Leaf Construction Cost Protocol notes Mason Heberling ([email protected]) First draft 2011 [revisions thru Sept 2017] A few definitions of leaf construction costs •   Measure of leaf resource need – a true cost of resource capture to complement those traits that provide information on species’ rates of resource capture. •   “…amount of fixed carbon required to provide C skeletons, reductant, and ATP for synthesizing all of the biochemical components in the leaf tissue. It does not include the energy required for maintenance or substrate transport (including mineral nutrition).” – Williams et al. (1989) •   “functional trait which summarized inorganic mineral content, calorific value and N need of a leaf carbon economy” – Osunkoya et al. (2010) •   “energy invested by plants to synthesize carbon skeletons and nitrogenous compounds” – Baruch & Goldstein (1999) •   cost of biosynthesis – essentially a proxy for growth respiration (carbon required to produce leaf) •   Both proxy measures below derive from the assumption that plant tissue is more reduced relative to that of the carbohydrates reactants (i.e. level of reduction of tissue = reducing power/energy needed for construction ~ leaf construction cost). This gives an estimate of costs (in glucose equivalents per g tissue) for providing carbon skeletons and reductant for leaf construction. •   Some references view SLA as a close measure of leaf construction cost and others suggest otherwise. Few studies directly plot measured SLA against CC. Popular methods to approximate CC based on biochemical assumptions and empirical tests 1) Williams et al. (1987) [as used in Baruch & Goldstein 1999; Funk & Vitousek 2008; Nagel & Griffin 2001; Osunkoya et al. 2010] – * preferred method in literature? Many variants of CC estimation! CCmass = [0.06968 HC – 0.065) (1-ash) + 7.5(k x Nmass/14.0067)]/0.87 CCarea= CCmass/SLA •   N content [Funk & Vitousek (2007) used total N using elemental analyzer rather than organic N; others used Kjeldahl N = total sum of organic N, ammonium, and ammonia; majority of ecological studies do not distinguish organic vs. inorganic N or oxidation state of dominant N uptake] •   Heat of Combustion (HC; using bomb calorimeter) •   Ash content (g ash g-1 dry matter - incinerate leaf sample of known mass in muffle furnace until white-grey residue remains; general protocol below) NEED: Leaf material for elemental analyzer (leaf N; 2-3 mg dry leaf tissue), bomb calorimeter (heat of combustion; _?_ g dry leaf tissue needed), and muffle furnace (ash content; about 0.5-1

g dry leaf tissue – note that this is a lot of material!; fewer than this, and you may have accuracy/precision issues with very small ash amounts to weigh) 2) Vertregt & Penning de Vries (1987) [as used in McDowell (2002)]; does NOT require heat of combustion. Uses carbon content (but Vertregt & Penning de Vries suggest more accurate formula using carbon content and ash content) CC = [5.39(Com) – 1191] / 1000 [as used in McDowell (2002) – C content measured with elemental analyzer] 2b) Boyd et al 2009 (modification to include ash content): CC = (-1.041 + 5.007C)(1-Min)+5.325Norg where C is carbon concentration from analyzer, Min is mineral concentration (used 0.67 *ashcontent), Norg is organic N concentration (but they used total N because they cite that if leaf N<29 mg/g, this substitution is appropriate- Funk & Vitousek (2007) uses total N as well; others calculate different CCs based on alternative scenarios of dominant N form uptake. Also see Feng et al. (2011). Note that equation listed in Methods section in Boyd et al. (2009) has small typo. This is method used in Heberling & Fridley (2013, 2016), Heberling et al. (2016) NEED (Method 2a,b): leaf material for autoanalyzer (leaf C) and muffle furnace (ash) 3) Other methods…more complicated biochemical lab procedures (e.g., biochemical assays, full elemental analysis, Navas et al 2003, …)] – not done often in ecological work but probably should be. Protocol for measuring Ash content (mineral content) in leaf tissue Ash (or Min) content: Combust carbon and inorganic, mineral nutrients remain (white powder residue) Mineral content calculated by various means such as multiply by a literature-derived constant [0.67 as in Boyd et al (2009)] – note some equations use min while others use ash content. -Range of ash content values [from Larcher (1980) ecophys text] Grasses (6-10% of leaf dry weight) Herbs (6-18%) Trees (3-4%)

Note that combustion of dried leaves oxidizes organic matter, and you are left with minerals plus nitrate oxids and organic acids. These acids then become carbonates. This is why some protocols account for this through determining ash alkalinity. The protocol below does not do this, but instead estimates mineral content as 0.67*ash. See http://prometheuswiki.org/tikiindex.php?page=Determination+of+ash+content+and+ash+alkalinity for more. General protocol: 1.   Dry leaf tissue upon collection (as for SLA) in drying oven at 60° C for at least 48 hrs. 2.   Grind leaves into powder. Use Wiley mill or mortar and pestle. Grinding is important to homogenize sample, especially if several leaves are in one sample. At minimum, grinding to some degree is necessary to fit in small crucibles. 3.   Place small, clean (and preferably rinsed with distilled water) crucibles with lids in drying oven. It is important to weigh the crucibles and leaf mass straight from oven (or place into dessicator). Sample and crucible will pick up moisture from air and affect weights. 4.   Record weights of crucible (no lid). Note: It is critical to label/number crucibles. This can be done with graphite pencil or crayon. I also made a map in notebook of arrangement of crucibles on trays in drying oven (leaving one corner empty for reference). 5.   Tare balance. Add dry leaf powder (minimum 0.5-1 g, depending on precision of balance). Record weight of sample AND record weight of sample+crucible no lid (or add together). Note that the more sample the better (as remaining ash mass might only be a few percent of initial leaf mass). But you may be limited by amount of sample and you may not want to combine many leaves (especially if they are different cohorts). Poorter & Villar (1997) recommend at least 0.1 g 6.   Burn sample in 400º C muffle furnace for 6 hrs (Nagel & Griffin 2001) – 8hrs (Boyd et al 2009); 500º C muffle furnace for 4 hrs (Williams et al. 1989; Osunkoya et al. 2010); 550º C muffle furnace for 6 hrs (Feng et al. 2011). 7.   Ash will be white residue. Weigh/record Ash+Crucible (no lid). Be sure to do so when sample is warm from oven or ensure it is completely dry. -Ash content = ash mass / initial sample mass (g g-1) Leaf N AND C (%): use elemental analyzer – largely replaced older Kjeldahl method (1-2 mg leaf sample – Nagel & Griffin 2001) (2-3 mg – Frank lab) See leaf N, C measurement protocol on Fridley lab website. Could calculate nitrate content as well. Ash-free heat of combustion (kJ g-1):

Use bomb calorimeter (one in chem. Department; T. Volk at ESF has one) Heat released per g of ash-free sample mass (combusted sample mass – ash mass)

6-20 mg dry mass per sample (?) Integrative traits using CC: PEUE (instantaneous photosynthetic energy use efficiency): Amax/CC Many others… see references. Maintenance costs: Can also measure leaf maintenance costs. See Nagel et al. (2002) and refs therein. Additional references: Griffin (1994). Calorimetric estimates of construction cost and their use in ecological studies. J Ecol 8: 551-562 (Review paper on the use of CC measures – excellent overview) http://www.science.poorter.eu/HS33_index.html - H. Poorter’s site with good advice References cited (plus some that weren’t cited above) Baruch, Z. & Goldstein, G. (1999) Leaf construction cost, nutrient concentration, and net CO 2 assimilation of native and invasive species in Hawaii. Oecologia, 121, 183–192. Baruch, Z. & Gomez, J.A. (1996) Dynamics of energy and nutrient concentration and construction cost in a native and two alien C4 grasses from two neotropical savannas. Plant and Soil, 181, 175–184. Boyd, J.N., Xu, C.-Y. & Griffin, K.L. (2009) Cost-effectiveness of leaf energy and resource investment of invasive Berberis thunbergii and co-occurring native shrubs. Canadian Journal of Forest Research, 39, 2109–2118. Caplan, J.S., Wheaton, C. & Mozdzer, T.J. (2014) Belowground advantages in construction cost facilitate a cryptic plant invasion. AoB PLANTS, plu020-. Chapin III, F.S. (1989) The cost of tundra plant structures: evaluation of concepts and currencies. American Naturalist, 133, 1–19. Chazdon, R. (1987) Determinants of photosynthetic capacity in six rainforest Piper species. Oecologia, 73, 222–230. Chiariello, N.R., Mooney, H.A. & Williams, K. (1989) Growth, carbon allocation and cost of plant tissues. Plant Physiological Ecology: field methods and instrumentation pp. 327–365. Coste, S., Roggy, J.-C., Schimann, H., Epron, D. & Dreyer, E. (2011) A cost-benefit analysis of acclimation to low irradiance in tropical rainforest tree seedlings: leaf life span and payback time for leaf deployment. Journal of experimental botany, 62, 3941–55. Durand, L.Z. & Goldstein, G. (2001) Photosynthesis, photoinhibition, and nitrogen use efficiency in native and invasive tree ferns in Hawaii. Oecologia, 126, 345–354. Eamus, D., Myers, B., Duff, G. & Williams, R. (1999) A cost-benefit analysis of leaves of eight Australian savanna tree species of differing leaf life-span. Photosynthetica, 36, 575–586. Feng, Y.-L., Fu, G.-L. & Zheng, Y.-L. (2008) Specific leaf area relates to the differences in leaf construction cost, photosynthesis, nitrogen allocation, and use efficiencies between invasive and noninvasive alien congeners. Planta, 228, 383–90. Feng, Y.-L., Li, Y.-P., Wang, R.-F., Callaway, R.M., Valiente-Banuet, A. & Inderjit,  . (2011) A quicker return energy-use strategy by populations of a subtropical invader in the non-native

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