle Morphology and Shoot Architecture ........................................................ 18 Light Acclimation ................................................................................................. 19 Summary ................................................................................................................... 19 Literature Cited ......................................................................................................... 25 CHAPTER ONE BUDBREAK AND WINTER INJURY IN EXOTIC F IRS Abstract ......................................................................................................................... 34 Introduction ................................................................................................................... 35 Materials and Methods .................................................................................................. 38 Results ........................................................................................................................... 41 Discussion ..................................................................................................................... 43 Literature Cited ............................................................................................................. 59 CHAPTER TWO CHLOROPHYLL FLUORESCENCE, PHOTOSYNTHESIS, GROWTH, AND FOLIAR NUTRIENT CONCENTRATION OF ABIES IN RESPONSE TO SOIL pH Abstract ......................................................................................................................... 64 Introduction ................................................................................................................... 64 Materials and Methods .................................................................................................. 66 Results ........................................................................................................................... 71 Discussion ..................................................................................................................... 73 Literature Cited ............................................................................................................. 90 iv CHAPTER THREE NEEDLE MORPHOLOGY, SHOOT ARCHITECTURE, AND NET PHOTOSYNTHETIC RESPONSE IN ABIES SPECIES. NEEDLE MORPHOLOGY, SHOOT ARCHITECTURE, AND NET PHOTOSYNTHETIC RESPONSE IN ABIES SPECIES. Abstract ........................................................................... . .............................................. 9 6 Introduction ................................................................................................................... 96 Materials and Methods ................................................................................................ 100 Results ........................................................................................................................ 106 Discussion ................................................................................................................... 112 Literature Cited ........................................................................................................... 143 APPENDIX Project Summary ......................................................................................................... 148 LIST OF TABLES TABLE LITERATURE REVIEW 1 Fourty-six species in 10 Abies sections as defined by Farjon (1990). 2 List of species tolerant or intolerant of extreme winter temperatures, drought, and soil pH. CHAPTER ONE 1 List of Abies species planted at four locations in Michigan. 2 Thirty-year climate summary and USDA plant hardiness zones for four Abies plantings in Michigan. 3 Soil properties of four Abies planting sites in Michigan. 4 Budbreak date of 17 Abies species grown at four locations in Michigan in 2004 and 2005. 5 Mean growing degree days required before budbreak in 17 Abies species grown at four location in Michigan in 2004 and 2005. 6 Pearson correlation coefficients for budbreak of 17 Abies at four locations in Michigan. 7 Mean Fv/Fm value of four Abies species following controlled freeze tests to -44 °C. 8 Mean needle damage ratings of four Abies species following controlled freeze tests to -44 °C. 9 Pearson’s correlation coefficients for winter damage in four Abies species growing in Michigan in March 2005 following controlled freeze tests. CHAPTER TWO 1 List of Abies species planted at four locations in Michigan. 2 Thirty-year climate summary and USDA plant hardiness zones for four Abies planting sites in Michigan. 3 Soil properties of four Abies planting sites in Michigan vi PAGE 21-22 23 49 50 50 51 52 53 53 54 54 77 78 78 Soil nutrient concentrations and cation exchange capacity (CEC) of 79 four Abies test plots in Michigan. ' Mean foliar nutrient levels in 17 Abies species growing at four 80 locations in Michigan in 2004. . Pearson’s correlation coefficient for leader height, soil pH, 81 photosynthesis, chlorophyll fluorescence and 11 foliar nutrient elements of 17 Abies species at four locations in Michigan sampled in October 2004. Mean growth, chlorophyll fluorescence (Fv/Fm), and net photosynthesis 82 (PNAmx) in 17 Abies species grown at four locations in Michigan in 2004. Mean foliar nutrient of four Abies test plots in Michigan in 2004. 83 Mean leader growth, chlorophyll fluorescence, and photosynthetic 83 values (umol COz-m'z-s'l) with needle area expressed as total needle area of four Abies test plots in Michigan in 2004. CHAPTER THREE List of Abies species planted at four locations in Michigan 116 Thirty-year climate summary and USDA plant hardiness zones for 117 four Abies planting sites in Michigan. Soil properties of four Abies planting sites in Michigan 117 Mean volumetric soil moisture content at four Abies test plots in 2003 118 and 2004 using a portable TDR device. Net photosynthesis expressed using projected shoot area (PSAmax) of 118 seven Abies subsections grown at four locations in Michigan in 2003 and 2004. Gas exchange was measured in late July 2003, and late June, July, and early September 2004. Mean photosynthetic rates expressed using projected needle area 119 (PNAmx) of 17 Abies species grown at four locations in Michigan in 2004. Gas exchange was measured in late June 2004, late July 2004, and early September 2004. vii 7 10 ll 12 13 14 15 16 17 Net photosynthesis expressed using projected needle area (PNAmax) of seven Abies sections and subsections grown at four locations in Michigan in 2004. Gas exchange was measured in late June, late July, and early September 2004. Pearson’s correlation coefficients for net photosynthesis (PSAmax, PNAmax, TNAmax), water use efficiency (WUE), needle morphology, carbon isotope discrimination (A), and shoot architecture in 17 Abies species grown at four locations in Michigan. Net photosynthesis expressed using projected needle area (PNAmax) of seven Abies sections and subsections grown at four locations in Michigan in 2004. Gas exchange was measured in late June, late July, and early September 2004. Mean carbon isotope discrimination (A) in 10 Abies species grown at four lOcations in Michigan in 2004. Mean carbon isotope discrimination (A) in four Abies subsections grown at four locations in Michigan in 2004. Needle morphology traits for 17 Abies species growing at four locations in Michigan in 2004. Needle morphology traits for seven Abies sub-sections grown at four locations in Michigan in August 2004. Mean projected shoot to total needle surface area (PSA/TN A), projected needle to projected shoot area (PNA/PSA), and total needle to projected needle area (TNA/PNA) ratios of 17 Abies species growing at four locations in Michigan in 2004. Mean projected shoot to total needle surface area (PSA/TNA), projected needle to projected shoot area (PNA/PSA), and total needle to projected needle area (TNA/PNA) ratios of seven Abies sections and subsections grown at four locations in Michigan in August 2004. Mean dark respiration (Rd), net photosynthesis expressed using projected shoot area (PSAmax), apparent quantum efficiency (4)), and light compensation point (LCP) in six Abies species grown at four locations in Michigan in 2004. Mean dark respiration (Rd), net photosynthesis expressed using total needle area (TNAmax), apparent quantum efficiency ((1)), and light compensation point (LCP) and in six Abies species grown at four locations in Michigan in 2004. viii 120 121 122 123 124 125 126 127 128 129 130 18 19 20 21 Mean dark respiration (Rd), net photosynthesis expressed using projected needle area (PNAmax), apparent quantum efficiency (4)), light compensation point (LCP), and light saturation point (LS) in six Abies species grown at four locations in Michigan in 2004. Mean respiration (R), net photosynthetic rate expressed using projected shoot area (PSAmax), and apparent carboxylation efficiency (CE) of six Abies species grown at four locations in Michigan is 2004. Mean respiration (R), net photosynthetic rate expressed using projected needle area (PNAmax), and apparent carboxylation efficiency (CE) of six Abies species grown at four locations in Michigan is 2004. Mean respiration (R), net photosynthetic rate expressed using total needle area (TNAmx), and apparent carboxylation efficiency (CE) of six Abies species grown at four locations in Michigan is 2004. APPENDIX Analysis of variance of the date of budbreak and growing degree days (GDD) accumulated at budbreak in 17 Abies species at four locations in Michigan in 2004 and 2005. Analysis of variance of chlorophyll fluorescence values as a measure of cold hardiness in four Abies species near East Lansing, MI. Equations depicting the relationship between net photosynthesis (PNAmax) and soil pH (Chapter 2, Figure 3). Equations depicting the relationship between soil pH and A)N, C) P, and E) K and net photosynthesis (PNAmax) and B) N, D) P, and E) K (Chapter 2, Figure 5). Equations depicting the relationship between leader growth and foliar K (Chapter 2, Figure 6). Pearson’s correlation coefficients for soil pH, chlorophyll fluorescence, and 11 foliar nutrient elements of 17 Abies species at four locations in Michigan. Samples from October 2003 and 2004 were combined. Pearson’s correlation coefficient for soil pH, chlorophyll fluorescence, and 11 foliar nutrient elements of 13 Abies species at four locations in Michigan sampled in October 2003. ix 131 132 133 134 150 150 151 151 151 152 153 10 11 12 13 14 15 16 17 18 Tolerance of Abies species to soil pH levels at four test plots in Michigan 154 in 2004. Tolerance based on decline in net photosynthesis with increasing soil pH. Mean photosynthetic rates expressed using total needle area (TNAmax) of 155 17 Abies species grown at four locations in Michigan in 2004. Gas exchange was measured in late June 2004 and early September 2004. F-values of PSAmax ratio of 17 Abies species grown at four locations in 156 Michigan in 2003 and 2004. F-values of PNAmax of 17 Abies species grown at four locations in 156 Michigan in 2004. F -values of TNAmalx of 17 Abies species grown at four locations in 156 Michigan in 2004. Analysis of variance for carbon isotope discrimination (A) and water use 157 efficiency (WUE) of several Abies subsections grown at four locations in Michigan in 2004. F -va1ues of PSA/TNA ratio of 17 Abies species grown at four locations in 157 Michigan in 2004. F-values of TNA/PNA ratio of 17 Abies species grown at four locations in 157 Michigan in 2004. F-values of PNA/PSA ratio of 17 Abies species grown at four locations in 158 Michigan in 2004. F-values of photosynthetic light response curve parameters [dark 158 respiration (Rd), apparent quantum efficiency ((b), net photosynthesis expressed using projected shoot area (PSAmax), and the light compensation point (LCP)] of six Abies species grown at four locations in Michigan in 2004. F-values of photosynthetic light response curve parameters [dark 159 respiration (Rd), apparent quantum efficiency (4)), net photosynthesis expressed using projected needle area (PNAmax), and the light compensation point (LCP)] of six Abies species grown at four locations in Michigan in 2004. 19 20 21 22 23 24 25 F-values of photosynthetic light response curve parameters [dark 159 respiration (Rd), apparent quantum efficiency ((b), net photosynthesis expressed using total needle area (TNAmax), and the light compensation point (LCP)] of six Abies species grown at four locations in Michigan in 2004. F-values of A/Ci curve parameters [respiration (R), apparent 160 carboxylation efficiency (CE), and net photosynthesis expressed using projected shoot area (PSAmax)] of six Abies species grown at four locations in Michigan in2004. F-values of A/Ci curve parameters [respiration (R), apparent 160 carboxylation efficiency (CE), and net photosynthesis expressed using projected needle area (PNAmax)] of six Abies species grown at four locations in Michigan in 2004. F-values of A/Ci curve parameters [respiration (R), apparent 160 carboxylation efficiency (CE), and net photosynthesis expressed using projected needle area (PNAmax)I of six Abies species grown at four locations in Michigan in 2004. Pearson’s correlation coefficients for light response parameters [dark 161 respiration (Rd), apparent quantum efficiency ((1)), net photosynthesis expressed using projected shoot area (PSAmax), and light compensation point (LCP)] and soil pH, photosynthetic efficiency (Fv/Fm), 10 soil nutrients, shoot architecture, and carbon isotope discrimination (A). Response parameters are from six Abies species grown at four locations in Michigan is 2004. Pearson’s correlation coefficients for light response parameters [dark 162 respiration (Rd), apparent quantum efficiency (4)), net photosynthesis expressed using projected needle area (PNAmax), and light compensation point (LCP)] and soil pH, photosynthetic efficiency (Fv/Fm), 10 soil nutrients, shoot architecture, and carbon isotope discrimination (A). Response parameters are from six Abies species grown at four locations in Michigan is 2004. Pearson’s correlation coefficients for light response parameters [dark 163 respiration (Rd), apparent quantum efficiency (4)), net photosynthesis expressed using total needle area (TNAmax), and light compensation point (LCP)] and soil pH, photosynthetic efficiency (EV/Fm), 10 soil nutrients, shoot architecture, and carbon isotope discrimination (A). Response parameters are from six Abies species grown at four locations in Michigan in 2004. xi 26 27 28 Pearson’s correlation coefficients for A/Ci response parameters 164 [respiration (R), apparent carboxylation efficiency (CE) and net photosynthesis (PSAmax)] expressed using projected shoot area and soil pH, photosynthetic efficiency (Fv/Fm), 10 soil nutrients, shoot architecture, and carbon isotope discrimination (A). Response parameters are from six Abies species grown at four locations in Michigan in 2004. Pearson’s correlation coefficients for NC, response parameters 165 [respiration (R), apparent carboxylation efficiency (CE) and net photosynthesis (PNAmax)] expressed using projected needle area and soil pH, photosynthetic efficiency (Ev/Fm), 10 soil nutrients, shoot architecture, and carbon isotope discrimination (A). Response parameters are from six Abies species grown at four locations in Michigan is 2004. Pearson’s correlation coefficients for A/Ci response parameters 166 [respiration (R), apparent carboxylation efficiency (CE) and net photosynthesis (TNAmax)] expressed using total needle area and soil pH, photosynthetic efficiency (Fv/Fm), 10 soil nutrients, shoot architecture, and carbon isotope discrimination (A). Response parameters are from six Abies species grown at four locations in Michigan in 2004. xii LIST OF FIGURES FIGURE PAGE LITERATURE REVIEW 1 Sample light response curve for Abies koreana x balsamea. 24 CHAPTER 1 1 Location of four Abies trials in Michigan. 1) Kellogg Research Forest 55 (KRF), 2) Clarksville Horticulture Experiment Station (CHES), 3) Horticulture Teaching and Research Center (HTRC), 4) Northwest Michigan Horticultural Research Station (N WHRS). 2 Growing degree day accumulation since January 1 in A) 2004 and 56 B) 2005 at four Abies test plots in Michigan. 3 Comparison of average F v/F m at -44 °C and mean days to budbreak 57 in 2004 of four Abies species at the HTRC in December 2004 and January and March 2005. 4 Frost damage to recently emerged shoots at the Kellogg Research 58 Forest, 5 May 2004. CHAPTER 2 1 Location of four Abies test plots in Michigan. 1) Kellogg Research 84 Forest (KRF), 2) Clarksville Horticulture Experiment Station (CHES), 3) Horticulture Teaching and Research Center (HTRC), 4) Northwest Michigan Horticultural Research Station (N WHRS). 2 Relationship between average foliar A) N, B) P, C) Ca, and D) Mg in 85 six Abies species grown at four locations in Michigan in 2004. 3 Relationship between PNAmax and soil pH in four Abies species grown 86 at four locations in Michigan in 2004. 4 Relationship between photosynthetic efficiency (Fv/Fm) and A) N 87 and B) K in 17 Abies species grown at four locations in Michigan in 2004. 5 Relationship between A) N, C) P, E) K, and soil pH and PNAmax and 88 B) N, D) P, and F) K in 17 Abies species grown at four locations in Michigan in 2004. xiii Relationship between leader growth and foliar K concentration in four Abies species grown at four locations in Michigan in 2004. CHAPTER THREE Two contrasting needle architecture arrangements. Left. Flat arrangement, (A. veitchii). Right. Bottlebrush needle arrangement (A. procera). Location of four Abies trials in Michigan. 1) Kellogg Research Forest (KRF), 2) Clarksville Horticulture Experiment Station (CHES), 3) Horticulture Teaching and Research Center (HTRC), 4) Northwest Michigan Horticultural Research Station (N WHRS) Needle cross-section displaying maximum needle width (horizontal), maximum needle thickness (vertical), and perimeter measurements. Needle are measured as projected shoot area (lefi), projected needle area (center) when needles are plucked and scanned, and total needle area (right) when the perimeterzwidth ratio of cross-sections is multiplied by the projected needle area. Relationship between water use efficiency and A in 10 Abies species grown at four locations in Michigan in 2004. Relationship between A) PSAmax, B) PNAmax, and C) TNAM and needle width of 17 Abies species grown at four locations in Michigan in 2004. 89 135 136 137 137 138 139 Relationship between the projected shoot to total needle area (PSA/TNA) 140 ratio and carbon isotope discrimination (A). Photosynthetic light response curves for six Abies species expressed using 141 A) projected shoot area, B) projected needle area, and C) total needle area and grown at four locations in Michigan in 20004. A/Ci curves for six Abies species expressed using A) projected shoot area, B) projected needle area, and C) total needle area and grown at four locations in Michigan in 2004. xiv 142 LITERATURE REVIEW LITERATURE REVIEW Introduction Michigan has a large nursery industry of which conifers represent a sizable portion. In the year 2000, coniferous trees produced in Michigan resulted in sales of over $35 million (USDA, 2001). Colorado blue spruce (Picea pungens var. glauca Engelm.), Norway spruce (Picea abies (L.) Karstens), white pine (Pinus strobus L.), and Douglas- fir (Pseudotsuga menziesii (Mirb.) Franco) are used extensively in the landscape, to the point where they are overplanted. This lack of diversity has resulted in increased disease problems and insect pressures (McCullough et al., 1999; McCullough et al., 1998). True firs (Abies spp. Mill.) are generally underutilized in landscapes. In general, Abies prefer cool, moist, well-drained sites with acidic soil. They have been used primarily for Christmas trees, with A. fiaseri grown in the eastern United States, while A. procera and A. nordmanniana are grown in the Pacific Northwest. In cooler regions of the United States, A. concolor is commonly planted in the landscape. However, the use of additional species and varieties has been limited due to their intolerance to varying site conditions. The number of Abies species has been debated, reportedly ranging from 39 (Liu, 1971) to 46 (Farjon, 1990) to 55 (Rushforth, 1987). Abies species are found only in the northern hemisphere at higher latitudes or at lower latitudes at higher elevations. In their natural habitat, Abies are considered late-successional trees (Kohyama, 1984) as they are not among of the first plant community that successfully colonizes disturbed sites. Rather, they are slow growing and shade tolerant replacing pioneer species as shade levels increase. Abies have been fragmented by glaciation and have adapted to a wide range of site conditions. As a result, species ranges vary greatly. Abies nebrodensis, for example, consists of only several dozen trees spread over a few square kilometers (Parducci et al., 2001). In contrast, the range of A. sibirica stretches for thousands of square miles. Species such as A. balsamea and A. fiaseri are thought to be closely related but isolated due to glaciation (Jacobs et al., 1984; Myers Jr. and Bormann, 1963). F arjon (1991) clustered the species into 10 sections based on cone characteristics, flower color, needle structure, pollen grains, geography, and fossil records (Table l). Abies naturally hybridize easily within and between groups (Liu, 1971) and where native ranges overlap (Isoda et al., 2000). Hybrids often display increased vigor (Klaehn, and Winieski, 1962). Crichfield (1988), however, suggests that artificial crosses between sections are more difficult than crosses within sections. Perhaps genetic relationships need to be investigated and considered in future classification efforts. In past introductions, adaptive traits varied greatly among species and provenances. These different characteristics and can be used to screen future introductions. The following literature review will focus on the different adaptive characteristics that are important in Michigan and the upper Midwest with respect to temperature, water relations, nutrition, and light response in the genus Abies. Temperature Tolerance to temperature extremes are an important adaptive characteristic for future introductions. In Michigan, temperatures vary considerably. During winter, temperatures below -20 °C are common and have reached -45 °C. Temperatures ranging from 25 to 30 °C are not unusual during summer. Cold Hardiness In the Upper Midwest, cold hardiness is an important requirement for quality trees. Cold hardiness develops as trees pass through the following three stages: 1) short days cause the cessation of growth, 2) freezing temperatures cue a metabolic reorganization of macromolecules resistant to severe dehydration, and 3) extreme low temperatures increase cold hardiness through dehydration resistance or supercooling (Weisner, 1970). When days become shorter and temperatures drop in the fall, trees accumulate sugars and starches in their cells, lower their freezing point (Sutinen et al., 2001; Weisner, 1970). Trees reach their maximum cold hardiness in mid-winter and then gradually become less cold hardy as temperatures warm in the spring (Ritchie, 2003). Gradume rising temperatures in the spring lead to decreasing cold hardiness. Trees can regain cold hardiness when colder temperatures return; however, cold hardiness is still lost more quickly than it is regained (Strimbeck et al., 1995). Temperature fluctuation in the spring can lead to late winter injury and damage to buds, needles, roots, and cambial tissue (van der Kamp and Worrall, 1990). Conditioning to low temperatures effects the physiological responses to temperature extremes. Abies procera seedlings exposed to temperatures ranging from 24 to 35 °C experienced more damage when exposed to sub-fieezing temperatures than those grown at warm (18 to 27 °C) or cool (12 to 19 °C) temperatures (Owston and Kozlowski, 1981). Branches of field-grown A. Iasiocarpa trees were cold hardy to temperatures below -60 °C in mid-winter. Branches from the same tree exposed to 20 °C for 132 hours and were cold hardy only to temperatures near -20 °C (Gordon-Kamm, 1980) Under field conditions, snow cover often insulates plant material from colder air temperatures. In cold regions receiving heavy snowfall, snow can keep needle temperatures near 0 °C and prevent them fiom acclimating to colder conditions. Abies sachaliensis grown in areas with heavy snow cover were more prone to frost injury than trees grown in areas with less snow cover (Eiga and Sakai, 1987). These results support the concept that trees can lose cold hardiness when exposed to prolonged warming periods. Trees adapt to different temperature extremes found in their native ranges. Abies spectabilis, native to Nepal, is cold hardy to -25 °C while A. balsnmea, native to Canada and A. sibirica, native to Siberia, are cold hardy to -70 °C (Sakai, 1982). Christmas tree growers found A. fiaseri, native to North Carolina, and A. concolor, native to the Rocky Mountains, were less cold hardy than A. balsamea (N icholls and Palmer, 1985). Likewise, varying levels of cold hardiness are also found within species. A lO-year provenance test in British Columbia showed A. grandis from coastal areas were more susceptible to frost damage than trees from inland seed sources (Xie and Ying, 1993). Average air temperatures can influence the maximum cold hardiness of trees. Air temperatures in dry air cool at the adiabatic lapse rate of 3 °C per 300 m increase in elevation. Abies are common to many mountainous regions in the higher latitudes of the northern hemisphere. Abies sachalinensis is native to the Hokkaido islands in Japan where their maximum cold hardiness increases as elevation increases (Eiga and Sakai, 1984; Xie and Ying, 1993). Snow cover keeps branches covered by more snow closer to freezing and insulates them from extreme freezing temperatures. In trees a chilling requirement and a thermal time requirement exists during winter and restricts tree growth (Howe et al., 2003; Campbell and Sugano, 1979). Cold hardiness can be lost and growth can occur once the thermal time requirement is met (Gordon-Kamm, 1980; Perry and Wu, 1960). Prolonged warming spells followed by dramatic temperature drops result in the loss of cold hardiness and damage to trees. In British Columbia, temperatures were above normal for much of December 1988 and January 1989. This warm period was followed by a sudden drop in temperatures to -30° C. In A. amabilis and A. lasiocarpa buds on lateral branches above the snow level were killed following a sudden drop to temperatures to -30 °C; however terminal buds remained undamaged (van der Kamp and Worrall, 1990). Once the temperature threshold has been met, trees accumulate growing degree days (GDD). Growing degree days are used to quantify temperatures above a threshold specific to the tree and their duration. In Abies, GDD influence budbreak. Frequently a base temperature of 50 °C is used to calculate growing degree days (Dickson et al., 2000). Trees growing at higher elevations are exposed to fewer warm days. Because trees at higher elevations are exposed to cooler temperatures, they take longer to surpass their GDD requirement than trees at lower elevations or coastal regions. In response, trees grown at higher elevations have a reduced GDD requirement and break bud only after sufficient warming has occurred. For example, Worrall (1983) found A. Iasiocarpa growing at high elevations have a shorter temperature threshold than provenances growing at lower elevations or coastal areas, suggesting adaptation to a shorter growing season. In provenance tests and species trials, trees are often grown in areas with climates different from their native regions. Worrall (1983) found that A. amabilis broke bud later than A. lasiocarpa and thus species differ in their GDD requirement for bud break. Trade—offs exist as trees breaking bud early are at increased risk from late spring frosts (Hansen and Larsen, 2004). Different tree organs have varying levels of cold hardiness. Roots are insulated from extreme winter temperatures by the soil. However, soil temperatures can still fall below freezing and injure roots during winter months (Bigras et al., 2001). Maritime species (A. amabilis) and continental species (A. lasiocarpa) did not differ at the temperature in which root damage was 50% (Coleman et al., 1992). While different species may have similar cold hardiness levels in their roots, they can differ in the degree of cold hardiness present in other organs. Coleman et al. (1992) found A. lasiocarpa withstood needle injury at lower temperatures than A. amabilis. In A. koreana, primordial shoots were cold hardy to -40 °C while needles and twigs were cold hardy to - 70 °C (Sakai, 1982). High Temperature Temperature is an important environmental condition affecting net carbon gain. Gross photosynthetic increases with increasing temperatures, plateaus and declines rapidly as high temperatures begin to degrade cell processes (Berry and Bjorkman, 1980). Net photosynthesis is the difference between gross photosynthesis and respiration. As temperatures rise, respiration increases exponentially and eventually causes net photosynthesis to decline. The temperatures where net photosynthesis is greatest represents a peak range of optimal temperatures. Plants have an optimal temperature range where photosynthesis occurs at maximum rates. Maximal quantum yield of photosystem 11 (P811) increased in A. alba with increasing temperature reaching a maximum at 26 °C and declining steadily after 32 °C (Robakowski et al., 2002). In Abies, species differ in their optimal temperature range. In cooler regions, plants have evolved methods to maximize their leaf temperature and reach their Optimal temperature range more quickly. In conifers needle packing is an adaptation that results in increased needle temperature to levels higher than air temperature. Needle temperatures rise as the number of needles-cm'l of shoot length increase due to boundary layer effects (Smith and Carter, 1988; Martin et al., 1999, Smith, 1980). In A. lasiocarpa, needle packing increased morning temperatures by 8 °C to temperatures near the optimal temperature range for photosynthesis resulting in increased carbon gain (Smith and Carter, 198 8). The boundary layer is a layer of relatively calm air that exists due to the friction above a surface. This layer of calm air can help insulate needles and keep them warmer than surrounding air temperatures. Needles of conifers are much coarser than broadleaves and hence create more friction with air. This increased fiiction results in enhanced boundary conductance layer and increases needle temperatures in A. amabilis (Martin et al., 1999) and A. lasiocarpa (Smith and Carter, 1988). Temperature gain from needle packing in A. lasiocarpa decreased in the afiemoon as wind speed increased (Smith and Carter, 1988). Increases in wind speed decreases the boundary layer existing above the needle surface, reducing the insulating effect. In contrast, elevated needle temperature resulted in no increased photosynthetic response in A. amabilis, which has a much larger range of optimal photosynthetic temperatures, and thus reached its optimal temperature range more easily (Martin et al., 1999). Populations adapt and function with lower optimal photosynthetic temperatures in cooler regions. In New Hampshire, the optimal temperature for photosynthetic C02 uptake declined by 2.7 °C for a 305 m increase in elevation in A. balsamea (Fryer and Ledig, 1972). This suggests adaptations occurring at high and low elevations and resulting in changes to optimum temperature. Water Relations In Michigan, periods of water stress are common throughout the growing season and can limit tree growth. Identifying species capable of tolerating drought conditions is an important criterion for future tree introduction in Michigan. Drought tolerance Conifers respond to both soil drought and atmospheric drought. Abies species and provenances differ in their response to drought. Drought reduced the number of shoot intemodes and intemode elongation in A. magnifica and A. concolor (Hallgren and Helms, 1998). In Switzerland, A. alba shed needles in response to drought conditions (Webster et al., 1996). Inland and higher elevation provenances of A. grandis survived planned dry-down treatments better than coastal provenances (Scholz and Stephan, 1982) Stomata regulate water lost during transpiration and are particularly important as humidity decreases. At low hurrridity, the water vapor pressure deficit increases and results in more plant water loss if not regulated. Increases in water vapor pressure deficit reduced photosynthetic gain in A. nordmanniana (Guehl et al., 1989). Stomatal conductance decreases as humidity decreases and results in lower photosynthetic rates in several Mediterranean Abies species (Guehl et al., 1991). Stomatal response to increased water vapor pressure deficits was delayed in A. alba and resulted in excess transpiration loss and eventually decreased photosynthetic gain. However, A. alba provenances differed in their photosynthetic gain and transpiration levels under favorable and drought conditions (Guehl and Aussenac, 1987). ’ Species also respond differently to soil drought conditions. In A. bornmulleriana, stomata closed quickly and the photosynthetic rate was reduced at low soil water potentials. Under the same soil conditions, A. cephalonica exhibited a higher photosynthetic rate than A. bornmulleriana. Guehl et a1. (1991) suggest A. bornmulleriana adapts to avoid internal water deficits while A. cephalonica adapts to tolerate drought. In mountainous areas, lower temperatures can reduce moisture stress. During winter and spring months, colder temperatures increase snowfall, limit snowrnelt, and improve summer soil-moisture levels at higher elevations. Adequate snowfall improved summer soil moisture levels and increased growth in A. lasiocarpa. F urtherrnore, cooler temperatures reduce evapotranspiration in the summer and reduce moisture stress in A. lasiocarpa (Peterson etal., 2002). Water use efliciency and Carbon isotope discrimination Water use efficiency (WUE) is the amount of C02 fixed per unit of water lost and is often used to compare drought tolerance between species or provenances. Other factors being equal, high WUE indicates better drought tolerance. Under normal growing conditions, A. alba provenances from southern Italy (warmer, drier regions) maintained higher photosynthetic values in one- and two-year-old needles than provenances from central and eastern Europe (cooler, wetter regions) in which 2-year—old needles declined 10 in photosynthetic gain (Larsen and Mekic, 1990). This resulted in higher WUE in the provenances from southern Italy indicating increased drought tolerance. Carbon isotope discrimination (A) may be used to investigate WUE and drought stress. 12C and 13C represent 98.9% and 1.1% of atmospheric carbon respectively. C3 plants, which include conifers, discriminate against ’3 C while fixing carbon and thus have a lower ratio of 13C/ 12C (5’3C) than the atmosphere (F arquhar et al., 1989). However, as stomata close, intercellular C02 levels decline due to photosynthetic assimilation. As a result, discrimination against l3C decreases resulting in a higher 8’3 C ratio. Tissues with lower A (higher 613C) values indicate periods of reduced stomatal conductance. Increased water use efficiency is frequently correlated with reduced A (Farquhar et al., 1989; Masle and Farquhar, 1988). Several studies have documented environmental response of A in Abies. In A. spectabilis, increased relative humidity and precipitation were positively correlated with A in tree rings (Xiaohong et al., 2003). Similarly Guehl et a1. (1991)showed A increased (lower 6’3 C) due to more conductance with increased rainfall. Other environmental factors such as pollution reduce stomatal conductance and lower A (increase 8’3 C) values in tree rings (Sakata and Suzuki, 2000). In the mountainous areas of the western United States, precipitation patterns can vary greatly. Alexander et a1. (1990) describe the native range of A. lasiocarpa to include regions receiving less precipitation than A. magnifica (Laacke, 1990a) and A. procera (Franklin, 1990) and more precipitation than A. concolor (Laacke, 1990b) and A. grandis (F oiles et al., 1990). Instantaneous WUE measurements in A. lasiocarpa were higher early and late in the day than another subalpine conifer, Pinus albicaulis Engelm., which 11 shares a similar native range; however, WUE levels declined and were lower than P. albicaulis at midday. Conversely, needles fi'om the previous year show A. lasiocarpa had a lower 8’3C than P. albicaulis and thus a lower WUE over time (Sala et al., 2001). When compared to five other conifers native to the northern Rocky Mountains, A values were significantly higher (lower WUE) in A. lasiocarpa (Pifiol and Sala, 2000) suggesting that while A. lasiocarpa may be moderately drought tolerant among other Abies species in the Western United States; it still less tolerant of dry sites when compared to other conifers sharing its native range. Soil Conditions Different nutrients are required for many physiological processes by trees. Even if soil nutrient levels are high, the availability of these nutrients may be regulated by soil pH (Lucas and Davis, 1961). In New Hampshire, A. balsamea showed little correlation between soil and foliar nutrient levels, suggesting that another factor such as soil pH influenced nutrient availability (Bruns, 1973). In Michigan, soil pH levels vary greatly from more acidic in coniferous forests to more alkaline in grassland regions. Therefore, tolerance to varying soil pH and nutrient availability is necessary in future plant introductions in Michigan. Nutrition Abies from around the world have adapted to survive under varying soil pH levels, although they generally prefer acidic soils. As a result, species differ in their soil pH tolerances. Soil pH levels influence the plant nutrients available to the plant and can lead to nutrient deficiencies (Lucas and Davis, 1961), which can ultimately hinder physiological processes. Seedling grth in A. fiaseri was greatest in soil pH ranging 12 from 4.2 to 4.5 (Bryan et al., 1989) while A. balsamea grows best on sites with soil pH levels between 6.5 and 7.0 (Bakuzis and Hansen, 1965). In A. nordmanniana, needle chlorosis developed in higher pH soils (Khalil et al., 1989). Nutrient deficiencies disrupt tree physiological processes, which can lead to abnormalities such as needle chlorosis and stunted growth. Decreasing soil pH levels tend to increase the Mn, Fe, Mg, and P that is available to the tree (Lucas and Davis, 1961). Increasing soil pH in A. alba led to Mn deficiencies and needle chlorosis ultimately resulting in tree decline (Hiltbrunner and Fliickiger, 1996). In several Abies species, photosynthetic efficiency and foliar Mn, B, K, Zn, and Cu levels declined with increased soil pH levels (Cregg et al., 2004). Decreasing levels of these nutrients were also strongly correlated with a decreased variable fluorescence to maximum fluorescence (FV/Fm) ratio and decreased chlorophyll concentration in the needles. Nitrogen, Mg, Mn, and Cu are several nutrients that are important to the photosynthetic process. In A. fiaseri, higher foliar N and P levels were characteristic of increased visual quality while higher foliar Ca, Mg, and Fe levels were found in trees with lower visual quality. In the same study, foliar nutrient ratios (eg. NzCa, NzMg, N:F e, PzCa, PzMg, and P:Fe) were important indicators of increased visual quality in A. fraseri (Rothstein and Lisuzzo, 2003). Needle chlorosis developed in A. nordmanniana with decreased foliar Fe, Mg, Mn, N, and S levels (Khalil et al., 1989). However, other site factors can influence foliar nutrient levels. F oliar P and Ca levels were highly variable in A. grandis, possibly in response to shade conditions (Moore et al., 2004). Chlorophyll concentration increased following NPK fertilization in A. balsamea (Lavigne 13 et al., 2001). In A. balsamea var. phanerolepis F em. N, K, and Ca foliar nutrient levels were significantly correlated with tree height and needle length (Brown, 2000). Nitrogen is frequently applied in conifer nurseries to improve tree color (Bruns, 1973, Rothstein and Lisuzzo, 2003), increase lateral bud development (Timmer et al., 1977), and increase tree grth (Hawkins et al., 1998) in several Abies species. Applying excess N can result in luxury consumption. Luxury consumption is continued N uptake by the tree with no increase in growth. Increased N fertilization in A. balsamea resulted in increased needle dry weight. Fertilizing beyond a foliar N content of 2.3%, however, resulted in luxury consumption (Tirnmer and Stone, 1978). Conifers keep their needles for several growing season. Some nutrients are mobile and can move from older tissue to younger tissues while other nutrients cannot. Younger branches tend to produce more photosynthate than older branches (Larsen and Mekic, 1990; J ach and Ceulemans, 2000). As a result, trees alter nutrient levels and needle quantities in older needles to maintain the productivity of younger shoots. Hinesley and Wright (1989) found that in A. fraseri N, P, and K concentrations were higher in younger branches than branches 4- to 5-years-old. Abies amabilis are slow growing trees and tend to keep their needles for a several growing seasons. Due to the increased importance of its older needles, little N and P are exported to newly emerging shoots in the spring (Hawkins et al., 1998). However, any exported nutrients were partially replaced later in the grong season. Lignin concentrations increase as trees age. Hinesley and Wright (1989) also found that Ca levels increase in older A. fiaseri branches as lignin levels increase and productivity declines. Chlorophyll and Nitrogen 14 Chlorophyll and nitrogen are two important components of the photosynthetic process. Trees vary their concentrations in response to contrasting light environments. Nitrogen concentrations increased in A. amabilis (Brooks et a1, 1994; Stenberg et al., 1998) and A. alba (Robakowski et al., 2003) as irradiance increased. However in A. balsamea, the N concentrations in sun and shade shoots were not different. Richardson (2004) suggests this contradiction could be due to sun shoots undergoing photoinhibition where additional N would not increase photosynthetic output or due to shade shoots capturing light more efficiently than sun shoots. According to Grassi and Bagnaresi (2001), shade-tolerant plants commonly have a higher chlorophyll/mass ratio than plants acclimated to high light environments, but chlorophyll/area is unaffected by the light environment. Increased irradiance resulted in lower chlorophyll/mass in A. alba (Grassi and Bagnaresi, 2001; Robakowski et al., 2003). In A. amabils (Brooks et al., 1994) chlorophyll/area increased with increasing irradiance while it was unchanged in A. alba (Grassi and Bagnaresi, 2001; Robakowski et al., 2003). As irradiance decreased, chlorophyll b concentrations increased more than chlorophyll a and resulted in a decline in the chlorophyll a/b ratio in A. alba (Brooks et al., 1994). Since both N and chlorophyll are important components of photosynthesis, their increasing concentrations in sun foliage results in higher photosynthetic output in sun shoots than shade shoots (Brooks et al., 1994; Carter and Smith, 1985; Robakowski et al., 2003). Lime Lime (CaCO3) application is a broadly accepted practice to raise soil pH levels. Using lime to raising soil pH levels reduces aluminum toxicity, improves nutrient 15 availability, returns Ca lost by tree harvest to the soil (Jablanczy, 1971). In A. balsamea, lime application improves tree quality (J ablanczy, 1971). However, other studies question the effectiveness of lime applications. Lime applications did not improve growth in the 2 years following application in A. balsamea on sites with a pH of 3.7 (Timmer et al., 1977). Rothstein and Lisuzzo (2003) found high foliar Ca levels in A. fiaseri imply nutrient imbalances. Light In the landscape, trees are often planted on exposed sites only to become shaded over time. Abies species have adapted to acclimate differently to varying light environments. In Japan, under moderate light, A. veitchii has a higher growth rate than A. mariesii. Under low light however, A. mariesii regenerates more successfirlly than A. veitchii (Kohyama, 1984). Changing shoot dynamics can increase the light harvested by the needles while reducing maintenance costs. Trees can alter their morphological and chemical characteristics to maximize their light harvest in response to changing light environments. Tolerance and adaptation to a wide range of light conditions would be beneficial in future tree introductions. Biomass Allocation Changes in light environment can lead to changes in biomass allocation. Tree crowns are more conical in high light environments and more globose in low light environments (King, 1997, Kohyarna, 1980). Under low light conditions, both A. amabilis and A. lasiocarpa displayed reduced height growth and increased caliper growth and resulted in increased lateral shoot growth and increased light interception (Klinka et al., 1992). King (1997) however, found that light increases branch biomass allocation l6 while limiting stem biomass allocation under low light conditions. In A. balsamea biomass allocation to foliage, branches, stems, and total shoot remained constant at 13, 25, 45, and 100% light intensities, while total biomass accumulation was highest at 45% light. In the same study, root mass increased with increasing light intensity in 4-year-old A. balsamea seedlings (Logan, 1969). Life-spans for needles on shade shoots were longer than life-spans for needles on sun shoots thus compensating for increased branch biomass costs (Kohyama, 1980, Mori and Takeda, 2004). Mori and Takeda (2004) found little difference in shoot length between sun and shade trees in A. veitchii while sun shoots were longer than shade shoots in A. mariesii. Photoinhibition Many conifers, including Abies, maintain their needles year-round and have limited photosynthetic capacity during winter months when warm temperatures occur (Schaberg et al., 1998). Snow cover is an important insulator of plant material during cold winter months. However, snow has a high albedo and increases. the amount of reflected light. Increased reflection raised light intensity levels and resulted in needle photodamage when exposed to low temperatures (Yamazaki, et al., 2003). Light Response Light response curves are used to distinguish changes in photosynthetic response at different light intensities and include the following: dark respiration, the light compensation point, apparent quantum efficiency, light saturation point, and maximum photosynthetic gain (Amu) (Figure 1). Dark respiration is the amount of respiration when no light is present. The light compensation point is the light intensity where dark respiration equals the gain due to photosynthesis and results in a net carbon gain of zero. 17 Apparent quantum efficiency is the change in photosynthesis for change in incident photosynthetically active radiation (PAR). With increasing light intensities, shoots eventually reach A“rum at the light saturation point. At varying light levels, trees alter their photosynthetic response to maximize their photosynthetic. gain. In A. alba, dark respiration, quantum efficiency, and the light compensation point increased with increased irradiance (Grassi and Bagnaresi, 2001). Brooks et a1. (1994) found Amax was higher in sun shoots than shade shoots in A. amabilis. Needle Morphology and Shoot Architecture Light plays an important role by influencing the needle morphology of conifers. In A. amabilis, needle weight, needle thickness, and leaf mass/area increased with increased irradiance (Brooks et al., 1994; Sprugel et al., 1996). Leaf area and needle weight increase with higher light levels in A. amabils and A. lasiocarpa (Klinka et al., 1992). Conifers will change their shoot architecture to limit self-shading in shade conditions, while producing shoots to maximize light harvest in high irradiance conditions. Shoots growing in full light tend to have more needles than shoots grown in the shade. Smith and Carter (1988) found that in A. lasiocarpa needle density (needles'cm") was 50% higher in sun shoots than shade shoots. Needles of A. lasiocarpa growing at high light exposure were more vertically oriented, while trees growing at reduced light exposure were more horizontally oriented to maximize light harvest and reduce self-shading (Gernrino and Smith, 1999). In A. amabilis, leaf mass/shoot silhouette area increases linearly as canopy openness increases suggesting trees in full sun spread incoming light over more area than trees growing in shade (Sprugel et al., 18 1996). Shade shoots undergo less self-shading than sun shoots and therefore increase their light-capturing efficiency (Stenberg, et al., 1998). Increased fertilization resulted in improved growth leading to increased self-shading of older needles in A. grandis, which eliminated a positive carbon balance in those shoots and caused needle abscission (Balster and Marshall, 2000). By altering shoot architecture, sun and shade shoots can maximize their area for light harvest while limiting maintenance costs. Light Acclimation As trees age, sun shoots can eventually become shade shoots and trees can acclimate in response to this change in irradiance. After applying shade to sun shoots, A. amabilis reduced its photosynthetic production at light saturation, chlorophyll-to-nitrogen ratio, and chlorophyll azb ratio to levels similar to shoots that were naturally shaded, while chlorophyll content increased (Brooks et al., 1994). Even though chlorophyll azb ratio declined, both chlorophyll a and chlorophyll b levels increased with decreased irradiance. Needle thickness was unchanged as shoots became shaded but needle weight decreased suggesting internal changes and needle abscission. Summary Abies use in the landscape and by the Christmas tree industry has been rather limited; however, different species and provenances have adapted to varying site conditions (Table 2). Several adaptive characteristics warrant further investigation to identify potential species and hybrids worthy of consideration for use in Michigan. Considerable variation exists among species and provenances in their level of cold hardiness and time to budbreak. Shoot adaptations result in needle temperatures above that of the surrounding air to improve their photosynthetic output in some species. 19 Species and provenances may respond differently to drought and vary in drought tolerance. Foliar nutrient levels are largely dependent on soil pH; however, different species show tolerance to varying soil pH levels. Varying light environments elicit different responses exhibited by biomass allocation, shoot architecture, light response characteristics, and photosynthetic elements. 20 Table 1. F ourty-six species in 10 Abies sections as defined by Farjon (1990). Section Species Abies Amabilis Balsamea Bracteata Momi alba Mill. cephalonica Loud. cilicica (Ant. et Kotschy) Carriére nebrodensis (Loj ac.) Mattei nordmanniana (Stev.) Spach. amabilis (Dougl.) Forbes mariesii Mast. balsamea (Linn.) Mill. fiaseri (Pursh.) Poir. kawakamii (Hay.) Ito koreana Wils. lasiocarpa (Hook) Nutt. nephrolepis (Tratv.) Maxim. sachalinensis (Fr. Schm.) Mast. sibirica Ledeb. veitchii Lindl. racteata D. Don ex Poiteau beshanzuensis Wu chensiensis Van Tiegh. firma Sieb. et Zucc. holophylla Maxim. homolepis Sieb. et Zucc. pindrow (Lamb.) Royle recurvata Mast. ziyuanensis Fu et Mo 21 Figure 1 Cont’d. Section Species Grandis Nobilis Oiamel Piceaster Pseudopicea concolor (Gord. et Glend.) Lindl. durangensis Mart. grandis (Dougl.) Forbes guatemalensis Rehd. magnifica A. Murr. procera Rehd. hickeli Flous et Gauss religiosa (H.B.K.) Schlect. et Charn. vejari Mart. numidica De Lann. pinsapo Boiss. densa Griff. fabric (Masters) Craib fanjingshanensis Huang, Tu et Fang fargesii French. forrestii C. Coltrn. Rogers spectabilis (D.Don) Spach chengii Rushforth delavayi Van Tiegh. squamata Masters yuanbaoshanensis Lu et Fu 22 Table 2. List of species tolerant or intolerant of extreme winter temperatures, drought, and soil pH. Cold Hardiness Less Cold Hardv (Temps 2 -20 °C) A. spectabilis Medium Cold Hardy (Temps -21 to -39 °C) ' A. firma A. homolepis A. veitchii A. procera A. sachalinensis More Cold Hardv (Temps S —40 °C) A. koreana A. balsamea A. sibirica A. lasiocarpa Drought tolerance Poor Water Use Efficiency A. alba - Eastern European provenances A. lasiocarpa Soil Chemistry Tolerant of high pH soils A. balsamea A. veitchii A. lasiocarpa Intolerant of high pH soils A. alba A. borisii regis A. sibirica A. sachalinensis Sakai, 1982 Sakai, 1982 Sakai, 1982 Sakai, 1982 Sakai, 1982 Sakai, 1982 Sakai, 1982 Sakai, 1982 Sakai, 1982 Gordon-Kamm, 1980 Guehl and Assenanc, 1987 Pifiol and Sala, 2000 Bakuzis and Hansen, 1965 Cregg et al., 2004 Cregg et al., 2004 Hiltbrunner and Fltickiger, 1996 Cregg et al., 2004 Cregg et al., 2004 Cregg et al., 2004 23 sl umol CO2 m’2 .............................................................................................. Quantum Efficiency A 1 Light Saturation Point N r O & 5 3 Dark Respiration ¢ -2 z I I I I 0 500 1000 1500 2000 PPFD Light Compensation Point Figure 1. Sample light response curve for Abies koreana x balsamea. 24 Am _>6 q ......................................................................................... Quantum Efficiency 4” 4 - Light Saturation Point E N O 0 3 2 . E =- L o L%s\\r 3 Dark Respiration \Z -2 1 r a ‘ . . O 500 1000 1 500 2000 PPFD Light Compensation Point Figure 1. Sample light response curve for Abies koreana x balsamea. 24 Literature Cited Alexander, R.R., R.C. Shearer, and W.D. Shepperd. 1990. Subalpine fir. In: Russell M. Burns and Barbara H. Honkala (tech coords.). Silvics of North American: 1. Conifers. USDA For. Serv., Agr. Hdbk. 654. Aussenac, G. 2002. Ecology and ecophysiology of circum-Mediterranean firs in the context of climate change. Ann. For Sci. 59: 823-832. Bakuzis, E.V. and H.L. Hansen. 1965. Balsam fir-a monographic review. University of Minnesota Press, Minneapolis. 445p. Balster, NJ. and J .D. Marshall. 2000. Decreased needle longevity of fertilized Douglas- fir and grand fir in the northern Rockies. Tree Physiol. 20: 1191-1197. 3394087 - 304 Berry, J. and O. Bjorkman. 1980. Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant Physiol. 31: 491-543. Bigras, F .J ., A. Ryyppo, A. Lindstrom, and E. Stattin. 2001. Cold acclimation and deacclirnation of shoots and roots of conifer seedlings, p. 57-88. In: F.J. Bigras and SJ. Colombo (eds.). Conifer cold hardiness. Kluwer Academic Publishers, Norwell, Mass. Brooks, J.R., T.M. Hinckley, and DO. Sprugel. 1994. Acclimation responses of mature Abies amabilis sun foliage to shading. Oecologia. 100: 316-324. Brown, J .H. 2000. Survival and growth of trees of a Canaan Valley, West Virginia, seed source in relation to varying soil/site conditions. Ohio State Univ. Ext. Spec. Circ. 175- 00. Bruns, PE. 1973. Cultural practices, fertilizing and foliar anaylsis of balsam fir Christmas trees. N.H. Agric. Exp. Stn. Bull. 501, Durham, NH. Bryan, J.A., J .R. Seiler, and RD. Wright. 1989. Influence of growth medium pH on the growth of container-grown fraser fir seedlings. J. Environ. Hort. 7(2) 62-64. Campbell, R.K. and AI Sugano. 1979. Genecology of bud-burst phenology in Douglas- fir: response to flushing temperature and chilling. Bot. Gaz. 140(2): 223-231. Carter, GA. and WK. Smith. 1985. Influence of shoot structure on light interception and photosynthesis in conifers. Plant. Physiol. 79: 103 8-1043. Coleman, M.D., T.M. Hinckley, G. McNaughton, and BA. Smit. 1992. Root cold hardiness and native distribution of subalpine conifers. Can. J. For. Res. 22: 932-938. 25 Cregg, B.M., M.W. Duck, C.M., Rios, D.B. Rowe, and MR. Koelling. 2004. Chlorophyll fluorescence and needle chlorophyll concentration of fir (Abies sp.) seedlings in response to pH. Hort Sci. 39(5): 1121-1125. Critchfield, W.B. 1988. Hybridization of the California firs. For. Sci. 34(1): 139-51. Dickson, R.L., G.B. Sweet, N.D. Mitchell. 2000. Predicticing Pinus radiata female strobilus production for seed orchard site selection in New Zealand. For. Ecol. Mgt. 133: 197-21 5 . Eiga, S. and A. Sakai. 1984. Altitudinal variation in freezing resistance of Saghalien fir (Abies sachalinensis). Can. J. Bot. 62: 156-160. Eiga, S. and A. Sakai. 1987. Regional variation in cold hardiness of Sakhalin fir (Abies sachalinensis Mast.) in Hokkaido, Japan. In Li (ed.). Plant cold hardiness. Liss, New York, NY. F arjon, A. 1990. Pinaceae drawings and descriptions of the genera Abies, Cedrus, Psudolarix, Keteleeria, Nothotsuga, Tsuga, Cathaya, Pseudotsuga, Larix, and Picea. Champaign, IL. Foiles, M.W., R.T. Graham, and DR Olson, Jr. 1990. Grand fir. In: Russell M. Burns and Barbara H. Honkala (tech coords.). Silvics of North American: 1. Conifers. USDA For. Serv., Agr. Hdbk. 654. Franklin, J .F. 1990. Noble fir. In: Russell M. Burns and Barbara H. Honkala (tech coords.). Silvics of North American: 1. Conifers. USDA For. Serv., Agr. Hdbk. 654. Fryer, J .H. and RT. Ledig. 1972. Microevolution of the photosynthetic temperature optimum in relation to the elevational complex gradient. Can. J. Bot. 50: 1231-1235. F arquhar, G.D., J .R. Ehleringer, and KT. Hubick. 1989. Carbon isotope discrimination and photosynthesis. Annu. Rev. Plant Physiol. And Plant Mol. Biol. 40: 503-537. Gordon-Kamm, W.J. 1980. Freezing tolerance of several conifers in a western Washington forest community. Western Washington Univ. Bellingham, MS. Thesis. Gerrnino, MJ. and WK. Smith. 1999. Sky exposure, crown architecture, and low- temperature photoinhibition in conifer seedlings at alpine treeline. Plant Cell Environ. 22: 407-415. Grassi, G. and U. Bagnaresi. 2001. F oliar morphological and physiological plasticity in Picea abies and Abies alba saplings along a natural light gradient. Tree Physiol. 21: 959- 967. 26 Guehl, J .M. and G. Aussenac. 1987. Photosynthesis decrease and stomatal control of gas exchange in Abies alba Mill. in response to vapor pressure difference. Plant Physiol. 83: 3 16-3 22. Guehl, J.M, G. Aussenac, J. Bouachrine, R. Zimmerman, J .M. Pennes, A. Ferhi, and P. Grieu. 1991. Sensitivity of leaf gas exchange to atmospheric drought, soil drought, and water-use efficiency in some Mediterranean Abies species. Can. J. For. Res. 21: 1507- 1515. Guehl, J.M., J. Bouachrine, R. Zimmermann, and E. Dreyer. 1989. Responses of photosynthesis and stomatal conductance to atmospheric humidity in some Mediterranean Abies species. Ann. Sci. For. 46(S): 4015-4058. Hallgren, SW. and IA. Helms. 1988. Control of height growth components in seedlings of California red and whit fir by seed sources and water stress. Can. J. For. Res. 18: 521- 529. Hansen, J .K. and J .B. Larsen, 2004. European silver fir (Abies alba Mill.) provenances for Calabria southern Italy: 15-year results from Danish provenance field trials. Eur. J. For. Res. 123: 127-138. Hawkins, B.J., G. Henry, and S.B.R. Kiiskila. 1998. Biomass and nutrient allocation in Douglas-fir and amabilis fir seedlings: influence of growth rate and nutrition. Tree. Physiol. 18: 803-810. Hiltbrunner, E. and W. Flfickiger. 1996. Manganese deficiency of silver fir trees (Abies alba) at a reforested site in the Jura mountains, Switzerland: aspects of cause and effect. Tree Physiol. 16:963-975. Hinesley, LE. and RD. Wright. 1989. Biomass and nutrient accumulation in fraser-fir Christmas trees. HortScience 24(2): 280-282 Howe, G.T., S.N. Aitken, D.B. Neale, K.D., Jermstad, N.C. Wheeler, and T.H.H. Chen. 2003. From genotype to phenotype: unraveling the complexities of cold adaptation in forest trees. Can. J. Bot. 81: 1247-1266. Isoda, K., S. Shiraishi, S. Watanabe, and K. Kitamura. 2000. Molecular evidence of natural hybridization between Abies veitchii and A. homolepis (Pinaceae) revealed by chloroplast, mitochondrial and nuclear DNA markers. Mol. Ecol. 9: 1965-1973. Jablanczy, A. 1971. Use of lime for tree cultivation. Am. Christmas Tree J. 15: 21-23. Jach, ME. and R. Ceulemans. 2000. Effects of season, needle age and elevated atmospheric C02 on photosynthesis in Scots pine (Pinus sylvestris). Tree Physiol. 20: 145-157. 27 Jacobs, B.F., C.R. Werth, and S.I. Guttrnan. 1984. Genetic relationships in Abies of eastern United States: an electrophoretic Study. Can. J. of Bot. 62: 609-616. Johnson, D.M., M.J. Germino, and WK. Smith. 2004. Abiotic factors limiting photosynthesis in Abies lasiocarpa and Picea engelmannii seedlings below and above the alpine timberline. Tree Physiol. 24: 377-3 86. King, DA. 1997. Branch growth and biomass allocation in Abies amabilis saplings in contrasting light environments. Tree Physiol. 17: 251-258. Klaehn, F.U. and J.A. Wirrieski. 1962. Interspecific hybridization in the genus Abies. Silvae Genet. 11: 130-140. Klinka, K., Q. Wang, G]. Kayahara, R.E. Carter, and BA. Blackwell. 1992. Light- growth response relationships in Pacific silver fir (Abies amabilis) and subalpine fir (Abies lasiocarpa). Can. J. Bot. 70: 1919-1930. Kohyarna, T. 1980. Growth pattern of Abies mariesii saplings under conditions of open- growth and suppression. Bot. Mag. Tokyo. 93: 13-24. Kohyarna, T. 1984. Regeneration and coexistence of two Abies species dominating subalpine forests in central Japan. Oecologia. 62:156-161. Laacke, R.J. 1990a. California red fir. In: Russell M. Burns and Barbara H. Honkala (tech coords.). Silvics of North American: 1. Conifers. USDA For. Serv., Agr. Hdbk. 654. Laacke, R.J. 1990b. White fir. In: Russell M. Burns and Barbara H. Honkala (tech coords.). Silvics of North American: 1. Conifers. USDA For. Serv., Agr. Hdbk. 654. Larsen, J .B. and F. Mekic. 1991. The geographic variation in European silver fir (Abies alba Mill.). Silvae Genet. 40(5/6): 188-198. Lavigne, M.B., C.H.A. Little, and J .E. Major. 2001. Increasing the sink:source balance enhances photosynthetic rate of 1-year-old balsam fir foliage by increasing allocation of mineral nutrients. Tree Physiol. 21: 417-426. Liu, TS. 1971. A monograph of the genus Abies. Taipei, Taiwan. Logan, K.T. 1969. Growth of tree seedlings as affected by light instensity IV. black spruce, white spruce, balsam fir, and eastern white cedar. Can. For. Serv. Publ. No. 1256. Lucas, RE. and J .F. Davis. 1961. Relationships between pH values of organic soils and availabilities of 12 Plant Nutrients. Soil Sci. 92: 177-1 82. 28 Martin, T.A., T.M. Hinckley, F.C. Meinzer, and D.G. Sprugel. 1999. Boundary layer conductance, leaf temperature and transpiration of Abies amabilis branches. Tree Physiol. 19:435-443. Masle, J. and GD. Farquhar. 1988. Effects of soil strength on the relation of water-use efficiency and growth to carbon isotope discrimination in wheat seedlings. Plant Physiol. 86: 32-38. McCullough, D.G., S.A. Katovich, D.L. Mahr, D.D. Neumann, C.S. Sadof, M.J. Raupp. 1999. Biological control of insect pests in forested ecosystems: a manual for foresters, Christmas tree growers and landscapers. Mich. State Ext. Bull. E-2679. McCullough, D.G., S.A. Katovich, M.E. Ostry, and J. Cummings-Carlson. 1998. Christmas tree pest manual. 2“(1 ed. Mich. State Ext. Bull. E-2676. Moore, J .A., PC. Mika, T.M. Shaw, and MI. Garrison-Johnston. 2004. F oliar nutrient characteristics of four conifer species in the interior northwest United States. Western J. Appl. For. 19(1): 13-24. Mori, A. and H. Takeda. 2004. Functional relationships between crown morphology and within-crown characteristics of understory saplings of three codominant conifers in a subalpine forest in central Japan. Tree Physiol. 24: 661-670. Myers Jr., 0. and RH. Bormann. 1963. Phenotypic variation in Abies balsamea in response to altitudinal and geographic gradients. Ecology. 44(3): 429-436. Nicholls, T.H., and MA. Palmer. 1985. Christmas tree winter injury in the lake states. Amer. Christmas tree J. 29: 21-25. 0wston, P.W. and T.T. Kozlowski. 1981. Growth and cold hardiness of container grown Douglas-fir, noble fir, and Sitka spruce seedlings in simulated greenhouse regimes. Can. J. For. Res. 11:465-474. Parducci, L., A.E. Szmidt, A. Madaghiele, M. Anzidei, and G. G. Vendramin. 2001. Genetic variation at chloroplast microsatellites (cpSSRs) in Abies nebrodensis (Lojac.) Mattei and three neighboring Abies Species. Theor. Appl. Genet. 102:733-740. Pifiol, J and A. Sala. 2000. Ecological implications of xylem cavitation for several Pinaceae in the Pacific Northern USA. Funct. Ecol. 14: 53 8-545. Perry, T0. and W.C. Wu. 1960. Genetic variation in the winter chilling requirement for date of dormancy break for Acer rubrum. Ecology. 41(4): 790-794. Peterson, D.W., D.L. Peterson, and G.J. Ettl. 2002. Grth responses of subalpine fir to climatic variability in the Pacific Northwest. Can. J. For. Res. 32(9): 1503-1517. 29 Richardson, AD. 2004. Foliar chemistry of balsam fir and red spruce in relation to elevataion and the canopy light gradient in the mountains of the northeastern United States. Plant and Soil. 260: 291-299. Ritchie, GA. 2003. Root physiology and phenology: the key to transplanting success. In: Riley L.E., R.K. Dunrroese, and TD. Landis (tech. coords.). National proceedings: Forest and conservation nursery associations -2002. Ogden, UT: USDA For. Serv., Rocky Mountain research station. Proceedings RMRS-P-28: 98-104. Robakowski, P., P. Montpied, and E. Dreyer. 2003. Plasticity of morphological and physiological traits in response to different levels of irradiance in seedlings of silver fir (Abies alba Mill). Trees. 17: 431-441. Robakowski, P., P. Montpied, and E. Dreyer. 2002. Temperature response of photosynthesis of silver fir (Abies alba Mill.) seedlings. Ann. For. Sci. 59: 163-170. Rothstein, RE. and N. Lisuzzo. 2003. Optimal nutrition of fiaser fir Christmas trees in Michigan, p. 19-21. In: D. Brown-Rytlewski and J. O’Donnell (eds.). Nursery, Landscape and Christmas Tree Research Projects and Educational Programs. Michigan State Univ. Extension, East Lansing, MI. Rushforth, K.D. 1987. Conifers. Christopher Helm. London. Sakata, M. and K. Suzuki. 2000. Evaluating possible causes for the decline of Japanese fir (Abies firma) forests based on {BBC records of annual growth rings. Environ. Sci. Technol. 34: 373-376. Sakai, A. 1970. Mechanism of desiccation damage of conifers wintering in soil-frozen areas. Ecology. 51(4): 657-664. Sakai, A. 1982. Extraorgan fi'eezing of primordial shoots of winter buds of Conifers, p. 199-209. In P.H. Li and A. Sakai (eds.). Plant cold hardiness and freezing stress mechanisms and crop implications v. 2. Academic Press, New York, NY. Sala, A., E. V. Carey, R.e. Kaene, and RM. Callaway. 2001. Water use by whitebark pine and subalpine fir: potential consequences of fire exclusion in the northern Rocky Mountains. Tree Physiol. 21: 717-725. Schaberg, P.G., J .B. Shane, P.F. Cali, J .R. Donnelly, and GR. Strimbeck. 1998. Photosynthetic capacity of red spruce during winter. Tree Physiol. 18: 271-276. Scholz, F. and B.R. Stephan. 1982. Growth and reaction to drought of 43 Abies grandis provenances in a greenhouse study. Silvae Genet. 31(1): 27-35. Smith, W.K. and GA. Carter. 1988. Shoot structure effects on needle temperatures and photosynthesis in conifers. Amer. J. Bot. 75(4): 496-500. 30 Smith, W.K. 1980. Importance of aerodynamic resistance to water use efficiency in three conifers under field conditions. Plant Physiol. 65: 132-135. Sprugel, D.G., J .R. Brooks, and T.M. Hinckley. 1996. Effects of light on shoot geometry and needle morphology in Abies amabilis. Tree Physiol. 16: 91-98. Stenberg, P., H. Smolander, D. Sprugel, and S. Smolander. 1998. Shoot structure, light interception, and distribution of nitrogen in an Abies amabilis canopy. Tree Physiol. 18: 759-767. Strimbeck, G.R., Schaberg, P.G., DeHayes, D.H., Shane, J .B., and G.J. Hawley. 1995. Midwinter dehardening of montaine red spruce during a natural thaw. Can. J. For. Res. 25: 2040-2044. Sutinen, M.L., R. Arora, M. Wisniewski, E. Ashworth, R. Strimbeck, and J. Palta. 2001. Mechanisms of frost survival and freeze-damage in nature, p. 89-120. In: F .J . Bigras and SJ. Colombo (eds.). Conifer cold hardiness. Kluwer Academic Publishers, Norwell, Mass. Tirnmer, V.R., E.L. Stone, and D.G. Embree. 1977. Growth response of young balsam fir fertilized with nitrogen, phosphorus, potassium, and lime. Can. J. For. Res. 7: 441- 446. Timmer, V.R. and EL. Stone. 1978. Comparative foliar analysis of young balsam fir fertilized with nitrogen, phosphorus, potassium, and lime. Soil Sci. Soc. Am. J. 42: 125- 1 30. United States Department of Agriculture. 2001. Nursery crops 2000 summary. 13 Feb 2005. < http://usda.mannlib.comell.edu/reports/nassr/other/nursery/nurserO1.pdf‘> van der Kamp, B.J. and J. Worrall. 1990. An unusual case of winter bud damage in British Columbia interior conifers. Can. J. For. Res. 20: 1640-1647. Webster, R., A. Rigling, and L. Walthert. 1996. An analysis of crown conditions of Picea, F agus and Abies in relation to environment in Switzerland. Forestry 69: 347-355. Weiser, C]. 1970. Cold resistance and injury in woody plants. Science. 169: 1269- 1278. Worrall, J. 1983. Temperature - Bud-burst relationships in amabilis and subalpine fir provenance tests replicated at different elevations. Silvae Genet. 32:203-9. Xiaohong, L., Q. Dahe, S. Xuemei, C. Tuo, and R. Jiawen. 2003. Climatic significance of stable carbon isotope in tree rings of Abies spectabibis in southeastern Tibet. Chinese Sci. Bul. 48(18): 2000-2004. 31 Xie, CY. and CC. Ying. 1993. Geographic variation of grand fir (Abies grandis) in the Pacific coast region: lO-year results from a provenance trial. Can. J. For. Res. 23: 1065- 1072. Yamazaki, J ., A. Ohashi, U. Hashimoto, E. Negishi, S. Kumagai, T. Kubo, T. Oikawa, E. Maruta, and Y. Kamimura. 2003. Effect of high light and low temperature during harsh winter on needle photodamage of Abies mariesii growing at the forest limit on Mt. Norikura in Central Japan. Plant Sci. 165: 257-264. 32 CHAPTER ONE BUDBREAK AND WINTER INJURY IN EXOTIC F IRS For submission to HortScience 33 BUDBREAK AND WINTER INJURY IN EXOTIC F IRS Additional index words. Cold Hardiness, chlorophyll fluorescence, frost damage Abstract I Fir (A bies spp.) trees are occasionally used as landscape trees but are more commonly grown as Christmas trees. Recently, the Michigan State University Department of Forestry and the Michigan Christmas tree industry initiated a field test of exotic firs for potential use as Christmas trees. In the present study we expanded the evaluation of these exotic fir species to include their suitability as landscape trees and characterize their tolerance to cold damage. In spring 2004 and 2005, trees were surveyed weekly for budbreak and late spring fiost damage. Freeze tests were conducted on four species growing at the Horticulture Teaching and Research Center to determine cold hardiness levels during winter. Species differed significantly in their days to budbreak at all locations. Trees that had already broken bud were more prone to late spring frost damage than trees yet to break bud. Chlorophyll fluorescence, bud damage, and needle damage differed among species at -44 °C. Bud, foliar, and cambiurn damage was correlated with chlorophyll fluorescence following freeze tests. Budbreak and cold hardiness were correlated with species breaking bud earlier displaying greater mid-winter cold hardiness than species breaking bud later. Selection criteria for future Abies introductions to the upper Midwest should include identifying species with late budbreak. 34 Introduction The genus Abies Mill. (true firs) consists of 46 species (Farjon, 1990) found only in the northern hemisphere at higher latitudes or at higher elevations in lower latitudes. They generally prefer sites with cool temperatures, adequate moisture, well-drained soil, and low pH soils. Expanded use of Abies in landscapes has been limited by their intolerance of many site conditions. However, Abies may be more tolerant of environmental conditions than originally thought as species differ in their response to soil pH (Cregg et al., 2004), drought (Guehl et al., 1991), light (Kohyarna, 1984), and freezing temperatures (Sakai, 1982). Collectively, Abies cover large elevational and latitudinal ranges (Liu, 1971). Langlet (1963) suggests large species distribution ranges increase the likelihood of genetic adaptation to diverse external factors. Several Abies species are native to North America, but their use in the landscape, Christmas tree, and forestry industries has been limited to a few speCies. Abies concolor is common to the landscape in cooler regions. For Christmas tree production, A. fraseri is commonly grown in the eastern United States, while A. procera and A. nordmanniana are commonly grown in the Pacific Northwest. Forestry use has been limited to A. amabilis, A. grandis, and A. procera in the Pacific Northwest. Recent efforts to increase conifer diversity have sparked increased interest in planting exotic conifers. Much of this interest has centered on exotic firs and their hybrids. Throughout the Midwest, planting of conifers has been typically limited to a few types of trees. Trees like Douglas-fir [Pseudotsuga menziesii (Mirbel.) Franco], Norway spruce [Picea abies (L.) Karst.], Colorado blue spruce [Picea pungens Englem.], Scotch 35 pine (Pinus sylvestris L.), Austrian pine (Pinus nigra Arnold), and eastern white pine [Pinus strobus L.] are frequently used to the point of overplanting. The resulting lack of diversity has led to increased disease problems and insect pressures (McCullough et al., 1999; McCullough et al., 1998). A For future evergreen conifer introductions in the upper Midwest, tolerance of freezing temperatures is a necessary characteristic. Trees with adequate cold hardiness are required as average winter temperatures range from —20 to —42 °C in the upper Midwest (USDA Plant Hardiness Zone Map, 1990). Species also need to break bud in late spring to reduce damage from late spring frosts. In conifers, cold hardiness levels increase in the late fall, reach a maximum in rrridwinter, and decrease as temperatures rise in late winter (Ritchie, 2003). In A. lasiocarpa (Gordon-Karnm, 1980; van der Kamp and Worrall, 1990), A. procera (Owston and Kozlowski, 1981), and A. amabilis (van der Kamp and Worrall, 1990) cold hardiness was reduced following periods of warm temperatures. Maximum cold hardiness also varies among species. For example, A. spectabilis is cold hardy to -25 °C while A. balsamea and A. sibirica are cold hardy to -70 °C (Sakai, 1982). Considerable intra-specific variation has also been documented for provenances of various species of Abies including A. grandis (Xie and Ying, 1993) and A. sachalinensis (Eiga and Sakai, 1984; Eiga and Sakai, 1984). Provenance variation in cold hardiness may be related to latitude, elevation, and winter snowfall. The direct effects of freezing temperatures result in the failure of cell biological functions (Binder and Fielder, 1996; Yordanov, 1992) and cause secondary limitation to the photoharvesting system (Adams and Perkins, 1993). Chlorophyll fluorescence is 36 used to measure the efficiency by which photosystem II captures light and is often expressed as the ratio of variable fluorescence to maximum fluorescence (Fva) (Bjorkman and Demming, 1987). Chlorophyll fluorescence is useful in comparing plant stress (Cregg et al., 2004; Maxwell and Johnson, 2000; Toivonen and Vidaver, 1988; Ritchie and Landis, 2005) and quantifying cold hardiness (Binder and Fielder, 1996; Adams and Perkins; 1993). Budbreak is under strict genetic control (Worrall, 1983) and is an adaptive response to climate conditions at the population’s origin (Campbell and Sugano, 1979). First a chilling requirement and then a growing degree day (GDD) requirement must be satisfied for budbreak to occur (Howe et al., 2003; Campbell and Sugano, 1979). Northern provenances have a longer chilling requirement than southern provenances (Perry and Wu, 1960). Provenances from colder regions have a reduced GDD requirement for budbreak than provenances from warmer regions (Campbell and Sugano, 1979). For example, Picea mariana (Mill.) B. S. P. provenances from northern Canada broke bud 7 days earlier than provenances from the northern United States when grown at the same location (Morgenstem, 1978). Species that break bud earlier in the spring are more susceptible to late spring frosts than those breaking bud later in the spring (Hansen and Larsen, 2004; Leege, 2002). Timing of budbreak differs among provenances for A. alba (Hansen and Larsen, 2004), A. amabilis (Worrall, 1983), A. grandis (Scholz and Stephan, 1982), and A. lasiocarpa (Hansen et al., 2004; Worrall, 1983). High elevation provenances have reduced threshold temperatures and thus break bud earlier than lower elevation provenances, suggesting adaptation to a shorter growing season (Worrall, 1983). 37 The goal of this project is to identify Abies species tolerant of the Michigan climate, promote increased plant diversity, and thus reduce disease problems and insect pressures. Objectives in this paper are to 1) compare the date of budbreak between species, 2) identify differences in winter injury, and 3) test the hypothesis that rrridwinter cold hardiness is inversely related to budbreak. Materials and Methods Site Locations Over 1100 trees representing 38 species, hybrids, provenances, and varieties of Abies were part of a true fir species and provenance trial initiated at the Kellogg Research Forest (KRF), Augusta, Michigan, in early 1991. In the fall of 2002 and spring of 2003, 246 trees representing 21 species and hybrids (Table 1) were transplanted to three locations in Michigan: Clarksville Horticulture Experiment Station, Clarksville, MI (CHES); Horticulture Teaching and Research Center, East Lansing, MI (HTRC); and Northwest Michigan Horticulture Research Station, Traverse City, MI (N WMHRS) (Figure 1). These three locations along with the KRF represent different climate regions (Table 2) and soil environments (Table 3) in Michigan. Trees were dug and ball and burlapped as 60 to 76 cm root balls in accordance with American nursery standards (American Nursery and Landscape Association, 2004) In the fall of 2003, 57 additional trees were transplanted from the KRF to replace 37 trees that died due to transplant stress during the previous year and add three new species and varieties to the study. At least four trees of each species or hybrid were planted at each location with the exception of the HTRC where one to four more trees of 38 each species were planted when available. Trees were planted in a complete randomized design at each location. Trees were planted at approximately 4.5 m intervals. Fertilization In spring 2004, sites were fertilized with 21-0-0 ammonium sulfate at a rate of approximately 133 g per tree, to lower soil pH by 0.5 and insure that nitrogen was not limiting. Fertilizer was applied at CHES on 29 April, HTRC on 22 April, and NWI-IRS on 11 May. On 7 July 2004, 46-0-0 urea was applied at CHES at a rate of 91 g per tree. Trees at KRF were fertilized every fall with 21-0-0 at 85 g per tree. Trees exceeding 0.9 m received 28 g of fertilizer for each additional 0.3 m in height. Budbreak Beginning 16 March, 2004, each of the three outlying sites and the KRF were surveyed for budbreak, considered to have occurred once one bud broke its bud scale. Trees were inspected weekly until all trees at each location had broken bud. For each inspection date, growing degree days (GDD) were calculated using a base temperature of 10 °C (Dickson et al., 2000) and the numerical integration method using the Michigan Automated Weather Network (MAWN) web-site (http://www.agweather.geo.msu.edu/mawn/). Frost damage Temperatures reached -2.2 and -2.4 °C on 3 and 4 May 2004 at the KRF after trees had begun to break bud. Trees were visually rated on 5 May 2004 using the following 0-4 scale: 0= no shoots damaged, 1: 1-25%, 2= 26-50%, 3= 51-75%, and 4= 76-100% of shoots damaged. Shoots were considered damaged if they were brown in color or were dropping. All trees in each species block (n=7-42) were inspected for frost 39 damage, in addition to the four individuals previously selected at random for the budbreak study. Cold Hardiness Four species were chosen to measure cold hardiness and represent trees in four different bud break groups: A. balsamea var. phanerolepis; A. chensiensi; A. nephrolepis; and A. veitchii. Shoots from the current year’s growth were collected fi'om three trees of each species from HTRC. Samples were collected on 22 Nov 2004, 13 Dec 2004, 24 Jan 2005, and 7 Mar 2005 with freeze tests beginning 1-3 days later. Twelve samples for each temperature (4 spp x 3 rep) treatment were laid on moist cheese cloth, covered with aluminum foil, and rolled into bundles. A thermocouple was inserted into the stem of one sample in each bundle to measure stem temperature. Bundles were then placed into a freezer (ScienTemp, Adrian, MI) and stored at 2 °C until the test began. Temperatures were lowered at 3 °C-hr" and a bundle was removed at each targeted temperature until completion of the run. A control bundle was kept in a walk-in cooler at 2 °C where remaining bundles were allowed to thaw following removal from the freezer. In the 22 Nov 2004 test, a bundle was removed at the following temperatures; 2, - 6, -9, -12, -15, -18, -21, -24, -27, -30, -33 and -36 °C. For both the 13 Dec 2004 and 24 Jan 2005 tests, a bundle was removed at the following temperatures: 2, -l 8, -21, -24, -27, -30, -33, -36, -39, -42, and -44 °C. In the 7 Mar 2005 test, a bundle was collected at the following temperatures: 2, -6, -9, -12, -15, -l8, -21, -24, -27, -30, -33, -36, -39, —42, -44, and -78 °C. Bundles were placed in a walk-in cooler and allowed to thaw at 2 °C for 2-3 days and then placed in a high humidity chamber at room temperature (25 °C) for 4-5 days. Then samples were visually rated for needle damage, bud damage, and cambium 40 damage using the following 0-2 scale: 0= no damage; 1= partial browning of the tissue; 2=dead tissue. Chlorophyll fluorescence (F v/F m) was measured using two needles from every sample in each temperature treatment using a portable chlorophyll fluorescence system (Plant Efficiency Analyzer, Hansatech Instruments Ltd., Norfolk, England). Samples were clipped and dark-acclimated for 15 min before readings were taken. An index of injury percentage was calculated for each species comparing the F "/F m at each temperature to the Fv/Fm of the control. Statistical Analysis Species effects on budbreak and cold hardiness damage were determined using PROC MD(ED (SAS Inc., Cary, NC). When significant differences were indicated, means were separated using Tukey’s Studentized test (Sexton, 1998). Species and year differences for required growing degree day to budbreak were determined using PROC GLM (SAS Inc.) and means were separated using the Tukey’s Studentized test. Damage ratings were analyzed using non-parametric measures and means were separated by comparing two species using Kruskal-Wallis. Correlation between tissue damage, Fv/Fm, and the mean date of budbreak were identified using PROC CORR (SAS Inc.). Results Budbreak The date of budbreak varied with planting location (p<0.0001), species (<0.0001), and year (p<0.03) (Table 4). Budbreak began in mid to late April in both years, lasting between 29-49 days in 2004 and 36-49 days in 2005. Trees at southern sites began and finished breaking bud earlier than northern sites. All trees had broken bud by 17 June 41 2004 and 9 June 2005. In both years, A. nephrolepis, A. bifolia, A. holophylla, and A. lasiocarpa were among the first to break bud at all four locations, while A. veitchii and A. homolepis were two of the last species to break bud at all four locations. The GDD required for budbreak differed among species (p50.0001), locations (p50.0001), and years (p50.04). Trees at the southern locations accumulated GDD faster (Figure 2) and required more GDD for budbreak than at northern locations (Table 5). GDD accumulation was initially slower in 2005 but by early June GDD accumulation was nearly equal to 2004. Fewer GDD were required at NWHRS for budbreak than at the other locations. Budbreak was ranked among species at each location and a strong location: location correlation existed (Table 6) suggesting budbreak among species was generally related at each location. Location x species interaction for both days to budbreak and GDD was significant (p50.0001), indicating the rank order of some species changed among locations. For example, A. koreana was in the last group to break bud at CHES, HTRC, and NWHRS while being one of the first species to break bud at KRF. In A. koreana x veitchii and A. fraseri x homolepis, budbreak was not closely related to the parent species, while budbreak for A. koreana x balsamea was similar to its parents’. Mean days to budbreak at the HTRC were correlated (R2 = 0.38, p=0.033) with average PV/Fm at -44°C (Figure 3). Trees breaking bud earlier had higher Fv/Fm values than trees breaking bud later. Abies veitchii had the lowest F v/F m of the species included in the cold hardiness study. Late Frost damage 42 Late frost damage following the May 2004 freeze was related to the date of budbreak. Trees breaking bud early displayed more damage from late spring frosts than those breaking bud later. At KRF, A. nephrolepis, A. bifolia, A. holophylla, and A. lasiocarpa displayed a high percent of frost damage in a large number of trees (Figure 4). In contrast, A. procera, A. koreana, A. chensiensis, A. nordmanniana ssp. equi-trojani, A. fiaseri x homolepis displayed no frost damage. At the time of frost, the following species had not completed budbreak: A. fiaseri x homolepis, A. koreana, A. koreana x balsamea, A. homolepis, A. procera, and A. veitchii. In both A. homolepis and A. veitchii late fi'ost damage occurred to some trees not included in the budbreak survey but surveyed for late frost damage. Cold Hardiness Cold hardiness varied among species and by test date. Chlorophyll fluorescence values differed at different temperatures (p50.001). F vIFm values decreased as temperatures were lowered during controlled freeze tests in A. chensiensis and A. veitchii but remained constant in A. balsamea var. phanerolepis and A. nephrolepis as temperatures reached -44 °C (Table 7). Species differed for tests during December 2004 (p50.002) and January 2005 (p50.001), but not March (p=0.10). Needle damage differed among species at -44 °C in all tests (p50.05) (Table 8). Needle damage was greatest in A. chensiensis during all tests. Damage to stem tissue and buds was not significant among species during any tests. Damage to needles, stem tissue, and buds were highly correlated (p<0.001) with declining in Fv/Fm values (Table 9). Discussion 43 In each species, the date of budbreak was similar in both years; however, GDD at the time of budbreak differed in both years for each species. Also, rank correlations between locations suggest budbreak is under strong genetic control which is supported by previous studies (Worral, 1983). Meeting a chilling requirement and accumulating a set number of GDD are necessary for trees to break bud (Howe et al., 2003). In both years, budbreak first occurred at southern locations in mid April and began at NWHRS in late April. The difference between the first and last species to break bud ranged between 29- and 49-days depending on location suggesting late bud breaking species such as A. koreana and A. veitchii require more GDD to break bud than species with earlier budbreak. Within species, some variation in budbreak occurred at different locations and was most prevalent in hybrids; although several species were influenced by unknown location factors. Abies balsamea is native to Michigan and can serve as a point of reference for the species included in this study. At all three transplanted locations, the following species broke bud following A. balsamea: A. procera; A. fraseri x homolepis; A. fraseri; A. chensiensis; A. nordmanniana ssp. equi-trojani; A. homolepis; A. koreana; and A. veitchii. In A. balsamea, a Michigan native, late spring frost damage is a problem (Lantagne and Koelling, 2004). Trees breaking bud afier A. balsamea showed no evidence of late frost damage with the exception of A. homolepis and A. veitchii. Damage in these two species was limited to trees located on top of a hill which received more thermal time than those on the slope or at the base of the hill. The mean number of GDD required to break bud was different (p30.0001) at each location. At the NWHRS, trees required fewer GDD to break bud than more southern 44 locations, implying that another factor in addition to thermal time may be influencing budbreak. There is some evidence that photoperiod has some influence on budbreak (Partanen et al., 1998); although its effect is debated (Worral, 1983). In 2004, daylength for trees at the NWHRS were nearly l-hour longer when the last tree broke bud than at KRF, which perhaps can explain the difference in the GDD required for budbreak. In P. menziesii var. menziesii (Mirb.) Franc. populations from regions with similar winter temperatures, trees from regions with the largest moisture deficit broke bud earlier than the average (Campbell and Sugano, 1979). This suggests that trees from regions frequented by summer drought break bud early in the spring to complete stem elongation and set bud before summer drought conditions begin (Kaya et al., 1994). Both A. koreana and A. veitchii, two of the last species to break bud in this study, are native to regions with increased summer precipitation (Farj on, 1990) so perhaps their late budbreak is an adaptive response to a mild climate and adequate summer precipitation. Within species, provenances can vary greatly in time of budbreak (Hansen and Larsen, 2004; Scholz and Stephen, 1982; Worral, 1983) and cold hardiness levels (Dolnicki and Kraj, 1998; Eiga and Sakai, 1984; Eiga and Sakai, 1987, Xie and Ying, 1993). One of the limitations of the current study is that provenance information for each species is unknown. Moreover, it is unknown if the parent trees of the hybrids were from the same provenance as the straight species included in this study. For example, the balsam parent of the Korean x Balsam hybrid is not necessarily from the same seed source as the straight balsam species included in this study, which could explain some of the inconsistencies in the budbreak between the parents and their hybrids. Also, variation within a species 45 is not accurately represented because each species is represented by a single provenance. Chlorophyll fluorescence was a good indicator of cold injury during controlled freeze tests as Fv/Fm values declined with increasing temperatures. These results paralleled increasing needle, stem, and bud damage, which is consistent with previous studies (Adams and Perkins, 1993; Binder and Fielder, 1996). Cold hardiness between different plant organs differs in the temperature at which damage occurs (Coleman et al., 1992; Sakai, 1982). The temperature where damage occurred was different for buds, stems, and needles. However, damage variables and Fv/Fm values were strongly correlated suggesting that while the temperatures that damage different organs may vary, relative cold hardiness is related. Many studies show differences in cold hardiness among species and provenances (Sakai, 1982; Xie and Ying, 1993; Eiga and Sakai, 1987; Eiga and Sakai, 1984). As expected, trees included in the cold hardiness study also varied in the temperature at which they displayed damage to freezing temperatures. One limitation of the current study was the inability of the freezer to be lowered beyond -44 °C. From a practical stand point, this is near to the lowest annual temperatures in the coldest regions of the upper Midwest. In most years, species showing no signs of damage should be able to survive most winters if given the necessary time to acclimate. The degree of cold hardiness in trees reaches a maximum during mid-winter and with a gradual acclimation and de-acclirnation period before and after respectively (Ritchie, 2003). Bud damage was the greatest in the March test. Some GDD accumulation had begun by then, internal development processes related to budbreak may 46 have begun, and warmer temperatures likely reduced cold hardiness levels leading to increased freeze damage. Needle damage ratings were the lowest in January. Typically January temperatures were the lowest so more conditioning lead to greater cold hardiness. Increased damage in December and March was likely due to incomplete acclimation and the start of de-acclirnation leading to less cold hardiness. Stem damage ratings were not different suggesting cambium tissue was adequately insulated at -44 °C, the lowest temperature possible in our controlled freeze test. F v/F m declined progressively between each test suggesting that repairs to cold damage did not begin until growth began again in the spring. Fv m at -44 °C differed significantly among species in December and January, but not March. In the March test, species breaking bud had the highest F‘,/Flm values suggesting they may have already begun recovery from cold damage that occurred during the winter. Other factors being equal, trees from colder regions are cold hardy at lower temperatures (Sakai, 1982) and break bud earlier in a common site, due to a reduced chilling and heat accumulation requirement, than trees from warmer regions. Worral (1983) suggests this may be an adaptation allowing trees to complete their growth before fall hosts in cold regions with short growing seasons. In the present study, date of budbreak and Fv/Fm readings at -44 °C were strongly correlated for the individual trees included in the cold hardiness experiment. Trees with maximum cold hardiness levels were among the first species to break bud in the spring while species with reduced cold hardiness were among the last, suggesting trade-offs between mid-winter cold hardiness and the timing of budbreak in the spring. Interestingly, A. chensiensis had the lowest 47 Fv/Fm while being the third of the four species in the study to break bud, but its subsequent cause is unknown. In summary, species varied in their tolerance of freezing temperatures and in the date they broke bud. Strong correlations existed between the temperatures different plant tissues showed visual signs of damage. Trees that were among the first to break bud in the spring withstood colder winter temperatures than trees breaking bud later. Species breaking bud early in the spring were more likely to be damaged by late spring frosts. Budbreak should continue as an important selection criterion for conifer species introduced to the landscape and Christmas tree industries in the upper Midwest. Species such as A. homolepis, A. koreana, and A. veitchii were among the last species to break bud at all locations and should be considered for future introduction. Additional studies should focus on larger provenance tests could be conducted to select provenances species such as A. bifolia, A. lasiocarpa, and A. nephrolepis which break bud later, yet display desirable ornamental characteristics. 48 83885, .2 23885 .2 5:33: a 3383 .V 328.83 a 8533 .v. 25»: 88> a annex 2.53m Ema—mm x 523— - - 23385. .2 finesse a 588 a 2.5? 982 a seem 5:30 Ho 2680 émflom 3» t CV OONN l Goa: sop—E3622 xm .35 Ho .muo:0m 8 - 8 88 - e8 32 .882 38.6 383%: .w e 223 5:88 8 - 8 88 - 8e 8e. .85 a... .85 misuse: .e. a 982 8 - 8 8: - o 2.2 .882 3333 .e. a 832 8 - 8 88 - 8.: a? .83 SSE .e. e 823. 8 - mm 88 - 88 a5. .82... 5> masseuse .w a seam 20V 883 ea 8:85 :35 28880 eaaz seesaw usaz eeaseo 8&332 S 80:32 .38 8 v8.33 83on 8.533 85 ._ 03m... 49 Table 2. Thirty-year climate summary and USDA plant hardiness zones for four Abies planting sites in Michigan. Location Average Average Annual Annual Growing USDA January July Precipitation Snowfall Season Hardiness Low High (cm) 1 (cm) ’ (days) 1 Zone2 (°C)‘ (°C)‘ HTRC -ll 28 78.5 99 150 5A NWHRS -10 27 85.1 244 135 5B CHES -10 28 90.7 145 147 5B KRF -9 28 89.4 135 149 5B 1 Illinois Dept. of Nat. Res., 2005. 2 USDA Plant Hardiness Map, 1990. Table 3. Soil properties of four Abies planting sites in Michigan. Location Sand (%)' Silt (%)l Clay (°/o)l Soil Type’ 2003 pH 2004 pH HTRC 83.1 8.7 9.3 Loamy Sand 6.25 5.72 NWHRS 83.3 7.6 9.1 Loamy Sand I 7.11 6.66 CHES 61.3 23.5 15.1 Sandy Loam 6.76 4.62 KRF 72.4 17.1 10.5 Sandy Loam 4.63 4.12 ' Analde at the MSU Soil and Plant Nutrition Laboratory, East Lansing, MI 50 .M2 8 38: Bob 8m 03288 203 $.88.— 02 88.3. .nodnd gauge 2.83288 8: 803 3:2 2:8 05 B 330:8 8538 5.23 382 o _ as: o 8 82 m 8 82 8 2 a: 8 8 us: o 2 a: E 8 8a: o 2 an: E82 .8 one 8 a: 28 2 32 we 8 82 8 t 2: see 8 82 one 8 52 E 8 as: o 2 2.2 388 .e. one 8 8: o8 : a: 228 2 82 88 2 8.2 one 8 a: 22 22 2 82 e 2 52 .888 .w on 8 82 8 8 a: 2o 8 .82 e 2 a: noose 8 e: o 2 8: Eu 2 a: o 2 8: finesse a one 2 52 one 2 a: 83 2 a: 8n 2 a: Bee 2 a: one 8 a: 82s 2 a: on 2 a: assuage .w one mm e82 when w 32 mono S ‘82 cups _ 82 one 2.. 282 one m ~32 five 3 CE 3 m_ .32 33.32.33 .98 298: .v. o 8 32 28 2 a: 28 2 82 88 o 82 88 2 82 on a 82 8.28 2 an: on 2 8: Eudora .w one 2 8: 82. a 8: 83 a .32 88 a 82 82. 8 a: one 8 a: mean a a: B 2 a: c.8228: a rose .8 3 mm 82 one N 82 mm on ‘82 tons o 82 o v 0:2. one m .32 _ em 82 one m an: 35:23 x 3833 .V 22 88 e a: 83 a a: one 8 =5. 22 22 88 m 82 so 8 8: sausages: .w one 2 a: one 8 8% 8e 8 a: 88 8 8%. 258 2 8: 22 82. m a: one 8 £2 38888 as Son .8 one 2 a: 8 8 Ear. Be 8 82 Bee 8 a: 8er 2 as: 8 8 Ear. 88 m a: one 8 a: .882 a 833 a one 2 an: . one 8 =5. one N as: ea 8 83 an 2 as: 22 one _ a: 8n 8 8.2 8588 .e. e 8 =5. e 8 Ear. no 8 Ex? 88 8 .23 one a: e 8 :23 an 8 ES 8 8 =.a< 8823 .w an m 82 a 8 E? a 8 E? e 8 =5. 8 e as: a 8 =.a< a 8 E? a 8 :22 8.238 .8 ea _ a: a 8 =5. e 8 E2 a 2 =5. e a 82 a 8 =8< ea 8 8%. a 8 E? 388 e. a 8 =2? e 8 8%. e 8 E? a 8 =.a< a a a: e 8 8%. Ba 8 =5. e 8 :5. 3838: .w 9532 a 0.2.: 850 2:32 .22 02...: 82:0 seam 88 88 .88 e5 88 5 e822: a 888. so. a seam sea... 82.. 2 co 3% 8285 .a 2.3 51 .M Z mm 83m: 808 .8 03282. 3.88.. oz .36 Nd 4:88.86 3.8888... 8.. 9.03 .88. 2:8 08 3 330:8 8828 55.3 88.2 o 88 o 8.. o .8 o 88 o 88 .. 88 8.. 8m 8 .8 88.8.. .8 Q8 8. 08 .8 8 o. m 2. 8 8 .8 8 m8 8 8.“ 8 :8 888 .V 8 8. 98 88 8.. .8 08 88 08 88 8,. 08 88 8.. .8 .888 .w 08 a8 2. 88 08 8.8 o 88 98 8. .. 8m 8.. W8 88 :m 8.8822. .w 8 8. 08 .8 88 a8 8 8 08 8. 8 ..8 8.. W8 88 88 8.8.88 .w 2. .8 08 .8 8.. 88 98 88 8 8. 8 :8 8.. 28 88.. 88 8888 .w 8 8. 8 .8 8 m8 08 8.8 8 .8 8 98 28 88 88.. 88 8.8852. x .888 .w 08 88 8 .8 88 .8 08 8. 08 z 8 8 .8 “.8 o. m 88.. .8 8808.58 88 .88 .w 8 8... a .... 8 8. a ..2 8 3. «z a S. 888 88 888.8 .w 08 88 a 88 8 o. m 08 .28 o 88 8 888 8 88 888 m8 828.8 x 888.. .w 08 .m. a E 8 8. 08 a. 8 8. a 8. 8 8. 88 8. 88.8; x 888 .w 08 8. a .8 8 8. 8 8. 08 R. «2 8 8. 88 8. 8.8.8888. 8., 8.. .8 «z 8 88 8 88 a 8. 82 82 8 88 88 8.: 838.888 8 a 2. .8. 8 8. 8 8. 8 o: 2.2 a 8. 08 8. 388.8. .w a 8. a 8. a a... 8 m2 8 8 a 82 a 2. 8 8. 8888.. .w a 8. m 8. 8 8. 8 m2 8 8 a 82 a 8. 8 2. 888 .w a 8.: 88. a 8. 8 m2 8 z. 2.2 28. 3... ”.8838: .w 3832 88. BE. 8...“. 98.32 80. SE. 8:0 8.8% 88 88 .38 8.3 vocm 5 5.35:2 E «.5882 .38 8 559% 3.8% .838 S 5 x8583 9.83 89:53.. 93.. oouwou maria» 5.22 .m 28F 52 Table 6. Pearson correlation coefficients for budbreak of 17 Abies species at four locations in Michigan. KRF NWHRS HTRC CHRS 0.61*** 053*" 0.71*** KRF 058*" 0.60*** NWHRS 0.71*** *, ", “" p50.05, 0.01, and 0.0001 respectively Table 7. Mean F wIF m value of four Abies species following controlled freeze tests to -44 °C. - FV/Fm Species December" January“ March A. baI. var. phanerolepis 0.627 a 0.657 a 0.462 ns A. nephrolepis 0.611 a 0.654 a 0.705 ns A. veitchii 0.375 a 0.492 a 0.291 us A. chensiensis 0.022 b 0.071 b 0.304 ns ‘, ", "" p50.05, 0.01, and 0.0001 respectively and indicates significant species effects for controlled freeze test that month. Means within a column followed by the same letter were not significantly different, a = 0.05, Tukey. 53 Table 8. Mean needle damage ratings of four Abies species following controlled freeze tests to -44 °C. Needle Damage Species December“ J anuary“ March" A. bal. var. phanerolepis 1.0 a 0.0 a 0.3 ab A. nephrolepis 1.0 a 0.0 a 0.0 a A. veitchii 1.7 a 0.7 a 1.7 bc A. chensiensis 2.0 b 2.0 b 2.0 c *, ", *" p50.05, 0.01, and 0.0001 respectively and indicates significant species effects for controlled freeze test that month. Means within a column followed by the same letter were not significantly different, or = 0.05, Kruskal- Wallis. Table 9. Pearson’s correlation coefficient for winter damage in four Abies species growing in Michigan in March 2005 following controlled freeze test. Needle Damage Stem Damage Bud Damage Fv/Fm -0.60*** -0.69*** -0.46*** Needle Damage 0.77:" 0.65“” Stem Damage 0.68“" *, ", "" p50.05, 0.01, and 0.0001 respectively 54 Figure 1. Location of four Abies trials in Michigan. 1) Kellogg Research Forest (KRF), 2) Clarksville Horticulture Experiment Station (CHES), 3) Horticulture Teaching and Research Center (HTRC), 4) Northwest Michigan Horticultural Research Station (N WHRS). 55 800 - 600 - 4co- Growing Degree Days 2004 200 1 600 - Growing Degree Days 2005 b 8 200 ~ W'T VVVVVV "Tr‘I'I IIIII I '''''' l ''''' I '''''' I'Tfrrtr' 3/7 3/21 4/4 4/18 5/2 5/16 5/30 6/13 Date Figure 2. Growing degree day accumulation since January 1 in A) 2004 and B) 2005 at four Abies test plots in Michigan. 56 0.8 0.6 - 0 3. *5 0.4 ~ E u. 3 LL “’ o 2 ~ , 8’ ' O A. balsamea var. phaneroleprs '03 O A. chensiensis E v A. nephrolepis O V A. veitchii O 0.0 1 p = 0.0328 R2 = 0.38 110 115 120 125 130 135 140 145 Days to mean budbreak Figure 3. Comparison of average Fv/Fm at -44 °C and mean day to budbreak in four Abies species at HTRC in December 2004 and January and March 2005. 57 80 -. 0 -25% Bud Damage 1:] 25-50 % Bud Damage 8 60 - a) to E as D m § 40 - I- “6 E § 3; 20 ~ 0 "' |I l I ' I l I l y l I" '11 &OQ‘“,°‘O\O 0% \069 130% \ 9‘00 \0 00®9*°‘00\ 50¢:9: 00 O:°:\S\ ‘9“o\ :‘og (0&0 0“ WV" '<°° t» 9 o0 a We“ “*0 \ 40‘. F’ «00 «050 5 v7" ,\«° . r t 1" 9° Species Figure 4. Frost damage to recently emerged shoots at the Kellogg Research Forest, 5 May 2004. 58 Literature Cited Adams, G.T. and TD. Perkins. 1993. Assessing cold tolerance in Picea using chlorophyll fluorescence. Environ Exp. Bot. 33(3): 377-3 82. American Standards for Nursery Stock. 2004. Amer. Nurs. Land. Asso. Washington, DC. Binder, W.D. and P. Fielder. 1996. Chlorophyll fluorescence as an indicator of frost hardiness in white spruce seedlings from different latitudes. New For. 11: 233-253. Bjorkman, O. and B. Demming. 1987. Photo yield of 02 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta. 170: 489-504. Campbell, R.K. and Al. Sugano. 1979. Genecology of bud-burst phenology in Douglas- fir: response to flushing temperature and chilling. Bot. Gaz. 140(2): 223-231. Coleman, M.D., T.M. Hinckley, G. McNaughton, and B.A. Smit. 1992. Root cold hardiness and native distribution of subalpine conifers. Can. J. For. Res. 22: 932-938. Cregg, B.M., M.W. Duck, C.M., Rios, D.B. Rowe, and MR. Koelling. 2004. Chlorophyll fluorescence and needle chlorophyll concentration of fir (Abies sp.) seedlings in response to pH. Hort Sci. 39(5): 1121-1125. Dickson, R.L., G.B. Sweet, and ND. Mitchell. 2000. Predicting Pinus radiata female strobilus production for seed orchard site selection in New Zealand. For. Ecol. Mgt. 133: 197-215. Dolnicki, A. and W. Kraj. 1998. Dynamics of frost resistance in various provenances of Abies grandis Lindl. Acta Societatis Botanicormn Poloniae. 67(1): 51-58. Eiga, S. and A. Sakai. 1984. Altitudinal variation in freezing resistance of Saghalien fir (Abies sachalinensis). Can. J. Bot. 62: 156-160. Eiga, S. and A. Sakai. 1987. Regional variation in cold hardiness of Sakhalin fir (Abies sachalinensis Mast.) in Hokkaido, Japan. In Li (ed.). Plant cold hardiness. Liss, New York, NY. F arjon, A. 1990. Pinaceae drawings and descriptions of the genera Abies, Cedrus, Psudolarix, Keteleeria, Nothotsuga, T suga, Cathaya, Pseudotsuga, Larix, and Picea. Champaign, IL. Gordon-Kamm, W.J. 1980. Freezing tolerance of several conifers in a western Washington forest community. Western Washington Univ. Bellingham, MS. Thesis. 59 Guehl, J .M, G. Aussenac, J. Bouachrine, R. Zimmerman, J .M. Pennes, A. Ferhi, and P. Grieu. 1991. Sensitivity of leaf gas exchange to atmospheric drought, soil drought, and water-use efficiency in some Mediterranean Abies species. Can. J. For. Res. 21: 1507- l 5 1 5. Hadley, J .L. and WK. Smith. 1987. Influence of krummholz mat microclimate on needle physiology and survival. Oecologia. 73: 82-90. Hansatech Instruments Ltd. 1997. Operating instructions for Plant Efficiency Analyzer (PEA) advanced fluorescence analysis. Hansatech Instruments Ltd., Norfolk, UK. Hansen, J .K. and J. B. Larsen. 2004. European silver fir (Abies alba Mill.) provenances from Calabria, southern Italy: 15-year results from Danish provenance field trials. Eur. J. For. Res. 123: 127-138. Hansen, O.K., U.B. Nielsen, QM. Edvardsen, B. Skulason, and J. Skage. 2004. Nordic provenance trials with Abies lasiocarpa and Abies lasiocarpa var. arizonica: Three-year results. Scand. J. For. Res. 19: 112-126. Howe, G.T., S.N. Aitken, D.B. Neale, K.D., Jermstad, N.C. Wheeler, and T.H.H. Chen. 2003. From genotype to phenotype: unraveling the complexities of cold adaptation in forest trees. Can. J. Bot. 81: 1247-1266. Illinois Department of Natural Resources. 2005. Midwest Regional Climate Center. Champaign, IL. 20 June 2005. http://sisyphus.sws.uiuc.edu/index.html. Kaya, Z., W.T. Adams, and R. K. Campbell. 1994. Adaptive significance of intermittent shoot growth in Douglas-fir seedlings. Tree Physiol. 14: 1277-1289. Kohyarna, T. 1984. Regeneration and coexistence of two Abies species dominating subalpine forests in central Japan. Oecologia. 62:156-161. Langlet, O. 1963. Patterns and terms of intra-specific ecological variability. Nature. 200: 347-348. Lantagne, D. and M. Koelling. 2004. Tree planting in Michigan. MSU Extension Publication. http://forestry.msu.edu/extension/ExtDocs/treeplnt.htm Leege, T. 2002. An evaluation of exotic true firs in the inland northwest. Amer. Christmas Tree J. 46: 49-51. Liu, TS. 1971. A monograph of the genus Abies. Taipei, Taiwan. Maxwell, K. and G.N. Johnson. 2000. Chlorophyll fluorescence — a practical guide. J. Exp. Bot. 51: 659-668. 60 McCullough, D.G., S.A. Katovich, D.L. Mahr, D.D. Neumann, C.S. Sadof, M.J. Raupp. 1999. Biological control of insect pests in forested ecosystems: a manual for foresters, Christmas tree growers and landscapers. Mich. State Ext. Bull. E-2679. McCullough, D.G., S.A. Katovich, M.E. Ostry, and J. Cummings-Carlson. 1998. Christmas tree pest manual. 2Ind ed. Mich. State Ext. Bull. E-2676. Morgenstern, E.K. 1978. Range-wide genetic variation of black spruce. Can. J. For. Res. 8: 463-473. 0wston, P.W. and T.T. Kozlowski. 1981. Growth and cold hardiness of container grown Douglas-fir, noble fir, and Sitka spruce seedlings in simulated greenhouse regimes. Can. J. For. Res. 11:465-474. Partanen, J ., V. Koski, and H. Hanninen. 1998. Effects of photoperiod and temperature on the timing of bud burst in Norway spruce (Picea abies). Tree Physiol. 18: 811-816. Perry, TC. and W.C. Wu. 1960. Genetic variation in the winter chilling requirement for date of dormancy break for Acer rubrum. Ecology. 41(4): 790-794. Ritchie, G.A. 2003. Root physiology and phenology: the key to transplanting success. In: Riley L.E., R.K. Dumroese, and TD. Landis (tech. coords.). National proceedings: Forest and conservation nursery associations -2002. Ogden, UT: USDA For. Serv., Rocky Mountain research station. Proceedings RMRS-P-28: 98-104. Ritchie, G. and TD. Landis. 2005. Seedling quality tests: chlorophyll fluorescence. For. Nurs. Notes. Publ. R6-CP-TP-ll-04. Winter: 12-16. Sakai, A. 1982. Extraorgan freezing of primordial shoots of winter buds of Conifers, p. 199-209. In P.H. Li and A. Sakai (eds.). Plant cold hardiness and freezing stress mechanisms and crop implications v. 2. Academic Press, New York, NY. Scholz, F. and ER. Stephan. 1982. Growth and reaction to drought of 43 Abies grandis provenances in a greenhouse study. Silvae Genet. 31( 1): 27-35. Saxton, A.M. 1998. A macro for converting mean separation output to letter groupings in Proc Mixed. In Proc. 23rd SAS Users Group Intl., SAS Institute, Cary, NC, pp1243- 1246. Nashville, TN, March 22-25. Toivonen, P. and W. Vidaver. 1988. Variable chlorophyll a fluorescence and C02 uptake in water-stressed white spruce seedlings. Plant Physiol. 86: 744-748. USDA Plant Hardiness Zone Map. 1990. USDA Misc. Publ. 1475. Washington DC. van der Kamp, B.J. and J. Worrall. 1990. An unusual case of winter bud damage in British Columbia interior conifers. Can. J. For. Res. 20: 1640-1647. 61 Worrall, J. 1983. Temperature — bud-burst relationships in amabilis and subalpine fir provenance tests replicated at different elevations. Silvae Genet. 32(5-6): 203-209. Xie, CY. and CC. Ying. 1993. Geographic variation of grand fir (Abies grandis) in the Pacific coast region: 10-year results from a provenance trial. Can. J. For. Res. 23: 1065- 1072. Yordanov, I. 1992. Response of photosynthetic apparatus to temperature stress and molecular mechanisms of its adaptation. Photosynthetica. 26(4): 517-531. 62 CHAPTER TWO CHLOROPHYLL FLUORESCENCE, PHOTOSYNTHESIS, GROWTH, AND FOLIAR NUTRIENT CONCENTRATION OF ABIES IN RESPONSE TO SOIL pH 63 CHLOROPHYLL FLUORESCENCE, PHOTOSYNTHESIS, GROWTH, AND F OLIAR NUTRIENT CONCENTRATION OF ABIES IN RESPONSE TO SOIL pH. Additional index words. Abies, foliar nutrition, growth, photosynthesis, FV/Fm, soil chemistry Abstract We measured foliar nutrition, maximum photosynthesis (Am), variable chlorophyll fluorescence (Fv/Fm) and leader grth in 17 Abies Mill. species, hybrids, and varieties grown under field conditions at four locations in Michigan. Sites represented soil pH ranging from 3.5 to 7.6. Increased soil pH significantly reduced foliar nutrient concentrations of N, P, K, S, B, Zn, Mn, Fe, and Cu. Fv/F"n decreased with increasing pH and was correlated with foliar N, P, K, and Cu. Amax declined as soil pH increased, although, the response varied by species (p30.05). Both A. lasiocarpa and A. homolepis were more tolerant of higher soil pH soils than A. balsamea var. phanerolepis. F oliar N, P, and K were negatively correlated with soil pH, explaining photosynthetic limitations in high pH soils. Leader growth was more closely related to foliar N, P, and K concentrations than Am“. Tolerance of high pH soil is an important selection criterion for future Abies introductions to the landscape and Christmas tree industry in the upper Midwest. Introduction Adequate foliar nutrition is required for most plant physiological processes to function properly. Soil pH may influence the availability of several plant nutrients such as K, P, Fe, Mn, B, Zn (Lucas and Davis, 1961). Cregg et a1. (2004) observed decreased foliar chlorophyll concentrations and foliar nutrient concentrations in response to 64 elevated soil pH levels in several Abies species. However, some species were more tolerant of higher pH soil. Throughout the upper Midwest and Great Lakes region, soil pH levels are quite variable. Coniferous forests tend to be acidic while grassland regions are more alkaline. Therefore, tolerance of Abies to varying soil pH and nutrient availability is necessary for future plant introductions in the upper Midwest. Chlorophyll fluorescence is a rapid and non-destructive method to measure the efficiency by which photosystem II captures light and is often expressed as the ratio of variable fluorescence to maximum fluorescence (Fv/Fm) (Bjorkman and Demming, 1987). The effects of various environmental plant stresses on plant health have been identified using chlorophyll fluorescence (Toivonen and Vidaver, 1988; Adams and Perkins, 1993; Sun et al., 2001). In Abies, photosynthetic efficiency declined due to the development of nutrient deficiencies caused as soil pH increased, making chlorophyll fluorescence an important means to identify nutrient stress (Cregg et al., 2004). The genus Abies Mill. (true firs) contains 46 species (F arjon, 1990) and covers large elevational and latitudinal ranges (Liu, 1971), which increase the probability of genetic adaptation to diverse external factors (Langlet, 1963). In A. fiaseri, seedling growth is highest at soil pH of 4.2 to 4.5 (Bryan et al., 1989), while A. balsamea grows best on sites with soil pH of 6.5 and 7.0 (Bakuzis and Hansen, 1965). Cregg et al. (2004) showed A. lasiocarpa and A. veitchii were more tolerant of soils with higher soil pH than A. sachalinensis Mast, A. sibirica Ledeb., or A. borisii regis Mattf. Soil that is more acidic or more alkaline than preferred ranges is problematic as well. Extreme alkaline conditions resulted in lower N and Mn concentrations in several conifers (Mandre et al., 1999). Increasing soil pH led to needle chlorosis, nutrient 65 deficiencies, and tree decline in A. alba Mill. (Hiltbrunner and F liickiger, 1996). Needle chlorosis developed in A. nordmanniana grown in high pH soils (Khalil et al., 1989). Conversely, extremely low soil pH led to increased A] toxicity, interfered with Mg uptake, and reduced specific needle mass in A. firma (N akatani et al., 2004; Igawa et al., 1 997). Nutrient deficiencies affect different plant physiological processes. Magnesium deficiencies led to needle chlorosis, reduced biomass accumulation, and decreased photosynthetic output (Sun and Payn, 1999; Laing et al., 2000; Sun et al., 2001). Excess K uptake inhibited Mg translocation from roots to foliage (Sun and Payn, 1999). Decreased needle growth, bud frequency, photosynthetic output, and stomatal conductance resulted from N deficiencies (Chandler and Dale, 1995; Tan and Hogan, 1995; Hinsley et al., 2000; Tirnmer et al., 1977). Phosphorous deficiencies reduced carboxylation of Rubisco and electron transport resulting in decreased Amax in Pinus pinaster Ait. (Loustau et al., 1999). ' The goal of this project is to identify Abies species tolerant of different edaphic conditions in the upper Midwest, to promote increased plant diversity, and thus reduce disease problems and insect pressures. The objective of this paper is to 1) identify the influence of soil pH on important macro- and micro-nutrients, 2) investigate how soil pH and nutrient deficiencies impede physiological processes, and 3) understand how nutrient deficiencies and reduced physiological processes affect plant growth. Materials and Methods Site Locations 66 Over 1100 trees representing 38 species, hybrids, provenances, and varieties of Abies were part of a true fir species and provenance trial initiated at the Kellogg Research Forest (KRF), Augusta, Michigan, in early 1991. In the fall of 2002 and spring of 2003, 246 trees representing 21 species and hybrids (Table 1) were transplanted to three locations in Michigan: Clarksville Horticulture Experiment Station, Clarksville, MI (CHES); Horticulture Teaching and Research Center, East Lansing, MI (HTRC); and Northwest Michigan Horticulture Research Station, Traverse City, MI (N WMHRS) (Figure 1). These three locations along with the KRF represent different climate regions (Table 2) and soil environments (Table 3). Trees were dug and ball and burlapped as 60 to 76 cm root balls in accordance with American nursery standards (American Nursery and Landscape Association, 2004) In the fall of 2003, 57 additional trees were transplanted from the KRF to replace 37 trees that died due to transplant stress during the previous year and add three new species and varieties to the study. At least four trees of each species or hybrid were planted at each location with the exception of the HTRC where one to four more trees of each species were planted when available. Trees were planted in a complete randomized design at each location. Trees were planted at approximately 4.5 m intervals. Fertilization In spring 2004, sites were fertilized with 21-0-0 ammonium sulfate at a rate of approximately 133 g per tree, to lower soil pH by 0.5 and insure that N was not limiting. Fertilizer was applied at CHES on 29 April, HT RC on 22 April, and NWHRS on 11 May. On 7 July 2004, 46-0-0 urea was applied at CHES at a rate of 91 g per tree. Trees at KRF 67 were fertilized every fall with 21-0-0 at 85 g per tree. Trees exceeding 0.9 m received 28 g of fertilizer for each additional 0.3 m in height. Soil pH Test Four evenly spaced soil samples (15 cm depth) were collected approximately 30 cm fiom the stem of each tree at the three transplant sites and KRF. Samples for each tree were combined and stored in a polyethylene bag. In 2003, samples were taken from 13 Abies species at the CHES on 21 Oct, HTRC on 10 Oct, KRF on 7 Oct, and NWHRS on 16 Oct. In 2004, after fertilization, samples were taken from 17 Abies species at the following locations: CHES on 7 Oct; HTRC on 5 Oct; KRF on 10 Oct; NWHRS on 8 and 9 Oct. Soil pH was measured using an Orion Soil pH Meter, Model 410 (Orion Research Incorporated, Boston, MA). An equal volume of soil and reverse osmosis water were mixed. Soil pH readings were taken after 15 minutes. In 2003, four samples from each location were chosen at random and a soil texture analysis was conducted at the Michigan State University Soil and Plant Nutrition Laboratory (East Lansing, MI) (Table 3). Soil Nutrition Samples In 2004, four soil samples from each site were chosen at random from excess soil not used in soil pH tests for a full soil nutrient and cation exchange capacity analysis. Chlorophyll Fluorescence Chlorophyll fluorescence (F v/F m) was measured on three needles from every tree at all four locations using a portable chlorophyll fluorescence system (Plant Efficiency Analyzer, Hansatech Instruments Ltd., Norfolk, England). Readings were taken on sunny days when temperatures were above 16 °C. Needles were dark-acclimated for 15 min 68 using the manufacturer’s plastic/foam clips before measurements were recorded. In 2003, readings were taken fiom 13 Abies species at CHES on 13 Oct, HTRC on 8 Oct, KRF on 7 Oct, and NWHRS on 11 Oct. In 2004, 17 Abies species were measured at CHES on 7 Oct, HTRC on 6 Oct, KRF on 10 Oct, and NWHRS on 9 Oct. F oliar Nutrient Samples Foliar nutrient samples were taken from current year needles selected at random from each tree at all four locations. In 2003, samples were collected from 13 Abies species at CHES on 13 Oct, HTRC on 8 Oct, KRF on 7 Oct, and NWHRS on 11 Oct. In 2004, samples were collected from 17 Abies species at CHES on 7 Oct, HTRC on 5, 11, and 12 Oct, KRF on 10 Oct, and NWHRS on 8 Oct. Samples were stored in paper bags and oven-dried at 60 °C for four days. Needles were stripped from branches, ground in a coffee grinder, and sifted through a #40 sieve. In both years, samples were sent to Waters Agricultural Laboratories, Inc. (Camilla, GA) for a full foliar nutrient analysis. Gas Exchange 9 Gas exchange was measured on current years growth using a portable photosynthesis system (LI-6400, Li-Cor, Lincoln, NE) fitted with a conifer chamber (LI- 6400-05, Li-Cor). The maximum photosynthetic rate (Amax) was measured from sun- exposed shoots in the upper one-third of the tree canopy on sunny days with PPF D greater than 1500 umolm'Z-s'l. Block temperature of the conifer chamber was maintained near 25 °C. The flow of air into the chamber was 500 umol-s". The reference C02 concentration was slightly above ambient at 400 umol COz-mol". Gas exchange was measured at CHES on 29 June, 6 August, and 30 August 2004, HTRC on 23 June, 28 July, and 2 September 2004, KRF on 1 July, 1 August, and 10 69 September 2004, and NWHRS on 15 July, 5 August, and 4 September 2004. Immediately afier gas exchange measurements, shoots used for gas exchange measurements were removed. Samples were transported in a cooler with ice and then transferred to a cooler at 2.5 °C. Needle area was deterrrrined in two ways: 1) projected shoot area and 2) projected needle area. To determine projected shoot area, samples were passed through a leaf area meter (LI-3000, Li-Cor). Projected needle area was measured by removing all the needles on each shoot and scanning them at 200 dpi using an Epson Perfection 2450 scanner (Seiko Epson Corp., Long Beach, CA). Images were analyzed using WinSeedle (Regent Instruments, Inc., Quebec, Canada) to determine projected needle area. Maximum photosynthetic rates are expressed in terms of projected shoot area (PSAmx) and projected needle area (PNAmax). Growth The topmost shoot of each tree was considered the leader. Current year leader heights were measured at CHES, HTRC, and NWHRS following the completion of growth in September 2004. Trees at KRF were sheared while they were still growing, so no shoot growth measurements were taken on those trees. Statistical Analysis PROC MIXED (SAS Inc., Cary, NC) was used for analysis of variance for location, species, year effects, interactions, and means were separated using Tukey’s Studentized test. PROC CORR (SAS) was used to identify Pearson’s correlation coefficients between leader growth, photosynthesis, soil pH, Fv/Fm, and the following foliar elements: N, P, K, Mg, Ca, S, B, Zn, Mn, Fe, and Cu. Species differences in response to pH were tested by Analysis of Covariance using PROC GLM. 70 Results Mean soil pH levels differed significantly among location (p50.001) and years (1350.001) but not species (pSO.46). Soil pH levels decreased in 2004 following ammonium sulfate application. In both years, mean soil pH was highest at NWHRS and lowest at KRF. Photosynthetic efficiency (Fv/F m) declined across sites in 2004 (p50.02). Mean F v m values were highest at NWHRS and lowest at HTRC in both years. Foliar nutrient concentrations of P, K, Zn, Cu, B, and S concentrations were higher (p30.05) in 2004 than 2003 and nutrient concentrations were generally higher in 2004 than 2003, except for Mn. In 2003, 95% confidence intervals indicated P was lower than suggested optimal ranges (Rothstein and Lisuzzo, 2003), while K, Mg, and Fe concentrations were higher than optimal. In 2004, 95% confidence intervals were higher than optimal ranges for K, Mg, Ca, S, and Fe. Soil concentrations of 11 essential mineral plant nutrients varied among locations (Table 4). KRF had lower Ca and Mg than the three horticultural stations where the sites had a history of crop production and presumed lime application. Species X location effects existed for all variables (p50.05). Species differed for foliar N, P, K, Mg, Ca, 8, Fe, Zn, B, Cu, leader growth, Fv/Fm, and PNAmax, but not foliar Mn (p=0.12) (Table 5). Foliar N was lower at HTRC than at other sites and A. balsamea var. phanerolepis had higher P and Ca at KRF than at other locations (Figure 3). Generally, nutrient concentrations were highest in A. nephrolepis. Soil pH was negatively correlated with PSAmax, N, P, K, S, B, Zn, and Cu in both 2003 and 2004 (p50.05). In addition, soil pH was negatively correlated with Mn, (Pearson’s r=-0.l9, p=0.0015) Fe, (Pearson’s r =-0.17, p=0.003) and PNAmx (Pearson’s 71 r=-0.40, p50.0001) in 2004 (Table 6). With the exception of Mg, all nutrients correlated with pH were also correlated with PSAmax when data for 2003 and 2004 were combined. Similarly, all nutrients correlated with pH were also correlated with PSAmax, with the exceptions of Ca in 2003 and Mn in 2004. Net photosynthesis (PNAmax) varied by species and location (pS0.0001) (Table 7) and was positively correlated with N, P, K, Mg, Ca, 8, B, Zn, Fe, Cu, and leader growth. However, a species >< location interaction was significant (p=0.046) In addition, PNAmax declined with increasing soil pH (Figure 2). Analysis of covariance indicated a significant interaction between species and pH (p=0.003). At the upper quartile of soil pH (pH=6.3 8), PNA“W was significantly higher in A. lasiocarpa than A. balsamea var. phanerolepis but at the lower quartile of soil pH (pH=4.28) species were not significantly different. Increased soil pH reduced available N, P, and K, which resulted in reduced photosynthesis (Figure 4). Photosynthetic efficiency (Fv/F m) differed significantly by species, year, and location (p50.05). However, in 2004 a species >< location interaction was significant (p=0.03 7). Photosynthetic efficiency (Fv/Fm) was correlated with foliar N, Ca, S, and Zn in 2003 and foliar N, P, K, Mn, Cu, leader growth, and PSAmax were in 2004. Soil pH and Fv/Fm were not correlated in 2003 (p50.77) or 2004 (p30. 10). Increased concentrations of foliar N (R2 = 0.39, p50.0001) and K (R2 = 0.22, p30.0001) resulted in increased Fv m values when all species were combined (Figure 3). Fv/Fm values were highest in A. fiaseri, A. fi'aseri x homolepis, and A. koreana x veitchii and lowest in A. chensiensis. In both A. koreana x balsamea and A. koreana x veitchii, Fv/Fm values were closely related to their A. koreana parent. 72 Leader growth differed by species and location in 2004 (p_<_0.0001) and was positively correlated with foliar N, P, K, Ca, S, Fe, Cu, PNAmx, and Fv/Fm, and negatively correlated with soil pH and foliar Mn (p50.05). However, a species >< location interaction was significant (p=0.026). While foliar N (Pearson’s r = 0.46, p50.0001), P (Pearson’s r = 0.35, p30.0001), and K (Pearson’s r = 0.56, p_<_0.0001) were closely related to increased leader growth (Figure 5). Analysis of covariance indicated significant interaction between species and foliar K (p=0.046). At the upper quartile of foliar K (K=1.04), leader growth was significantly higher in A. koreana x balsamea and A. koreana x veitchii than A. nordmanniana ssp. equi-trojani but at the lower quartile of foliar K (K=0.59) A. koreana x balsamea and A. nordmanniana ssp equi-trojani are not different (Figure 5). Discussion Soil pH levels declined at NWHRS, HTRC, and CHES in response to ammonium sulfate application in 2004. Soil nutrient and plant foliar nutrient concentrations differed suggesting an additional factor influenced nutrient availability in the trees. In this case, increased soil pH reduced most foliar nutrient concentrations but its influence was strongest for foliar N, P, K, and Cu concentrations. Foliar nutrient concentrations have been negatively correlated with soil pH in previous studies (Cregg et al., 2004; Lucas and Davis, 1961). Mean foliar nutrient concentrations were in the range of adequate nutritional guidelines proposed for A. fiaseri (FRA) by Rothstein and Lisuzzo (2003). However, foliar K, Mg, and Fe concentrations were above optimal guidelines. In our study, Mg was not correlated with soil pH, which contrasts with previous findings (Cregg et al., 73 2004; Lucas and Davis, 1961). Before being transplanted, trees were grown at KRF in low pH soils and supplied with annual fertilization. As a result, some antecedent effects, such as high foliar Mg concentrations, may have existed in the two years following transplanting. Immobile nutrient concentrations such as Mn and Fe declined in 2004 and were not correlated with soil pH in 2003 but were in 2004, further supporting this explanation. Little relation between soil pH and Fv/Fm was evident either year, which contradicts previous findings in a greenhouse study (Cregg et al., 2004). In the present study, trees were grown under field conditions and influenced by many factors such as drought stress, nutrient availability, soil type, and temperature among others. Adequate foliar nutrient concentrations and antecedent effects may have impeded the interaction between soil pH and F "/F m. Increased foliar N coincided with F,,/Fm in all species until N was no longer limiting. When foliar N was greater than 2%, F v m increased little. Rothstein (2005) found initial N application resulted in increased F v/F m. In the same study though, additional N, did not increase F "/F m but did cause nutrient imbalances and deficiencies. In the present study, foliar P and K were correlated with F‘,/Fm in 2004 following ammonium sulfate application. Following N application, foliar P and B concentrations increased in A. procera (Fletcher et al., 1998). In A. balsamea, foliar Ca, Mg, Na, Fe, and Zn concentrations increased following N fertilization, however, foliar P, K, and Mn concentrations declined (Czapowskyj et al., 1980; Tirnmer and Stone, 1978). Photosynthetic rates generally declined with increasing soil pH, although, some species were more tolerant of increasing soil pH than others. Declines in PNAmax were 74 due to the influence of soil pH on foliar N, P, and K, although other elements were influential to a lesser extent. Surprisingly, A. balsamea var. phanerolepis was the least tolerant species to increasing soil pH. Previously, A. balsamea var. phanerolepis was typically considered tolerant of higher soil pH (Brown, 2000; Frank, 1990). In the fall of 2003, A. balsamea var. phanerolepis was transplanted and establishment effects may have reduced its photosynthetic output in 2004. A. lasiocarpa maintained a higher PNAnmm rate at elevated soil pH, while A. homolepis was more insensitive to increased soil pH. Photosynthetic efficiency was maintained in A. lasiocarpa while declining in other Abies species as soil pH increased (Cregg et al., 2004). Field observations indicate A. lasiocarpa is tolerant of many soil conditions and a wide range of soil pH levels (Alexander et al., 1990). Leader growth differed among species. In A. koreana x balsamea and A. koreana x veitchii, leader growth increased more with foliar K than in A. nordmanniana ssp. equi- trojani. Hybrid species had the greatest leader growth and is evidence of hybrid vigor, which has been reported in other Abies hybrids (Klaehn, and Winieski, 1962). Leader growth was strongly correlated with N, P, and K. Also, leader growth was more strongly correlated with several nutrients than with PNAmax. Leader growth is but one growth parameter and biomass is allocated to shoot, stem, root, and reproductive growth. Trees growing under nutrient deficiencies, allocate more biomass to roots than shoots (Kaakinen et al., 2004). Both root and shoot growth increased in A. amabilis (Dougl.) Forbes when N and P were applied at optimal levels than when N or P was limiting (Hawkins et al., 1998). The leaf area index (LAI) and biomass accumulation per LAI increased following either fertilizer, irrigation, or both in 75 Pinus taeda L. (Albuagh et a1, 1998; Albaugh et al., 2004). Schoettle (1994) suggests increased foliar biomass per shoot compensates for decreased photosynthesis in P. contorta (Dougl.) Loud. We found that when multiple foliar nutrient concentrations were optimum, tree health improved and leader growth increased. In summary, soil pH influenced nutrient availability. Photosynthetic efficiency as indicated by F vIF m was not related to soil pH, although, site factors and antecedent nutrient effects influenced these results. Photosynthetic response differed among species to increasing soil pH suggesting photosynthesis of A. balsamea and A. balsamea var. phanerolepis is especially sensitive to soil pH, while A. nephrolepis and A. lasiocarpa are less sensitive to soil pH. Negative relationships between foliar N, P, and K and soil pH explained photosynthetic limitations in high pH soils. In many nutrients, concentrations were influenced by soil pH suggesting declined photosynthetic production was a result of multiple nutrient limitations. Hybrids crosses had the greatest leader grth and showed signs of hybrid vigor. Leader growth was influenced more by foliar nutrient concentrations than by photosynthetic output. 76 .52 .55 032:: a: a; 3% use? <2 <2 <2 «33:85.5: .2 $8.8.» a 9883 .V 3:32 coco> x 523— <2 <2 m_m<\:aotoE< .2 32:53 a 9823 .V 2532 Sam—mm x 523— <2 <2 2m umseéec .V E 5:50 am - mm 82 - o mucosa .z .52 2:3 assuage a. a seem an - mm cocm - com. «74 .85..— ESB.» .V E nozo> mm - mm ooom - com £3. .Equ czafib “ongoing .V E 323 SE35 cm - mm comm - coo £2 doom :0 53m “.3326: .V E 832 3. - mm cc: .. o £3 Eta—2 SRESE .V E o632 om - mm cocm - ooc_ £m< ES» 3823 .V E :3qu mm .. mm cocm - comm £3 .302. 5> £33,339 .V E “SE 96 38:3 95 cargo—m :35 029580 332 Egomum 0:82 56:80 dawmnoaz E 80:82 SE «a 322: 86on 83V? 5.3 ._ 038. 77 Table 2. Thirty-year climate summary and USDA plant hardiness zones for four Abies planting sites in Michigan. Location Average Average Annual Annual Growing USDA January July Precipitation Snowfall Season Hardiness Low High (cm) 1 (cm) ’ (days) 1 Zone2 (°C)‘ (°C)‘ HTRC -ll 28 78.5 99 150 5A NWHRS -10 27 85.1 244 135 5B CHES -10 28 90.7 145 147 5B KRF -9 28 89.4 135 149 5B 1 Illinois Dept. of Nat. Res., 2005. 2 USDA Plant Hardiness Map, 1990. Table 3. 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Location of four Abies test plots in Michigan. 1) Kellogg Research Forest (KRF), 2) Clarksville Horticulture Experiment Station (CHES), 3) Horticulture Teaching and Research Center (HTRC), 4) Northwest Michigan Horticultural Research Station (N WHRS) 84 Foliar N (g-kg") Foliar Ca (g-kg") 8 8 3 - — -- - - A. koreana x veitchii — - - -v~ - — -- A. nephrolepis ‘——o—- A.V9it0hii _ . —o— - — A. bal. var. phanerolepis —o— A. lasiocarpa A . ........ o ....... A komana HTRC NWHRS CHES KRF Location HTRC NWHRS CHES KRF Location Figure 3. Relationship between average foliar A) N, B) P, C) Ca, and D) Mg in six Abies species grown at four locations in Michigan in 2004. 85 Foliar P (g-kg") Foliar Mg (g-kg") 12 1o « '79» “E 8 . 'N O o B 6 ‘ E 3: <2 4. ._ E . a\._\ V —---O-—~- A. bal. var phanerolepis R2 = 0.70. P = 00001 "x. 2 - ._ —o — A. chensiensis R2 = 0.34, p = 0.0105 -------- v~~~ A. homolepis R2 = 0.49, p = 0.0004 —v—— A. lasiocarpa R2 = 0.32, p = 0.0139 0 l I I l 3 4 5 6 7 8 Soil pH Figure 3. Relationship between PNAmax and soil pH in four Abies species grown at four locations in Michigan in 2004. 86 0.9 0.8 T 0.7 - 0.6 - FJFm 0.5 - 0.4 i 0.3 T O y = 0.44 + 0.10 in (x-8.89) y = FV/Fm, x = N, R2 = 0.39, p=0.0001 0.2 5 10 15 20 25 30 35 Foliar N (g-kg-1) 0.9 0'8 l o ‘# yak o ,3. 0.7 - 0.6 - FJFm 0.5 - 0.4 - 0.3 - 0 y = 0.58 + 0.07 In (x-2.72) y = Fv/Fm, x = K, R2 = 0.21, p=0.0001 2 4 6 8 10 12 14 16 18 20 Foliar K (g-kg-1) 0.2 Figure 4. Relationships between photosynthetic efiiciency (Fv/Fm) and A) N and B) K in 17 Abies species grown at four locations in Michigan in 2004. 87 - -1 Foliar P (g-kg") Foliar N (g-kg ) Foliar K (g-kg") _l .55.: N OmeQO Soil pH Foliar K (g-kg“) Figure 5. Relationships between A) N, C) P, E) K, and soil pH and PNAmu and B) N, D) P, and F) K in 17 Abies species grown at four locations in Michigan in 2004. 88 3 Soil pH Foliar N (g-kg") B E . ° R’=0.12 .- R’=0.25 p = 0.0001 . . p = 0.0001 0 '0'? . 3 3 4 5 6 7 8 0 PNAM (pmol COZ-m'Z-s“) PNAmx (pmol COz-m'z-s") PNAmax (pmol COz-m'z-s'1) 80 —~-o.—--- A. kor. x bal. R2 = 0.77, p=0.0008 ------- OW A. kor. x veit. R2=0.36. p=0.0381 _ 4r. — A. homolepis R2=O.54, p=0.0008 60 . —v—— A. nord. ssp eq.-tr. R2=0.40. FPO-0496 .0 E 3 “E 9 40 1 (9 L- a) '0 m 3 20 " o q Foilar K (g-kg") Figure 6. Relationship between leader growth and fo liar K concentration in four Abies species grown at four locations in Michigan in 2004. 89 Literature Cited Adams, G.T. and TD. Perkins. 1993. Assessing cold tolerance in Picea using chlorophyll fluorescence. Environ. Exp. Bot. 33(3): 377-382. Albaugh, T.J., H.L. Allen, P.M. Dougherty, and K.H. JohnSen. 2004. Long term growth responses of loblolly pine to optimal nutrient and water resource availability. For. Ecol. Mgt. 192: 3-19. Albaugh, T.J., H.L. Allen, P.M. Dougherty, L.W. Kress, and J .S. King. 1998. Leaf area and above- and belowground growth responses of loblolly pine to nutrient and water additions. For. Sci. 44(2): 317-328. Alexander, R.R., R.C. Shearer, and W.D. Shepperd. 1990. Subalpine fir. In: R.M. Burns and B.H. Honkala (tech. cords). Silvics of North America: 1. Conifers. USDA For. Serv., Agr. Hdbk. 654. American Standards for Nursery Stock. 2004. Amer. Nurs. Land. Asso. Washington, DC. Bakuzis, E.V. and H.L. Hansen. 1965. Balsam fir—a monographic review. University of Minnesota Press, Minneapolis. 445p. Bj6rkman, O. and B. Demming. 1987. Photo yield of 02 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta. 170: 489-504. . Brown, J .H. 2000. Survival and growth of trees of a Canaan Valley, West Virginia, seed source in relation to varying soil/site conditions. Ohio State Univ. Ext. Spec. Circ. 175- 00. Bryan, J .A., J .R. Seiler, and RD. Wright. 1989. Influence of grth medium pH on the growth of container-grown fraser fir seedlings. J. Environ. Hort. 7(2) 62-64. Chandler, J.W. and J .E. Dale. 1995. Nitrogen deficiency and fertilization effects on needle growth and photosynthesis in Sitka spruce (Picea sitchensis). Tree Physiol. 15: 813-817. Cregg, B.M., M.W. Duck, C.M., Rios, D.B. Rowe, and MR. Koelling. 2004. Chlorophyll fluorescence and needle chlorophyll concentration of fir (A bies sp.) seedlings in response to pH. Hort Sci. 39(5): 1121-1125. Czapowskyj M.M., L.O. Safford, and RD. Briggs. 1980. Young nutrient status of young red spruce and balsam fir in a fertilized stand. USDA For. Ser. Res. Paper NE-467. 9O Farjon, A. 1990. Pinaceae drawings and descriptions of the genera Abies, Cedrus, Psudolarix, Keteleeria, Nothotsuga, Tsuga, Cathaya, Pseudotsuga, Larix, and Picea. Champaign, IL. Fletcher, R., C. Landgren, S. Webster, and M. Bondi. 1998. Fertilizing noble fir. Christmas Tree Lookout. 31(1): 21-13, 26-28. ' Frank, RM. 1990. Balsam fir. In: R.M. Burns and B.H. Honkala (tech. cords). Silvics of North America: 1. Conifers. USDA For. Serv., Agr. Hdbk. 654. Hansatech Instruments Ltd. 1997. Operating instructions for Plant Efficiency Analyzer (PEA) advanced fluorescence analysis. Hansatech Instruments Ltd., Norfolk, UK. Hawkins, B.J., G. Henry, and S.B.R. Kiiskila. 1998. Biomass and nutrient allocation in Douglas-fir and amabilis fir seedlings: influence of grth rate and nutrition. Tree. Physiol. 18: 803-810. Hiltbrunner, E. and W. Flilckiger. 1996. Manganese deficiency of silver fir trees (Abies alba) at a reforested site in the J ura mountains, Switzerland: aspects of cause and effect. Tree Physiol. 16:963-975. Hinesly, L.E., L.K. Snelling, C.R. Campbell, D.K. Roten, and J. Hartzog. 2000. Nitrogen increases fresh weight and retail value of fraser fir Christmas trees. HortScience. 35(3): 860-862. Hinesley, LE. and RD. Wright. 1989. Biomass and nutrient accumulation in Fraser-fir Christmas trees. HortScience. 24(2): 280-282. Igawa, M., H. Kameda, F. Maruyama, H. Okochi, and I. Otsuka. 1997. Effect of simulated acid fog on needles of fir seedlings. Environ. Exp. Bot. 38: 155-163. Illinois Department of Natural Resources. 2005. Midwest Regional Climate Center. Champaign, IL. 20 June 2005. http://sisyphus.sws.uiuc.edu/index.html. Kaakinen, S., A. Jolkkonen, S. Iivonen, and E. Vapaavuori. 2004. Growth, allocation and tissue chemistry of Picea abies seedlings affected by nutrient supply during the second growing season. Tree Physiol. 24: 707-719. Klaehn, F .U. and J .A. Winieski. 1962. Interspecific hybridization in the genus Abies. Silvae Genet. 11: 130-140. Khalil, N., C. Leyval, M. Bonneau, and B. Guillet. 1989. Influence du type de nutrition azotée sur le déclenchement de la chlorose du sapin de Nordmann (Abies nordmanniana, Spach, 1842) (Summary in English). Ann. Sci. For. 46: 325-343. 91 Laing, W., D. Greer, 0. Sun, P. Beets, A. Lowe, and T. Payn. 2000. Physiological impacts of Mg deficiency in Pinus radiata: growth and photosynthesis. New Phytol. 146: 47-57. Langlet, O. 1963. Patterns and terms of intra—specific ecological variability. Nature. 200: 347-348. ' Liu, TS. 1971. A monograph of the genus Abies. Taipei, Taiwan. Loustau, D., M. Ben Brahim, J .P. Gaudillére, and E. Dreyer. 1999. Photosynthetic responses to phosphorus nutrition in two-year-old maritime pine seedlings. Tree Physiol. 19: 707 -71 5. Lucas, RE. and J .F. Davis. 1961. Relationships between pH values of organic soils and availabilities of 12 Plant Nutrients. Soil Sci. 92:177-182. Mandre, M., J. K16§eiko, K. Ots, and L. Tuulmets. 1999. Changes in phytomass and nutrient partitioning in young conifers in extreme alkaline grth conditions. Environ. Poll. 105: 209-220. Nakatani, N., A. Kume, T. Kobayashi, T. Hirakawa, and H. Sakugawa. 2004. Needle morphology related to chemical contents in the needles of Japanese Fir (Abies firma) trees subjected to acidic depositions at Mt. Oyama, Eastern Japan. Water Air Soil Poll. 152: 97-110. Rothstein, DE. 2005. Nitrogen management in a fraser fir (Abies fraser [Pursh] Poir.) Christmas tree plantation: effects of fertilization on tree performance and nitrogen leaching. For. Sci. 51(2): 175-184. Rothstein, DE. and N. Lisuzzo. 2003. Optimal nutrition of fraser fir Christmas trees in Michigan, p. 19-21. In: D. Brown-Rytlewski and J. O’Donnell (eds.). Nursery, Landscape and Christmas Tree Research Projects and Educational Programs. Mich. State Univ. Ext., East Lansing, MI. Schoettle, A. 1994. Influence of tree size on shoot structure and physiology of Pinus contorta and Pinus aristata. Tree Physiol. 14: 105 5-1 068. Sun, O.J., G.J.H.P. Gielen, R. Sands, C.T. Smith, and A.J. Thorn. 2001. Growth, Mg nutrition and photosynthetic activity in Pins radiata: evidence that NaCl addition counteracts the impact of low Mg supply. Trees: Structure and Function. 15: 335-340. Sun, OJ. and T.W. Payn. 1999. Magnesium nutrition and photosynthesis in Pinus radiata: clonal variation and influence of potassium. Tree Physiol. 19: 535-540. Tan, W. and GD. Hogan. 1995. Limitations to net photosynthesis as affected by nitrogen status in jack pine (Pinus banksiana Lamb.) seedlings. J. Exp. Bot. 46: 407-413. 92 Tirnmer, V.R., E.L. Stone, and D.G. Embree. 1977. Growth response of young balsam fir fertilized with nitrogen, phosphorus, potassium, and lime. Can. J. For. Res. 7: 441- 446. Tirnmer, V.R. and EL. Stone. 1978. Comparative foliar analysis of young balsam fir fertilized with nitrogen, phosphorus, potassium, and lime. Soil Sci. Soc. Am. J. 42: 125- 1 30. Toivonen, P. and W. Vidaver. 1988. Variable chlorophyll a fluorescence and C02 uptake in water-stressed white spruce seedlings. Plant Physiol. 86: 744-748. USDA Plant Hardiness Zone Map. 1990. USDA Misc. Publ. 1475. Washington DC. 93 CHAPTER THREE NEEDLE MORPHOLOGY, SHOOT ARCHITECTURE, AND NET PHOTOSYNTHETIC RESPONSE IN ABIES SPECIES. 94 List of Abbreviations Abbreviation Name Unit of Measurement Amax Net Photosynthesis umol C02-m'2-s‘l gw Needle Conductance mol H20-m'2-s'l WUE Water Use Efficiency mol COz~mol HZO’l Ci Intercellular C02 umol COz A Carbon Isotope Discrimination per mil PSA Projected Shoot Area cm2 PNA Projected Needle Area cm2 TNA Total Needle Area cm2 PSAM Net Photosynthesis - Projected Shoot Area umol COz°m'2-s'l PNAmax Net Photosynthesis - Projected Needle Area mol C02-m'2-s'l TNAmx Net Photosynthesis - Total Needle Area 1111101 COz-m'2°s" CE Carboxylation Efficiency umol m'Z-s'l R Respiration umol COz-m'Z-s'l Incoming Radiation mol-s”l ¢ Quantum Efficiency umol CO;»,-p.mol'l Rd Dark Respiration umol COz-m'z-s'l k Convexity Coefficient Unitless STAR Shoot Silhouette Area : Total Needle Area Unitless PNA/TNA Projected Needle Area : Total Needle Area Unitless PSA/PNA Projected Shoot Area : Projected Needle Area Unitless 813C Stable Isotope Ratio Unitless 95 NEEDLE MORPHOLOGY, SHOOT ARCHITECTURE, AND NET PHOTOSYNTHETIC RESPONSE IN ABIES SPECIES. Addition index words: Net photosynthesis, carbon isotope discrimination, needle morphology, shoot architecture, water use efficiency, phot0synthetic light response curves, A/Ci curves, Abies. ‘ Abstract We measured gas exchange, carbon isotope discrimination (A), needle morphology, shoot architecture, photosynthetic light response curves, and NC, curves in 17 Abies species and hybrids grown at four locations in Michigan. Net photosynthesis (PSAmax, PNAmax, and TNAmax) varied among species and taxonomic subsections. Carbon isotope discrimination (A) differed among species and subsections, was negatively correlated with instantaneous water use efficiency, and was positively correlated with PSAmx, PNAmax, and TNAmx. Needle morphology and shoot architecture differed among species and subsections and was correlated with A. Photosynthetic light response and NC; curves indicate main species differences were in net photosynthesis and not apparent quantum efficiency, apparent carboxylation efficiency, or dark respiration. F oliar P and K were strongly correlated with increased PSAmax, PNAmax, and TNAmax from photosynthetic light response and A/Ci curves. Introduction Plant production is dependent on the energy produced during photosynthesis (Kfimer, 1991). Site conditions and morphological and physiological characteristics impact net photosynthesis (Amax) rates in conifers. Improved nutrition increased Amax rates and improved Rubisco capacity in Picea mariana Mill. B.S.P. (Paquin et al., 2000). Atmospheric and soil drought conditions reduce Amax although species respond 96 differently (Cregg, 1994; Silim et al., 2001; Cregg and Zhang, 2001). Increased light intensity affects dark respiration, quantum efficiency, and the light compensation point in A. alba Mill. (Grassi and Bagnaresi, 2001). In sun shoots, Amax rates were higher than shade shoots in A. amabilis Doug]. ex Forb. (Brooks et al., 1994). Photosynthetic gas exchange characteristics vary among conifer species. Net photosynthesis is closely related to stomatal conductance (gw) (Teskey et al., 1986; Cregg, 1994; Monson and Grant, 1989). Genotypes from dry climates frequently have fewer stomata than genotypes from climates with more precipitation (Knauf and Bilan, 1974: Cregg et al., 2000). Stomatal conductance decreases as water vapor pressure deficits increase (Guehl et al., 1991). Stomatal conductance and transpiration rate are closely related when compared to xylem potentials, but increased vapor pressure deficits can cause transpiration to increase while stomatal conductance decreases (Teskey et al., 1986). The relationship between Amax and gW is frequently used to describe the water use efficiency (WUE) between genotypes or environmental treatments (Silim et al., 2001; Cregg and Zhang, 2001). . Stomata conductance influences the internal C02 (Ci) levels inside the leaf (F arquahr and Sharkey, 1982). Air contains three carbon isotopes, of which 12C and 13 C represent 98.9% and 1.1% of atmospheric carbon respectively. C3 plants discriminate against '3 C, however, lower Ci levels reduce the plants ability to discriminate against isotopes (F arquhar et al., 1989). Increased WUE is frequently correlated with reduced carbon isotope discrimination (A) and used to quantify drought adaptation (F arquhar et al., 1989; Masle and Farquhar, 1988; Farquhar and Richards, 1984). 97 Periods of decreased gw from drought or pollutants are often correlated with decreased A (Guehl etal., 1991; Sakata and Suzuki, 2000). In Pinus sylvestris L., genotypes from drier regions had less discrimination than genotypes from wetter regions (Cregg and Zhang, 2001). Reduced A resulted in decreased Amax and dry matter accumulation in two Picea species (Silim et al., 2001). Conifers respond to drought conditions in several ways. Shoot intemode number and length were reduced under drought conditions in A. magnifica A. Murr. and A. concolor (Gord. and Glend.) Lindl. (Hallgren and Helms, 1988). In Switzerland, A. alba shed needles in response to drought conditions (Webster et al., 1996). To avoid drought conditions species such as A. cephalonica Loud. have optimized stomatal regulation of gas exchange while species like A. bornmulleriana Mattf. avoid internal water stress (Guehl et al., 1991). Needle morphology influences many physiological processes in conifers. Increased needle packing or needles per unit of shoot area can increase boundary layer effects and shoot temperatures (Martin et al., 1999 and Smith and Carter, 1988). More commonly, the relationship between needle morphology and light harvesting is investigated. Sun exposed shoots in A. lasiocarpa were more vertically oriented than shade shoots (Germino and Smith, 1999). In A. mariesii Mast. and A. veitchii, needle packing (needles-cm'l of shoot) was less dense in shoots from shade seedlings than fiorn sun seedlings (Mori and Takeda, 2004). This suggests acclimation to reduce self-shading occurs in sun shoots and to maximize light capture in shade shoots. Needle morphology characteristics in Pinus monticola (Dougl.) D. Don seedlings from the same genetic background varied when grown at different locations suggesting environmental factors 98 such as soil pH, precipitation, and temperature can affect needle morphology (Woo et al., 2002). Needle growth was reduced in trees growing on nutrient-poor sites (Ishii et al., 2003). Needle area of single needle conifers is frequently expressed as projected needle area (PNA), shoot silhouette area (SSA) or total needle area (TNA). Photosynthetic rates can vary by species depending upon the method used to express needle area (Ishii et al., 2003). PNA tends to over-estimate light interception by needles while SSA accounts for self-shading with-in shoots (Smith et al., 1991). Ratios such as STAR (the SSA to TNA ratio) and TNA/PNA are used to investigate shoot architecture (Stenberg et al., 1999: Ishii et al., 2003) and vary among Abies species (Figure 1). Ratios such as TNA/PNA are related to cross-sectional needle measurements such as needle thickness (Sellin, 2000). Shade shoots have increased STAR values (Stenberg, 1996). However, STAR values had little impact on photosynthetic rates in A. balsamea (Bemier et al., 2001). I Photosynthetic response to varying light intensities and C02 concentrations provides insight to physiological limitations and adaptations in conifers. Light saturation was lower in A. balsamea shade seedlings, while dark respiration, quantum efficiency, and the light compensation point increased with light intensity in A. alba Mill. (Landhfiusser and Lieffers, 2001; Grassi and Bagnaresi, 2001). In A. amabilis, Amam was higher in sun shoots than shade shoots (Brooks et al., 1994). A/Ci curves provide good ways to study stomatal limitations to photosynthesis (F arquahr and Sharkey, 1982). For example, in Picea abies, A/Ci curves show apparent carboxylation efficiency (CE) and 99 light respiration were higher in sun shoots than in shade shoots (Sprtova and Marek, 1 999). The genus Abies Mill. (true firs) contains 46 species (Farjon, 1990). However, research has been generally limited to a few species. Recent efforts to increase conifer diversity in the upper Midwest have created interest in additional Abies species. The following paper contains two parts. In experiment one we characterize gas exchange, needle morphology, shoot architecture, and carbon isotope discrimination in 17 Abies species and hybrids. In experiment two we characterize photosynthetic light response curves and A/Ci curves for a subset of species used in experiment one. Materials and Methods Site Locations Over 1100 trees representing 38 species, hybrids, provenances, and varieties of Abies were part of a true fir species and provenance trial initiated at the Kellogg Research Forest (KRF), Augusta, Michigan, in early 1991. In the fall of 2002 and spring of 2003, 246 trees representing 21 species and hybrids and seven taxonomic sections or subsections (Table l) were transplanted to three locations in Michigan: Clarksville Horticulture Experiment Station, Clarksville, MI (CHES); Horticulture Teaching and Research Center, East Lansing, MI (HTRC); and Northwest Michigan Horticulture Research Station, Traverse City, MI (NWMHRS) (Figure 2). These three locations along with the KRF represent different climate regions (Table 2) and soil environments (Table 3). Trees were dug and ball and burlapped as 60 to 76 cm root balls in accordance with American nursery standards (American Nursery and Landscape Association, 2004) 100 In the fall of 2003, 57 additional trees were transplanted from the KRF to replace 37 trees that died due to transplant stress during the previous year and add three new species and varieties to the study. At least four trees of each species or hybrid were planted at each location with the exception of the HTRC where one to four more trees of each species were planted when available. Trees were planted in a complete randomized design at each location. Trees were planted at approximately 4.5 m intervals. Fertilization In spring 2004, sites were fertilized with 21-0-0 ammonium sulfate at a rate of approximately 133 g per tree, to lower soil pH by 0.5 and insure that nitrogen was not limiting. Fertilizer was applied at CHES on 29 April, HTRC on 22 April, and NWHRS on 11 May. On 7 July 2004, 46-0-0 urea was applied at CHES at a rate of 91 g per tree. Trees at KRF were fertilized every fall with 21-0-0 at 85 g per tree. Trees exceeding 0.9 m received 28 g of fertilizer for each additional 0.3 m in height. E_xperiment One Needle Morphology and Shoot Architecture Three needles were collected at random from sun-exposed shoots in the upper one-third of the tree canopy twice during the growing season. Shoots were sampled at CHES on 29 June and 30 August 2004, HTRC on 23 June and 31 August 2004, KRF on 1 July and 1 September 2004, and NWHRS on 15 July and 3 September 2004. Needle length was measured using a digital caliper (Mitutoyo Corp., Aurora, IL). Free-hand cross-sections were cut from the center of each needle and photographed using a digital camera (Micropublisher 3.3, QImaging, Burnaby, BC) and software (Qcapture Suite, QImaging) under a lab microscope at 30x magnification and saved as a TIF image. 101 Images were hand traced using a pen tablet (Graphire3, Wacom Technology, Vancouver, WA) and cross-sectional area, needle width, thickness, perimeter, and shape factor (Figure 3) were measured with image analysis software (SigrnaScan Pro 5.0, SPSS Inc., Chicago). Shape factor is a determination of the roundness of a needle cross-section where a value of l is a circle and 0 is a flat line. The shape factor was calculated as: Shape factor = M2— (Sigma Scan Pro 5.0). perimeter Shoot architecture was examined for all trees. The ratio of shoot silhouette area to total needle area (STAR) was calculated as PSA/TN A. The ratio of TNA to PNA was also calculated. Gas Exchange Gas exchange was measured on current year growth using a portable IRGA (LI- 6400, Li-Cor, Lincoln, NE) fitted with a conifer chamber (LI-6400-05, Li-Cor). Sun- exposed shoots were measured in the upper one-third of the tree canopy on sunny days with PPF D greater than 1500 umol m'2 3". Block temperature of the conifer chamber was maintained near 25 °C. The flow of air into the chamber was 500 mol 3". The reference C02 concentration was slightly above ambient at 400 mol C02 mol". Shoots were sampled at CHES on 29 July 2003 and 29 June, 6 August, and 30 August 2004, HTRC on 25 July 2003 and 23 June, 28 July, and 2 September 2004, KRF on 1 July, 1 August, and 10 September 2004, and NWHRS on 30 July 2003 and 15 July, 5 August, and 4 September 2004. Shoots were harvested immediately after gas exchange measurements and transported in a cooler with ice and then stored at 2.5 °C until needle area was measured. Needle area was determined in three ways: 1) projected shoot area (PSA), 2) projected needle area (PNA), and 3) total needle area (TNA). To determine 102 PSA, samples were passed through a leaf area meter (LI-3000, Li-Cor). For determining PNA, all the needles on each shoot were removed and scanned at 200 dpi using an Epson Perfection 2450 scanner (Seiko Epson Corp., Long Beach, CA). Images were analyzed using WinSeedle Regent Instruments, Inc., Quebec, Canada) to determine PNA. TNA was determined by multiplying the projected needle area by the average perimeterzneedle width ratio of three needle cross-sections calculated using SigmaScan (SPSS Inc.). Maximum photosynthetic rates are expressed in terms of projected shoot area (PSAmax), projected needle area (PNAmax), and total needle area (TNAmax) (Figure 4). Soil Moisture Volumetric soil water content was measured using a portable TDR unit (Trase 1, Soil Moisture Equipment Corp., Santa Barbara, CA) on the days gas exchange was measured. Due to an equipment problem, volumetric soil moisture was not measured during gas exchange measurements in late August/ early September 2004. Water Use efliciency Intrinsic water use efficiency (WUE) was calculated from gas exchange data. WUE was expressed as the ratio of photosynthesis (Amu)vto needle conductance (g). Ratios were calculated from gas exchange measurements collected on the dates listed in the previous section. Carbon Isotope Discrimination Carbon isotope discrimination was measured in shoots from current year growth from select species at all four locations. Species were selected to represent a range of shoot morphologies, geographic ranges, and all species hybrids. Species sampled include: A. balsamea, A. lasiocarpa, A. fiaseri, A. fi'aseri x homolepis, A. koreana, A. 103 koreana x balsamea, A. koreana x veitchii, A. holophylla, A. nephrolepis, and A. veitchii. Shoots were harvested on the following days: CHES on 30 August 2004, HTRC on 2 September 2004, KRF on 1 September 2004, and NWHRS on 4 September 2004. Samples were oven-dried at 60 °C for four days. Needles were stripped from branches, ground in a coffee grinder, and sified through a #40 sieve. Between 2-3 mg of each sample were weighed into 5x9 mm tin capsules and placed in a plastic, 96-well sample tray. Relative abundance of ‘3 C and 12C was determined with a gas phase isotope ratio mass spectrometer at the Center for Stable Isotope Biogeochenristry, University of 13 California at Berkeley. The stable carbon isotope ratio (613C) was expressed as the Tz—C— ratio relative to PeeDee Belemnite (limestone) (Craig, 1957). The resulting 813C values were used to estimate isotope discrimination (A) as zq-g where 8,, is the isotopic composition of the plant material and 8, is that of the air (assumed to be -8%o, Farquhar et al., 1989). Experiment Two Photosynthetic Light Response and A/C; Curves Photosynthetic light response and A/Ci curves were measured in five Abies species and one hybrid: A. balsamea, A. bifolia, A. holophylla, A. koreana, A. koreana x balsamea, and A. nephrolepis. A/Ci curves were measured on the following days: CHES on 20, 21, and 22 September 2004, HTRC on 7, 8, and 9 September 2004, KRF on l8, l9, and 21 September 2004, and NWHRS on 4 and 5 September 2004. Light response was measured on the following days: CHES on 20, 21 , and 22 September 2004, HTRC 104 on 13, 14, and 15 September 2004, KRF on l7, l8, and 19 September 2004, and NWHRS on 11 and 12 September 2004. Trees were blocked by species to reduce time of day effects. Shoots were selected at random from sun-exposed shoots in the upper one-third of the tree canopy to be measured. Shoots were illuminated with two halogen light bulbs (Philips Halotone 50MR16/SP10, Phillips Electronics, New York, NY) on each side of the conifer chamber. Lights were powered by a 12V deep cycle marine battery. While measuring A/Ci curves, lights were repositioned to maintain PPFD near 2000 umol CO; 2'3’1 and monitored while measuring each tree. om. Gas exchange was measured at increasing C02 values of 0, 50, 125, 250, 400, 600, 900, and 1200 pmol COz-mofl by manipulating the C02 concentration flowing into the conifer chamber. Needle area was expressed as projected needle area and response curves were fitted to the follow equation = (CE*C1 *Amax)__R (CE*C,+AM) using Photosyn Assistant software (Dundee Scientific, Dundee, Scotland, UK) where A is needle photosynthesis, CE is the apparent carboxylation efficiency, C, is intercellular C02, R is respiration, and Amax is photosynthesis as the saturation of C02. Light response was measured at decreasing light intensities. Incoming light was filtered using a combination of mesh screens, black tinted plexiglass, and black plastic to reach pre-determined light intensities of 2000, 1500, 1000, 800, 600, 400, 250, 150, 100, 50, 20, and 0 umol-m'zs'l. Needle area was expressed as project needle area and response curves were fitted to the following equation _ ¢Q+A.... -\[(¢Q+ AMY —4¢QkA... — 2k - A Rd 105 using Photosyn Assistant (Dundee Scientific) where Q is the light level, ¢ is the apparent quantum efficiency, Amax is light saturated maximum photosynthesis, k is the convexity, Rd is dark respiration, and A is shoot photosynthesis (Prioul and Chartier, 1977). After determining light response values, Amax was estimated for each tree by calculating the light response equation at light intensities of 2000 umol m'2 5". Statistical Analysis Species effects for needle morphology, shoot architecture, gas exchange, and carbon isotope discrimination were determined using PROC MIXED (SAS Inc., Cary, NC). When significant differences were indicated, means were separated using Tukey’s Studentized test (Saxton, 1998). Correlation among needle morphology, gas exchange, shoot architecture ratios, and carbon isotope discrimination were identified using PROC CORR and linear regression was analyzed using PROC REG (SAS Inc.). Results Qtperiment One Net Photosynthesis When all species were combined, mean Amax differed among methods used to express needle area (p50.0001). Rates were 10.97, 7.25, and 3.06 mol COz-m'z-s'I for PSAmx, PNAmax, and TNA...am respectively. Photosynthetic rates varied widely among species and locations. Volumetric soil moisture content was lowest at HTRC and NWHRS (Table 4). Photosynthesis expressed using projected shoot area (PSAmax) varied by species and location during all dates (p50.0001). However, location X species interactions were significant (p30.03) during measurements in late July and early September 2004. When PSAmax was compared among taxonomic sections and sub- 106 sections, location and subsection were significant (p50.01), while location x subsection effects were not (Table 5). In late July, PSA"mm was highest in A. balsamea and A. balsamea var. phanerolepis at KRF, but among the lowest. of all species at the other three locations. Net photosynthesis (PSAmax) was consistently low in A. veitchii across all locations and times. Net photosynthesis (PSAmax) was generally greatest in subsection Laterales and section Nobilis and lowest in subsections Homolepides and Medianae in 2004. In 2003, PSA“...x was significantly (p30.0001) lower at HTRC than at CHES or HTRC. While PSAmax varied among locations during all measurements, location differences were less pronounced during later experiments. Net photosynthesis expressed using projected needle area (PNAmx) varied by location and species during all experiments (p50.0001) (Table 6). However, species X location interaction was significant (p50.001) in late July 2004. Again, PNAmax was highest in A. balsamea and A. balsamea var. phanerolepis at KRF in late July, while they were among the lowest at all other locations. Net photosynthesis (PNAmax) was higher in A. nordmanniana ssp. equi-trojani and lower in A. nordmanniana compared to other species at KRF in June 2004 than found at other locations. When PNAmax was considered by taxonomic sections and sub-sections, PNAmax differed among location and subsection (p50.01), however, a location X subsection effect was significant (p50.05) in late August and early September 2004 (Table 7). Net photosynthesis (PNAmax) was generally greatest in the section Nobilis and lowest in subsection Homolepides in 2004. Throughout 2004, PNAmax increased progressively with additional measurements at all locations with the exception of the NWHRS in late July. The greatest increase occurred at CHES between late July and early September 2004. 107 Both PNAmax and TNAmax were strongly correlated (Pearson’s r = 0.99, p50.0001) and species and location trends were similar throughout all experiments (Table 8). When TNA...ax was considered by taxonomic sections and sub-sections, TNAmx differed among location and subsection (p50.01) in June 2004 but only differed among locations in late August and early September 2004 (Table 9). Net photosynthesis (TNAmax) was greatest in subsections Laterales and Holophyllae and lowest in subsections Homolepides and Medianae. Water Use Efliciency and Carbon Isotope Discrimination Water use efficiency (WUE) differed (p50.01) among locations during all measurements. However, WUE did not vary by species, except for early September 2004 (pS0.0I), or subsection. In June 2004, WUE was greatest at HTRC. In A. procera, WUE was lower among all species at HTRC than at other locations during late July and early September 2004. Carbon isotope discrimination (A) varied among locations (p50.0001), species (p50.01) (Table 10), and subsection (p50.05) (Table 11). However, location >< species interaction (pS0.01) was significant. Mean A was greatest at KRF and least at HTRC and NWHRS. At HTRC, A in A. balsamea was less compared to other species than among other locations. Generally A was greatest in the hybrid crosses, although some variation by location occurred. Both WUE and A were closely related (R2 = 0.19, pS0.0001) (Figure 5). Needle Morphology Needle cross-sectional area, width, thickness, perimeter, and shape factor varied among species and location (p50.01) during experiments in late June and early September 2004 (Table 12). However, species >< location effects were significant for 108 needle thickness (p50.04) in September and shape factor (p_<,0.01) during both June and September. Needle morphology parameters varied among taxonomic section and subsection classification (p_<_0.001) in both late June and early September 2004 (Table 13). Cross-sectional area, needle width, and perimeter were generally the lowest at NWHRS. All needle morphology parameters increased during the growing season except needle width and perimeter. The increases were generally the greatest at CHES. Shape factor, needle thickness, and cross-sectional area were correlated with PSAmx, PNAW, and TNA...“ (p50.0001) but not needle width. Increasing needle thickness was related to higher PSA...ax (R2 = 0.11, pS0.0001), 1811.41,...x (R2 = 0.23, p50.0001), and TNAmax (R2 = 0.21, pS0.0001) (Figure 6). When species were combined, cross-sectional area, major diameter, minor diameter, and perimeter were strongly correlated. Shape factor was the only needle morphology parameter correlated with A (Pearson’s r = -0.l9, p_<_0.02). Show Architecture Shoot architecture ratios (PSA/TNA, TNA/PNA, and PSA/PNA) differed among species and locations (p50.0001) (Table 14). However, species >< location interactions were significant for PSA/TNA and TNA/PNA (p50.05) and for PNA/PSA (p50.0001) in early September. Shoot architecture ratios differed among taxonomic sections and subsections and locations (p50.05) in June and September 2004 (Table 15). Throughout the growing season PSA/TNA and TNA/PNA increased, while PNA/PSA remained constant. Both TNA/PNA and PSA/PNA were lowest at KRF. Carbon isotope discrimination (A) was negatively correlated with PNA/PSA (Pearson’s r = -0.28, p50.001). However, A increased with PSA/TNA (R2 = 0.10, p30.0001) (Figure 7). 109 E_xperimen_t Two Photosynthetic Light Response Location and species affected photosynthetic light response parameters. Dark respiration, PSAmax, and the LCP differed (p30.01) among species and location when PSA was used to express photosynthetic light response curves (Table 16). When TNA was used to express photosynthetic light response curves, TNAmax and LCP varied among species and location, while Rd and 0 did not (Table 17) (Figure 8). When curves were expressed using projected needle area, Rd, PNAmax, and the LCP varied among locations (pS0.0001) (Table 18). When using all three methods of expressing needle area, Rd was greatest at NWHRS and lowest at KRF. However, LCP was lowest at KRF and highest at NWHRS. Midday xylem potential during light response measurements was lowest at the NWHRS (-l .3 MPa) and highest at KRF (-0.9 MPa). Apparent 4) did not vary by location or species. Light response measurements indicate foliar P was correlated with PSAmax (Pearson’s r = 0.38, p50.0001), PNAmam (Pearson’s r = 0.48, p50.0001), and TNAmax (Pearson’s r = 0.43, p30.0001). Similar results existed for foliar K and net photosynthesis expressed using all three methods of expressing needle area. Carbon isotope discrimination (A) was correlated with PSAmax (Pearson’s r = 0.52, p50.0001), PNAmax (Pearson’s r = 0.58, p_<.0.0001), and TNAmax (Pearson’s r = 0.59, p50.0001). Dark respiration was negatively correlated with Mg and soil pH and positively correlated with Mn using all three methods of expressing needle area for photosynthetic light response curves. 110 When using PNA to generate curves, species differed in PNAmax (p50.0001) and light compensation point (p50.05). The LCP was lowest in A. nephrolepis and highest in A. balsamea. Abies koreana x balsamea had a lower LCPthan either of its parents, however, it was more closely related to A. koreana. Dark respiration, PNAmax, and light saturation were the highest at KRF, however, the light compensation point was also the lowest. Abies lasiocarpa had the highest PNAmax and A. balsamea and A. holophylla had the lowest. A/C; Curves In A/Ci experiments, apparent CE and Amax differed among species and location (p50.05) for all three methods of expressing needle area, while respiration differed among species only when using PSA (Figure 9). In A/Ci curves expressed using projected shoot area, R, apparent CE, and PSA,“ax differed (p50.05) among species, although, a locations X species effect was significant for PSAM (Table 19). When PNA was used to express curves, apparent CE and PNA.mm differed (p30.0’5) among species, while respiration did not. Respiration was negatively correlated with apparent CE (Pearson’s r = -0.52, p50.0001) (Table 20). Apparent CE was lowest in A. holophylla when all three methods of expressing needle are were used. When PNA was used to generate curves, apparent CE increased with PNA/PSA (R2 = 0.22, p50.0001). In A/Cg curves expressed using TNA, respiration, apparent CE, and TNAmax did not differ among species, although location effects were significant for apparent CE and TNAmax (1350.01) (Table 21). When all three methods of expressing needle area were used to generate A/C; curves, apparent CE was negatively correlated with foliar N (p50.05). Foliar P was 111 correlated with PSAmax (Pearson’s r = 0.23, pS0.0S), PNAmax (Pearson’s r = 0.26, p30.01), and TNAmax (Pearson’s r = 0.20, p50.05). Similar results occurred between foliar K and PSAmax (Pearson’s r = 0.24, p50.05), PNAmax (Pearson’s r = 0.32, p50.01), and TNAmax (Pearson’s r = 0.29, p50.05). Carbon isotope discrimination (A) was strongly correlated with PSAmax (Pearson’s r = 0.28, p50.01), PNAmax (Pearson’s r = 0.31, p_<_0.01), and TNAmax (Pearson’s r = 0.35, p50.01). Discussion Multiple nutrient limitations and soil moisture deficits led to lower PSAmax at the HTRC in 2003 (refer to chapter two). Although PSAmax, PNAmx, and TNAmax increased progressively during the growing season at all locations, the greatest increases were at CHES and resulted from additional N application in July. Nitrogen application increased photosynthesis, and gas exchange measurements, and chlorophyll content in several tree species (Chandler and Dale, 1995; Warren et al., 2004). Transplant effects in A. balsamea and A. balsamea var. phanerolepis may have contributed to species X location variation. Trees were transplanted from KRF in 2003 and Amax was higher at KRF than other locations when compared among all species. Net photosynthesis was strongly related to taxonomic relationships. Both Laterales and Medianae are two subsections of the section Balsaemea (F arj on, 1990), yet PSAmm PNAmx, and TNAmax differed between these subsections. Grouping species by section and subsection also reduced the frequency of significant location X species interaction. Water use efficiency was greatest at locations with sandy soils suggesting soil moisture was an influencing factor. Needle stomatal conductance declines before Amam or 112 as water becomes limiting, resulting in increased WUE (Cregg, 1994). In our study, WUE was closely related to A, and consistent with previous findings (Amdt et al., 2001; Cregg and Zhang, 2001; Farquhar and Richards, 1984; Silim et al., 2001). In hybrid crosses, WUE was lower and A was higher than in other species and subsections. In addition, hybrids had increased leader growth suggesting a trade-off between drought tolerance and increased foliar growth. Needle cross-sectional thickness, width, and shape factor increased between late June and early September indicating needles were still growing during the first measurement. Needle thickness and shape factor were greatest at CHES and HTRC. Both sites are not shaded during the day, as opposed to KRF and NWHRS which are shaded either in the morning or late aftemoon. Needle morphology parameters were closely related to section and subsection classification, which was expected given the importance of needle morphology to systematic classification (Liu, 1971 , F arjon, 1990). Needle width and thickness increased with increased light exposure in several conifer species (Stenberg et al., 1999; Sprugel et al., 1996). However, in the present study needle width was greatest at KRF. Needle thickness and shape factor increased at CHES following N application in July 2004. Needle thickness was correlated with increased N and P, while shape factor was correlated with foliar Mg and Ca, however N application increased both at CHES. We found net photosynthesis (PSAmax, PNAmax, and TNAmax) increased with needle thickness and shape factor. In conifers, needle thickness was related to needle dry mass to projected area and photosynthesis increased as needle thickness increased 113 (N iinemets, 1999). Needle mesophyll increased with needle thickness resulting in more area to harvest light (Lin et al., 2001; Sprugel et al., 1996). Carbon isotope discrimination (lower ‘35) increased with PSA/TNA among all trees in our study. Shoots with a higher PSA/TNA ratio had fewer needles-cm'l than shoots with a lower PSA/TN A ratio (Carter and Smith, 1985). Increased needle packing has resulted in boundary layer effects in conifers under calm conditions (Smith and Carter, 1988; Martin et al., 1999). Increased aerodynamic resistance resulting in boundary layer effects resulted in reducing transpiration more than photosynthesis and increasing WUE in A. lasiocarpa (Smith, 1980). Increased stand density in A. amabilis resulted in lower CO; concentrations and lower discrimination (more ’3 C) (Buchmann et al., 1998). Similar effects on a smaller scale may explain the increased A (less l38) as shoots became more vertically arranged. Dark respiration from photosynthetic light response measurements was greatest at NWHRS which also had the lowest midday water potential. Block effects were highly significant (p30.0001) suggesting that time of day influenced Rd. This is logical since respiration increases rapidly with temperature (Zha et al., 2001) and measurements were carried out throughout the day. Net photosynthesis was closely correlated with foliar P and K and was highest at KRF, which coincided with the most foliar P and K of the four trial sites. Foliar N was not as closely correlated with PSAmax, PNAmax, and TNAmax from photosynthetic light response measurements. This is presumably due to adequate foliar N from resulting from spring N fertilization. Net photosynthesis (PSAmax, PNAM, and TNAmax) in A balsamea was lowest at all locations except KRF. Reduced Amax in A. balsamea at other sites may be from pH intolerance (refer to chapter two) or from 114 transplanting stress. Species selected for photosynthetic light response measurements represent trees from diverse geographical origins and with varying needle morphology and shoot architecture. Yet, net photosynthesis and the light compensation point were the only parameters to consistently vary among species when comparing all three methods of expressing needle area. Net photosynthesis (PSAmax, PNAmax, and TNAmax) was greatest in A. lasiocarpa, A. koreana and A. nephrolepis when external C02 levels were increased to generate A-Ci curves. In both, needle shape factor was greater than the other four species included in the study. Low apparent CE corresponded with low PSAmax and PNAmax in A. holophylla, however the relationship varied among other species and locations. Similar to photosynthetic light response curves, foliar P and K were strongly correlated with PSAmax, PNAmaX, and TNAmax in NC; curves while foliar N was not correlated presumably to adequate foliar N from spring fertilization. In summary, net photosynthesis (PSAmax, PNAmx, and TNAmax) varied among species and taxonomic subsections. Instantaneous water use efficiency did not differ among species, however, carbon isotope discrimination (A) did differ among species and subsections. Strong relationships existed between PSAmax, PNAmx, TNAmax and A. Both needle morphology and needle architecture differed among species and subsection. Needle thickness was correlated with increased net photosynthesis. Differences in shoot architecture appear to influence boundary layer effects and increase carbon isotope discrimination. Photosynthetic light response and NC, curves differed among species, however main differences were to Am“. 115 .555. .55.:55: - - 0.5<\...50..0E< .Z 2...»... 530...? 5. 0.80.8. .V 2...»... :0..0> x 550.3. - - 5.5<\..00..0:.< .Z 2...»... 3.553 x 3.80.8. .V 2.5.5.. 8.55.55. 5. 5.0.3. - - 253550.085. .7. 2.5.5.. 5.3.0.053 x 208% .V 2.5.5.. 82.7. x .055... 55 - 55 5555 - 555. 555.555.55.552 55.5... Boxers? .555 555.585.5555.. .5 5.. 55.0.55 35 - o: comm - cc... 5505:3283. 5.0.2.... 55.555.555.588: .5. .... 56.0.07. we - mm comm - o 02.08.... .7. 50.0.0.3 nuggets. .V .... 0:.5..<.....m we - .v . comm - co. 00..0:.< .2 5.5.302 9.009% .V .... 0.5.07. mm - mm co.m - 8.. 52.08.... .2 0023.303. 205.5% .55 .m. .050... an - mm co... - c 52.0.95. .7. 50.50.53 SENS .V. .... V155.350 on .. mm com. - o 50..0:.< .7. 50.9.0.3 5.&0.o.0=§Q 55> 3033...: .V .... 55:50 on - am .55. - o 50..0:.< .Z 5.0.9.031. 550.5553: .5. .... 8.55.3. mm - mm cocm - com. 5.5.5. 0555530: 530.50.. .5 .... :0..0> mm - mm cocm - 85 5.5.5.. 0.5.8.8}. 5.30.9530: .55 .... 8...? 5.05% 55 - 55 5555 - 555 5.5.... 555.55.55.55. 5.8.5.555 .5. 5.. 8552 55 - 55 555. - 5 5.5.5. 5555.55.55. 5.35.5.5. .5 5.. 5.555... em - mm ccom - o8. 5.5.5.. 0.555.550: 6.80.3 .V .w. 5.0.3. 55 - 55 5555 - 5555 5.5.... 555558.55. 55.55.555.55 .5. 5.. .555. .:o..0055...m AZ. 0.53:... A5. :o..5>0.m. Ewto 0.....0.w000 Bozoom 08.57. 0.....:0.0m 0.557. 58:80 .:..m...0..>. :. 5:03.500. .30.. .5 3.55... 50.00% 503.55.? .5... .. 0.5.0... 116 Table 2. Thirty—year climate summary1 and USDA plant hardiness zones2 for four Abies planting sites in Michigan. For location descriptions see Figure 1. Location Average Average Annual Annual Growing USDA January July Precipitation Snowfall Season Hardiness Low High (cm) I (cm) 1 (days) 1 Zone2 (°C)‘ (°C)‘ HTRC -11 28 78.5 99 150 5A NWHRS -10 27 85.1 244 135 5B CHES -10 28 90.7 145 147 SB KRF -9 28 89.4 135 149 SB 1 Illinois Dept. of Nat. Res., 2005. 2 USDA Plant Hardiness Map, 1990. Table 3. Soil properties of four Abies planting sites in Michigan Location Sand (%) Silt (%) Clay (%) Soil Type 2003 pH 2004 pH HTRC 83.1 8.7 9.3 Loamy Sand 6.25 5.72 NWHRS 83.3 7.6 9.1 Loamy Sand 7.11 6.66 CHES 61.3 23.5 15.1 Sandy Loam ' 6.76 4.62 KRF 72.4 17.1 10.5 Sandy Loam 4.63 4.12 117 Table 4. Mean volumetric soil moisture content at four Abies test plots in 2003 and 2004 using a portable TDR device. % Moisture Location July 2003*" June 2004*" July 2004*" October 2004’" CHES 16.38 a 24.4 b ’ 15.36 b 10.15 c HTRC 7.19 b NA 12.76 c 6.26 d KRF NA 28.86 a 18.05 a 16.10 a NWHRS 16.38 a 11.19 c 6.78 d 12.73 b Means within the same column and followed by the same letter are not significantly different a = 0.05, Tukey. *, ”, *** pS0.05, 0.01, and 0.001 respectively Table 5. Net photosynthesis expressed using projected shoot area (PSAmx) of seven A bies subsections grown at four locations in Michigan in 2003 and 2004. Gas exchange was measured in late July 2003, and late June, July, and early September 2004. PSA"... (umol C02-m'2-s°') Subsection July 2003" June 2004*” July 2004" September 2004*” Laterales 9.23 a 12.72 a 12.04 ab 14.16 ab Homolepides 7.23 ab 7.78 c 11.50 ab 11.55 c Hybrid 7.00 b 11.8 ab 9.92 b ‘ 13.13 abc Medianae 6.24 b 9.55 be 10.82 ab 13.02 be Holophyllae 6.16 b 10.31 b 11.66 ab 12.00 c Nobih's NA 13.72 ab 13.59 a 16.34 a Abies NA 10.66 ab 11.20 ab 12.09 bc Location July 2003 June 2004" July 2004"" September 2004‘" CHES 9.93 12.76 a 13.13 a 16.06 a HTRC NA 9.75 b 11.80 a 12.58 b KRF NA 10.64b 11.65 a 12.71 b NWHRS 9.50 10.59 b 9.55 b 11.40 b Means within the same column and followed by the same letter are not significantly different a = 0.05, Tukey. *, ", “* p50.05, 0.01, and 0.001 respectively 118 Table 6. Mean photosynthetic rates expressed using projected needle area (PNAmx) of 17 A bies species grown at four locations in Michigan in 2004. Gas exchange was measured in late June 2004, late July 2004, and early September 2004. PNAm (umol COz'm'z-s'l) June July 2004" September 2004*" 2004 m Location Species CHES HTRC"* KRF NWHRS“I A. bal. var. 5.05 cdef 5.88 a 3.17 c 10.31 a 2.44 c 7.28 b phanerolepis A. balsamea 6.86 abcdef 6.21 a 3.44 be 7.99 a 3.58 be 7.39 b A. bifolia 9.21 a 8.12 a 6.82 a 8.78 a 9.89 a 9.55 a A. chensiensis 7.08 abcde 8.68 a 8.35 a 7.43 a 7.26 abc 9.10 ab A. fiaseri NA 6.44 a 6.51 abc 7.91 a 3.22 be 7.34 b A. fiaseri x 6.58 abcdef 7.88 a 8.02 a 5.99 a 6.23 abc 8.44 ab homolepis A. holophylla 7.08 abcd 8.65 a 7.16 a 9.31 a 5.49 abc 8.03 ab A. homolepis 4.56 ef 8.29 a 6.24 abc 7.06 a 5.43 abc 7.64 b A. koreana 4.77 def 8.62 a 5.34 abc 8.16 a 3.92 be 8.11 ab A. koreana x 6.15 bcdef 7.45 a 5.35 abc 7.27 a 5.35 abc 7.72 ab balsamea , A. koreana x 6.34 bcdef 6.60 a 7.86 a 7.23 a 4.74 abc 8.64 ab veitchii A. lasiocarpa 9.20 a 9.70 a 8.26 a 9.42 a 7.60 abc 9.27 ab A. nephrolepis 7.51 abc 6.95 a 6.03 abc 8.23 a 8.27 ab 9.15 ab A. nord ssp. equi- 6.94 abcdef 8.60 a 7.83 a 8.03 a 5.34 abc 8.19 ab trojani A. nordmanniana 8.75 ab 9.60 a 7.91 a 8.41 a 3.42 abc 7.71 ab A. procera 6.98 abcdef 10.29 a 9.44 a 8.16 a 6.24 abc 9.31 ab A. veitchii 4.46 f 6.52 a 6.28 abc 5.02 a 5.25 abc 7.29 b Means within the same column and followed by the same letter are not significantly different a = 0.05, Tukey. " Location x species was significant at p50.001. ‘3 "3 "* p50.05, 0.01, and 0.001 respectively 119 Table 7. Net photosynthesis expressed using projected needle area (PNAmx) of seven Abies sections and subsections grown at four locations in Michigan in 2004. Gas exchange was measured in late June, late July, and early September 2004. PNAM (umol C02-mas") Subsection June 2004*" ' July 2004“ September 2004" Laterales 7.73 a 7.10 ab 8.43 Homolepides 4.56 c 6.75 ab 7.64 Hybrid 6.37 ab 6.67 ab 8.26 Medianae 5.49 be 6.43 b 7.99 Holophyllae 7.07 a 7.73 a 8.56 Nobilis 6.98 ab 8.53 a 9.31 Abies 7.41 ab 7.48 ab 8.07 Location June 2004" July 2004‘" September 2004" CHES 7.35 a 8.33 a 10.36 HTRC 5.64 c 7.14 b 7.69 KRF 6.93 ab 7.86 ab 8.77 NWHRS 6.13 bc 5.65 c 6.47 Means within the same column and followed by the same letter are not significantly different on = 0.05, Tukey. ‘, ", "'” p50.05, 0.01, and 0.001 respectively " Sub-section and location were significant at p50.05, however Location x Sub-section effect was significant at p50.05. 120 262688. .83 .86 .8516 t. .: .. < Nod. ZSZF .vad. < species effect is significant at p500] 132 Table 20. Mean respiration (R), net photosynthetic rate expressed using projected needle area (PNAmax), and apparent carboxylation efficiency (CE) of six Abies species gown at four locations in Michigan is 2004. umol C02-m'2-s'l Species R PNAmax**"' CE*** A. koreana 3.39 43.5 ab 0.035 ab A. koreana x balsamea 3.65 36.3 ab 0.038 ab A. holophylla 3.63 39.2 ab 0.034 b A. nephrolepis 3.90 52.5 a 0.039 ab A. balsamea 3.99 33.5 b 0.036 ab A. bifoIia 3.97 44.7 ab 0.041 a Location R PNAmax*** CE*** CHRS 3.55 42.7 ab 0.031 b KRF 3.65 48.0 a 0.038 ab HTRC 3.76 36.0 b . 0.040 a NWHRS 4.06 39.7 ab 0.040 a Means within the same column and followed by the same letter are not significantly different a = 0.05, Tukey. t, in, our PEG-05’ 0.01, and 0.0001 respectively 133 Table 21. Mean respiration (R), net photosynthetic rate expressed using total needle area (TNAmax), and apparent carboxylation efficiency (CE) of six Abies species gown at four locations in Michigan is 2004. umol C02-m'2-s'l Species R TNAmax CE A. koreana 1.42 18.1 0.015 A. koreana x balsamea 1.53 15.0 0.016 A. holophylla 1.52 16.3 0.014 A. nephrolepis 1.57 20.3 0.016 A. balsamea 1.67 13.8 0.015 A. bifolia 1.56 17.6 0.016 Location R TNAmaxx CE" CHRS 1.45 17.2 0.013 b KRF 1.53 20.1 0.016 ab HTRC 1.50 13.6 . 0.016 a NWHRS 1.70 16.6 00.017 a Means within the same column and followed by the same letter are not significantly different a = 0.05, Tukey. ’, ", ’” [350.05, 0.01, and 0.0001 respectively " Significant at p50.05, however, location x species effect is significant at p50.01 134 a). 135 Figure 1. Two contrasting needle architecture arrangements. Lefi. Flat arrangement (A. veitchii). Right Bottlebrush needle arrangement (A. procer Figure 2. Location of four Abies trials in Michigan. 1) Kellogg Research Forest (KRF), 2) Clarksville Horticulture Experiment Station (CHES), 3) Horticulture Teaching and Research Center (HTRC), 4) Northwest Michigan Horticultural Research Station (N WHRS) 136 resin duct perimeter . thickness needle width Figure 3. Needle cross-section displaying maximum needle width (horizontal), maximum needle thickness (vertical), and perimeter measurements. Figure 4. Needle area measured as projected shoot area (left), projected needle area (center) when needles are plucked and scanned, and total needle area (right) when the perimeterzwidth ratio of cross-sections is multiplied by the projected needle area. 137 WUE (umol co, umol H20") 160 R2 = 0.19, p=0.0001 140 - 0 120 - 100 - 80- 60- 40 I I l I Figure 5. Relationship between water use efficiency and A in 10 Abies species gown at four locations in Michigan in 2004. 138 A 25. R2=0.11 . o 0 p=0.0001 0'. o PSA__ (umol cozcm'ls") PNAM (umol co,-m"-s") TNA", (umol coz-m'2-s") Needle Thickness (mm) Figure 6. Relationship between A) PSAmax, B) PNAmax, and C) TNAmax and needle width of 17 Abies species grown at four locations in Michigan in 2004. 139 24 R2 = 0.09 = ' o p 0.0003 . . 22 -« 20 . < 18 - 16 ~ 14 l T I T 0.1 0.2 0.3 0.4 PSA/T NA Figure 7. Relationship between the projected shoot to total needle area (PSA/TNA) ratio and carbon isotope discrimination (A). 140 12 A. holophylla //—-——6 9.. w . .E _.—::_:‘-:-"“"" ON 6 . ,. ~-~--—o— A. balsamea o x/ ——o— A. bifolia 6 4 4 . . ......... v ........ Akoreana g 2 -——v—-- A. kor.xbal. e? O. PNA,“m (umol COz-m'z-s") 7'2» ‘1‘ .5 N o O 6 E 3; Z l.- -1 r 1 . . r 0 500 1000 1 500 2000 PPFD Figure 8. Photosynthetic light response curves for six Abies species expressed using A) projected shoot area, B) projected needle area, and C) total needle area and gown at four locations in Michigan in 2004. 141 PSAM (umol COz-m‘z-s") PNAmaul (pmol COz-m‘z-s") 4j .. -—-- A. balsamea _ —e- -— A. bifolia ...... .....'..... A. koreana _——v—-- A. kor. xbal. - -—I— — A. holophylla . —°— A. nephrolepis TNAm (umol C02-m'2-s°') N I T 0 200 400 600 800 1000 1200 1400 co2 Figm'e 9. MG curves for six Abies species expressed using A) projected shoot area, B) projected needle area, and C) total needle area and gown at four locations in Michigan is 2004. 142 Literature Cited American Standards for Nursery Stock. 2004. Amer. Nurs. Land. Asso. Washington, DC. Arndt, S.K., S.C. Clifford, W. Wanek, H.G. Jones, and M. Popp. 2001. Physiological and morphological adaptations of the fi'uit tree Ziziphus rotundifolia in resonse to progessive drought stress. Tree Physiol. 21: 705-715. Bernier, P.Y., F. Rauleier, P. Stenberg, and C. Ung. 2001. Importance of needle age and shoot structure on canopy net photosynthesis of balsam fir (Abies balsamea): a spatially inexplicit modeling analysis. Tree Physiol. 21: 815-830. Brooks, J .R., T.M. Hinckley, and D.G. Sprugel. 1994. Acclimation responses of mature Abies amabilis sun foliage to shading. Oecologia. 100: 316-324. Buchmann, N., T.M. Hinckley, and J .R. Ehleringer. 1998. Carbon isotope dynamics in Abies amabilis stands in the Cascades. Can. J. For. Res. 28: 808-819. Carter, G. A. and WK. Smith. 1985. Influence of shoot structure on light interception and photosynthesis in conifers. Plant Physiol. 79: 103 8-1043. Chandler, J .W. and J .E. Dale. 1995. Nitrogen deficiency and fertilization effects on needle gowth and photosynthesis in Sitka spruce (Picea sitchensis). Tree Physiol. 15: 81 3-8 1 7. Crafis-Brandner, SJ. and ME. Salvucci. 2000. Rubisco activase constrains the phosynthetic potentialof leaves at high temperature and C02. Proc. Natl. Acad. Sci. USA. 97(24): 13430-13435. Craig, H. 1957. Isotopic standards for carbon and oxygen and correlation factors for mass-spectrometric analysis of carbon dioxide. Geochim. Cosmochim Acta, 12: 133- 149. Cregg, BM. 1994. Carbon allocation, gas exchange, and needle morphology of Pinus ponderosa genotypes known to differ in gowth and survival under imposed drought. Tree Physiol. 14: 883-898. Cregg, B.M., J .M. Olives-Garcia, and T.C. Hennessey. 2000. Provenance variation in carbon isotope discrimination of mature ponderosa pine trees at two locations in the Great Plains. Can. J. For. Res. 30: 428-439. Cregg, B.M. and J .W. Zhang. 2001. Physiology and morphology of Pinus sylvestris seedlings from diverse sources under cyclic drought stress. For. Ecol. Mngt. 154: 131- 139. 143 Faljon, A. 1990. Pinaceae drawings and descriptions of the genera Abies, Cedrus, Psudolarix, Keteleeria, Nothotsuga, T suga, Cathaya, Pseudotsuga, Larix, and Picea. Champaign, IL. Farquhar, G.D. and TD. Sharkey. 1982. Stomatal conductance and photosynthesis. Ann. Rev. Plant Physiol. 33: 317-345. ' Farquhar, G.D. and RA. Richards. 1984. Isotopic composition of plant carbon correlates with Water-use efficiency of wheat genotypes. Aust. J. Plant Physiol. 11: 539- 52. Farquhar, G.D., J .R. Ehleringer, and KT Hubick. 1989. Carbon isotope discrimination and photosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol., 40: 503-537. Germino, M.J. and WK. Smith. 1999. Sky exposure, crown architecture, and low- temperature photoinhibition in conifer seedlings at alpine treeline. Plant, Cell, and Envir. 22: 407-415. Pm‘rmffl Grassi, G. and U. Bagnaresi. 2001 . Foliar morphological and physiological plasticity in Picea abies and Abies alba saplings along a natural light gadient. Tree Physiol. 21: 959- 967. Guehl, J .M., G. Aussenac, J. Bouachrine, R. Zimmermann, J .M. Pennes, A. F erhi, and P. Grieu. 1991. Sensitivity of leaf gas exchange to atmospheric drought, soil drought, and water-use efficiency in some Mediterranean Abies species. Can. J. For. Res. 21: 1507- 1 5 1 5. - Hallgen, SW. and J .A. Helms. 1988. Control of height gowth components in seedlings of California red and whit fir by seed sources and water stress. Can. J. For. Res. 18: 521- 529. Ishii, H., M. Ooishi, Y. Maruyama, and T. Koike. 2003. Acclimation of shoot and needle morphology and photosynthesis of two Picea species to differences in soil nutrient availability. Tree Physiol. 23: 453-461. Knauf , TA. and M.V. Bilan. 1974. Needle variation in loblolly pine from mesic and xeric seed sources. For. Sci. 20(1): 88-90. Kéirner, C. 1991. Some often overlooked plant characteristics as determinants of plant gowth: a reconsideration. Funct. Ecol. 5: 162-173. Landhausser, S.M., and V.J. Lieffers. 2001. Photosynthesis and carbon allocation of six boreal tree species gown in understory and open conditions. Tree Physiol. 21: 243-250. Lin, J ., M.E. Jach, and R. Ceulemans. 2001. Stomatal density and needle anatomy of Scots pine (Pinus sylvestris) are affected by elevated C02. New Phytol. 150: 665-674. 144 Martin, T. A., T.M. Hinckley, F. C. Meinzer, and D.G. Sprugel. 1999. Boundary layer conductance, leaf temperature and transpiration of Abies amabilis branches. Tree Physiol. 19: 435-443. Masle, J. and GD. Farquhar. 1986. Effects of soil strength on the relation of water-use efficiency and gowth to carbon isotope discrimination in wheat seedlings. Plant Physiol. 86: 32-38. Monson, R.K. and M.C. Grant. 1989. Experimental studies of Ponderosa pine. III. Differences in photosynthesis, stomatal conductance, and water-use efficiency between two genetic lines. Amer. J. Bot. 76(7): 1041-1047. Mori, A. and H. Takeda. 2004. Functional relationships between crown morphology and within-crown characteristics of understory saplings of three codominant conifers in a subalpine forest in central Japan. Tree Physiol. 24: 661-670. Niinemets, U. 1999. Components of leaf dry mass per area - thickness and density — alter leaf photosynthetic capacity in reverse direction in woody plants. New. Phytol. 144: 35-47. Paquin, R., H.A. Margolis, R. Doucet, and MR. Coyea. 2000. Physiological response of black spruce layers and planted seedlings to nutrient addition. Tree Physiol. 20: 229- 237. Prioul J .L. and P. Chartier, 1977. Partitioning of transfer and carboxylation components of intracellular resistance to phosynthetic C02 fixation — Critical analysis of methods used. Ann. Bot. 41(174): 789-800. Sakata, M. and K. Suzuki. 2000. Evaluating possible causes for the decline of Japanese fir (Abiesfirma) forests based on 613 C records of annual gowth rings. Environ. Sci. Technol. 34: 373-376. Saxton, A.M. 1998. A macro for converting mean separation output to letter goupings in Proc Mixed. In Proc. 23rd SAS Users Group Intl., SAS Institute, Cary, NC, pp1243- 1246. Nashville, TN, March 22-25. Sellin, A. 2000. Estimating the needle area from geometric measurements: application of different calculation methods to Norway spruce. Trees. 14: 215-222. SignaScan Pro 5.0 Users guide. 1999. SPSS Inc. Chicago, IL. Silim, S.N., R.D. Guy, T.B. Patterson, N.J. Livingston. 2001. Oecologia. Plasticity in water-use efficiency of Picea sitchensis, P. glauca and their natural hybrids. 145 Smith, W. K. and G. A. Carter. 1988. Shoot structural effects on needle temperatures and photosynthesis in conifers. Amer. J. Bot. 75(4): 496-500. Smith, W.K., A.W. Schoettle, and M. Cui. 1991. Importance of the method of leaf measurement to the interpretation of gas exchange of complex shoots. Tree Physiol. 8: 121-127. ' Smith, W. K. 1980. Importance of Aerodynamic resistance to water use efficiency in three conifers under field conditions. Plant Physiol. 65: 132-135. Sprtova, M. and M.V. Marek. 1999. Response of photosynthesis to radiation and intercellular C02 concentrations in sun and shade shoots of Norways spruce. Photsynthetica. 37(3): 433-445. Sprugel, D.G., J .R. Brooks, and T.M. Hinckley. 1996. Effects of light on shoot geometry and needle morphology in Abies amabilis. Tree physio]. 16: 91-98. Stenberg, P., T. Kangas, H. Smolander, and S. Linder. 1999. Shoot structure, canopy openness, and light interception in Norway spruce. Plant, Cell, Environ. 22: 1133-1142. Stenberg, P. 1996. Simulations of the effects of shoot structure and orientation on vertical gradients in intercepted light by conifer canopies. Tree Physiol. 16: 99-108. Teskey, R.O., J .A. Fites, L.J. Samuelson, and BC. Bongarten. 1986. Stomatal nonstomatal limitations to net photosynthesis in Pinus taeda L. under different environmental conditions. Tree Physiol. 2: 131-142. Warren, C.R., N.J. Livingston, and DH. Turpin. 2004. Photosynthetic responses and N allocation in Douglas-fir needles following a brief pulse of nutrients. Tree Physiol. 24: 601 -608. Webster, R., A. Rigling, and L. Walthert. 1996. An analysis of crown conditions of Picea, Fagus and Abies in relation to environment in Switzerland. Forestry 69: 347-355. Woo, K., L. Fins, G.I. McDonald, D. L. Wenny, and A. Eramian. 2002. Effects of nursery environment on needle morphology of Pinus monticola Dougl. and implications for tree improvement progams. New For. 24: 113-129. Zha, T., A. Ryyppéi, K. Wang, and S. Kellomaki. 2001. Effects of elevated carbon dioxide concentration and temperature on needle gowth, respiration and carbohydrate status in field-gown Scots pines during the needle expansion period. Tree Physiol. 21: 1279-1287. 146 APPENDIX 147 Project Summary Cold Hardiness The date of budbreak and gowing degee days (GDD) necessary for budbreak differed by nearly a month among some species. Species native to colder regions such as A. lasiocarpa, A. bifolia, and A. nephrolepis were among the first to break bud when gown at a common site and were more prone to late frost damage. Early budbreak indicates a reduced GDD requirement in these species. Species such as A. koreana and A. veitchii were among the last to break bud when gown at a common site. Control fieeze tests show species with geater mid-winter cold hardiness require fewer GDD to break bud than less cold hardy species. Future tree improvement research should identify provenances exhibiting late budbreak yet maintaining adequate mid-winter cold hardiness in species possessing other desirable ornamental characteristics and environmental tolerances such as A. lasiocarpa and A. bifolia. Future research should further investigate the relationship between mid-winter cold hardiness and GDD required for budbreak. Nutrition Tolerance of high pH soils varied widely among the species included in this study. Species such as A. lasiocarpa, A. bifolia, and A. nephrolepis were more tolerant of high pH soils than species such as A. balsamea or A. balsamea var. phanerolepis. Overall, net photosynthesis and foliar N, P, and K declined in trees gown in high pH soils as multiple nutrient deficiencies developed. Adequate foliar N was maintained following N fertilizer application in the spring. However, foliar P and K levels were strongly correlated with photosynthetic output indicating these nutrients became most limiting when foliar N requirements were met. Leader gowth was geatest in the hybrid crosses, A. 148 nephrolepis, and A. homolepis. In all species, foliar N, P, and K were more important to leader gowth than net photosynthesis. Future tree improvement efforts should continue to include tolerance of high pH soils as a selection criterion. Needle Momhology, Shoot Architecture, and Gas Exchange Species with similar needle morphology (cross-sectional area, needle thickness, width, perimeter, and roundness), shoot architecture, and net photosynthesis shared similar taxonomic classification (sections and subsections). Net photosynthesis increased with needle thickness and roundness. Species with vertical shoot arrangements were less drought tolerant presumably due to reduced boundary layer effects. Instantaneous water use efficiency was negatively correlated with carbon isotope discrimination (A). Hybrids had more leader gowth than other species and subsections, yet were less drought tolerant (increased carbon isotope discrimination) suggesting trade-offs occur. Photosynthetic light response curves (light reactions) and A/Ci curves (dark reactions) were measured to further investigate differences in net photosynthesis among a subsample of five diverse species one hybrid. Curves differed depending on the method of expressing needle area, however, in all curves main differences were in net photosynthesis and not other curve parameters. Future tree improvement efforts should continue select species and provenances with increased drought tolerance. In addition, close relationships among species with similar taxonomic classification (subsections) should be used by plant collectors to identify species with potentially desirable characteristics for future introduction to the landscape industry. 149 Table 1. Analysis of variance of the date of budbreak and gowing degee days (GDD) accumulated at budbreak in 17 Abies species at four locations in Michigan in 2004 and 2005. - Date of Budbreak GDD Source df P Value F Value Species 16 7339*" 54.15"” Location 3 101.33*** 33.68183”: Year 1 5.41 ** 4.27* SprLocation 48 2.57*** 266*" SprYear 16 1.57 2.62" LocationXYear 3 435*" 7.51:" SprLoc> 5.0 A. balsamea A. balsamea var. phanerolepis A. homolepis Intolerant of soils with pH > 5.5 A. procera Intolerant of soils with DH > 6.0 A. koreana x balsamea A. koreana x veitchii A. lasiocarpa A. chensiensis Intolerant of soils with pH > 6.5 A. fraseri Tolerant of soils with pH < 6.5 A. bifolia A. nephrolepis 154 A F.J...AAt-un“ E Table 9. Mean photosynthetic rates expressed using total needle area (TNAmax) of 17 Abies species grown at four locations in Michigan in 2004. Gas exchange was measured in late June 2004 and early September 2004. TNAmax (umol C02-m'2-s'1) ‘ Time Species June 2004*" September 2004" A. lasiocarpa 3.76 abc 3.78 abc A. nordmanniana 3.73 ac 3.37 abcd A. bifolia 3.64 ac 3.72 abcd A. nephrolepis 3.09 abcd 3.87 ab A. chensiensis 3.02 abcd 3.82 a A. nord. ssp. equi-trojani 2.94 abcdef 3.46 abcd A. procera 2.91 abcde 3.80 ab A. fiaseri x homolepis 2.77 abcdef 3.51 abcd A. koreana x veitchii 2.65 defg 3.68 abcd A. koreana x balsamea 2.62 bdefg 3.17 abcd A. balsamea 2.53 defg 3.10 bed A. bal. var. phanerolepis 2.16 defg 2.98 d A. koreana 2.01 efg 3.45 abcd A. homolepis 1.98 fg 3.26 abcd A. veitchii 1.95 efg 3.12 bcd A. fi'aseri NA 3.06 bcd A. holophylla NA 3.32 abcd Means within the same column and followed by the same letter are not significantly different a = 0.05, Tukey. *, ", "" Significant at ps0.05, 0.01, or 0.0001, respectively. 155 Table 10. F-values of PSAmax ratio of 17 Abies species grown at four locations in Michigan in 2003 and 2004. July 2003 June 2004 July 2004 Sept 2004 Source df F-Value df F -Value F-Value F-Value Loc 2 175.30 *** 3 913*" 838*" 2528*" Species 13 6.78*** 16 5.71 *** 4.37*** 6.63*** SXL 22 1.50 48 1.15 1.82" 1.53“ *, **, "* p_<_0.05, 0.01, and 0.0001 respectively Table 11. F-values of PNAmax of 17 Abies species grown at four locations in Michigan in 2004. June 2004 July 2004 September 2004 Source df F -Value F-Value F-Value Location 3 9.07*** 2159*" 4390*" Species 16 7.36*** 492*" 357*” LXS 48 1.01 1.95M 1.37 *, **, *** p 0.05, 0.01 and 0.0001 respectively Table 12. F-values of TNA“mix of 17 Abies species grown at four locations in Michigan in 2004. June 2004 September 2004 Source df F-Value F-Value Location 3 1250*“ 4004*" Species 16 798*” 3.66" LXS 48 1.35 1.46 *, **, **"‘ p 0.05, 0.01 and 0.0001 respectively 156 Table 13. Analysis of variance for carbon isotope discrimination (A) and water use efficiency (WUE) of several Abies sub-sections grown at four locations in Michigan in 2004. A - WUE Source df F-Value df F-Value Location 3 6599*" 3 21.66"" Sub-section 3 2.97* 6 l .78 L X S 9 0.55 18 0.78 Means within the same column and followed by the same letter are not significantly different a = 0.05, Tukey. *, **, *** p 0.05, 0.01 and 0.0001 respectively ‘3! Fa" -_‘l’lfll&.~ Table 14. F-values of PSA/TNA ratio of 17 Abies species grown at four locations in Michigan in 2004. June 2004 September 2004 Source df F-Value F-Value Location 3 1381*" 1769*" Species 16 12.26 **"' 1769*“ LXS 48 1.47 * 1.66" *, **, *"’* p 0.05, 0.01 and 0.0001 respectively Table 15. F-values of TNA/PNA ratio of 17 Abies species grown at four locations in Michigan in 2004. June 2004 September 2004 Source df F -Value F-Value Location 3 725*" 3590*" Species 16 3788*" 2504*" LXS 48 1.53* 1.59* *, ”, *** p 0.05, 0.01 and 0.0001 respectively 157 Table 16. F-values of PNA/PSA ratio of 17 Abies species grown at four locations in Michigan in 2004. June 2004 July 2004 September 2004 Source df F -Value F -Value F—Value Location 3 832*“ 3.32:” 20.11...” Species 16 1521*" 964*" 17.43": L X S 48 1.41 1.46 2.43m” *, **, *** p 0.05, 0.01 and 0.0001 respectively M. Table 17. F-values of photosynthetic light response curve parameters [dark respiration (Rd), apparent quantum efficiency (4)), net photosynthesis expressed using projected shoot area (PSAmax), and the light compensation point (LCP)] of six Abies species grown at four locations in Michigan in 2004. Source df Rd 4, PSAmax LCP Block(loc) 16 326*" 2.10* 2.12* 2.35" Species 5 3.86” * 1.04 6.10* *‘l’ 4.46M Location 3 27.12*** 3.95“ 2554*" 10.27"” SXL 15 1.07 1.36 1.60 0.95 *, **, *** p=0.05, 0.01, and 0.0001 respectively 158 Table 18. F -values of photosynthetic light response curve parameters [dark respiration (Rd), apparent quantum efficiency (4)), net photosynthesis expressed using projected needle area (PNAmax), and the light compensation point (LCP)] of six Abies species grown at four locations in Michigan in 2004. Source df Rd 4, PNAM LCP Block(loc) 16 4.51* ** 1.77 2.76M 2.76" Species 5 1.87 2.24 8.76“ * 3.05"“ Location 3 25.98“” 0.93 2162*” 1207*“ SXL 15 1.40 1.74 1.36 0.99 *, **, "'** p=0.05, 0.01, and 0.0001 respectively Table 19. F-values of photosynthetic light response curve parameters [dark respiration (Rd), apparent quantum efficiency (4)), net photosynthesis expressed using total needle area (TNAmax), and the light compensation point (LCP)] of six Abies species grown at four locations in Michigan in 2004. Source df Rd 4) TNAmax LCP Block(loc) 16 4.59*** 1.82* 2.40“? 1122*" Species 5 1.53 1.60 7.79* ** 7.45" "' * Location 3 2554*" 0.66 2918*" 9.61 ”* SXL 15 1.00 1.81 1.52 1.45 ‘, **, *** p=0.05, 0.01, and 0.0001 respectively 159 Table 20. F-values of A/Ci curve parameters [respiration (R), apparent carboxylation efficiency (CE), and net photosynthesis expressed using projected shoot area (PSAm)] of six Abies species grown at four locations in Michigan in 2004. Source df R ‘ CE PSAmax Block(loc) 16 3.30" 2.14* 0.99 Species 5 688*” 1323*" 3.19“ Location 3 9.98*** 1468*" 3.00* SXL 15 0.91 0.67 2.25“ *, **, *** p=0.05, 0.01, and 0.0001 respectively Table 21. F-values of A/Ci curve parameters [respiration (R), apparent carboxylation efficiency (CE), and net photosynthesis expressed using projected needle area (PNAmax)] of six Abies species grown at four locations in Michigan in 2004. Source df R CE PNA.mm Block(loc) 16 2.04”“ 3.40"“ * 1 .07 Species 5 l .04 3.49* * 3 .03 * Location 3 1.20 4.26“ "‘ 2.75* SXL 15 1.40 1.38 1.79 *, **, *** p=0.05, 0.01, and 0.0001 respectively Table 22. F -va1ues of A/Ci curve parameters [respiration (R), apparent carboxylation efiiciency (CE), and net photosynthesis expressed using total needle area (TNAmax)] of six Abies species grown at four locations in Michigan in 2004. Source df R CE TNAm.ix Block(loc) 16 1 .95" 5.40* ** 0.66 Species 5 0.59 1.55 1.83 Location 3 1.52 4.18” 4.13" S>