AJB Advance Article published on September 23, 2013, as 10.3732/ajb.1300123. The latest version is at http://www.amjbot.org/cgi/doi/10.3732/ajb.1300123 American Journal of Botany 100(10): 000–000. 2013. SPECIAL INVITED PAPER BRANCH ARCHITECTURE IN GINKGO BILOBA: WOOD ANATOMY AND LONG SHOOT–SHORT SHOOT INTERACTIONS1 STEFAN A. LITTLE2,3,6, BROOKE JACOBS2, STEVEN J. MCKECHNIE2, RANESSA L. COOPER4, MICHAEL L. CHRISTIANSON5, AND JUDITH A. JERNSTEDT2 2Department of Plant Sciences, One Shields Avenue, University of California, Davis, California 95616, USA; 3Centre for Forest Biology, Department of Biology, University of Victoria, Victoria, British Columbia, V8W 3N5, Canada; 4Biology Department, Hillsdale College, 33 E. College St., Hillsdale, Michigan 49242, USA; and 5Department of Plant and Microbial Biology, University of California, Berkeley, California 94704, USA • Premise: Ginkgo, centrally placed in seed plant phylogeny, is considered important in many phylogenetic and evolutionary studies. Shoot dimorphism of Ginkgo has been long noted, but no work has yet been done to evaluate the relationships between overall branch architecture and wood ring characters, shoot growth, and environmental conditions. • Methods: Branches, sampled from similar canopy heights, were mapped with the age of each long shoot segment determined by counting annual leaf-scar series on its short shoots. Transverse sections were made for each long shoot segment and an adjacent short shoot; wood ring thickness, number of rings, and number of tracheids/ring were determined. Using branch maps, we identified wood rings for each long shoot segment to year and developmental context of each year (distal short shoot growth only vs. at least one distal long shoot). Climate data were also analyzed in conjunction with developmental context. • Key results: Significantly thicker wood rings occur in years with distal long shoot development. The likelihood that a branch produced long shoots in a given year was lower with higher maximum annual temperature. Annual maximum temperature was negatively correlated with ring thickness in microsporangiate trees only. Annual minimum temperatures were correlated differently with ring thickness of megasporangiate and microsporangiate trees, depending on the developmental context. There were no significant effects associated with precipitation. • Conclusions: Overall, developmental context alone predicts wood ring thickness about as well as models that include temperature. This suggests that although climatic factors may be strongly correlated with wood ring data among many gymnosperm taxa, at least for Ginkgo, correlations with climate data are primarily due to changes in proportions of shoot developmental types (LS vs. SS) across branches. Key words: branch architecture; climate; Ginkgo biloba; Ginkgoaceae; mean annual maximum temperature; mean annual minimum temperature; mean annual precipitation; shoot dimorphism; wood development. Ginkgo biloba L. (Ginkgoaceae) is the single extant species of the order Ginkgoales with a fossil record that extends into the Permian (Seward and Gowan, 1900; Sprecher, 1907; Zhou, 1991, 2009; Zhou and Zheng, 2003; Zhou et al., 2007; Feng et al., 2010). The modern genus is present in the Lower Cretaceous and is often deemed a “living fossil” because it has undergone little apparent morphological change over 100 million years (Royer et al., 2003; Zheng and Zhou, 2004; Feng et al., 2010). As the only ginkgoalean representative of the five main extant seed plant lineages, and because of its central placement in seed plant phylogeny, studies of Ginkgo provide key insights into the development and evolution of seed plants. Our work follows in the extensive tradition of investigations into the distinctive vegetative morphology of Ginkgo (Seward and Gowan, 1900; Sprecher, 1907; Tupper, 1911; Sakisaka, 1928; Gunckel and Wetmore, 1946a, b; Gunckel et al., 1949; Gunckel and Thimann, 1949; Arnott, 1959; Critchfield, 1970; Hoddinott and van Zinderen Barker Jr., 1974; Mundry and Stutzel, 2004; Del Tredici, 1991; Christianson and Jernstedt, 2009; Christianson and Niklas, 2011; Leigh et al., 2011; Niklas and Christianson, 2011; Rudall et al., 2012). Many examinations of the vegetative morphology of Ginkgo have focused on the characteristic shoot dimorphism: long shoots with spaced nodes that extend the canopy, and primarily unbranched short shoots lacking internode elongation and with axillary reproductive structures. Some of the earliest observations of Ginkgo recorded anatomical differences between shoot types (Seward and Gowan, 1900; Sprecher, 1907; Coulter and Chamberlain, 1917; Sakisaka, 1928; Chamberlain, 1935). Short shoots have more primary ground tissue and less dense xylem with more parenchymatous cells. The suite of traits that characterize short shoots, 1 Manuscript received 1 April 2013; revision accepted 7 August 2013. A portion of this work was presented at the Katherine Esau Research Symposium, “Integrating Plant Structure with Function, Development and Evolution,” March 29, 2012, University of California, Davis. We thank Professor Patrick von Aderkas (University of Victoria) for helpful comments on this manuscript, Ellen Roundey for assistance with wood ring observations, Megan Saunders for electron microscopy assistance, and Angie Girdham for plant collection of Ginkgo from Hillsdale, Michigan. We acknowledge support from the Katherine Esau Postdoctoral Fellowship (SAL) and the Grady L. Webster Memorial Research Fund (JAJ). 6 Author for correspondence (e-mail: [email protected]) doi:10.3732/ajb.1300123 American Journal of Botany 100(10): 1–13, 2013; http://www.amjbot.org/ © 2013 Botanical Society of America 1 Copyright 2013 by the Botanical Society of America 2 AMERICAN JOURNAL OF BOTANY [Vol. 100 Fig. 1. Schematic diagram of an ultimate branch segment of Ginkgo biloba. Short shoots are often wider in diameter than the subtending long shoot. The blue boxes represent short shoots, and box lengths represent relative shoot ages. The thick green line represents long shoot segments in a branch. Long shoot segments are typically separated by zones of previous short shoot growth. including suberized leaf bases, little to no periderm production, wide pith, manoxylic-like xylem (low density of tracheids/abundant parenchyma) and pachycaulous form (unbranched, often tubular to obconic), led several botanists to suggest that the Ginkgo short shoot was anatomically similar to a cycad main axis (Sakisaka, 1928; Chamberlain, 1935; Christianson and Jernstedt, 2009). Later developmental studies compared the shoot apical meristem organization in Ginkgo with that of cycads, finding that both fit Foster’s (1938) zonate model later considered typical of all extant gymnosperms (Gunckel and Wetmore, 1946a; Gunckel and Wetmore, 1946b; Gunckel et al., 1949; Gunckel and Thimann, 1949). These studies concluded that the developmental features examined (i.e., apical organization and origin of primary tissues) were fundamentally similar in long and short shoots. This subsequent downplaying of the cycad-like peculiarity of short shoots has continued generally and may be explained in part by the assignment of Ginkgo as a coniferophyte in the dominant phylogenetic hypotheses prevailing since the mid-1900s (Crane, 1985; Doyle and Donoghue, 1992; Rothwell and Serbet, 1994; Doyle, 2006; Hilton and Bateman, 2006; Rydin and Korall, 2009; Mathews, 2009; Mathews et al., 2010). More recently, there has been renewed interest in characterizing differences between short and long shoots, mostly with regard to leaf traits (Critchfield, 1970; Hoddinott and van Zinderen Barker, 1974; Mundry and Stutzel, 2004; Guo et al., 2005; Christianson and Niklas, 2011; Leigh et al., 2011; Niklas and Christianson, 2011). In particular, specific leaf area, the scaling of specific leaf area, and modeled leaf hydraulics differ significantly between shoot types and between mega- and microsporangiate trees (Christianson and Niklas, 2011; Niklas and Christianson, 2011). Compared with the long history of research on shoots of Ginkgo, investigation of larger scale branch architecture remains relatively uninvestigated (Del Tredici, 1991), and to date, there have been no attempts to integrate morphological, anatomical, and developmental data in the context of branch architecture. The aim of this work is to analyze morphological and anatomical observations of shoot dimorphism and their relationship with branch architecture using a quantitative approach. We are interested in understanding whether the occurrence and proximity of pachycaulous short shoots (Seward and Gowan, 1900; Sakisaka, 1928) is related to wood ring thickness in proximal long shoot wood, and what relationship, if any, climate data has with wood ring thickness in long shoots. Our work addresses the following questions: (1) How does wood ring thickness differ between years in which branch growth is allocated to canopy expansion (long shoot development) or canopy maintenance (short shoot growth only)? (2) What is the relationship of interannual climatic variation with the production of long and short shoots in branches from micro- and megasporangiate trees? (3) What is the relationship between interannual variation in climate data and patterns of branch wood ring thickness? Our study shows that proximal long shoot wood ring thickness differs between years in which there is distal short shoot growth only and years with at least one distal long shoot. These differences in shoot developmental type and branch architecture influence long shoot wood deposition. Interannual variation in temperature interacts in a complex way with these structural and developmental differences, and mega- vs. microsporangiate trees show differential relationships of wood ring thickness with climate data. Fig. 2. Two short shoots of Ginkgo biloba showing external features that allow age determination of the axis independent of wood anatomy. White arrows indicate examples of persistent outer bud-scales; blue arrowheads indicate examples of the seam-like zone where ephemeral bracts abscised in spring; plain white lines indicate ovulate stalk scars. The photographs also highlight the persistence of short shoots, indicated by the presence of lichen on the basal regions shown. Eight and nine annual cycles of growth are visible for the shoot on the right and left, respectively. October 2013] LITTLE ET AL.—GINKGO BRANCH WOOD 3 MATERIALS AND METHODS Plant material—Structured sampling for statistical analyses involved the collection of 28 branches in June and July of 2011. Branches were sampled from six trees growing on the campus of the University of California, Davis (UCD): three trees growing near Hunt Hall (two microsporangiate trees and one megasporangiate tree), and three trees adjacent to Haring Hall (one microsporangiate tree and two megasporangiate trees). Each branch was collected with pole-pruners at approximately 3 m above ground, near the periphery of the canopy, and contained at least two long shoot segments per major branch axis. Additional branches were sampled from four trees on the Hillsdale College campus (Hillsdale, MI), although these supplemental branches were not included in statistical models of ring thickness. Instead, they were used to corroborate some of the observations made on samples collected at UCD including instances where the age of axis and wood ring number were not equal. Morphology and anatomy of mapped branches—According to our sampling design, each branch had a minimum of two long shoot segments along the major axis of the branch, although some branches had up to six long shoot segments. The diameters of the long and short shoots were measured using digital calipers for each mapped branch segment (Fig. 1). The age of each long shoot segment was determined by counting the annual cycles of leaf production by observation of the short shoot exterior (Sprecher, 1907; Christianson and Jernstedt, 2009). Three criteria were used to determine the boundaries of annual leaf cycles on a short shoot: (1) the presence of outer bud-scales which do not always abscise completely, (2) the seam-like line where the ephemeral bracts abscise in spring (these tend to abscise with pollen strobili toward the end of April in Davis), and (3) the disruption in parastichies in the phyllotaxy of the suberized leaf scars (Figs. 2, 3). These criteria were used to determine the age of the short shoots occurring along each long shoot segment, thus providing an assessment of the age of each long shoot axis bearing them, independent of the observation of wood rings. Age determinations were verified by counting leaf production cycles of multiple short shoots in each long shoot segment. This mapping of branches and age determination for each long shoot segment is possible because all axils on a long shoot form buds the same year that the long shoot forms. The only exception is the basal most one to three (typically two) axillary positions in the elongate portion of the long shoot, just above the basal pseudo-whorl of preformed leaves (Fig. 3; Critchfield, 1970; Christianson and Jernstedt, 2009). We have observed that these basal most axillary positions will form shoots, but only if there is damage to the long shoot. It is also important to note that the first formed (basalmost) foliar scar series in a short shoot will not be complete because axillary buds formed the same year as long shoot formation are proleptic, bearing only the scales and leaves for the following year’s growth. Anatomical observations were made approximately midway along each long shoot segment and on one short shoot adjacent to each long shoot transverse section (Figs. 3–5). All transverse sections were stained with 0.01% toluidine blue O, or phloroglucinol-HCL (Ruzin, 1999), mounted on a slide with coverslip and water, and observed with an Olympus BH-2 compound microscope (Olympus Optical, Tokyo, Japan) with a 10× objective lens. Images were captured using an Olympus MicroFire digital camera (Olympus America, Melville, New York, USA). Ring thickness and tracheid counts were measured in Adobe Photoshop CS5 (Adobe Systems, San Jose, California, USA). Larger scale images were captured with a Nikon D50 digital SLR camera (Nikon Corp., Tokyo, Japan), and on a dissecting scope also mounted with the MicroFire digital camera. All images were processed in Adobe Photoshop CS5, with linear adjustments applied across the whole image (i.e., sharpening, levels, color balance). Some composite images were assembled using Photoshop autoalign and auto-blend algorithms. Line figures were created directly in Adobe Illustrator CS5, or were exported from JMP (version 10.0.0 , SAS Institute, Cary, North Carolina, USA) and modified in Adobe Illustrator CS5. Fig. 3. Branch segments of Ginkgo biloba. Left, outer surface of branch segment, showing annual growth increments on short shoots and previous terminal short shoot; the annual increments were used to determine the age of the branch segments; note that the basalmost axil did not grow to form a short shoot. Right, phloriglucinol-HCL-stained median longitudinal cut surface shows wide pith of current and previous short shoots and contiguous piths between short shoots and their subtending long shoots. Scale bar = 5 mm. 4 AMERICAN JOURNAL OF BOTANY [Vol. 100 The range of ages for the branch segments observed was 1–39 yr. An age of 1 year indicated that the given long shoot had formed earlier in the spring of the sampling period (June and July of 2011). The 28 branches together represented 169 long shoot segments, with each having one or more rings, representing 884 long shoot wood rings. Of the 169 long shoot segments, 30 were shoots established the year of sampling, which had yet to establish well-developed axillary short shoots. Thus, only 136 short shoots were described. Using the maps of each branch made prior to anatomical observations, we determined the identity (short shoot or long shoot) of the distal stem increment at the time of formation for each long shoot wood ring (designated as developmental context). Each wood ring was categorized based on the presence of at least one long shoot having developed distal to that position in that year (LS) or the presence of only short shoot growth that year (SS). A ring produced the year after distal long shoot development was also classified for developmental context (ALS; after distal long shoot development). In some cases, the number of rings observed did not equal the independently determined age of the axis; these long shoot wood segments with “missing rings” could not be scored for developmental context. The exception to this case was on terminal branch segments; in these cases, the innermost ring produced was scored LS (first year formed as a long shoot), and all subsequent rings were recorded as SS years. Further characterization of branch anatomy with missing rings involved quantification of the radial number of tracheids in xylem. We counted radial tracheid numbers in a subsample of 56 short shoots with a single ring, all long shoots with missing rings (48), and a subsample of 76 long shoot segments with regular, annual rings. The number of tracheids per ring was estimated by counting cells that intercepted a radial transect through a given wood ring. The number of tracheids in a given transect was then divided by the age of the shoot to give a rough estimate of the number of tracheids produced over the shoot life span (tracheids/year). This value (tracheids/year) did not take into account differences in LS, ALS, or SS years. Scanning electron microscopy (SEM)—Several branches collected from trees in Michigan were observed using SEM. Transverse sections of Ginkgo shoots were adhered to aluminum stubs and sputter-coated with (gold (60 s at 30 mA) using a Cressington 108 auto sputter coater (Cressington Scientific Instruments, Watford, UK). The sections were then examined at 5 kV using a JEOL JSM-5510 scanning electron microscope (JEOL USA, Peabody, Massachusetts, USA). Statistical analyses—To characterize the relationship between variation in climate data and long shoot wood ring thickness in G. biloba, we downloaded 50 yr of annual maximum and minimum temperature and precipitation data from the United States Historical Climatological Network (US HCN; website http://www.ncdc.noaa.gov/oa/climate/research/ushcn/). The US HCN data provided mean monthly maximum and minimum temperatures, as well as total monthly precipitation for a weather station in Davis, CA (DAVIS 2 WSW EXP), within approximately 2 km of the trees sampled. Monthly means were averaged to produce mean annual temperature variables; precipitation was summed. For each year in which a ring was measured, we recorded the mean maximum and minimum temperature and total annual precipitation. Variation in mean long shoot wood ring thickness between tree type (microsporangiate vs. megasporangiate trees) and developmental context (LS, ALS, or SS)—The developmental context of individual long shoot wood rings (LS, ALS, or SS) could be definitively assigned for at least one long shoot segment in each of the 26 branches. The mean thickness of the wood rings in each category (LS, ALS, or SS) for each branch was included in this analysis. Terminal long shoot segments with missing rings were included, since the innermost ring represented the year that the axis developed into a long shoot (LS), and the subsequent rings could only represent years of distal short shoot growth (SS). Internal branch segments with missing rings could not be included in the analysis. Fig. 4. Longitudinal section of a terminal short shoot of Ginkgo biloba, stained with phloriglucinol-HCL. Overall short shoot diameter, as well as pith diameter, increases toward the apex, with a slightly narrower subapical diameter. Leaf traces have not been sealed off by subsequent wood cambium (vascular cambium) development, even at base of the short shoot (oldest part of the axis). Scale bar = 2 mm. October 2013] LITTLE ET AL.—GINKGO BRANCH WOOD 5 Fig. 5. Series of transverse sections of a Ginkgo biloba short shoot adjacent to that shown in Fig 4. Sections stained with phloriglucinol-HCL. Each transverse section is from a relative location (A, near apex of short shoot; F, at base of short shoot). Diameter decreases toward the base of the short shoot, both in overall diameter and pith diameter. Leaf traces (e.g., at arrows) have not been sealed off by subsequent vascular cambium development, even at base of short shoot (F, oldest part of the axis). Scale bar = 2 mm. A mixed model ANOVA was used to test for differences in mean branch wood ring thickness between megasporangiate and microsporangiate trees and among shoot developmental contexts (proc GLM, SAS 2011). In this model, the mean long shoot wood ring thickness for each developmental context (LS, ALS, SS) per branch was included as the dependent variable (N = 75). The main fixed effects of tree type (mega- or microsporangiate tree), developmental context (LS, ALS, SS), and the interaction between tree type and developmental context were included as fixed effects. The main effect of branch (nested within tree type) was included as a random effect. When a significant main effect of tree type, developmental context, or an interaction was detected, post hoc Tukey’s tests were used to compare means. Effect of annual variation in climate data and tree type on the likelihood of long shoot production—Two logistic regression models were used to investigate the effect of tree type (megasporangiate tree or microsporangiate tree) and variation in climate data on the likelihood that G. biloba will produce a long 6 AMERICAN JOURNAL OF BOTANY [Vol. 100 vs. microsporangiate). In a separate model, we tested for a relationship between proportion of shoot types produced (LS vs. SS) in each year, total annual precipitation, and tree type. In both analyses, years following long shoot production (ALS) were classified as short shoot years (SS). For this analysis, we simplified the classification of developmental context because this test was used to determine whether variation in climate data was a significant determinant of the probability that a given branch would have invested more in canopy maintenance (short shoot growth) or in canopy expansion (production of long shoots). Fig. 6. Relationship between mean shoot diameter and shoot age for long shoots (blue diamonds and blue regression line) and short shoots (green squares and green regression line) of Ginkgo biloba. Means were calculated for each age class (long shoots N = 29, based on 169 axes; short shoots N = 27, based on 139 axes). Long shoot relationship, R2 = 0.56; short shoot relationship, R2 = 0.52. shoots in branches (proc LOGISTIC, SAS 2011). We tested for a relationship between proportion of shoot types produced (LS vs. SS) in each year, the annual maximum temperature, minimum temperature, and tree type (megasporangiate Effect of interannual variation in climate data, tree type, and developmental context on ring thickness—It was necessary to assign precise ages to each wood ring observed in the analyses examining the relationship of variation in climate data with ring thickness. Because it was not possible to assign a year for each ring of long shoot branch segments determined to have missing rings, climate data values could not be assigned in these branches. Thus, 18 branches and 565 total wood ring observations were included in these models. Two mixed model ANCOVAs were used to assess the effect of interannual variation in precipitation data (first model) and maximum and minimum temperature data (second model) on branch wood ring thickness (proc GLM, SAS 2011). We included total annual precipitation in a separate model because it was highly correlated with both maximum (r = −0.6655) and minimum temperature (r = 0.6502). We conducted follow up analyses of the relationship between wood ring thickness and precipitation using monthly precipitation records. We chose to include average annual precipitation in the model after less conservative simple linear regression analyses of the relationship between wood ring thickness and average monthly precipitation for each month did not reveal significant relationships. Maximum and minimum temperatures were not highly correlated (r = −0.3030) and were included as covariates in a second ANCOVA model. In both models, Fig. 7. Transverse sections of Ginkgo biloba long shoots and short shoots. A, B. Phloriglucinol-HCl-stained free-hand sections. C, D. SEM images of transverse cut faces. Each pair represents a set of branch segment observations. (A, C) Long shoots with growth rings. (B, D) Short shoots showing a single ring of xylem. Both pairs are from the same branch segment and are thus the same age; A, B = 8-yr-old axes; C, D = 15-yr-old axes, determined from annual leaf scar series on short shoots on the branch segment. Note: C and D are from a branch segment with nine missing rings in the long shoot wood. Scale bars = 100 µm. October 2013] LITTLE ET AL.—GINKGO BRANCH WOOD Fig. 8. Box plots of radial tracheid density for Ginkgo biloba long shoots and short shoots. The first three box plots represent the number of tracheids along a stem radius, divided by age of the axis. The last box plot (far right) represents the number of radial tracheids in the single ring of short shoot xylem. Short shoots produce more radial tracheids per single ring than annual long shoot wood rings. Long shoots with missing rings have fewer radial tracheids/year than long shoots with annual rings. The number of radial tracheids/year in short shoots is similar to that of long shoots with missing rings. Missing rings are deduced by comparing observed ring number to the age of the branch segment as determined by counting annual leaf scar series of adjacent short shoots (N = 76, annual rings long shoot; 48, long shoot missing rings; 56 short shoots). 7 Fig. 9. Mean wood ring thickness in branches from megasporangiate (red) and microsporangiate (blue) trees of Ginkgo biloba in years when at least one long shoot was produced distally (LS), the year after a distal long shoot was produced (ALS), and years in which only short shoots were produced distally (SS) on an individual branch. Different letters indicate significant differences in mean wood ring thickness. Least square means and standard errors are shown. (ANOVA, R2 = 0.88, model df = 31, error df = 633, F = 9.99, P < 0.0001). Description of shoots across mapped branches— Plots of shoot diameter vs. shoot age corroborated preliminary observations that in parts of branches, short shoots may be thicker than their subtending long shoots (Figs. 3–6). Long shoots tend to have narrower diameters than short shoots in their first 1–10 yr. Axis age is a reasonable predictor of shoot diameter in both shoot types (Fig. 6), but short shoots only gradually increase in diameter over their life spans. Branch segments that represented previous short shoot growth (Figs. 1, 13) reflected the pattern expected from short shoots which have a single xylem ring over their life spans, namely, a single ring for the period of time as a short shoot with additional rings that correspond to the years as a long shoot. The rings in this “previous short shoot” zone of the branch consist of the innermost ring with N rings to the outside, where N = the age of the distal long shoot segment. As an example, consider the branch segment in Fig. 13 with the 15-yr-old previous short shoot. The subtending 19-yr-old branch segment existed for 15 yr, then the terminal short shoot grew out 4 yr ago. This previous short shoot, with 15 leaf scar cycles, will have “1+4” rings—the innermost ring produced during the 15-yr short shoot growth period and four distinct rings from the 4 yr of long shoot growth. We determined the number of radial tracheids in (1) long shoot wood with the full complement of annual rings as deduced from overall branch architecture, (2) from long shoot wood with fewer rings than indicated from counting short shoot annual leaf scar series (missing rings), and (3) in the single ring of short shoots (Figs. 7, 8). The single ring of xylem in short shoots had more tracheids than the number of tracheids/year in both long shoots with annual rings and those with inferred missing rings (Fig. 8). Long shoots with annual rings had more tracheids/year than long shoots with missing rings, whereas the number of tracheids/year in the single xylem ring of short shoots was similar to that of long shoots with missing rings (Fig. 8). TABLE 1. TABLE 2. the thickness of each long shoot ring was included as the dependent variable. Tree type, developmental context (LS, ALS, SS), and their interaction were included as fixed effects. Branch samples were nested within tree type and included as a random effect. To test for variation in the relationship between wood ring thickness and climate, we included all possible two- and three-way interactions between/among tree type, developmental context, and covariates in both models. RESULTS Mixed model ANOVA of mean long shoot wood ring thickness of Ginkgo biloba. Tree type (mega- vs microsporangiate tree), developmental context (distal long shoot growth vs. short shoot growth only), and their interaction were analyzed as fixed effects (R2 = 0.88, model df = 31, error df = 633, F = 9.99, P = <0.0001). Effect Tree type Developmental context Branch(tree type) Tree type × shoot type Logistic regression model used to test for the response of developmental context (distal long shoot growth = 0; short shoot growth only = 1) in relation to tree type (mega- vs. microsporangiate tree) and annual maximum (max) and minimum (min) temperatures (temp) in Ginkgo biloba. Num. df Den. df Type 3 SS F P Parameter df Estimate SE Wald χ2 P 1 2 26 2 62.135 633 633 633 0.0451 4.7421 2.2409 0.1942 1.10 112.60 4.09 4.61 0.2980 <0.0001 <0.0001 0.0103 Intercept Max temp Min temp Tree type 1 1 1 1 8.0087 −0.3278 −0.0467 0.4197 3.4182 0.1233 0.2233 0.0931 5.4896 7.0729 0.0438 20.3072 0.0191 0.0078 0.8342 <0.0001 8 AMERICAN JOURNAL OF BOTANY TABLE 3. Logistic regression model used to test for the relationship between developmental context (distal long shoot growth = 0; short shoot growth only = 1), tree type (mega- or microsporangiate tree), and total annual precipitation in Ginkgo biloba. Parameter df Estimate SE Wald χ2 P Intercept Precipitation Tree type 1 1 1 −0.3034 0.0003 0.4385 0.3892 0.0008 0.0906 0.6078 0.1432 23.4533 0.4356 0.7051 <0.0001 Some short shoots had more than one xylem growth ring. Of the total number of short shoots observed (136), 127 displayed only one growth ring, five displayed two growth rings, three had three growth rings, and one short shoot had four growth rings. However, these short shoots did not have their leaf-traces closed off by wood, such that there was still ground tissue continuity between pith and cortex (Figs. 3–5, 7C, 7D). Variation in mean long shoot wood ring thickness among tree types and developmental contexts— We observed significant differences in mean wood ring thickness among developmental contexts (at least one distal long shoot, LS; year after distal long shoot growth ALS; distal short shoot growth only, SS) and tree types (mega- vs. microsporangiate trees) (R2 = 0.88, model df = 31, error df = 633, F = 9.99, P < 0.0001). Megasporangiate trees had thicker wood rings, on average, than microsporangiate trees (significant main effect of tree type, Table 1). Megasporangiate wood ring thickness was greatest in years when a branch had at least one distal long shoot (LS) and was smallest in years when the branch had only short shoot growth distally (SS). Thickness was intermediate in the year following long shoot development (ALS) in megasporangiate trees. However, microsporangiate trees produced uniformly thinner rings in years when no long shoots were formed (ALS and SS; significant tree type by developmental context interaction (Fig. 9). Fig. 10. Relationship between wood ring thickness and annual maximum temperature in branches of Ginkgo biloba from megasporangiate (red diamonds, dashed regression line) and microsporangiate trees (blue squares, solid regression line). The relationship between ring thickness and maximum temperature was significant in microsporangiate trees (slope = −0.0264, P = 0.0020), but not in megasporangiate trees (slope = 0.0073, P = 0.1268). [Vol. 100 Effect of tree type and variation in annual climate data on branch development—Not all long shoot segments with missing rings could be included in the statistical models that included climatic covariates because such individual rings could not be assigned a year (Figs. 7C, 7D, 8). The number of missing rings per long shoot segment ranged from 2 to 27. Missing rings tended to occur in proximal branch segments (closer to the trunk) and locations of prolonged short shoot development. Long shoot transverse sections with missing rings represented 48 of the total 169 long shoot segments on 28 branches (Fig. 8). Annual variation in both temperature and differences between tree type had significant effects on the likelihood a branch will allocate growth to canopy expansion (produce long shoots) or to canopy maintenance (only short shoots growth). Overall, megasporangiate tree branches had 0.4196 greater likelihood of producing a long shoot than microsporangiate trees (Table 2). In both mega- and microsporangiate trees, every one degree increase in annual maximum temperature was correlated with a 0.3535 decrease in the likelihood of a branch having produced a long shoot (Table 2). In contrast, annual variation in total precipitation did not have a significant effect on the likelihood that an individual branch had allocated growth to canopy expansion or canopy maintenance (Table 3). Relationship between interannual variation in climate data and wood ring thickness in microsporangiate and megasporangiate trees of Ginkgo biloba— The previous analyses demonstrated significant effects of developmental context (LS, ALS, SS) and tree type (mega- vs. microsporangiate) on ring thickness and that temperature data predicted the likelihood that a branch will have invested in canopy expansion (produced long shoots) or canopy maintenance (short shoot growth only). In light of these combined results, we used ANCOVA to determine whether interannual variation in climate data had a significant effect on ring thickness and whether it improved the overall ability to describe the variation in long shoot wood ring thickness we observed. Both annual maximum and minimum temperature had a significant predictive effect on wood ring thickness, which varied among developmental contexts (LS, ALS, SS) and between tree types (whole model R2 = 0.45, model df = 33, error df = 531, F = 12.95, P < 0.0001; Appendix S1; see Supplemental Data with the online version of this article). Thinner rings were correlated with higher maximum temperatures across all developmental contexts in microsporangiate trees (slope = −0.0264, P = 0.0020), but no significant relationship of higher maximum temperature with ring thickness was detected in megasporangiate trees (slope = 0.0073, P = 0.1268, significant interaction of max temp, and tree type; Fig. 10). Increasing minimum temperatures were correlated with thicker rings in megasporangiate trees when distal long shoot development occurred (LS; slope = 0.0748, P = 0.0003) and the year after long shoots developed (ALS; slope = 0.0584, P = 0.0009), but there was no significant relationship when growth was allocated to maintain short shoots (SS; slope = −0.0132, P = 0.6422; Fig. 11). In contrast, there was no significant relationship between minimum temperature and microsporangiate tree ring thickness when distal long shoot development occurred (LS; slope = −0.0303, P = 0.2676) nor the year after (ALS; slope = −0.0053, P = 0.7950; Fig. 11). Instead, we observed a positive relationship between minimum temperature and ring thickness (slope = 0.0241, P = 0.0258) when growth was allocated to maintain short shoots (SS; significant interaction of October 2013] LITTLE ET AL.—GINKGO BRANCH WOOD 9 relationship with wood ring thickness in either tree type (Appendix S2). Although we observed a significant effect of interannual variation in temperature on wood ring thickness, the inclusion of climate data in our models did not substantially improve the proportion of variance explained by the model. The R2 values of the ANCOVA models with annual maximum and minimum temperature (R2 = 0.45) or total annual precipitation (R2 = 0.41) were all relatively low. Furthermore, these values were only subtly greater than the R2 value in an ANOVA on the raw data without climate included as a covariate (R2 = 0.39). Combined, the relatively low model R2 values and inspection of the data points in subsequent simple linear regression analyses revealed substantial scatter (Figs. 10–12). Fig. 11. Relationship between wood ring thickness and annual minimum temperature in megasporangiate trees of Ginkgo biloba. Years when only short shoots were produced (SS, blue triangles and blue regression line), years when at least one long shoot was produced (LS, green squares and green regression line), and the year after a long shoot was produced (ALS, red diamonds and red, dashed regression line). The relationship between ring thickness and minimum temperature was significant in megasporangiate trees only when distal long shoot development occurred (LS; slope = 0.0748, P = 0.0003) and the year after long shoots developed (ALS; slope = 0.0584, P = 0.0009). There was no significant relationship in years when growth was allocated to maintain short shoots (SS; slope = −0.0132, P = 0.6422). minimum temperature, tree type, and developmental context; Fig. 12). While wood ring thickness varied significantly among branches (R2 = 0.41, model df = 27, error df = 537, F = 13.62, P < 0.0001; Appendix S2, see online Supplemental Data), total annual precipitation showed no significant predictive Fig. 12. Relationship between wood ring thickness and annual minimum temperature in Ginkgo biloba microsporangiate trees. Years when only short shoots were produced (SS, blue triangles and blue regression line), years when at least one long shoot was produced (LS, green squares and green regression line), and the year after a long shoot was produced (ALS, red diamonds and red, dashed regression line). The relationship between microsporangiate tree ring thickness and minimum temperature was significant only in years when growth was allocated to maintain short shoots (SS, slope = 0.0241, P = 0.0258). In years when distal long shoot development occurred (LS, slope = −0.0303, P = 0.2676), and the year after (ALS, slope = −0.0053, P = 0.7950), there was no significant relationship between ring thickness and minimum temperature. DISCUSSION This work was motivated by the stark shoot dimorphism in branches of Ginkgo biloba. We were interested in determining whether the occurrence of pachycaulous short shoots vs. leptocaulous long shoots is related to proximal wood ring thickness and whether climate data are also predictive of wood ring thickness. Pachycaulous axes are relatively thick-stemmed and unbranched, whereas leptocaulous axes are relatively slender and highly branched. The following questions are addressed by this study: (1) How does wood ring thickness differ between years in which branch growth is allocated to canopy expansion (long shoot development) or canopy maintenance (short shoot growth only)? (2) What is the relationship of interannual climatic variation with the production of long and short shoots in branches from micro- and megasporangiate trees? (3) What is the relationship, if any, between interannual variation in climate data and patterns of branch wood ring thickness? Overall, our results show that within the architecture of branches, there is a complex interaction involving developmental context and temperature in both the likelihood of a branch producing long (LS) vs. short shoots (SS) and the wood ring thickness of long shoot wood. Developmental context is defined for this study as the occurrences of distal shoot types on a branch for a given annual wood ring. Further, some of these effects differed between mega- and microsporangiate trees. Mapped branches, with age determinations based on external observation of short shoot growth cycles, provided us with a natural experiment that allowed us to test hypotheses based on development and climate across branch architecture (Fig. 13). These tests were made possible due to both temporal and spatial information made available by the mapping procedure. Effect of developmental context on branch wood ring anatomy— Qualitative comparisons of wood anatomy between long shoot wood and short shoot xylem revealed significant differences in the average number of tracheids produced (Fig. 8). In short shoots, the single ring of xylem is made up of about twice the average annual radial number of tracheids as in long shoots (Fig. 7). In long shoot wood with missing annual wood rings, the number of radial tracheids/year was about a third of “normal” long shoot wood in which the number of annual rings corresponded with the number determined by counting adjacent short shoot annual leaf scar series. The number of radial tracheids/year of the single short shoot ring is similar to that of long shoots with missing rings. Taken as a whole, these comparisons suggest that short shoots have a larger initial investment in the 10 AMERICAN JOURNAL OF BOTANY [Vol. 100 Fig. 13. Schematic diagram of how a branch of Ginkgo biloba is mapped and ages determined for each segment. As in Fig. 1, the blue boxes represent short shoots, and box lengths represent relative shoot ages. Thick green lines represent long shoot units in a branch; their lengths are established by the elongation growth that occurred the year each long shoot developed. Long shoot segments are typically separated by zones of previous short shoot growth. Ages were independently determined, prior to anatomical study, using the scar series from annual leaf flushes on short shoots. Circled numbers represent the age of the adjacent long shoot segment; numbers without circles represent the age of adjacent previous short shoots. development of the single ring of xylem. The explanation of missing rings may be that growth increments are cryptic due to environmental factors (Reukema, 1959; Colenutt and Luckman, 1991; Parent et al., 2002; Wilmking et al., 2012). However, the low to fractional number of tracheids/year in long shoot wood with missing rings suggests that wood development greatly slows and may even stop for periods of time. It appears that a predominance of short shoots on a branch, with their stable xylem width over their lifespans, reduces xylem production requirements of subtending long shoot segments. Significantly thicker long shoot wood rings occurred in years that had canopy expansion (distal long shoot development) than in years of canopy maintenance (SS years). Branches of megasporangiate trees produced significantly thicker rings in the year following canopy expansion (after distal long shoot development, ALS), whereas ALS rings in microsporangiate trees were not different from those of rings produced in years of canopy maintenance (SS; Fig. 9). The complex relationship between developmental context and tree type may be related to the differences in reproductive biomass investment between mega- and microsporangiate trees. Megasporangiate branches bear substantially larger, heavier, and more persistent reproductive structures (the seeds) than microsporangiate branches and thus are exposed to greater mechanical stress throughout their lifespans. Similar relationships between branch thickness and reproductive structure size or abundance have been observed in various taxa, both gymnosperms and angiosperms (Obeso, 2002; Chen et al., 2009). Any given megasporangiate tree branch investing in canopy expansion must be, or become, more robust to accommodate increasing numbers of seeds produced by short shoots. This hypothesis could be tested in the future using a detailed analysis of fertile vs. nonfertile regions of megasporangiate trees, with the possible inclusion of prefertile juvenile trees. We predict differences in ring thickness between fertile and nonfertile megasporangiate branches, especially in light of the fact that specific leaf area also varies between fertile and nonfertile short shoots (Christianson and Niklas, 2011; Niklas and Christianson, 2011); presumbably these differences will also be seen in the subtending woody axes. Effect of interannual climatic variation on canopy investment patterns and wood ring thickness— Incorporation of information on interannual variation in temperature revealed an additional layer of complexity in our understanding of factors that influence Ginkgo branch development and anatomy. Using our developmental maps of Gingko branches, we were able, in most cases, to determine the calendar year when each observed wood ring was produced. A logistic regression analysis revealed that branches of both mega- and microsporangiate trees were substantially more likely to have produced a long shoot in years with cooler maximum temperatures than warmer years (Table 2). Furthermore, megasporangiate trees were more likely to have invested in canopy expansion (long shoot development) in a given year than in microsporangiate trees (Table 2), providing indications that both temperature and tree type can influence branch growth patterns. Xiong et al. (2000) reported, however, that wood-ring–climate relationships in trunk wood of Ginkgo could not be determined in the Three Gorges region of China. We suspect that the situation of missing wood rings as we deduced for branches may also occur in main trunk wood. This October 2013] LITTLE ET AL.—GINKGO BRANCH WOOD would make ring cross-dating (year determinations for each ring) difficult, thereby confounding the detection of wood climate relationships. Interannual variation in temperature influences both branch development (LS vs. SS formation) and branch wood anatomy in mega- and microsporangiate trees. Although trees of both types were less likely to have produced long shoots in years with higher maximum temperatures, only branches from microsporangiate trees produced thinner wood rings in response to increasing maximum temperature. Wood ring thickness in branches from megasporangiate trees was not correlated with variation in maximum temperature. Similarly, when microsporangiate tree growth was allocated to canopy maintenance (SS years), branches had significantly thinner wood rings in years with higher minimum temperatures (Fig. 12). In contrast, we did not observe a significant relationship between wood ring thickness of megasporangiate branches and interannual variation in minimum temperature in SS years. Instead, increased annual minimum temperatures were correlated with greater megasporangiate tree wood ring thickness in years when branch growth was allocated to canopy expansion and the following year (LS, ALS; Fig. 10). It is important to note that the observed relationship between interannual variation in temperature and wood ring thickness did not have substantial explanatory power in our models. It is likely that the close proximity of the trees sampled to summer irrigation on the UC Davis campus confounded our ability to detect a relationship between wood ring thickness and precipitation. It is also possible that other unmeasured variables interact strongly with wood ring thickness and branch developmental patterns. In general, resource availability is expected to modify biomass allocation across organs (Poorter et al., 2012). For example, it is likely that branch location in relation to light interception (shaded vs. sunlit) and overall tree resource availability also play an important role in determining whether branches allocate growth to canopy expansion or maintenance. In addition, the thicker major branches that arise from the main trunk will probably have different responses in wood formation and thickness compared to shoots at the canopy periphery. Differences in wood ring characteristics are found in conifers from base to tip of trees, including a preponderance of missing rings in lower trunk regions (Novak et al., 2011; Kerhoulas and Kane, 2012; van der MaatenTheunissen and Bouriaud, 2012). Our results suggest that missing rings in wood are a consequence of the short shoot abundance (or low levels of long shoot growth) in the canopy of Ginkgo trees. Because our samples were taken from similar canopy positions, hydraulic effects are not a likely explanation for variation in wood ring thickness. Hydraulic effects due to canopy height are known to influence leaf structure in Ginkgo (Leigh et al., 2011). Additional considerations— Our results show that the development of long and short shoots influences wood production along branches and that this effect differs between microsporangiate trees and megasporangiate trees. Whole plant size often differs between the pollen- and seed-producing trees of woody dioecious species (Obeso, 2002), although we know of no obvious overall tree size differences recorded for microsporangiate vs. megasporangiate trees of Gingko biloba. Rather, our results suggest more cryptic differences in resource allocation between tree types rather than a direct wood growth response to climate that is different between plant “sexes” (Rozas et al., 2009; Gao et al., 2010). It would be productive to test 11 more detailed hypotheses about megasporangiate branch wood production by investigating potential differences in fertile vs. nonfertile regions of the canopy, although this could be confounded by interannual variation in location of ovulate axes in the canopy. Several features of Ginkgo short shoots are reminiscent of cycad main axes, and we hypothesize that the distinct morphology of Ginkgo short shoots is due to primary thickening meristems (PTMs). The presence of PTMs in cycad axes and a variety of other plants (e.g., palms, Isoetes, Stigmaria) results in a pachycaulous morphology (Stevenson, 1980, 1988; Rothwell, 1984; Niklas et al., 2006). Short shoots of Ginkgo and cycads are characterized by ring(s) of low density xylem (abundant parenchyma cells; or manoxylic) that do not have leaf-traces closed off by a fully formed wood cambium, wide piths, large amounts of primary ground tissues, and more-or-less cylindrical forms over their lifespan (inverted conical morphology occurs as well). These observations suggest that short shoots are essentially (semi)herbaceous axes born on a scaffold of pycnoxylic branches (long shoots). This “semi-herbaceous” anatomy and development could facilitate movement of phytohormones, potentially synchronizing or enhancing axillary development of reproductive structures, which are restricted to short shoots. The strong effect of distal long shoots vs. short shoots on branch wood formation indicates that the fundamentally different developmental syndrome of short shoots is detected by the proximal parts of the tree branches. Our results as a whole suggest that dominance of short shoots in a branching system greatly reduces, or may even halt, wood development in a given branch. One hypothesis for the influence of short shoots in the reduction of branch wood growth is that the thick, single ring of xylem, in conjunction with abundant parenchyma, provides low weight additions to branches with hydraulic redundancy, thereby reducing the need for additional subtending wood production for both structural and hydraulic support. If this pattern is consistent across the whole tree (i.e., in the main trunk), then our study provides a developmental cause for missing rings prevalent in trunks of gymnosperms, as opposed to a direct effect of climate or environmental stress (Reukema, 1959; Colenutt and Luckman, 1991; Colenutt and Luckman, 1995; Parent et al., 2002; Novak et al., 2011; Wilmking et al., 2012; van der Maaten-Theunissen and Bouriaud, 2012; Kerhoulas and Kane, 2012). As some Pinaceae (e.g., Larix, Cedrus) are noted for similar strong shoot dimorphisms (Remphrey and Powell, 1987) and have been described with similar growth architectural models such as Massart’s model (Halle et al., 1978; Del Tredici, 1991), we suggest that future investigations focus on Pinaceae to apply our method to test current concepts of limiting resources or environmental stress as the primary causes of reduced wood growth and missing rings. The evolutionary implications of short shoot/pachycaul influence on development in other parts of branches may be broad, given that a diverse set of groups have shoot dimorphism, including extinct seed plant lineages. In particular extinct, Paleozoic seed plants are a compelling group to investigate because many compression localities preserve branches with paired short shoots and other localities preserve permineralized trunks and branches (Bomfleur and Kerp, 2010; Bomfleur et al., 2011). Further, many large branches and trunks have been noted to bear “holes” in their wood, often in a phyllotactic pattern (Taylor and Ryberg, 2007; Decombeix et al., 2010a, 2010b). If the anatomical relationships between long and short shoots of Ginkgo are applicable to such fossils, then there may be good 12 AMERICAN JOURNAL OF BOTANY evidence of long-term short shoot maintenance in extinct seed plants, with pith to pith connections over the life span of short shoots (Figs. 3, 4). Incorporating information about stages and patterns of development may be possible by including fossils from localities with well-preserved plant remains that have articulated branches and trunks (Taylor and Ryberg, 2007). We expect such information could enrich existing studies of wood rings from the fossil record. Our study corroborates historical work on branch anatomy of Ginkgo and provides additional information about interactions between shoot types (short vs. long) and wood development and structure. Whether the shoot dimorphism as seen in Ginkgo is a pleisiomorphic syndrome in seed plants or is derived independently in multiple lineages, investigations of developmental effects of pachycauly/PTM derived morphologies across a plant body (Niklas et al., 2006) are expected to play an important role in understanding the evolution of plants. The gene duplicationderived rooting of seed plant phylogeny (Mathews et al., 2010) emphasizes the pivotal position of Ginkgo and also places cycads as the extant sister to angiosperms. Bennettitales are also placed on this lineage leading to angiosperms in most phylogenetic trees that include fossils (Doyle, 1996; Hilton and Bateman, 2006); many fossil cycads and fossil bennettitalean remains have pachycaulous main axes or have terminal pachycaulous axes born on long shoots (Rothwell and Stockey, 2002; Zheng et al., 2005; Zhang et al., 2006; Rothwell et al., 2009; Pott et al., 2012). 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