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8-2014 Unexpected Effects of Chitin, , and Addition on Soil Dynamics in a Wet Tropical Forest Ann E. Russell Iowa State University, [email protected]

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This Article is brought to you for free and open access by the Natural Resource Ecology and Management at Iowa State University Digital Repository. It has been accepted for inclusion in Natural Resource Ecology and Management Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Unexpected Effects of Chitin, Cellulose, and Lignin Addition on Soil Dynamics in a Wet Tropical Forest

Abstract Decades of studies on the role of in (C) and (N) cycling have focused on organic matter (OM) of plant origin. Despite potentially large inputs of belowground OM from fungal walls and invertebrate , studies of the decomposability of their major constituent, chitin, are scarce. To explore effects on soil C dynamics of chitin, in comparison with two plant-derived chemicals, cellulose and lignin, I conducted a field-based chemical-addition experiment. The design contained three chemical treatments plus a control, with four replicates in each of two species of tropical trees grown in plantations. The chemicals were added in reagent-grade form at a rate that doubled the natural detrital C inputs of 1000 g C m−2 y−1. Despite its purported recalcitrance, chitin was metabolized quickly, with soil respiration (R soil) increasing by 64% above the control within days, coupled with a 32% increase in soil extractable ammonium. Cellulose, which was expected to be labile, was not readily decomposed, whereas lignin was rapidly metabolized at least partially in one of the forest types. I examined effects of stoichiometry by adding to all treatments ammonium nitrate in a quantity that adjusted the C:N of cellulose (166) to that of chitin (10), using both field and in vitro experiments. For cellulose, CO2 release increased more than five- to eightfold after N addition in root-free soil incubated in vitro, but only 0–20% in situ where roots were intact. By the end of the 2-year-long field experiment, fine-root biomass tended to be higher in the chitin treatment, where R soil was significantly higher. Together these findings suggest that soil N availability limited cellulose decomposition, even in this Neotropical forest with high soil N stocks, and also that trees successfully competed for N that became available as chitin decomposed. These results indicate that the major constituent of cell walls of soil fungi, chitin, can decompose rapidly and release substantial N that is available for plant and microbial growth. As a consequence, soil fungi can stimulate soil OM decomposition and N cycling, and thereby play a disproportionate role in ecosystem C and N dynamics.

Keywords cellulose, chitin, decomposition, lignin, nitrogen, organic matter stoichiometry, fungi

Disciplines Botany | Environmental Chemistry | Natural Resources Management and Policy | Organic Chemistry | Plant Biology | Soil Science

Comments This is a manuscript of an article from Ecosystems 17 (2014): 918, doi:10.1007/s10021-014-9769-1. Posted with permission.

This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/nrem_pubs/94 This is a manuscript of an article from Ecosystems 17 (2014): 918, doi:10.1007/s10021-014-9769-1. Posted with permission.

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ArticleTitle Unexpected Effects of Chitin, Cellulose, and Lignin Addition on Soil Dynamics in a Wet Tropical Forest Article Sub-Title Article CopyRight Springer Science+Business Media New York (This will be the copyright line in the final PDF) Journal Name Ecosystems Corresponding Author Family Name Russell Particle Given Name Ann E. Suffix Division Department of Natural Resource Ecology and Management Organization Iowa State University Address Ames, Iowa, 50011, USA Email [email protected]

Received 9 October 2013 Schedule Revised Accepted 20 March 2014 Abstract Decades of studies on the role of decomposition in carbon (C) and nitrogen (N) cycling have focused on organic matter (OM) of plant origin. Despite potentially large inputs of belowground OM from fungal cell walls and invertebrate exoskeletons, studies of the decomposability of their major constituent, chitin, are scarce. To explore effects on soil C dynamics of chitin, in comparison with two plant-derived chemicals, cellulose and lignin, I conducted a field-based chemical-addition experiment. The design contained three chemical treatments plus a control, with four replicates in each of two species of tropical trees grown in plantations. The chemicals were added in reagent-grade form at a rate that doubled the natural detrital C inputs of 1000 g C m−2 y−1. Despite its purported recalcitrance, chitin was metabolized quickly, with soil respiration (R soil) increasing by 64% above the control within days, coupled with a 32% increase in soil extractable ammonium. Cellulose, which was expected to be labile, was not readily decomposed, whereas lignin was rapidly metabolized at least partially in one of the forest types. I examined effects of stoichiometry by adding to all treatments ammonium nitrate in a quantity that adjusted the C:N of cellulose (166) to that of chitin (10), using both field and in vitro experiments. For cellulose, CO2 release increased more than five- to eightfold after N addition in root-free soil incubated in vitro, but only 0–20% in situ where roots were intact. By the end of the 2-year-long field experiment, fine-root biomass tended to be higher in the chitin treatment, where R soil was significantly higher. Together these findings suggest that soil N availability limited cellulose decomposition, even in this Neotropical forest with high soil N stocks, and also that trees successfully competed for N that became available as chitin decomposed. These results indicate that the major constituent of cell walls of soil fungi, chitin, can decompose rapidly and release substantial N that is available for plant and microbial growth. As a consequence, soil fungi can stimulate soil OM decomposition and N cycling, and thereby play a disproportionate role in ecosystem C and N dynamics. Keywords: (separated by '-') cellulose - chitin - decomposition - lignin - nitrogen - organic matter stoichiometry - fungi Footnote Information Electronic supplementary material The online version of this article (doi:10.1007/s10021-014-9769-1) contains supplementary material, which is available to authorized users. Metadata of the article that will be visualized in OnlineAlone

Electronic supplementary Below is the link to the electronic supplementary material. material MOESM1: Supplementary material 1 (DOCX 28 kb). Ecosystems DOI: 10.1007/s10021-014-9769-1 Ó 2014 Springer Science+Business Media New York

1

2 Unexpected Effects of Chitin,

3 Cellulose, and Lignin Addition F

4 on Soil Dynamics in a Wet

5 Tropical Forest

6 7 Ann E. Russell*

Author Proof 8 9 Department of Natural Resource Ecology and Management, Iowa State University, Ames, Iowa 50011, USA 10 PROO 11 ABSTRACT 12 Decades of studies on the role of decomposition in ammonium nitrate in a quantity that adjusted the 36 13 carbon (C) and nitrogen (N) cycling have focused C:N of cellulose (166) to that of chitin (10), using 37 14 on organic matter (OM) of plant origin. Despite both field and in vitro experiments. For cellulose, 38 15 potentially large inputs of belowground OM from CO2 release increased more than five- to eightfold 39 16 fungal cell walls and invertebrate exoskeletons, after N addition in root-free soil incubated in vitro, 40 17 studies of the decomposability of their major con- but only 0–20% in situ where roots were intact. By 41 18 stituent, chitin, are scarce. To explore effects on soil the end of the 2-year-long field experiment, fine- 42 19 C dynamics of chitin, in comparison with two rootTED biomass tended to be higher in the chitin 43 20 plant-derived chemicals, cellulose and lignin, I treatment, where Rsoil was significantly higher. 44 21 conducted a field-based chemical-addition experi- Together these findings suggest that soil N avail- 45 22 ment. The design contained three chemical treat- ability limited cellulose decomposition, even in this 46 23 ments plus a control, with four replicates in each of Neotropical forest with high soil N stocks, and also 47 24 two species of tropical trees grown in plantations. that trees successfully competed for N that became 48 25 The chemicals were added in reagent-grade form at available as chitin decomposed. These results indi- 49 26 a rate that doubled the natural detrital C inputs of cate that the major constituent of cell walls of soil 50 27 1000 g C m-2 y-1. Despite its purported recalci- fungi, chitin, can decompose rapidly and release 51 28 trance, chitin was metabolized quickly, with soil substantial N that is available for plant and micro- 52 29 respiration (Rsoil) increasing by 64% above the bial growth. As a consequence, soil fungi can 53 30 control within days, coupled with a 32% increase stimulate soil OM decomposition and N cycling, 54 31 in soil extractable ammonium. Cellulose, which and thereby play a disproportionate role in eco- 55 32 was expected to be labile, was not readily decom- system C and N dynamics. 56 33 posed, whereas lignin was rapidly metabolized at 34 least partially in one of the forest types. I examined Key words: cellulose; chitin; decomposition; lig- 57 35 effects of stoichiometry by adding to all treatments nin; nitrogen; organic matter stoichiometry; fungi. 58 59

60 INTRODUCTION 61 Received 9 October 2013; accepted 20 March 2014 Decomposition of organic matter (OM) and its ac- 62 Electronic supplementary material: The online version of this article crual and persistence in soil are processes that 63 (doi:10.1007/s10021-014-9769-1) contains supplementary material, 64 which is availableUNCORREC to authorized users. influence a variety of ecosystem services, including *Corresponding author; e-mail: [email protected] soil fertility, water availability, resistance to erosion, 65

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66 and mitigation of climate change. The effect of the simultaneously. Potentially confounding factors 121 67 chemistry of OM inputs is integral to our concep- include quantity and allocation of detrital inputs, 122 68 tual framework for understanding how plant spe- nutrient availability unrelated to plant tissue 123 69 cies composition influences soil organic matter quality, and unknown or unmeasured chemical 124 70 (SOM) dynamics. It has long been recognized that constituents, other litter traits such as toughness, 125 71 plant species differ in various aspects of their litter and plant–mycorrhizal associations that influence 126 72 chemistry, which in turn may influence the decay plant access to limiting nutrients, as hypothesized 127 73 process (Waksman 1929; Minderman 1968). Litter by Ha¨ttenschwiler and others (2011). Also,F this 128 74 stoichiometry, especially C:N, is expected to drive plant-based focus ignores effects on SOM decom- 129 75 decomposition, owing to trade-offs in delivery of an position of the potentially large OM inputs from 130 76 energy source (C compounds) and nutrients, mycorrhizal fungi and their decomposibility. 131 77 especially N. Because the C:N of plant litter can The goal of this study was to address the question 132 78 vary widely, from 14 to 87, and it is generally of whether different carbon compounds influenced 133 79 higher than the C:N of and fungi, 5–17, soil C dynamics differently. To examine the effect 134 80 plants can vary in the extent to which they limit of OM chemistry alone on soil, I applied a set of 135 81 nutrients needed for growth and activity of pure chemicals that vary in C:N and structural 136 82 microbial decomposers (Cleveland and Liptzin complexity and then measured the effects on soil C 137 83 2007). Fungi are relatively under-studied, but dynamics. Decomposition has been described as a 138 84 139 Author Proof Koide and Malcolm (2009) found that decomposi- two-stage process: comminution, the breakdown of 85 tion in ectomycorrhizal (EM) fungi was also cor- organic matter into smallerPROO pieces by detritivores; 140 86 related with tissue N concentration. and chemical breakdown by bacteria and fungi 141 87 Decomposition is not always predicted from (Aerts 1997). I focused on this latter stage by 142 88 stoichiometric ratios of plant litter (Aerts 1997; applying the chemicals in powder or flake form and 143 89 Cornwell and others 2008); however, secondary selected three compounds that are abundant in 144 90 compounds such as lignin or phenol, as well as nature. 145 91 specific leaf area, are often correlated with Cellulose and lignin are the second and third 146 92 decomposition (Meentemeyer 1978;Ha¨ttenschw- most abundant classes of biochemicals in plants. 147 93 iler and Vitousek 2000; Cornwell and others 2008). Chitin is the basic unit of cell walls of many fungi 148 94 Thus, molecular complexity of plant litter is also and exoskeletons of . Cellulose and 149 95 expected to influence decomposition. These con- chitinTED are homopolymers of in which the 150 96 cepts, based on studies of decomposition of fresh glucose units are linked by b-(1-4)-glycosidic 151 97 litter from plant species that differ in tissue chem- bonds, but chitin differs in that one hydroxyl group 152 98 istry, have informed our historical view of soil C on each is replaced with an acetyl 153 99 cycling. That is, stabilized SOM consists mainly of group. This allows for increased bonding 154 100 decomposed plant litter and microbially synthe- between adjacent , giving the chitin– 155 101 sized humic substances possessing a chemical matrix increased strength. The hydrolytic 156 102 complexity and composition that is resistant to enzymes involved in decomposition of cellulose 157 103 decomposition by microbes (Schmidt and others and chitin are highly substrate specific in that their 158 104 2011). Decomposition of fresh plant litter is not catalytic efficacy is based on the structural 159 105 always correlated with these plant traits (for arrangement/chemical bonds in the substrates 160 106 example, Ha¨ttenschwiler and others 2011; Freschet (Caldwell 2005). In contrast structurally, lignin is a 161 107 and others 2012); however, recent advances have high molecular, three-dimensional 162 108 revealed that persistence of SOM involves a com- consisting of phenyl propane units (Ko¨ gel-Knabner 163 109 plex interplay among an array of biological and 2002). The oxidoreductases involved in their min- 164 110 environmental factors (Schmidt and others 2011). eralization, such as lignin peroxidase, tend to be 165 111 Part of the problem in evaluating the role of less substrate specific. The reactivity of these het- 166 112 compound chemistry alone lies in the fact that eropolymers is determined by the identity of their 167 113 multiple traits vary simultaneously within a single heterogeneous and the linkages con- 168 114 plant species. As such, comparison of litter necting these monomers (Nierop and others 2006; 169 115 decomposition among multiple species—the main Schweitzer and others 2008). Lignin’s structure is 170 116 in vivo approach which has informed our theoret- thus generally considered to be the basis for its 171 117 ical constructs to date—constitutes an uncontrolled recalcitrance. However, its macromolecular struc- 172 118 observational study with respect to plant traits and ture and cross-linkages determine the redox po- 173 119 their controlUNCORREC over decomposition. That is, plant tential within the macromolecule, which in turn 174 120 factors other than the measured factor may co-vary influences its decomposability (Wong 2009). Thus, 175

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176 peroxidases could have substrate-specific catalytic In comparing the control with three model 231 177 abilities, depending on how the redox potential is compounds applied as treatments (cellulose, chitin, 232 178 influenced by the macromolecular structure. and lignin), with and without N addition, I tested 233 179 The objective of this study was to compare the the following alternate hypotheses: 234 180 effects of these three compounds on soil C dynamics 235 181 (1) Rapidity of degradation would range from and to gain insight into the mechanisms driving 236 182 fastest in cellulose to slowest in lignin, by the these effects. To that end, I applied the compounds 237 183 rationale that structural complexity regulates in their pure forms, with and without NH4NO3, 238 184 decomposition. Therefore, soil respirationF both in vitro and in a field experiment conducted 239 185 (Rsoil) was expected to be highest in cellulose for 2.5 years within an established long-term 240 186 and lowest in lignin. Added lignin that did not experiment at La Selva Biological Station in Costa 241 187 decompose would therefore accrue as soil C to Rica. This wet lowland tropical forest site offered 242 188 a greater extent than additions of cellulose or several advantages for achieving the objectives. (1) 243 189 chitin. Thus, soil C would increase the most Conditions are ideal for plant growth and OM 244 190 under lignin addition by the historical concepts decomposition, given the site’s warm climate with 245 191 of soil OM persistence described above. abundant rainfall. Thus, results for decomposition 246 192 (2) Lignin would degrade faster in Pentaclethra then studies come quickly, given that both C and N cy- 247 193 in Vochysia. With higher lignin concentrations cling are rapid and soil C and N stocks are very high 248 194 in Pentaclethra tissues, microbial communities Author Proof (Russell and others 2007; Raich and others 2009; more specialized in lignin decomposition were 249 195 Russell and others 2010; Russell and Raich 2012). expected to be presentPROO in this species. 250 196 (2) Tropical Oxisols are widely believed to be de- (3) Stoichiometry, in particular the C:N, would not 251 197 pleted in phosphorus (P), and soil extractable P in have a significant effect on decomposition of 252 198 this particular site is low (Russell and others 2007). the model compounds, by the reasoning that 253 199 Thus, I anticipated that P, rather than N, would be soil N stocks are very high and rates of N cy- 254 200 more limiting to decomposition, such that differ- cling are extremely rapid in this site (Russell 255 201 ences in the C:N of the model compounds would and Raich 2012). Thus, N was not expected to 256 202 not have an impact. (3) The tropical setting could limit decomposition in any of the compounds, 257 203 provide a broader perspective on lignin decompo- given the substantial N release from the natural 258 204 sition, given that the expected higher diversity of background of decomposing detritus, which 259 205 microbes would increase the likelihood of lignin TEDincludes both litter and SOM. 260 206 specialists that may not occur in higher latitudes. 261 207 (4) The pre-existing experimental setting allowed 262 208 for comparisons of the model compounds within 209 two forest types which differed only in the domi- 210 nant tree species. Set within the context of a larger METHODS 263 211 randomized complete block experiment, edaphic Site Description 264 212 factors were similar across the experimental units of 213 subplots used for chemical additions that were sit- I conducted two chemical-addition experiments in 265 214 uated within the main plots. The two native tree subplots within experimental plantations situated 266 215 species in this study differ in the chemistry of their at La Selva Biological Station in northeastern Costa 267 216 detrital inputs, but not in the quantity (see Rica (10°26¢N, 83°59¢W). The mature forest on this 268 217 Appendix 1 in Supplementary material). Litter and land had been felled in 1955, after which pasture 269 218 foliar N concentrations are relatively high and was established and grazed until abandonment in 270 219 similar in the two species, Pentaclethra macroloba 1987. In 1988 mono-dominant plantations of 11 271 220 (Willd.) Kunth. (Leguminosae, nodulated) and Vo- tree species were planted in this abandoned pasture 272 221 chysia guatemalensis Donn. Sm. (Vochysiaceae, not in a randomized complete block design with four 273 222 nodulated) (Raich and others 2007). Foliar d15N blocks (Fisher 1995). Thus, only the dominant tree 274 223 data indicate that both species promote biological N species differs among the experimental plantations, 275 224 fixation however (Russell and Raich 2012); thus, which all have similar parent material, soil type, 276 225 neither species was expected to be N limited. Newly climate, and topography, and thus minimal differ- 277 226 senesced leaves and fine roots of these species differ ences in other state factors sensu Jenny (Amund- 278 227 primarily in their carbon chemistry, with lignin son and Jenny 1997). 279 228 higher in Pentaclethra and water-soluble C com- The study site has a mean annual temperature of 280 229 pounds higherUNCORREC in Vochysia (Raich and others 2007; 25.8 °C. Mean annual rainfall is 4000 mm, with 281 230 Russell and others 2007). rainfall generally greater than 100 mm in all 282

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283 months (Sanford and others 1994). The soils in this Subplot Establishment 333 284 site are deep, volcanically derived, and classified as Within the main 50 9 50 m plot of each tree spe- 334 285 Oxisols (Mixed Haplic Haploperox, Kleber and cies in each replicate, four 1 9 1 m subplots were 335 286 others 2007). Despite the assumptions that La Selva randomly located within the relatively flat areas of 336 287 soils are greater than 1 Ma old and were formed the plots on hilltops. To avoid complications 337 288 in situ, these soils have relatively higher levels of resulting from overland flow or flooding, sloping 338 289 rock-derived Sr and macronutrient concentrations areas and toes of slopes were excluded from con- 339 290 (including P), in comparison with similarly aged sideration in siting the subplots. Chemical treat-F 340 291 Hawaiian and other continental soils (Porder and ments were randomly assigned among the four 341 292 others 2006). subplots which were situated within a 10-m radius 342 of each other within the main plot. In January 343 293 Field and Laboratory Methods 2008, each subplot was framed with PVC tubing, 344 with the corners firmly anchored with metal stakes. 345 294 Approach Each subplot was equipped in the center with one 346 295 The concept of these chemical-addition experi- 20-cm diameter PVC soil respiration collar, for a 347 296 ments was to apply model compounds in sub-plots total of 32 collars. The sharpened collar edge was 348 297 within the pre-existing experiment, with the inserted to a depth of only about 1 cm, deep en- 349 298 quantity of compounds applied large enough to ough to maintain a seal, but not to impede fine- 350 Author Proof 299 create measurable changes in response variables. root growth substantively. Comparisons of fine- 351 300 Doubling the OM inputs was thus the goal, to be root biomass in the mainPROO part of the plot (outside 352 301 achieved by adding 1000 g C m-2 y-1 of the model collars) confirmed this. The structure of this forest 353 302 compound to the natural background detrital in- soil derived from volcanic parent material is such 354 303 puts of approximately 1000 g C m-2 y-1 (mean that this soil is exceptionally well-drained on hill- 355 304 across species, Russell and others 2010). The tops. Thus, despite high rainfall, flooding did not 356 305 quantity of chemical required for delivering occur in the collars in this experiment. 357 306 1000 g C m-2 y-1 to the soil was calculated using For Experiment 2, a second soil respiration collar 358 307 the compound’s molecular formula. The response was installed within each of the 32 subplots for 359 360 308 variables measured included Rsoil, change in soil C addition of the model compound plus NH4NO3 (+N 309 and N stocks, extractable inorganic soil N, and fine- treatment), whereas the remainder of the subplot 361 310 root biomass; sampling depth was 0–15 cm. receivedTED only the former (0 N treatment). Thus 362 311 The experimental design of Experiment 1 con- there were n = 64 soil respiration collars. This 363 312 sisted of four treatments, three chemical amend- experiment lasted 6 months. 364 313 ments plus a control: Chemical Application 365 2 Tree Species  4 Chemical Treatments Following a 4-month calibration period after 366  4 Replicates ¼ 32 unitsðÞ subplots : establishment of the subplots for Experiment 1, the 367 315315 The formulae, C:N, and Sigma-Aldrich catalog # of chemical amendments began. The calibration per- 368 316 the compounds added were the following: cellu- iod was 3 months in Experiment 2. The same 369 370 317 lose: [C12H8O10], S3504; chitin: [C8H13O5N1], application procedure was used in both experi- 371 318 C7170; and lignin: [C10H12O2], 471003. Their ments. First, the litter layer was removed and 319 respective C:N values were 165.7, 9.6, and 244.6, as stored temporarily on a plastic sheet nearby. The 372 320 determined from analyses of the purchased chem- litter layer within the soil respiration collar was 373 321 icals by dry combustion (Thermo-Finnigan EA stored separately for its return there. The pre- 374 322 Flash Series 1112, CE Elantech, Lakewood, NJ). weighed dose of chemical was spread evenly over 375 323 Experiment 1 was conducted for two years, after the litter-free subplot, with a pre-weighed dose 376 324 which Experiment 2 was initiated in which an N allocated for the soil respiration collar, and another 377 325 treatment was added within each subplot of the dose for the remainder of the plot. The compound 378 326 four ongoing chemical treatments in Experiment 1, was worked into the soil to a depth of about 1 cm 379 380 327 for n = 64 units. Ammonium nitrate (NH4NO3) briefly with a fork. The litter layer was then re- 328 was added to all four chemical treatments in the placed, with the collar litter returned to its appro- 381 329 quantity required to adjust cellulose C:N to 9.6, priate collar. The control was given this same 382 330 that of chitin. This is hereafter referred to as the treatment, except that no chemical was added. Half 383 331 ‘‘+N’’ treatment,UNCORREC whereas treatments not receiving of the annual chemical addition was added 384 332 N are referred to as ‘‘0 N.’’ approximately every six months, except for a delay 385

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386 due to inclement weather in Nov–Dec 2008. To the calibration period, daily for the first week 441 387 the effect of dose size, chemicals were added at 1-, immediately following addition of chemicals; 1–2 442 388 2- and 3-month intervals, with the doses adjusted times per week for the week following the chemical 443 389 accordingly, during the last 6 months (that is, addition, and every 2 weeks thereafter. Cumulative 444 390 Experiment 2). Thus, chemicals were added on annual Rsoil was calculated by multiplying the mean 445 391 May 21, 2008, Jan 14, 2009, and April 22, 2009, value between one time point and the next by the 446 392 May 24–25, 2010, June 28–29, 2010, and August time elapsed between the two times and summing 447 393 30–31, 2010. The masses of chemicals added in these values over an annual time period. F 448 394 total were: cellulose 40 kg; chitin, 38 kg; and lig- Extractable inorganic N was measured at the end 449 395 nin, 24 kg. of Experiment 1, in February 2010 on soil samples 450 396 Soil organic carbon (SOC) and total N were (0–15 cm) also collected for measurement of SOC 451 397 sampled in the 0–5 and the 0–15 cm layers before and total N. As soon as samples were collected, they 452 398 chemicals were applied using a 3.2-cm push-tube were placed in Ziploc bags, brought to the lab on 453 399 sampler at randomly selected locations within the ice, and processed immediately. Extractions of ni- 454 - + 400 subplots. Eight sub-samples per subplot were trate (NO3 ) and ammonium (NH4 ) were con- 455 401 bulked to provide a single sample per subplot. To ducted with 2 M potassium chloride (KCl). Soil 456 402 determine changes in soil C and N stocks (DSOC, solutions with a 1:5 dry soil/KCl ratio were shaken 457 403 DSoil N, 0–15 cm), I re-sampled soil in February for 30 min, allowed to settle for 30 min, and then 458 404 459 Author Proof 2010 at the end of Experiment 1. Carbon and N filtered through No. 42 Whatman . Two 405 stocks were determined from these concentration blanks were also extractedPROO at every sample time to 460 406 data in combination with bulk density data as in account for contaminant nitrate and ammonium in 461 407 Russell and others (2007). Changes in stocks were the filters and vial. Filtrates were kept frozen and 462 408 calculated as the difference in SOC (or soil N) be- transported on dry ice to Iowa State University 463 409 tween final and initial values. Soil samples were where they were analyzed colorimetrically for 464 - + 410 analyzed for total C and N by dry combustion as NO3 –N and NH4 –N using an automated ion 465 411 described above for the model compounds. These analyzer (QuickChem 4100, Lachat Instruments 466 412 soils do not contain carbonates, so SOC equals total Division, Zellweger Analytics, Inc., Milwaukee, 467 413 soil C. WI). Field-moist subsamples of 5 g were dried at 468 414 To evaluate whether overland flow of the added 105°C for 48 h to convert measurements to a dry- 469 415 chemicals had occurred, soil was re-sampled in the weightTED basis. 470 416 0–5 cm layer 6 weeks after the first chemical 417 addition. , including cellulose-de- Fine-Root Biomass 471 418 rived glucose, were measured through extraction of Within each subplot, a single soil core, 15-cm deep, 472 419 soil with methanesulfonic acid, separation by anion was collected from the center of each soil collar at 473 420 exchange chromatography, and detection by the end of the first experiment. Soil samples were 474 421 pulsed amperometry using a Dionex DX 500 anion extracted using a hand-operated slide-hammer 475 422 chromatograph equipped with a CarboPac PA-10 metal corer, 5.37 cm inner diameter. Initial sepa- 476 423 column (2 mm 9 250 lm). Soil glucose measure- ration of roots from soil was done within 24 h of 477 424 ments corroborated findings and visual Rsoil collection, using a hydropneumatic elutriation 478 425 inspection of the white cellulose powder; all indi- system (mesh size of 530 lm; Smucker and others 479 426 cations were that the added cellulose was still re- 1982), as in Russell and others (2004). Roots were 480 427 tained in the soil six weeks after its addition. Thus, separated from detritus by hand and live roots were 481 428 loss of chemicals via erosion was deemed negligi- distinguished from dead roots, based on morpho- 482 429 ble. logical characteristics. Samples were dried at 65°C. 483 430 Soil respiration (R ) was measured with an soil Only live roots were included in biomass totals. 484 431 LI-8100 automated soil CO2 flux system and 8100- 432 Ò 102 (20-cm diameter) chamber (LI-COR Biosci- Lab Incubation Experiment 485 433 ences, Lincoln, Nebraska, USA). Chambers were 486 434 monitored for 240 s after closure; fluxes were cal- To assess CO2 respired from soil without live roots, I 435 culated based on the final 150 s of measurements. conducted an in vitro experiment, measuring po- 487 436 Soil respiration was measured at various frequen- tential C mineralization in field-moist soil (0–5 cm) 488 437 cies; the timing was determined with the goal of from the control subplots of Vochysia and Pentacle- 489 438 capturing peak degradation of the model com- thra in March 2010. One 50-g sample from Block 1 490 439 pounds, butUNCORREC also avoiding over-sampling so as not to in each of the two species was collected and 491 440 compact the soil. We sampled every 2 weeks during transported to ISU on ice. Roots and detritus were 492

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493 removed and each sample was homogenized well. chysia and Pentaclethra, respectively (Figure 1A). 541 494 Gravimetric moisture content was measured and With the addition of the first dose of chemicals, Rsoil 542 495 found to be similar in the two samples, so no responded immediately to chitin and lignin, but not 543 496 moisture adjustments were made. From each of the to cellulose addition (Figure 1, right panels provide 544 497 two homogenized samples, 24 2-g subsamples were magnified view). Within one day of lignin addition, 545 498 weighed and mixed with the model compound(s), Rsoil was 69 and 33% higher in the two species, 546 499 with treatment assignments randomized and the peaking at 10.37 (±0.51) and 6.23 (±0.42) lmol 547 500 chemical treatments the same as in field Experi- Cm-2 s-1 in Vochysia and Pentaclethra, respectively.F 548 501 ment 2. With three lab replicates for each treat- The response tapered off quickly in the lignin 549 502 ment, the design was: treatment to fall below rates in the control. With 550 chitin addition, soil respiration peaked at 30 days in 551 2 Species  4 Chemical Treatments -2 Vochysia when Rsoil was 16.76 (±1.32) lmol C m 552  2N Treatments  3 Replicates ¼ 48: s-1 and in Pentaclethra at 9.66 (±0.86) lmol C m-2 553 -1 554 504504 s 17 days after addition. By the third addition, Rsoil The soil plus chemical amendment(s) were placed ± 555 505 under chitin addition peaked at 26.87 ( 1.23) and in 2.5 cm-diameter incubation tubes and sealed ± -2 -1 556 506 23.94 ( 3.01) lmol C m s in Vochysia and Pen- tightly with butyl rubber septa. Incubated samples 557 507 ° taclethra, respectively. were kept at 21 C. Over a 30-day-period, the rate 558 508 Cumulative annual Rsoil was highest in chitin, of CO2–C released was measured periodically (be- 559 Author Proof but the extent of this response differed between the 509 fore CO concentrations reached 4%) by flushing 2 two species, such that the species 9 chemical 560 510 the flask headspace through an infrared gas ana- PROO interaction was significant (P < 0.0001). In Vo- 561 511 lyzer (LI-820, LI-COR Biosciences, Inc., Lincoln, -2 -1 chysia, Rsoil of 4604 (±290) g C m y in chitin 562 512 NE). The incubation-tube atmosphere was kept - was significantly higher than 2363 (±330) g C m 563 513 hydrated by piping the air supply through water. - 2 y 1 in the control and all other treatments 564 565 514 Statistical Analysis (P = 0.0001) (Figure 2). In Pentaclethra, Rsoil of 2748 (±186) in chitin was greater than 2200 566 515 Statistical analyses were conducted using the SAS (±318) g C m-2 y-1 in the control and the other 567 516 System (Version 9.3, SAS Institute 2002–2010). treatments (P = 0.0002). Lignin’s Rsoil of 1482 568 517 Differences in responses were tested using a mixed (±81) was significantly lower than in the control. 569 518 model in PROC MIXED, with tree species, chemical TED 519 compound and N-addition treated as fixed and the Soil Carbon and Fine Roots 570 520 block or replicate as random effects (Littell and 571 521 The effects of the added chemicals on changes in others 1996). A split-plot design was used for 572 522 soil C and N stocks (0–15 cm) were similar in the chemical treatments (subplots) applied within tree 573 523 two tree species, with no significant species or species (main plots) (n = 32), and a split–split–plot 9 574 524 species chemical interaction effects (Appendix 1 design for N addition (sub-subplots) within subplots D 575 525 in Supplementary material). Both responses, soil of chemical treatments (n = 64). I tested for homo- D 576 526 C and soil N, were significantly higher in the geneity of variances and normality of distributions. chitin treatment (Figure 3). The chemicals also had 577 527 For the response variable in the laboratory incuba- similar effects in both species with respect to 578 528 tion experiment (CO –C respired), a natural log 2 extractable inorganic soil N. Mean extractable soil 579 529 transformation was required and statistical test re- NH -N in chitin was significantly higher than in the 580 530 sults reflect analyses of the transformed variable. 4 other three treatments, by 32% (Figure 4). 581 531 Differences among individual means were compared Extractable soil NO –N was highly variable, such 582 532 using least squares means with a ‘‘pdiff’’ statement. 3 that so significant differences were detected. 583 533 Pearson’s correlation analysis was used to evaluate Fine-root biomass tended to be higher in Vochysia 584 534 the relationship between and fine-root biomass. Rsoil than in Pentaclethra, and within species it was 585 generally higher in the chitin treatment (Figure 5). 586 535 RESULTS At the end of Experiment 1, the positive correlation 587 of fine-root biomass with the cumulative annual 588 536 Chemical Addition Effects Rsoil was significant (P = 0.0046). 589 537 Soil Respiration 590 538 During the 4-month-calibration period at the Nitrogen Addition Effects 539 591 beginning ofUNCORREC the experiment, Rsoil averaged 6.12 In laboratory incubations, the response to N addi- 540 (±0.12) and 4.67 (±0.09) lmol C m-2 s-1 in Vo- tion was complex, with significant chemical 9 N 592

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Author Proof PROO

Figure 1. Soil respiration response to cellulose, chitin, and lignin addition. A Data over entire 2-year-period for planta- tions of Vochysia guatemalensis (upper panel) and Pentaclethra macroloba (lower panel). Arrows indicate date of chemical addition. B Detailed data immediately following chemical application on three dates within a species (three panels across) for each of the two species. Data are normalized by subtracting valuesTED within a plot from the Control (red line = 0). In all figures, error bars represent standard errors of the mean.

Figure 3. Change in soil C and N stocks (0–15 cm) after Figure 2. Cumulative annual soil respiration under four doubling C inputs with additions of CEllulose, CHitin, chemical addition treatments in tree plantations of the and LIgnin in comparison with no additions (COntrol). two species, Vochysia guatemalensis and Pentaclethra mac- Positive change denotes accumulation and negative de- roloba. Lowercase letters denote differences among treat- notes loss. Effect of tree species was not significant so ments. values are means within treatments across two tree species for N = eight replicates per chemical. Lowercase 593 interactions and differing trends between the two letters denote differences among treatments for both DSoil 594 species (see Appendix 2 in Supplementary materi- C and DSoil N because results were the same for both 595 als). Nevertheless,UNCORREC there was a clear response in variables.

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Figure 4. Soil extractable inorganic N (ammonium, NH4–N and nitrate, NO3–N) under the different chemical treatments. Chemical treatments were: COntrol; CEllu- lose; CHitin; and LIgnin. N = four replicates per chemical Figure 6. Soil CO2–C respired over 30 days during within each plantation. Soil data are for the 0–15 cm in vitro chemical-addition experiment. Ammonium-ni- depth. Effect of tree species was not significant so values trate N (denoted by ‘‘+’’ and cross-hatched bars) was added are means within treatments across two tree species for to each of the four chemicals, in comparison with no addition (denoted by ‘‘0’’). Chemicals are identified by

Author Proof N = eight replicates per chemical. Different lowercase letters their first two letters: COntrol; CEllulose; CHitin; and signify differences in NH4–N among the four chemicals. LIgnin. Means are for PROON = three lab replicates per chemical within each of the two tree species, Vochysia guatemalensis and Pentaclethra macroloba. Different lowercase letters signify differences in responses among the four chemicals with no N addition; uppercase letters are for the +N treatment; asterisks denote differences between N treatments within chemicals. TED

Figure 5. Soil respiration increased significantly with fine-root biomass. All data are shown, with N = four replicates per chemical within each plantation. Chemi- cals added were: COntrol; CEllulose; CHitin; and LIgnin. Pema Pentaclethra macroloba, Vogu Vochysia guatemalensis, DM dry matter.

Figure 7. Soil respiration following additions of four 596 both species on one aspect: N addition stimulated 597 chemicals in the field, with and without ammonium ni- mineralization of cellulose more than eightfold in trate. Chemicals are identified by their first two letters: 598 Vochysia and more than fivefold in Pentaclethra, COntrol; CEllulose; CHitin; and LIgnin. Nitrogen-amen- 599 increasing CO2–C release rates relative to those in ded treatment is denoted by ‘‘+’’ (cross-hatched bars) and 600 the chitin treatment (Figure 6). Nitrogen also sig- unamended by ‘‘0.’’ Letters denote significant differences 601 nificantly depressed degradation of lignin in both among chemical treatments. There were four replicates 602 species. Although N addition increased respiration per treatment within each tree plantations of the two 603 in the Vochysia control, the effect was not signifi- species, Vochysia guatemalensis and Pentaclethra macroloba. 604 cant in Pentaclethra. 605 In the field experiment, the effect of N addition was added to cellulose (Figure 7). In the field, ad- 609 606 was also complex (Appendix 2 in Supplementary ded N significantly increased Rsoil in all chemical 610 607 materials). TheUNCORREC results differed from the in vitro treatments in Vochysia, by 20% in chitin, whereas 611 608 experiment in the lack of a strong response when N there was no significant effect in Pentaclethra in any 612

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613 treatment. As in the first field experiment, Rsoil however, this suggests that reducing the C:N of 665 614 continued to be highest in the chitin treatment in lignin does not trump its structural complexity and 666 615 both species, and significantly lower in lignin in thus expose it to decomposition by new groups of 667 616 Pentaclethra. microbes, at least not to groups that were present in 668 this study site. 669 617 DISCUSSION Role of Fine Roots in Soil C and N 670 618 Compound Chemistry and Dynamics 671 619 Decomposition F Fine roots have the potential to regulate decom- 672 620 In general, the two aspects of compound chemistry position via two main effects on the microbial and 673 621 considered—structural complexity and stoichiom- fungal production of extracellular enzymes that 674 622 etry—were not very predictive of the model com- decompose and release nutrients from SOM. First, 675 623 pound’s decomposition. Cellulose, the most fine roots influence substrate availability, a major 676 624 structurally simple compound, was not readily constraint for microbial production of the enzymes 677 625 metabolized either in the laboratory or the field. in soil that depolymerize N from proteinaceous 678 626 This is consistent with Ko¨ gel-Knabner’s (2002) compounds, and thus increase N availability 679 627 observation that cellulose degrades slowly. Chitin, (Schimel and Weintraub 2003). Detrital inputs of 680 628 also a long-chain homopolymer, was expected to aboveground litter and dead fine roots both supply 681 Author Proof 629 be less labile than cellulose as a result of its in- substrates to microbes, but isotopic and biomarker 682 630 creased hydrogen bonding between adjacent poly- studies reveal the dominancePROO of root-derived sub- 683 631 mers, but it degraded rapidly. In contrast, the most strates in soil (Mendez-Millan and others 2010) 684 632 structurally complex compound—lignin—was ex- and soil microorganisms (Kramer and others 2010). 685 633 pected to decompose slowly, but instead very In addition, live root exudates supply energy-rich 686 634 quickly stimulated Rsoil in the field after all appli- and easily accessible C compounds that stimulate 687 635 cations in Vochysia and in two of three in Pentacle- microbial breakdown of SOM (Fontaine and others 688 636 thra (Figure 1, see normalized values magnified at 2003; Kuzyakov and others 2000; Blagodatsky and 689 637 right). The soil-respiration response to lignin was others 2010). 690 638 one fourth that of chitin addition, but nevertheless Second, fine roots influence soil nutrient con- 691 639 some decomposers responded rapidly to lignin. centrations and can successfully compete with 692 640 Interestingly, the response to lignin was minimal in microorganismsTED for these nutrients, owing to the 693 641 Pentaclethra, even though this species has higher capacity of fine roots to grow, form associations 694 642 foliar and fine-root lignin concentrations, such that with mycorrhizal fungi, and intercept and take up 695 643 decomposers adapted for lignin decomposition nutrients over space and time (Schimel and Ben- 696 644 were expected to be present. Apparently, Vochysia nett 2004). These plant-driven effects on soil 697 645 has modified soil conditions for decomposers, such nutrients influence microbial dynamics because 698 646 that Rsoil responses were higher under all treat- microbial enzyme-producing potential is linked to 699 647 ments, including lignin. A limited group of the stoichiometry of their nutrient demands (Sin- 700 648 decomposers, the white-rot fungi, are purported to sabaugh and others 2008). Differences among plant 701 649 be capable of complete decomposition of lignin, species in these root dynamics, coupled with their 702 650 although soft rot and brown rot fungi may whole-plant sink for nutrients and promotion of 703 651 accomplish partial decomposition (Ko¨ gel-Knabner biological N fixation, drive differences among spe- 704 652 2002; Floudas and others 2012). A consortium of cies in their regulation of soil nutrients. I hypoth- 705 653 microbial decomposers, however, is believed to esize that these differences drive variability among 706 654 mediate decomposition within soil (Haider 1992). species in the capacity to reduce soil nutrient 707 655 Given the structural similarity between cellulose availability for microbes and thereby regulate 708 656 and chitin, the results of the in vitro experiment decomposition. Ha¨ttenschwiler and others (2011) 709 657 indicate that stoichiometry explained differences in hypothesized that poor litter quality in tropical 710 658 decomposition of these two compounds. This is forests enforces starvation of decomposers. These 711 659 inferred because C mineralization in the cellu- two hypothesized mechanisms for regulation of 712 660 lose + N treatment (in which the C:N of 9.6 decomposition by tropical plants are not mutually 713 661 equaled that of chitin) was similar to rates in the exclusive; rather, both poor litter quality and 714 662 chitin treatment and represented a five- to eight- reduction of available nutrients in soil could oper- 715 663 fold increaseUNCORREC over rates in cellulose alone. Addition ate simultaneously to slow decomposition. Wein- 716 664 of N to lignin did not stimulate its decomposition; traub and others (in press) found that another 717

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718 microbially mediated process, nitrous oxide (N2O) which would in turn reduce fine-root biomass, 773 719 production, was negatively correlated with fine- thereby decreasing Rsoil and soil C accrual. Thus in 774 720 root production in this site at La Selva, with faster- the cellulose and lignin treatments, the lack of 775 721 growing trees having reduced NO3 substrate avail- positive DSOC reflects that the experimental addi- 776 -1 722 able and lower N2O emissions. tions of 1000 g C y were offset by reductions in 777 723 Fine roots also influence soil C and N stocks as a OM inputs from fine roots. Unfortunately, the d15N 778 724 result of their detrital inputs, with C and N stocks and d13C of the purchased model compounds did 779 725 changing in direct proportion to fine-root produc- not differ significantly from natural backgroundF 780 726 tivity if decomposition remains unchanged. Fine- values, but in future experiments if a distinctive 781 727 root productivity would also be expected to influ- isotopic label or biomarker could be incorporated 782 728 ence Rsoil, given that the majority of CO2 respired in into chitin, cellulose, lignin, and ammonium ni- 783 729 forest soils comes from roots (Raich and others trate, the fate of the N constituents, whether to 784 730 2014). In this site, mean Rsoil across tree species was roots or microbes, could be traced. 785 731 1920 g C m-2 y-1, of which 55% was attributed to The role of N dynamics in the degradation of the 786 732 root + rhizosphere respiration (Rrrh) (Russell and model compounds is supported by the finding that 787 733 others 2010). N addition increased cellulose decomposition 788 734 These various effects of fine roots on soil C and N in vitro, without roots present (Figure 6), indicat- 789 735 dynamics provide insight into the results presented ing that N was relatively limiting for cellulose 790 736 -2 791 Author Proof here. With the addition of 1500 g C m into each decomposition. Similarly, Barantal and others 737 of the three chemical treatments in Experiment 1 (2012) found in a P-poorPROO Neotropical forest that 792 738 over an 18-month-period, one might expect to decomposition increased with N availability be- 793 739 detect measurable effects on Rsoil and soil C stocks. cause N was required for a positive P effect. They 794 740 That is, degradation of the compounds was ex- also found that energy limitations of microbial 795 741 pected to result in increased soil and/or decompositions were co-limited by nutrient avail- 796 742 accrual of the microbial remains and/or the un- ability. 797 743 metabolized compound in soil. The results were The question in this study becomes: why was the 798 744 surprising in that only one treatment—chi- response to N addition in the field less than that in 799 745 tin—resulted in significant accrual of soil C the laboratory (Figures 6, 7)? The most likely 800 746 (647 ± 145 and 526 ± 219 g C m-2 in Vochysia explanation is that despite large soil N stocks in this 801 747 and Pentaclethra, respectively) (Figure 3). Further- site,TED fast-growing trees were not saturated with N, 802 748 more, Rsoil in the chitin treatment in Vochysia sur- such that N demand was high and roots were able 803 749 passed that in the control by 2241 g C m-2 y-1, to out-compete microbes for the added N. If in- 804 750 thus exceeding the amount of C added experi- creased uptake by roots of added N did not have 805 751 mentally by more than 1200 g (Figure 2)! In con- immediate metabolic costs, then Rsoil would not 806 752 trast, in the lignin treatment in Pentaclethra, Rsoil increase immediately, as found in this study. A 807 753 was significantly lower than the control, by 15% or sustained increase in Rsoil in response to N addition 808 754 343 g C m-2 y-1, with no significant change in soil would be expected later; however, as root biomass 809 755 C stocks. The most plausible explanation for these accrued. Unfortunately, the length of this second 810 756 findings is that addition of the model compounds experiment did not allow for the longer-term 811 757 influenced fine-root dynamics, which in turn measurements. Together, these results highlight 812 758 influenced both soil respiration and soil C stocks. the importance of considering all aspects of fine 813 759 In another wet tropical forest in Costa Rica, root dynamics in relation to soil C and N dynamics. 814 760 Cleveland and Townsend (2006) also found that 761 soil respiration responses to N fertilization were 815 762 Chitin Chemistry and its Implications for likely driven by effects on fine-root biomass. Sim- 816 763 ilarly, I hypothesize that the model compounds in N Cycling 764 my experiment had different effects on N avail- The tissues of fungi may contain high concentra- 817 765 ability, and thus differed in their influence on fine- tions of N (Clinton and others 1999; Langley and 818 766 root productivity. By this hypothesis, the concom- Hungate 2003), as a result of the high content of 819 767 itant release of N from chitin decomposition (Fig- chitin and in their cell walls (Cooke and 820 768 ure 4) stimulated fine-root growth, resulting in the Whipps 1993). Both ectomycorrhizal (EM) and 821 769 observed higher biomass (Figure 5), and thereby arbuscular mycorrhizal (AM, Glomer- 822 770 increased root and rhizosphere respiration and OM omycota) fungi can have significant belowground 823 771 inputs to soilUNCORREC C stocks. In contrast, the high C:N of biomass. In a Swedish conifer forest, Wallander and 824 772 cellulose and lignin would reduce N availability, others (2004) found that EM biomass was the same 825

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826 order of magnitude as root biomass. Arbuscular the same time, its structural arrangement and types 881 827 mycorrhizal fungi are estimated to act as a sink for of chemical bonds would promote catalytic efficacy 882 828 3–20% of host plant photosynthate (Jakobsen and for these enzymes. 883 829 Rosendahl 1990; Johnson and others 2002a, b). Chitin cycling, accompanied by its release of N, is 884 830 Nottingham and others (2010) found that AM thus hypothesized to play an important role in the 885 831 fungi contributed 1.4 ± 0.6 t C ha-1 y-1 or rapid N cycling and SOC accrual. In this tropical 886 832 14 ± 6% of total soil respiration in a moist tropical study site, these effects of N cycling were mediated 887 833 forest, and were thus an important component of via effects of N on fine roots. The findings inF this 888 834 the tropical forest C cycle. Hodge and Fitter (2010) study and that of Fernandez and Koide (2012) 889 835 discovered that AM fungi have a high demand and suggest that where soil fungi are abundant, fast 890 836 capacity to acquire N, despite the fact that as obli- decomposition of their major constituent—chi- 891 837 gate biotrophs, they lack the saprotrophic capabil- tin—provides a mechanism for promoting rapid N 892 838 ity. Thus, given their biomass and N acquisition cycling, which feeds back to stimulate C cycling. 893 839 capacity, fungi represent a global N pool on par The results from this experiment in which the OM 894 840 with fine roots. In wet tropical forests, fungal inputs were pure pinpoint more precisely the role 895 841 communities vary both spatially and temporally, that the chemistry of chitin plays in the ability of 896 842 conferring variability in their effects on nutrient soil fungi to stimulate C and N cycling. 897 843 availability (Lodge and Cantrell 1995). The binding 844 Author Proof of leaf litter by fungal connections in Basidiomy- 845 cetes can also influence nutrient cycling on tropical CONCLUSIONS 898 846 PROO forest slopes by reducing export of OM downslope In this first experimental test of pure chitin addition 899 847 (Lodge and Asbury 1988). in a lowland wet tropical forest, the surprising re- 900 848 The capacity of fungi to drive N dynamics would sult was that this compound was readily metabo- 901 849 also hinge on their decomposability. Fungal tissues lized, despite its purported recalcitrance in some 902 850 have been considered to be recalcitrant (for studies. More unexpected were the results that 903 851 example, Treseder and Allen 2000; Steinberg and cellulose was not readily decomposed in either 904 852 Rillig 2003; Driver and others 2005). Others, forest type, and that lignin was rapidly metabolized 905 853 however, have found fungal tissues to be relatively at least partially in one of the forest types. The latter 906 854 labile (Okafor 1966; Gould and others 1981). Fer- finding suggested that lignin decomposition can be 907 855 nandez and Koide (2012) also found that chitin rapidTED in the presence of lignolytic decomposers, 908 856 within the walls of EM fungi was relatively labile despite its structural complexity and high C:N. Soil 909 857 compared with other cell-wall constituents, such N stocks and N cycling in this site rank among the 910 858 that species-specific differences in decomposition highest published values for a non-agricultural site 911 859 were positively related to chitin concentration. In (Russell and Raich 2012), so the most unantici- 912 860 experimental studies, Cheng and others (2012) pated result was that N limited decomposition of 913 861 found that elevated CO2 provided a C source that cellulose and apparently also limited fine-root 914 862 stimulated degradation of SOC by AM fungi. In growth. I hypothesize that fast-growing trees with a 915 863 contrast, Langley and others (2006) demonstrated large whole-plant sink for N and high fine-root 916 864 that colonization by EM fungi slowed decomposi- growth can regulate decomposition by reducing 917 865 tion of fine roots in a Pinus edulis system. Perhaps available N pools, thereby starving decomposers. 918 866 confounding factors play a role in the disparity of These results highlight the importance of soil fungi 919 867 these findings. in N cycling in this Neotropical forest, owing to 920 868 Based on chitin’s molecular structure and C:N, high concentrations of chitin in fungal cell walls, 921 869 one would expect it to be labile in an N-limited the low C:N and lability of chitin, and thus its 922 870 setting. In my experiment, despite high rates of N capacity to release N available for uptake by plants. 923 871 cycling and high soil N stocks in this setting (Russell 924 872 and Raich 2012), inputs of pure chitin influenced ACKNOWLEDGMENTS 925 873 soil respiration within one day after its addition, 926 874 increasing annual Rsoil by 64% above the control, Jim Raich provided advice throughout all stages of 927 875 and extractable NH4–N by 32% (Figure 1). This this study and bench space for the laboratory 876 indicates that chitin is inherently extremely labile incubations, for which I am very grateful. I also 928 877 and that it supplies plant-available N (Figure 4). thank: Ricardo Bedoya, Flor Cascante, Eduardo 929 878 This supports the expectation based in chitin’s Paniagua, and Marlon Herna´ndez for assistance in 930 879 chemistry. ThatUNCORREC is, its low C:N would stimulate the field; Amy Morrow for conducting soil C and N 931 880 microbial production of hydrolytic enzymes and at analyses; Terry Grimard for the 932

Journal : 10021_ECO Dispatch : 3-4-2014 Pages : 13 9769 Article No. : h LE h TYPESET MS Code : ECO h44CP h DISK Ann E. Russell

933 analyses; and Dan Olk for interpretation of the Haider K. 1992. Problems related to the humification processes 992 934 in soils of the temperate climate. In: Bollag J-M, Stotky G, Eds. 993 carbohydrate data and comments on a previous 994 935 version of this manuscript. This material is based on Soil biochemistry, Vol. 7. New York: Marcel Dekker. p 55–94. Ha¨ttenschwiler S, Coq S, Barantal S, Handa IT. 2011. Leaf traits 995 936 work supported by the U.S. National Science 996 937 and decomposition in tropical rainforests: revisiting some Foundation (Grants DEB 0703561 and 1119223). commonly held views and towards a new hypothesis. New 997 Phytol 189:950–65. 998 938 REFERENCES Ha¨ttenschwiler S, Vitousek PM. 2000. The role of in 999 terrestrial ecosystem nutrient cycling. Trends Ecol Evol 1000 939 Aerts R. 1997. Climate, leaf litter chemistry and leaf litter 15:238–43. F 1001 940 decomposition in terrestrial ecosystems: a triangular rela- Jakobsen I, Rosendahl L. 1990. Carbon flow into soil and 1002 941 tionship. Oikos 79:439–49. external hyphae from roots of mycorrhizal cucumber plants. 1003 942 Amundson R, Jenny H. 1997. On a state factor model of eco- New Phytol 115:77–83. 1004 943 systems. Bioscience 47:536–43. Johnson D, Leake JR, Ostle N, Ineson P, Read DJ. 2002a. In situ 1005 13 1006 944 Barantal S, Schimann S, Fromlin N, Ha¨ttenschwiler H. 2012. CO2 pulse-labelling of upland grassland demonstrates a ra- 945 Nutrient and carbon limitation on decomposition in an pid pathway of carbon flux from arbuscular mycorrhizal 1007 946 Amazonian moist forest. Ecosystems. doi:10.1007/s10021- mycelia to the soil. New Phytol 153:327–34. 1008 947 012-9564-9. Johnson D, Leake JR, Read DJ. 2002b. Transfer of recent pho- 1009 948 Blagodatsky S, Blagodatskaya E, Yuyukina T, Kuzyakov Y. 2010. tosynthate into mycorrhizal mycelium of an upland grassland: 1010 949 Model of apparent and real priming effects: linking microbial short-term respiratory losses and accumulation of C-14. Soil 1011 950 activity with soil organic matter decomposition. Soil Biol Biol Biochem 34:1521–4. 1012

Author Proof 951 Biochem 42:1275–83. Kleber M, Schwendenmann L, Veldkamp E, Ro¨ ßner J, Jahn R. 1013 952 Caldwell BA. 2005. Enzyme activities as a component of soil 2007. Halloysite versus gibbsite:PROO Silicon cycling as a pedoge- 1014 953 biodiversity: a review. Pedobiologia 49:637–44. netic process in two lowland neotropical rain forest soils of La 1015 1016 954 Cheng L, Booker FL, Tu C, Burkey KO, Zhou L, Shew HD, Rufty Selva, Costa Rica. Geoderma 138:1–11. 955 TW, Hu S. 2012. Arbuscular mycorrhizal fungi increase or- Ko¨ gel-Knabner I. 2002. The macromolecular organic composi- 1017 956 1018 ganic carbon decomposition under elevated CO2. Science tion of plant and microbial residues as inputs to soil organic 957 337:1084–7. matter. Soil Biol Biochem 34:139–62. 1019 958 Cleveland CC, Liptzin D. 2007. C:N:P stoichiometry in soil: is Koide RT, Malcolm GM. 2009. N concentration controls 1020 959 there a ‘‘Redfield ratio’’ for the microbial biomass? Biogeo- decomposition rates of different strains of ectomycorrhizal 1021 960 chemistry 85:235–52. fungi. Fungal Ecol 2:197–202. 1022 961 Cleveland CC, Townsend AR. 2006. Nutrient additions to a Kramer C, Trumbore S, Fro¨ berg M, Cisneros Dozal LM, Zhang D, 1023 962 tropical rain forest drive substantial soil carbon dioxide losses Xu X, Santos GM, Hanson PJ. 2010. Recent (<4 year old) leaf 1024 963 to the atmosphere. Proc Natl Acad Sci 103:10316–21. litter is not a major source of microbial carbon in a temperate 1025 TED 1026 964 Clinton PW, Buchanan PK, Allen RB. 1999. Nutrient composition forest mineral soil. Soil Biol Biochem 42:1028–37. 965 of epigeous fungal sporocarps growing on different substrates in Kuzyakov Y, Friedel JK, Stahr K. 2000. Review of mechanisms 1027 966 a New Zealand mountain beech forest. N Z J Bot 37:149–53. and quantification of priming effects. Soil Biol Biochem 1028 1029 967 Cooke RC, Whipps JM. 1993. Ecophysiology of fungi. Oxford: 32:1485–98. 968 Blackwell Scientific Publications. Langley JA, Hungate BA. 2003. Mycorrhizal controls on 1030 1031 969 Cornwell WK, Cornelissen JHC, Amatangelo K, Dorrepaal E, belowground litter quality. Ecology 84:2302–12. 970 Eviner VT, Godoy O, Hobbie SE et al. 2008. Plant species traits Langley JA, Chapman SK, Hungate BA. 2006. Ectomycorrhizal 1032 971 are the predominant control on litter decomposition rates colonization slows root decomposition, the post-mortem 1033 972 within biomes worldwide. Ecol Lett 11:1065–71. fungal legacy. Ecol Lett 9:955–9. 1034 973 Driver JD, Holben WE, Rillig MC. 2005. Characterization of Littell RC, Milliken GA, Stroup WW, Wolfinger RD. 1996. SAS 1035 974 glomalin as a hyphal wall component of arbuscular mycor- system for mixed models. Cary: SAS Institute. p 633. 1036 975 rhizal fungi. Soil Biol Biochem 37:101–6. Meentemeyer V. 1978. Macroclimate and lignin control of litter 1037 976 Fernandez CW, Koide RT. 2012. The role of chitin in the decom- decomposition rates. Ecology 59:465–72. 1038 977 position of ectomycorrhizal fungal litter. Ecology 93:24–8. Mendez-Millan M, Dignac MF, Rumpel C, Rasse DP, Derenne S. 1039 978 Fisher RF. 1995. Amelioration of degraded rain forest soils by 2010. Molecular dynamics of shoot vs. root biomarkers in an 1040 979 plantations of native trees. Soil Sci Soc Am J 59:544–9. agricultural soil estimated by natural abundance 13C labelling. 1041 1042 980 Floudas D, M, Riley R, Barry K, Blanchette RA, Henrissat Soil Biol Biochem 42:169–77. 981 B, Martı´nezAT et al. 2012. The Paleozoic origin of enzymatic Minderman G. 1968. Addition, decomposition and accumulation 1043 982 lignin decomposition reconstructed from 31 fungal genomes. of organic matter in forests. J Ecol 56:355–62. 1044 983 Science 336:1715–19. Nierop KGJ, Preston CM, Verstraten JM. 2006. Linking the B 1045 984 Fontaine S, Mariotti A, Abbadie L. 2003. The priming effect of ring hydroxylation pattern of condensed tannins to C, N and P 1046 985 organic matter: a question of microbial competition? Soil Biol mineralization. A case study using four tannins. Soil Biol 1047 986 Biochem 35:837–43. Biochem 38:2794–802. 1048 987 Freschet GT, Aerts R, Cornelissen JHC. 2012. A plant economics Nottingham AT, Turner BL, Winter K, van der Heijden MGA, 1049 988 spectrum of litter decomposability. Func Ecol 26:56–65. Tanner EVJ. 2010. Arbuscular mycorrhizal mycelia respiration 1050 1051 989 Gould WD, Bryant RJ, Trofymow JA, Anderson RV, Elliott ET, in a moist tropical forest. New Phytol 186:957–67. 990 Coleman DC.UNCORREC 1981. Chitin decomposition in a model soil Okafor N. 1966. Estimation of the decomposition of chitin in soil 1052 991 system. Soil Biol Biochem 13:487–92. by the method of carbon dioxide release. Soil Sci 102:140–2. 1053

Journal : 10021_ECO Dispatch : 3-4-2014 Pages : 13 9769 Article No. : h LE h TYPESET MS Code : ECO h44CP h DISK Effects of Chitin, Cellulose, and Lignin Addition on Soil Dynamics

1054 Porder S, Clark DA, Vitousek PM. 2006. Persistence of rock- Schmidt MWI, Torn MS, Abiven S, Dittmar T, Guggenberger G, 1086 1055 derived nutrients in the wet tropical forests of La Selva, Costa Janssens IA, Kleber M et al. 2011. Persistence of soil organic 1087 1056 Rica. Ecology 87:594–602. matter as an ecosystem property. Nature 478:49–56. 1088 1057 Raich JW, Lambers H, Oliver DJ. 2014. Respiration in terrestrial Sinsabaugh RL, Lauber CL, Weintraub MN, Ahmed B, Allison 1089 1058 ecosystems. In: Holland HD, Turekian KK, Eds. Treatise on SD, Crenshaw C, Contosta AR et al. 2008. Stoichiometry of 1090 1059 geochemistry, Vol. 10. 2nd edn. Oxford: Elsevier. p 613–49. soil enzyme activity at global scale. Ecol Lett 11:1252–64. 1091 1060 Raich JW, Russell AE, Bedoya-Arrieta R. 2007. Lignin and en- Smucker AJM, McBurney SL, Srivastava AK. 1982. Quantitative 1092 1061 hanced litter turnover in tree plantations of lowland Costa separation of roots from compacted soil profiles by hydro- 1093 1062 Rica. For Ecol Manag 239:128–35. pneumatic elutriation system. Agron J 74:500–3. 1094 1063 F 1095 Russell AE, Cambardella CA, Ewel JJ, Parkin TB. 2004. Species, Steinberg PD, Rillig MC. 2003. Differential decomposition of 1064 rotation, and life-form diversity effects on soil carbon in arbuscular mycorrhizal fungal hyphae and glomalin. Soil Biol 1096 1065 experimental tropical ecosystems. Ecol Appl 14:47–60. Biochem 35:191–4. 1097 1066 Russell AE, Raich JW, Valverde-Barrantes OJ, Fisher RF. 2007. Schweitzer JA, Madritch MD, Bailey JK, LeRoy CJ, Fischer DG, 1098 1067 Tree species effects on soil properties in experimental planta- Rehill BJ, Lindroth RL et al. 2008. From genes to ecosystems: 1099 1068 tions in tropical moist forest. Soil Sci Soc Am J 71:1389–97. the genetic basis of condensed tannins and their role in 1100 1069 nutrient regulation in a Populus model system. Ecosystems 1101 Russell AE, Raich JW, Bedoya R, Valverde-Barrantes O, Gon- 1102 1070 za´lez E. 2010. Impacts of individual tree species on carbon 11:1005–20. 1071 dynamics in a moist tropical forest environment. Ecol Appl Treseder KK, Allen MF. 2000. Mycorrhizal fungi have a potential 1103 1072 20:1087–100. role in soil carbon storage under elevated CO and nitrogen 1104 2 1105 1073 Russell AE, Raich JW. 2012. Rapidly growing tropical trees deposition. New Phytol 147:189–200. 1074 mobilize remarkable amounts of nitrogen, in ways that differ Waksman SA. 1929. Chemical and microbiological principles 1106 Author Proof 1075 surprisingly among species. Proc Natl Acad Sci 109:10398– underlying the decomposition of green manure in soils. J Am 1107 1076 402. Soc Agron 21:1–19. PROO 1108 1077 Sanford RL, Paaby P, Luvall JC, Phillips E. 1994. Climate geo- Wallander H, Go¨ ransson H, Rosengren U. 2004. Production, 1109 1078 morphology, and aquatic systems. In: McDade LA et al., Eds. standing biomass and natural abundance of 15N and 13Cin 1110 1079 La Selva: ecology and natural history of a neotropical rain ectomycorrhiza mycelia collected at different soil depths in 1111 1080 forest. Chicago: Univ. Chicago Press. p 19–33. two forest types. Oecologia 139:89–97. 1112 1081 Schimel JP, Bennett J. 2004. Nitrogen mineralization: challenges Weintraub, SR, Russell, AE, Townsend AR. in press. Native tree 1113 1082 of a changing paradigm. Ecology 85:591–602. species regulate nitrous oxide fluxes in tropical plantations. 1114 1115 1083 Schimel JP, Weintraub MN. 2003. The implications of exoen- Ecol Appl. 1084 zyme activity on microbial carbon and nitrogen limitation in Wong DWS. 2009. Structure and action mechanism of lignino- 1116 1085 soil: a theoretical model. Soil Biol Biochem 35:549–63. lytic enzymes. Appl Biochem Biotech 157:174–209. 1117 TED 1118

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Journal : 10021_ECO Dispatch : 3-4-2014 Pages : 13 9769 Article No. : h LE h TYPESET MS Code : ECO h44CP h DISK