Functional Ecology 2012, 26, 628–636 doi: 10.1111/j.1365-2435.2012.01986.x A petiole-galling herbivore decelerates leaf lamina litter decomposition rates

Christopher J. Frost†,1,2,3,4, Jennifer M. Dean1,4, Erica C. Smyers4, Mark C. Mescher1,4, John E. Carlson1,2,3,5, Consuelo M. De Moraes1,4 and John F. Tooker*,1,4

1Center for Chemical Ecology, Pennsylvania State University, University Park, Pennsylvania 16802, USA; 2School of Forest Resources, Pennsylvania State University, University Park, Pennsylvania 16802, USA; 3Schatz Center for Tree Molecular Genetics, Pennsylvania State University, University Park, Pennsylvania 16802, USA; 4Department of Entomol- ogy, Pennsylvania State University, University Park, Pennsylvania 16802, USA; and 5Department of Bioenergy Science and Technology (WCU), Chonnam National University, 333 Yongbongro, Buk-gu, Gwangju 500-757, Korea

Summary 1. Herbivore-mediated changes in leaf-litter chemistry are often considered responsible for alter- ing litter decomposition rates, but such chemical changes often co-occur with other factors such as physical alteration of leaf material that also influence decomposition rates. We attempted to disentangle these effects using the poplar petiole gall (Ectoedemia populella Brusk), which forms galls on petioles at the base of the leaf lamina but does not alter leaf morphology. Thus, differences in leaf decomposition rates between galled and ungalled leaves should be explained by gall-mediated changes in leaf chemistry. 2. Petiole galling decelerated leaf lamina litter decomposition in two Populus host species, but in temporally distinct ways. In Populus granidentata, galling decelerated decomposition by 7% after 4 months. After 12 and 18 months, Populus tremuloides litter decomposition rates were 12% and 17% lower, respectively, in lamina tissue whose petiole had been galled relative to ung- alled. On average, the petiole galler increased leaf lamina nitrogen concentrations by 17%, decreased tannin concentrations from 37% to 53% and decreased tannin-binding capacity by 11% and 37% in P. grandidentata and P. tremuloides, respectively. These changes would be expected to increase, rather than decrease, decomposition rates. 3. Unlike other insect herbivores guilds that have variable effects on litter decomposition in direction and magnitude, all gall studied to date have decelerated leaf-litter decomposi- tion. This consistent effect of galling on decomposition provides a framework for deciphering a fundamental aspect of insect herbivory on a critical ecosystem process. 4. We used a gall-inducing moth with a distinctive natural history to confirm the role of herbi- vore-mediated litter chemistry in leaf-litter decomposition dynamics. Moreover, we advance the hypothesis that gall-induced defensive manipulations that protect a host plant from injury by other herbivores lead to decelerated litter decomposition. Key-words: Ectoedemia, leaf chemistry, leaf-litter decomposition, plant–herbivore interactions, Populus

litter quality as a substrate can have demonstrable effects Introduction on decomposition processes and thus terrestrial nutrient The process of decomposing dead plant material facilitates availability. As many plant species presumably increase the recycling of mineral nutrients and organic matter essen- their Darwinian fitness by altering foliar quality in response tial for biological activity in most terrestrial ecosystems to herbivores (Karban & Baldwin 1997), the potential for (Parton et al. 2007). Senesced leaf litter is an abundant, herbivores to indirectly influence litter quality – and thus ubiquitous example of such material, and variation in leaf- decomposition rates – has long been considered plausible (Choudhury 1988). While a number of studies have shown *Correspondence author. E-mail: [email protected] clear effects of herbivores on litter decomposition (Findlay †Present address. Warnell School of Forest Resources, University of et al. 1996; Belovsky & Slade 2000; Chapman et al. 2003; Georgia, Athens, Georgia 30601, USA. Schweitzer et al. 2005b; Chapman, Schweitzer & Whitham

2012 The Authors. Functional Ecology 2012 British Ecological Society Galling effects on leaf-litter decomposition 629

2006; Crutsinger et al. 2008; Frost & Hunter 2008; Kurokawa (P. betae) or by radically altering leaf growth patterns & Nakashizuka 2008), constructing a more detailed theoret- (R. solidaginis) (Fig. S1, Supporting information). As a ical framework has been difficult because herbivory can result, these systems have shown clearly that gall insects have accelerate or decelerate decomposition depending in part on ecosystem-level effects, but neither system necessarily disen- whether secondary metabolites are repressed or augmented, tangles chemical and morphological factors influencing litter what type of physical damage occurs, the type of herbivory decomposition. suffered or the ecosystem in which the decomposition is Here, we describe how herbivory by larvae of the poplar measured. Moreover, determining the importance of her- petiole gall moth (Ectoedemia populella Busck.) influences bivory-induced changes in leaf chemistry for decomposition leaf-litter chemistry and decomposition in two species of Pop- rates is often complicated by the co-occurrence of other ulus. Ectoedemia are monotrysian, nepticulid that herbivore-related factors that can also alter litter decompo- include some of the smallest known lepidopterans; adults of sition. For example, herbivory may induce changes in leaf- E. populella have c. 6-mm wingspans (Wilkinson & Scoble drop phenology that results in herbivore-affected litter 1979). The family consists primarily of leaf miners; only a few entering the detrital system in a different condition than Ectoedemia species consume bark, buds, or – in the case of undamaged litter (Chapman et al. 2003). Of equal impor- E. populella – induce galls (Wilkinson & Scoble 1979). Leaf- tance, most foliar herbivores physically damage leaf tissue mining Ectoedemia trace at least to the mid-Cretaceous era during feeding, and such damage can alter access to the lit- 97 million years ago (Labandeira et al. 1994); gall-inducing ter substrate (Findlay et al. 1996; Cornelissen et al. 1999; Ectoedemia on Populus trace to the Miocene era, some 5– Pe´rez-Harguindeguy et al. 2000; Ostertag, Scatena & Silver 17 million years ago (Madler 1936). Importantly, E. populella 2003). Insect herbivore species that influence host-plant form galls at the junction of the petiole and the lamina caus- chemistry but only minimally alter other aspects of a host ing no observable morphological difference in the lamina tis- plant, such as some species of gall insects, offer an ideal sue itself (Fig. S2, Supporting information). Moreover, the opportunity to explore the effects of herbivore-induced leaf phenology of the moth is offset with its hosts such that the chemistry on leaf-litter decomposition apart from other adults emerge and oviposit in May after leaves of their hosts co-occurring effects. have fully expanded foliage (Wilkinson & Scoble 1979). Thus, Gall-inducing insects are herbivores that force their host E. populella galls have limited or no influence on lamina plants to produce a tumour-like growth that provides the growth and development, although they may influence lam- insect with food and shelter, usually at the expense of plant ina quality. Larvae emerge and pupate in October and over- growth and ⁄ or reproduction. These insects have evolved inti- winter in the soil, so larvae do not reside in decomposing mate relationships with their host plants and an unparalleled galled leaf litter. Little else is known about the ecology of ability to influence host-plant morphology and physiology E. populella, including the direct effects of the gall on leaf (Larson 1998; Stone & Schonrogge 2003). Gall insects com- lamina metabolite profiles associated with the galled petiole. monly modify host-plant chemistry, often altering concentra- However, there are at least two reasons to hypothesize that tions of plant secondary metabolites for their own purposes E. populella will influence decomposition possibly by altering (Weis & Abrahamson 1986; Nyman & Julkunen-Tiitto 2000; laminar chemistry. First, the gall envelopes the petiole, forc- Tooker, Koenig & Hanks 2002; Allison & Schultz 2005). For ing all assimilates and other transported materials through example, high concentrations of secondary metabolites such vasculature that has been altered by the gall. Second, E. pop- as phenolics tend to be localized in gall exteriors, where they ulella deposits its frass inside the gall in a tight packet that presumably provide protection against natural enemies, while remains in the gall after leaf senescence; frass, which is nutri- the inner nutritive tissue on which the gall insect feeds is lar- ent rich, is known to influence ecosystem processes in other gely or entirely devoid of such potentially toxic compounds systems (Frost & Hunter 2007; Madritch, Donaldson & (Nyman & Julkunen-Tiitto 2000; Allison & Schultz 2005). Lindroth 2007). Moreover, gall insects also variably influence the chemistry of Throughout its range, E. populella forms galls on big- plant tissue that is not part of the gall itself, although investi- toothed aspen (Populus grandidentata) and quaking aspen gations thus far have been confined to neighbouring tissues (Populus tremuloides) (Wilkinson & Scoble 1979). This (Tooker et al. 2008; Cooper & Rieske 2009); such systemic makes the Populus ⁄ Ectoedemia system well suited to explore effects on chemistry may also influence decomposition the independent effects of host species and galling. As plant dynamics of those tissues. diversity at different levels can affect decomposition rates A potential consequence of gall insect-mediated alterations (Madritch & Hunter 2002; Ball et al. 2008) – and the effects in host-plant chemistry is the modification of rates of decom- of herbivores on these rates (Schweitzer et al. 2005b) – we position of senesced or dead plant parts. Such effects have tested the hypothesis that E. populella galling influences leaf- been demonstrated using the aphid Pemphigus betae and the litter decomposition dynamics using naturally senesced midge Rhopalomyia solidaginis, two disparate gall-inducing leaves from P. grandidentata and P. tremuloides separately. taxa; these insect species induced changes that altered rates of This allowed us to examine variation of E. populella- leaf-litter decomposition (Schweitzer et al. 2005b; Crutsinger mediated changes to leaf-litter decomposition rates, chemis- et al. 2008). Yet, both of these species alter leaf morphology try and nutrient release dynamics between host species over to some degree by forming their gall on the leaf lamina the course of 18 months.

2012 The Authors. Functional Ecology 2012 British Ecological Society, Functional Ecology, 26, 628–636 630 C. J. Frost et al.

Materials and methods then weighed, and the lamina litter was separated from the gall body in the samples that were galled. The lamina litter tissue only was then Leaf litter from P. grandidentata and P. tremuloides was collected ground to a fine powder in a ball mill, and the resulting powder was separately from the ground during natural leaf senescence (October ) ) analysed for total C (mg g 1), total N (mg g 1), C ⁄ N ratios, tannins 2006) from three separate locations in Centre County, PA and pooled ) ) (mg g 1) and fibre content (lignin, cellulose, hemicellulose; mg g 1). by plant species. The two aspen species tend to drop their leaves over Subsamples were ashed (550 C, 5 h), and data are reported relative a window of generally <10 days (Fig. S3, Supporting information). to ash-free dry mass (AFDM). Thus, the chemical analyses represent We did not collect leaves until they naturally dehisced from the trees only the ungalled lamina portions of the leaf-litter tissue. to ensure natural levels of leaf senescence. However, we also used Total C and N were measured with a Carlo Erba 1500N total leaves that were freshly fallen and sitting on top of other leaf litter to CHN analyzer (Carlo Erba Instrumentazione, Milan, Italy). Fibre ensure that they had no contact with soil, to minimize any potential concentrations were determined by sequential acid digestions using ‘preprocessing’ of litters prior to the experiment. We observed no an ANKOM A200 Fiber Analyzer according to manufacturer’s apparent difference in leaf-drop phenology; we confirmed this by instructions (ANKOM Technology, Macedon, NY, USA). Tannin marking a number of leaves and monitoring their drop phenology, concentrations and protein-binding capacity were assayed colorimet- and there was no difference in leaf-drop timing between galled and rically following well-established methods (Bate-Smith 1977a,b) after ungalled leaves (Fig. S3, Supporting information). Litter from each three rounds of initial extraction with 70 ⁄ 30% acetone ⁄ water with species was sorted based on the presence of galls. Leaves were selected 1mM ascorbic acid and subsequent removal of the acetone under vac- that had little or no visible damage from chewing herbivores to the uum evaporation (Frost & Hunter 2008). Condensed tannin concen- leaf tissue itself, as such damage has been shown to affect decomposi- trations were assayed following acid depolymerization in a polar tion dynamics in Populus and other woody plant species (Findlay solvent (N-butanol) at 100 C (Hagerman & Butler 1980; Hagerman, et al. 1996; Belovsky & Slade 2000; Chapman et al. 2003; Chapman, Rice & Ritchard 1998), and hydrolyzable tannin concentrations were Schweitzer & Whitham 2006; Frost & Hunter 2008). However, we measured using the standard potassium iodate assay (Rossiter, cannot exclude the possibility that piercing ⁄ sucking herbivores such Schultz & Baldwin 1988). We also measured protein-binding capacity as aphids may have fed on the foliage during the growing season, of the leaf tannin extracts using the protein precipitatable phenolics although none was observed during sampling. assay, which provides a realistic assessment of the biological activity Litters were dried separately in the laboratory at room tempera- of the tannins. Briefly, protein-binding tannins form a precipitate ture, and then, subsamples were selected haphazardly, weighed and complex with the substrate protein bovine serum albumin; the precip- sealed in 15 · 15 cm screen bags (1 · 1-mm mesh-size) as previously itate is re-dissolved in triethanolamine ⁄ sodium dodecyl sulphate and described (Frost & Hunter 2008). We used c. 3 g of litter for each lit- tannins quantified (mg protein-binding tannin per g dry weight) spec- terbag. Based on the amount of material available, we filled 55–57 lit- trophotometrically after adding ferric chloride (Hagerman & Butler terbags with each of the P. grandidentata galled and ungalled litters 1978). Standards for all phenolic assays were generated from a pooled and P. tremuloides ungalled litters; 20 litterbags were made with mix of undecomposed leaf litter from all the individual experimental P. tremuloides galled litter owing to a limited amount of material. trees that was extracted exhaustively with 70 ⁄ 30% acetone ⁄ water Following previous convention (Schweitzer et al. 2005b; Crutsinger with 1 mM ascorbic acid similar to the individual samples (Hagerman et al. 2008), we removed petioles from all galled and ungalled laminas & Butler 1989; Hall et al. 2006; Ball et al. 2008; Frost & Hunter prior to filling the litterbags but did not remove galls from the lamina 2008). This pooled aqueous extract was then frozen and lyophilized, litter, and the Ectoedemia gall body was c. 8% of the total litter mass and an weighed aliquot of the resulting powder was resuspended in in galled samples. Importantly, different decomposition patterns water and used to generate standard curves for all tannin assays between the two Populus species and the magnitude of the observed (Madritch & Hunter 2002, 2004; Hunter & Forkner 2004; Hall et al. effects (see Results) suggest that the differences between the mass of 2006; Ball et al. 2008; Frost & Hunter 2008). galled and ungalled litters were not artifacts of the gall body itself. Data were analysed using PROC MIXED in SAS (Littell, Stroup & We then established a field plot in January 2007 in the Scotia Freund 2002) using tree species and gall presence as main effects and Range near State College, PA (40 47 0Æ56 N; 77 57 20Æ56 W). The ¢ ¢¢ ¢ ¢¢ plot as a random effect. Data reported on litter AFDM remaining site was a mixed-hardwood stand containing mature P. grandidentata were arcsine square root transformed, and all other data were log and P. tremuloides. The field site consisted of 15 replicated plots transformed prior to analysis to satisfy assumptions of ANOVA. (0Æ5 · 2 m per plot). We assigned randomly one litterbag per treat- ment per collection date to each plot. Thus, fifteen replicates per date were established for each of the P. grandidentata galled and ungalled Results and P. tremuloides ungalled litters. Five replicates per date were established for the P. tremuloides galled litters. For the initial sam- Presence of the petiole gall reduced decomposition rates of ples, there were 9–12 bags for P. grandidentata galled and ungalled lit- the big-toothed aspen and quaking aspen leaf litters, but in ters and P. tremuloides ungalled litters and five bags for the temporally distinct ways (Fig. 1). Both galling and tree spe- P. tremuloides galled litter. cies had significant main effects on rates of AFDM loss (spe-

One set of litterbags was collected immediately to measure initial cies F1,163 =12Æ90, P <0Æ001; gall F1,163 =7Æ62, litter quality, while the remaining litterbags were collected after 4, 12 P =0Æ006); however, only galling influenced AFDM loss and 18 months in the field. Litterbags containing initial litter samples over time (date*gall F3,149 =3Æ05, P =0Æ030; date*species were brought to the field, established at the site and then immediately F3,150 =2Æ19, P =0Æ091). Importantly, galling affected collected and returned to the laboratory. During collections, the stak- decomposition differently between tree species (date*spe- ing flag was carefully removed, debris on the outside of each collected cies*gall F =2Æ89, P =0Æ038): galling decelerated the bag was brushed from the surface and care was taken not to disturb 3,149 remaining litterbags. Litterbags were transported to the laboratory at ‘early’ stages of mass loss in P. grandidentata litter and the ambient temperatures and then dried at 65 C for 48 h. Samples were ‘late’ stages in P. tremuloides litter. After 4 months, AFDM

2012 The Authors. Functional Ecology 2012 British Ecological Society, Functional Ecology, 26, 628–636 Galling effects on leaf-litter decomposition 631

110 300 (a) (a) 100 250

90 200 6 DW)

80 –1 150 4

70 100 2 (mg g 0 60 Condensed tannins 50 41218 50 0

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(mg g 50 0 41218 Hydrolyzable tannins 60 0

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02468101214161820 250 25 20

Month DW) 200 15 –1 10 Fig. 1. Mass remaining (% ash-free dry mass) of Populus grandiden- 150 5 0 tata L. and Populus tremuloides L. leaf litters that had been galled by (mg g 100 the petiole galler Ectoedemia populella Brusk. In both panels, circles 41218 50 represent P. grandidentata, triangles represent P. tremuloides sam- Protein binding tannins ples; shaded symbols are leaf samples that were ungalled, open sym- 0 bols are leaf samples that had been galled by E. populella. 02468101214161820 (a) Means ± SE of each treatment group, with n = 5 for P. tremulo- Months ides galled samples and n = 15 for all others. (b) Regression analysis to determine the decomposition constant (k: slope of regression line) Fig. 2. (a) Condensed and (b) hydrolysable tannin concentrations for each group. Symbol identity are the same as panel a, and regres- and (c) protein-binding capacity of tannin extracts in initial and sion lines vary to distinguish the treatments: dash = P. tremuloides decomposing Populus tremuloides L. and P. grandidentata L. leaf lit- ungalled; solid = P. tremuloides galled; dot-dash = P. grandidenta- ters. Circles represent P. grandidentata, triangles represent P. tremu- ta ungalled; dot = P. grandidentata galled. loides samples; shaded symbols are leaf samples that were ungalled, open symbols are leaf samples that had been galled by the petiole gal- ler Ectoedemia populella Brusk. Inset graphs show details of time loss of the P. grandidentata galled leaf litter was 6% lower points at 4, 12 and 18 months not evident in the larger figures. See text than that of ungalled P. grandidentata litter (F1,26 =5Æ40, for Materials and methods. P =0Æ028); after 12 and 18 months, AFDM loss was 15% and 17% lower, respectively, in the galled P. tremuloides litter Conversely, hydrolyzable tannins were 37% and 53% lower relative to the ungalled P. tremuloides litter (F1,20 =4Æ99, in galled P. grandidentata and P. tremuloides litters (gall:

P =0Æ038; F1,18 =8Æ08, P =0Æ011; Fig. 1a). There were no F1,24Æ8 =19Æ41, P <0Æ001), respectively, and P. grandiden- significant effects of galling on AFDM loss of P. tremuloides tata litter had an overall greater hydrolysable tannin concen- litter at 4 months or P. grandidentata litter at 12 or tration than did P. tremuloides litter (species: F1,23Æ7 =55Æ40, 18 months. The changes to decomposition mediated by gall- P <0Æ001; Fig. 2b). The protein-binding capacity of the ini- ing and species altered the decomposition constant k over the tial litters was strongly correlated with hydrolysable tannin full-time course of the experiment (Fig. 1b). concentrations; galled P. grandidentata and P. tremuloides Galling had strong, species-specific effects on tannin con- litters had 11% and 35% lower protein-binding capacity than centrations and protein-binding activity in the initial did their respective controls (gall: F1,30 =10Æ13, P =0Æ003; senesced, undecomposed litter. Condensed tannin concentra- Fig. 2c). Moreover, the protein-binding capacity of ungalled tions were 46% lower in the P. tremuloides galled litter rela- P. grandidentata litters was 96% greater than that of ungalled tive to ungalled P. tremuloides litter (F1,14 =12Æ30, P. tremuloides litters (species: F1,30 =155Æ85, P <0Æ001). P =0Æ004), while galling had no such effect on P. grandiden- Time in the field dramatically reduced litter tannin concen- tata-condensed tannin concentrations (P =0Æ379; Fig. 2a). trations and protein-binding capacity (Fig. 2). After

2012 The Authors. Functional Ecology 2012 British Ecological Society, Functional Ecology, 26, 628–636 632 C. J. Frost et al.

4 months, condensed tannin concentrations were higher in cies (F1,28 =5Æ75, P =0Æ023; Fig. 3a), and P. grandidentata P. grandidentata litters relative to P. tremuloides litter had overall lower initial N concentrations than did P. tre-

(F1,36Æ5 =42Æ0, P <0Æ001), but both had dropped precipi- muloides litters (F1,28 =9Æ98, P =0Æ004). Tree species and tously (c. 4% and 0Æ2% of initial concentrations in the galling independently influenced litter N dynamics. Populus P. grandidentata and P. tremuloides litters, respectively). tremuloides accumulated N at a greater rate over time than

Consistently, tannin concentrations were very low in the 12- did P. grandidentata (date*species F3,153 =3Æ69, and 18-month litters. Not surprisingly, the protein-binding P =0Æ013). In addition, galling exerted a significant influ- capacity of litter extracts that had been decomposed in the ence on N concentrations over time independent of plant

field was essentially non-existent after 4 months. While there species (date*gall F3,152 =3Æ49, P =0Æ017). In contrast to were some statistically significant interactions over time (not N dynamics, litter total C was marginally influenced by shown), they are likely biologically meaningless considering galling (F1,156 =3Æ77, P =0Æ084; Fig. 3b). Total C was how little tannin remained in the litters. More importantly, consistently higher in P. grandidentata (F1,155 =11Æ75, the minimal tannin concentrations after 12 and 18 months in P <0Æ001). At 18 months, galled samples had higher total the field (Fig. 2) suggest that tannins exerted little if any influ- CinP. tremuloides litter and lower total C in P. grand- ence on decomposition after the first 4 months in the field. identata relative to the ungalled controls (species x gall

Gall effects on N and C concentrations and dynamics F1,43 =5Æ16, P =0Æ028); as a result, galling affected total also differed between Populus species. Leaf N was higher in C dynamics over time uniquely between species (date*spe- the galled litter before decomposition independent of spe- cies*gall F3,150 =2Æ88, P =0Æ038).

26 (a) 24

DW) 22 –1 20 P. grandidentata ungalled 18 P. grandidentata galled 16 P. tremuloides ungalled 14 12 P. tremuloides galled 10 8

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490 –1 130 DW)

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–1 18 250 16 200 150 14 100 Lignin:N ratio 12 Lignin (mg g 50 10 02468101214161820 02468101214161820 Month Month

Fig. 3. (a) Nitrogen [N], (b) carbon [C], (c) cellulose, (d) lignin and (e) hemicellulose concentrations, and (f) C ⁄ N and (g) lignin ⁄ Nratiosinini- tial and decomposing Populus tremuloides L. and P. grandidentata L. leaf litters. Circles represent P. grandidentata, triangles represent P. tremu- loides samples; shaded symbols are leaf samples that were ungalled, open symbols are leaf samples that had been galled by the petiole galler Ectoedemia populella Brusk. Only leaf lamina tissue is represented; that is, the physical gall was removed prior to analysis. Symbols represent means ± SE, with n = 5 for P. tremuloides galled samples and n = 15 for all others. See text for Materials and methods.

2012 The Authors. Functional Ecology 2012 British Ecological Society, Functional Ecology, 26, 628–636 Galling effects on leaf-litter decomposition 633

Concentrations of cell wall constituents were influenced by ter dramatically increases decomposition rates by facilitating galling and plant species. Cellulose and lignin concentrations access to the substrate (Madritch & Hunter 2003). Such in senesced, undecomposed leaf tissue were 12% and 29% substrate access increases the more a leaf is consumed by a higher in galled litters relative to ungalled litters, respectively, chewing herbivore, which causes physical damage to the leaf independent of plant species (cellulose: F1,28 =21Æ51, tissue (Cornelissen et al. 1999; Pe´rez-Harguindeguy et al.

P <0Æ001; lignin: F1,28 =22Æ97, P <0Æ001; Fig. 3c,d). 2000). The Ectoedemia ⁄ Populus system described here pro- During decomposition, cellulose concentrations segregated vides an opportunity to examine the role of herbivore-altered by plant species (species: F1,158 =39Æ95, P <0Æ001) and the plant chemistry apart from other factors associated with her- initial effect of galling were lost (gall: F1,159 =0Æ85, bivory because the galler does not physically manipulate the P =0Æ359). The two species also varied in the rate of cellu- lamina. Our results indicate that an insect herbivore that has lose loss (date*species: F3,149 =4Æ90, P =0Æ003). In con- little influence on leaf lamina development and does not trast, lignin concentrations and their rate of change in the physically damage the lamina can nonetheless clearly decomposing litter were influenced by galling (gall: influence litter decomposition and nutrient release rates.

F1,158 =8Æ27, P =0Æ005; date*gall: F3,149 =6Æ61, Herbivore-mediated changes in litter chemistry alone are suf- P <0Æ001). The date*gall interactive effect seemed driven by ficient to alter leaf-litter decomposition rates and, presum- a significant gall effect at 18 months (F1,32Æ6 =4Æ27, ably, other ecosystem processes responsive to those rates. P =0Æ047) that was not present at 4 or 12 months. Hemicel- Although decomposition dynamics differed between tree lulose concentrations were not affected by either tree species species, E. populella petiole galling consistently decelerated or galling (Fig. 3e). leaf lamina litter decomposition. Deceleration of decomposi- Nutrient ratios can play an important role in shaping litter tion theoretically results from low substrate quality; this typi- decomposition dynamics. The C ⁄ N ratios were driven by N cally implies relatively lower concentrations of beneficial concentrations and, P. tremuloides had lower C ⁄ Nratios components such as N and relatively higher concentrations of

(F1,161 =99Æ45, P <0Æ001) and a more rapid reduction in detrimental components such as tannins and lignin, each of

C ⁄ N ratios over time (date*species F3,152 =3Æ78, P =0Æ012) which has been shown to influence decomposition via effects than did P. grandidentata (Fig. 3f). In other words, assuming on decomposer organisms (Northup, Dahlgren & McColl that C ⁄ N ratios are predictive of litter quality, P. tremuloides 1998; Maie et al. 2003; Hattenschwiler & Gasser 2005). This was of higher quality to decomposers than was P. grandentata. general model held for the species main effect in our system: Galling had a relatively small effect on C ⁄ N ratios. In con- P. grandidentata litter had lower total N, higher tannin con- trast, the influence of galling on lignin ⁄ N ratios was pro- centrations and binding activity (but no difference in initial nounced (date*gall F3,147 =3Æ17, P =0Æ026; Fig. 3g). Even litter lignin) and decomposed more slowly than did P. tremu- though there was no statistically significant date*spe- loides litter. However, the presence of the gall confounded this cies*gall interaction (P =0Æ278), galling clearly affected lig- relationship. The relatively higher N concentrations in galled nin ⁄ N ratios in P. tremuloides more than in P. tremuloides and relatively lower tannin concentrations and P. grandidentata: there were significant species*gall interac- binding capacity in galled tissues would typically be predicted tions at 4 and 12 months (4 months F1,45 =6Æ91, to accelerate, not decelerate, decomposition. Evidently some

P =0Æ011; 12 months F1,35Æ4 =5Æ56, P =0Æ024). In fact, other metabolite(s) associated with galled lamina trumped lignin ⁄ N ratios were unchanged over time in galled P. tremu- the higher N concentrations. Moreover, the gall-mediated loides litters (Fig. 3g). reduction in initial tannin concentrations and binding capac- ity suggests that tannins are not involved. One possibility is that galling altered leaf toughness, which is has been shown to Discussion affect decomposition rates (Cornelissen et al. 1999; Pe´rez- A long-standing hypothesis in plant–herbivore ecology is that Harguindeguy et al. 2000). We did not measure leaf tough- herbivores influence rates of leaf-litter decomposition via ness directly, but P. grandidentata leaves were obviously changes in litter chemical quality (Grace 1986; Choudhury tougher than were P. tremuloides and, as expected, P. grand- 1988). While abiotic factors such as temperature and moisture identata decomposed more slowly. There were no such obvi- clearly drive decomposition dynamics across biomes (Trofy- ous phenotypic differences in leaf toughness in galled vs. mow et al. 2002; Wall et al. 2008), the influence of substrate ungalled litter within either species that would appear to quality is most pronounced on spatial scales where tempera- account for the differential decomposition rates. ture and moisture conditions have relatively minimal varia- The result that Ectoedemia galling decelerated lamina litter tion (Swift, Heal & Anderson 1979). That is, substrate quality decomposition is consistent with effects from other gall likely matters in most mixed-stand terrestrial forests. Yet, insects. In fact, gall insects from diverse lineages (i.e. a social herbivores can influence litter quality in a number of ways. In hemipteran, a cecidomyiid dipteran and the microlepidopter- all cases of chewing or piercing ⁄ sucking herbivores, physical an studied here) independently decelerate leaf-litter decompo- damage to the lamina tissue occurs regardless of herbivore- sition under field conditions (Schweitzer et al. 2005b; induced changes to lamina chemistry and may complicate the Crutsinger et al. 2008). These effects presumably result from effects of herbivore-induced chemistry per se. As a drastic modification of foliar chemical composition as our data sug- example of physical damage, experimental crushing of leaf lit- gest, but may also include morphological changes in the other

2012 The Authors. Functional Ecology 2012 British Ecological Society, Functional Ecology, 26, 628–636 634 C. J. Frost et al. systems. Indeed, gall insects may have a larger relative influ- higher initially in galled tissues, and this difference may have ence on decomposition dynamics than other insect–herbivore affected the slower decomposition after 4 months in the guilds because of their intimate host-plant associations and P. grandidentata litters. Yet, lignin dynamics over time did their ability to manipulate the molecular physiology of their not appear to explain the observed galling effects on decom- host (Schweitzer et al. 2005b). However, other herbivore position rates in P. tremuloides litters. In Solidago,lower guilds can also influence litter decomposition. In some cases, total N concentration in the midge-galled litters related to chewing and piercing-sucking herbivores have accelerated decelerated decomposition (Crutsinger et al. 2008); Ectoede- decomposition (Ritchie, Tilman & Knops 1998; Chapman mia-galled litters had higher N concentrations but nonethe- et al. 2003); in other cases, herbivory did not influence mass less exhibited decelerated decomposition. It is possible that loss despite significant differences in initial litter quality (Hall the form of N was different between the galled and ungalled et al. 2006; Frost & Hunter 2008). Based on the three galling litters and that this difference was important. In the cotton- systems studied, the magnitude of galling-herbivore effects on wood system, higher condensed tannin concentrations in decomposition relative to other insect–herbivore guilds may galled litters contributed to decelerated decomposition not be as important as their direction: galling modifies leaf (Schweitzer et al. 2005b). In our case, tannin concentrations quality in a manner that consistently decelerates leaf-litter and binding capacity were lower in galled tissue and were decomposition independent of the identity of the gall insect essentially lost after 4 months. Although these are com- or host-plant species. monly measured aspects of litter quality, they do not neces- Attributes that are common to gall insects may provide a sarily confer resistance against insect herbivores and do not theoretical framework for the consistently observed gall- appear to explain the decelerating effects of gall-mediated lit- mediated deceleration of litter decomposition that is based on ter chemistry on litter decomposition. A future line of modified litter chemistry. In forcing the host plant to con- enquiry will obviously be needed to explore in both Populus struct an enclosed domicile with a fortified exterior and nutri- species the gall-induced changes in chemistry that may be tive interior, many gall insects are less vulnerable to predators associated with delayed decomposition. yet have access to an ideal food source during development. Other aspects of E. populella life history might also influ- One limitation of galling is that the insect depends on the suc- ence leaf chemistry in ways that alter litter decomposition. cess of the host plant in a manner that is distinct from other For example, insect frass, which is N rich and remains in the herbivore guilds. While chewing and piercing-sucking herbi- gall after leaf senescence (See Fig. S1, Supporting informa- vores can often search for new suitable host plants, the sessile tion) and the higher initial concentration of N in the galled lit- nature of gall insects mandates that they have a vested interest ter may have resulted from N absorbed by the plant from the in protecting their host plants. This suggests that gall insects frass (Frost & Hunter 2007). This frass may therefore provide may manipulate their host plants to be less suitable to other a nutrient resource to decomposer microfauna and accelerate herbivores while evading host defenses themselves (Tooker & decomposition (Reynolds & Hunter 2001; Fonte & Schowalter De Moraes 2007; Stireman & Cipollini 2008; Tooker et al. 2005; Madritch, Donaldson & Lindroth 2007; Frost & Hun- 2008). Indeed, some gall insect activity can increase concen- ter 2008), although the consistent deceleration of decomposi- trations of defensive metabolites systemically in ungalled tion in galled tissue does not support this. Nevertheless, our parts of the plant, which presumably protects the resource results do not rule out a role of frass in mediating lamina from other forms of herbivory (Pascual-Alvarado et al. 2008; chemistry because frass can also contain high levels of plant Cooper & Rieske 2009; Tooker & De Moraes 2009). This defensive compounds (Kopper et al. 2002; Chen et al. 2007). leads to the hypothesis that gall-induced defensive manipula- With E. populella in particular, frass remains within the gall tions that protect a host plant from injury by other herbivores body and we assume has antimicrobial metabolites to pre- result in decelerated litter decomposition. That is, the same vent rampant bacterial or fungal growth in the enclosed gall chemical compounds in foliage that confer resistance to her- environment (Tooker and De Moraes 2006). Such metabo- bivores also decelerate decomposition in leaf litter. lites may have extended, indirect effects inhibiting microbial Although the life history of E. populella allows us to con- colonization of leaf litter that presumably would also decel- clude that changes in leaf chemistry play a central role influ- erate decomposition. encing leaf-litter decomposition, gall-mediated differences in As a final point, our experimental design isolated litters leaf quality that result in the observed decomposition from each tree species separately to investigate independent changes remain unclear. To complicate matters, the effects effects of galling on the two main host tree species, but litters of E. populella on leaf-litter decomposition in our study var- in natural mixed-stand forests obviously contain a mix of spe- ied between the two host Populus species (Fig. 1), which sug- cies. Litters from different tree species and genotypes decom- gests that the gall-induced chemistry that altered pose at different rates (Fig. 1a), but mixed-species litters can decomposition dynamics may differ between tree species. decompose in an non-additive manner that is not predicted Unfortunately, the gall-induced changes in litter chemistry by the decomposition rates of individual species (Madritch & in these species are not well known; we therefore focused on Hunter 2003, 2005; Schweitzer et al. 2005a; Ball et al. 2008). leaf chemistry parameters that are known to affect decompo- Herbivory may impose an additional factor on mixed-species sition (and had demonstrated effects between the two tree litter decomposition dynamics. Herbivory and geno- species). Lignin concentrations in our system were 29% typic ⁄ genetic variation are known to interact to affect litter

2012 The Authors. Functional Ecology 2012 British Ecological Society, Functional Ecology, 26, 628–636 Galling effects on leaf-litter decomposition 635 decomposition (Schweitzer et al. 2005b; Chapman, Schweit- Cornelissen, J.H.C., Perez-Harguindeguy, N., Diaz, S., Grime, J.P., Marzano, B., zer & Whitham 2006), and our results that Ectoedemia galling Cabido, M., Vendramini, F. & Cerabolini, B. (1999) Leaf structure and defence control litter decomposition rate across species and life forms in differentially affected decomposition dynamics in the two regional floras on two continents. New Phytologist, 143, 191–200. host Populus species provide further evidence for this. How- Crutsinger, G.M., Habenicht, M.N., Classen, A.T., Schweitzer, J.A. & Sanders, ever, it is unknown how herbivory interacts specifically with N.J. (2008) Galling by Rhopalomyia solidaginis alters Solidago altissima architecture and litter nutrient dynamics in an old-field ecosystem. Plant and non-additive effects of mixed-species litter decomposition. Soil, 303, 95–103. So, while the different decomposition dynamics between the Findlay, S., Carreiro, M., Krischik, V. & Jones, C.G. (1996) Effects of two Populus species in our study is not surprising, the differ- damage to living plants on leaf litter quality. Ecological Applications, 6, 269–275. ential effects of galling on decomposition may be altered Fonte, S.J. & Schowalter, T.D. (2005) The influence of a neotropical herbivore when the two leaf litters are mixed in in ways that may not be (Lamponius portoricensis) on nutrient cycling and soil processes. Oecologia, predicted by the galling effects on either species individually. 146, 423–431. Frost, C.J. & Hunter, M.D. (2007) Recycling of nitrogen in herbivore feces: In summary, we have used a model system with a unique plant recovery, herbivore assimilation, soil retention, and leaching losses. natural history to demonstrate that insect herbivore-mediated Oecologia, 151,42–53. changes in leaf chemistry can alter leaf-litter decomposition Frost, C.J. & Hunter, M.D. (2008) Insect herbivores and their frass affect Quer- cus rubra leaf quality and initial stages of subsequent litter decomposition. rates. Further, based on the results from this system and the Oikos, 117,13–22. other two gall systems, we develop the hypothesis that gall- Grace, J.R. (1986) The influence of gypsy moth on the composition and nutrient mediated deceleration in leaf-litter decomposition results content of litter fall in a Pennsylvania oak forest. Forest Science, 32, 855–870. Hagerman, A.E. & Butler, L.G. (1978) Protein precipitation method for quanti- from induced defensive changes that protect a host plant from tative determination of tannins. Journal of Agricultural and Food Chemistry, damage from other herbivores, thus increasing gall insect fit- 26, 809–812. ness. This hypothesis provides a basis for generalizing the Hagerman, A.E. & Butler, L.G. (1980) Condensed tannin purification and characterization of tannin-associated proteins. Journal of Agricultural and influence of a large guild of herbivores on a fundamental eco- Food Chemistry, 28, 947–952. system process and should contribute to the theoretical Hagerman, A.E. & Butler, L.G. (1989) Choosing appropriate methods and framework relating species interactions with ecosystem standards for assaying tannin. Journal of Chemical Ecology, 15, 1795–1810. Hagerman, A.E., Rice, M.E. & Ritchard, N.T. (1998) Mechanisms of protein ecology. precipitation for two tannins, pentagalloyl glucose and epicatechin 16 (4– >8) catechin (procyanidin). Journal of Agricultural and Food Chemistry, 46, 2590–2595. Acknowledgements Hall, M.C., Stiling, P., Moon, D.C., Drake, B.G. & Hunter, M.D. (2006) Ele- vated CO2 increases the long-term decomposition rate of Quercus myrtifolia We thank Jennifer Schweitzer for insightful comments, Janet Saunders for labo- leaf litter. Global Change Biology, 12, 568–577. ratory assistance, Peter Wilf and Conrad Labandeira for discussions on Ec- Hattenschwiler, S. & Gasser, P. (2005) Soil alter plant litter diversity toedemia evolutionary history, Patrick Abbot for Pemphigus photographs effects on decomposition. Proceedings of the National Academy of Sciences and discussion, Megan Marshall and Tom Richard for use of an ANKOM of the United States of America, 102, 1519–1524. fiber analyzer, and Gary Felton and Michelle Peiffer for use of a SpectraMax Hunter, M.D. & Forkner, R.E. (2004) Hurricane damage influences foliar poly- plate reader. Tom Maddox (Odum School of Ecology Analytical Laboratory) phenolics and subsequent herbivory on surviving trees. Ecology, 80, 2676– processed the C ⁄ N samples. The National Research Initiative of the USDA 2682. (#2007-35302-18087 [CJF] and #2006-01823 [JFT]) funded this work. Karban, R. & Baldwin, I.T. (1997) Induced Responses to Herbivory. University of Chicago Press, Chicago, IL. Kopper, B.J., Jakobi, V.N., Osier, T.L. & Lindroth, R.L. (2002) Effects of References paper birch condensed tannin on whitemarked tussock moth (: Lymantriidae) performance. Environmental Entomology, 31, 10–14. Allison, S.D. & Schultz, J.C. (2005) Biochemical responses of chestnut oak to a Kurokawa, H. & Nakashizuka, T. (2008) Leaf herbivory and decomposability galling cynipid. Journal of Chemical Ecology, 31, 151–166. in a Malaysian tropical rain forest. Ecology, 89, 2645–2656. Ball, B.A., Hunter, M.D., Kominoski, J.S., Swan, C.M. & Bradford, M.A. Labandeira, C.C., Dilcher, D.L., Davis, D.R. & Wagner, D.L. (1994) Ninety- (2008) Consequences of non-random species loss for decomposition dynam- seven million years of angiosperm-insect association: paleobiological ics: experimental evidence for additive and non-additive effects. Journal of insights into the meaning of coevolution. Proceedings of the National Acad- Ecology, 96, 303–313. emy of Sciences of the United States of America, 91, 12278–12282. Bate-Smith, E.C. (1977a) Astringent tannins of Acer species. Phytochemistry, Larson, K.C. (1998) The impact of two gall-forming on the photo- 16, 1421–1426. synthetic rates of their hosts. Oecologia, 115, 161–166. Bate-Smith, E.C. (1977b) Detection and determination of ellagitannins. Phyto- Littell, R.C., Stroup, W.W. & Freund, R.J. (2002) SAS for Linear Models.SAS chemistry, 11, 1153–1156. Institute, Inc., Cary, NC. Belovsky, G.E. & Slade, J.B. (2000) Insect herbivory accelerates nutrient cycling Madler, V.A.K. (1936) Eine Blattgalle an einem vorweltlichn Pappel-Blatt. and increases plant production. Proceedings of the National Academy of Sci- Natur und Volk, 66, 271–274. ences of the United States of America, 97, 14412–14417. Madritch, M.D., Donaldson, J.R. & Lindroth, R.L. (2007) Canopy herbivory Chapman, S.K., Schweitzer, J.A. & Whitham, T.G. (2006) Herbivory differen- can mediate the influence of plant genotype on soil processes through frass tially alters plant litter dynamics of evergreen and deciduous trees. Oikos, deposition. Soil Biology & Biochemistry, 39, 1192–1201. 114, 566–574. Madritch, M.D. & Hunter, M.D. (2002) Phenotypic diversity influences Chapman, S.K., Hart, S.C., Cobb, N.S., Whitham, T.G. & Koch, G.W. (2003) ecosystem functioning in an oak sandhills community. Ecology, 83, Insect herbivory increases litter quality and decomposition: an extension of 2084–2090. the acceleration hypothesis. Ecology, 84, 2867–2876. Madritch, M.D. & Hunter, M.D. (2003) Intraspecific litter diversity and nitro- Chen, H., Gonzales-Vigil, E., Wilkerson, C.G. & Howe, G.A. (2007) Stability gen deposition affect nutrient dynamics and soil respiration. Oecologia, 136, of plant defense proteins in the gut of insect herbivores. Plant Physiology, 124–128. 143, 1954–1967. Madritch, M.D. & Hunter, M.D. (2004) Phenotypic diversity and litter chemis- Choudhury, D. (1988) Herbivore induced changes in leaf-litter resource quality: try affect nutrient dynamics during litter decomposition in a two species mix. a neglected aspect of herbivory in ecosystem nutrient dynamics. Oikos, 51, Oikos, 105, 125–131. 389–393. Madritch, M.D. & Hunter, M.D. (2005) Phenotypic variation in oak litter influ- Cooper, W.R. & Rieske, L.K. (2009) Woody stem galls interact with foliage to ences short- and long-term nutrient cycling through litter chemistry. Soil affect community associations. Environmental Entomology, 38, 417–424. Biology & Biochemistry, 37, 319–327.

2012 The Authors. Functional Ecology 2012 British Ecological Society, Functional Ecology, 26, 628–636 636 C. J. Frost et al.

Maie, N., Behrens, A., Knicker, H. & Kogel-Knabner, I. (2003) Changes in the ings of the National Academy of Sciences of the United States of America, 99, structure and protein binding capacity of condensed tannins during decom- 15486–15491. position. Soil Biology & Biochemistry, 35, 577–589. Tooker, J.F., Rohr, J.R., Abrahamson, W.G. & De Moraes, C.M. (2008) Gall Northup, R.R., Dahlgren, R.A. & McColl, J.G. (1998) Polyphenols as regula- insects can avoid and alter indirect plant defenses. New Phytologist, 178, tors of plant-litter-soil interactions in northern California’s pygmy forest: a 657–671. positive feedback? Biogeochemistry, 42, 189–220. Trofymow, J.A., Moore, T.R., Titus, B.D., Prescott, C., Morrison, I., Siltanen, Nyman, T. & Julkunen-Tiitto, R. (2000) Manipulation of the phenolic chemis- M., Smith, S.M., Fyles, J., Wein, R., Camir, C., Duschene, L., Kozak, L.M., try of willows by gall-inducing sawflies. Proceedings of the National Academy Kranabetter, M. & Visser, S. (2002) Rates of litter decomposition over of Sciences of the United States of America, 97, 13184–13187. 6 years in Canadian forests: influence of litter quality and climate. Canadian Ostertag, R., Scatena, F.N. & Silver, W.L. (2003) Forest floor decomposition Journal of Forest Research, 32, 789–804. following hurricane litter inputs in several Puerto Rican forests. Ecosystems, Wall, D.H., Bradford, M.A., John, M.G.S., Trofymow, J.A., Behan-Pelletier, V., 6, 261–273. Bignell, D.D.E., Dangerfield, J.M., Parton, W.J., Rusek, J., Voigt, W., Wol- Parton, W., Silver, W.L., Burke, I.C., Grassens, L., Harmon, M.E., Currie, W.S., ters, V., Gardel, H.Z., Ayuke, F.O., Bashford, R., Beljakova, O.I., Bohlen, King, J.Y., Adair, E.C., Brandt, L.A., Hart, S.C. & Fasth, B. (2007) Global- P.J., Brauman, A., Flemming, S., Henschel, J.R., Johnson, D.L., Jones, scale similarities in nitrogen release patterns during long-term decomposi- T.H., Kovarova, M., Kranabetter, J.M., Kutny, L., Lin, K.C., Maryati, M., tion. Science, 315, 361–364. Masse, D., Pokarzhevskii, A., Rahman, H., Sabara, M.G., Salamon, J.A., Pascual-Alvarado, E., Cuevas-Reyes, P., Quesada, M. & Oyama, K. (2008) Swift, M.J., Varela, A., Vasconcelos, H.L., White, D. & Zou, X.M. (2008) Interactions between galling insects and leaf-feeding insects: the role of plant Global decomposition experiment shows soil impacts on decomposi- phenolic compounds and their possible interference with herbivores. Journal tion are climate-dependent. Global Change Biology, 14, 2661–2677. of Tropical Ecology, 24, 329–336. Weis, A.E. & Abrahamson, W.G. (1986) Evolution of host-plant manipulation Pe´rez-Harguindeguy, N., Dı´az, S., Cornelissen, J., Vendramini, F., Cabido, M. by gall makers – ecological and genetic-factors in the Solidago-Eurosta sys- & Castellanos, A. (2000) Chemistry and toughness predict leaf litter decom- tem. American Naturalist, 127, 681–695. position rates over a wide spectrum of functional types and taxa in central Wilkinson, C. & Scoble, M.J. (1979) The (Lepidoptera) of Argentina. Plant and Soil, 218, 21–30. Canada. Memoirs of the Entomological Society of Canada, 111,1–129. Reynolds, B.C. & Hunter, M.D. (2001) Responses of soil respiration, soil nutri- ents, and litter decomposition to inputs form canopy herbivores. Soil Biology Received 17 July 2011; accepted 15 February 2012 & Biochemistry, 33, 1641–1652. Handling Editor: Dan Hare Ritchie, M.E., Tilman, D. & Knops, J.M.H. (1998) Herbivore effects on plant and nitrogen dynamics in oak savanna. Ecology, 79, 165–177. Rossiter, M., Schultz, J.C. & Baldwin, I.T. (1988) Relationships among defolia- tion, red oak phenolics, and gypsy moth growth and reproduction. Ecology, 69, 267–277. Supporting Information Schweitzer, J.A., Bailey, J.K., Hart, S.C. & Whitham, T.G. (2005a) Nonaddi- tive effects of mixing cottonwood genotypes on litter decomposition and Additional supporting information may be found in the online ver- nutrient dynamics. Ecology, 86, 2834–2840. sion of this article. Schweitzer, J.A., Bailey, J.K., Hart, S.C., Wimp, G.M., Chapman, S.K. & Whitham, T.G. (2005b) The interaction of plant genotype and herbivory Figure S1. Photographs showing morphological and structural decelerate leaf litter decomposition and alter nutrient dynamics. Oikos, 110, changes to the leaf lamina by (A–B) Rhopalomyia solidaginis and (C) 133–145. Pemphigus betae. Stireman, J.O. & Cipollini, D. (2008) Stealth tactics of galling parasites and Figure S2. Gall induced on the petiole by Ectoedemia populella Brusk their potential indirect effects. New Phytologist, 178, 462–465. Stone, G.N. & Schonrogge, K. (2003) The adaptive significance of insect gall (A) in the field showing no apparent morphological changes to the morphology. Trends in Ecology & Evolution, 18, 512–522. leaf lamina, which was fully expanded before galling. (B–C) Close Swift, M.J., Heal, O.W. & Anderson, J.M. (1979) Decomposition in Terrestrial view of the gall exterior and interior. (D–E) E. populella larvae and Ecosystems. University of California Press, Berkeley, CA. frass pack. Photo credits: Christopher Frost. Tooker, J.F. & De Moraes, C.M. (2006) Jasmonate in lepidopteran larvae. Figure S3. Leaf drop timing in Populus grandidentata during active Journal of Chemical Ecology, 32, 2321–2326. Tooker, J.F. & De Moraes, C.M. (2007) Feeding by Hessian fly [Mayetiola litter fall. destructor (Say)] larvae does not induce plant indirect defences. Ecological As a service to our authors and readers, this journal provides Sup- Entomology, 32, 153–161. porting Information supplied by the authors. Such materials may be Tooker, J.F. & De Moraes, C.M. (2009) A gall-inducing caterpillar species re-organized for online delivery, but are not copy-edited or typeset. increases essential fatty acid content of its host plant without concomitant increases in phytohormone levels. Molecular Plant-Microbe Interactions, 22, Technical support issues arising from Supporting Information (other 551–559. than missing files) should be addressed to the authors. Tooker, J.F., Koenig, W.A. & Hanks, L.M. (2002) Altered host plant volatiles are proxies for sex pheromones in the gall wasp Antistrophus rufus. Proceed-

2012 The Authors. Functional Ecology 2012 British Ecological Society, Functional Ecology, 26, 628–636