Herbivorous Prairie Insects Can Be Co-Limited by Macronutrients and Sodium

Ecology Letters, (2018) 21: 1467–1476 doi: 10.1111/ele.13127 LETTER Seeking salt: herbivorous prairie insects can be co-limited by macronutrients and sodium

Abstract Chelse M. Prather,1,2* The canonical factors typically thought to determine herbivore community structure often explain Angela N. Laws,3,4 only a small fraction of the variation in herbivore abundance and diversity. We tested how Juan F. Cuellar,3,† macronutrients and relatively understudied micronutrients interacted to inﬂuence the structure of Ryan W. Reihart,2 insect herbivore (orthopteran) communities. We conducted a factorial fertilisation experiment Kaitlin M. Gawkins2 and manipulating macronutrients (N and P, added together) and micronutrients (Ca, Na and K) in 9 2 Steven C. Pennings3 large plots (30 30 m ) in a Texas coastal prairie. Although no single or combination of micronutrients affected herbivore communities in the absence of additional macronutrients, macronutrients and sodium added together increased herbivore abundance by 60%, richness by 15% and diversity by 20%. These results represent the ﬁrst large-scale manipulation of single micronutrients and macronutrients in concert, and revealed an herbivore community co-limited by macronutrients and Na. Our work supports an emerging paradigm that Na may be important in limiting herbivore communities.

Keywords Acrididae, grassland, micronutrient, nutrient limitation, prairie, sodium, tettigonidae.

Ecology Letters (2018) 21: 1467–1476

(Haddad et al. 2001; Joern et al. 2012). However, some of the INTRODUCTION intricacies of these factors have not been fully examined. For An understanding of the factors that determine herbivore instance, most research examining the role of plant nutritional abundance and diversity is of fundamental ecological interest. quality has only considered herbivore limitations by either N It is also necessary for the control of some herbivore species or P; N is a key component of amino acids (and proteins) that cause damage (Hulme 1994; Branson et al. 2006) and to while P is a deﬁning element in nucleic acids and phospho- support those species that are important purveyors of ecosys- lipids. Following others (Kaspari & Powers 2016; Bonoan tem services (McNaughton et al. 1997; Prather et al. 2012). et al. 2018), here we refer to N and P as ‘macronutrients’, and Several canonical factors are typically thought to largely regu- elements that are rarer in living tissues as ‘micronutrients’. In late herbivore communities, including top-down consumers the same way that multi-nutrient studies have shed new (Spiller & Schoener 1990; Dial & Roughgarden 1995) and bot- insight into limitations on plant diversity and abundance tom-up resources, typically characterised as plant biomass (Harpole et al. 2016), experiments that incorporate multiple (Lawton 1983; McNaughton et al. 1989; Lewinsohn et al. nutrients to examine the possibility of multiple resource limi- 2005; Wimp et al. 2010), plant diversity (Hutchinson 1959; tation (co-limitation) or that manipulate micronutrients may May 1990; Siemann et al. 1998; Pakeman & Stockan 2014), provide a better understanding of the relative importance of and plant nutritional quality (White 1984; Fagan et al. 2002; the factors that structure herbivore communities. Huberty & Denno 2006; Joern et al. 2012). Although manipu- The historical focus on single factor limitation arose lations of any one these factors has usually resulted in some through the application of a parsimonious and logical con- change to herbivore communities, our ability to explain herbi- cept: Leibig’s law of the minimum (van der Ploeg & Kirkham vore abundance and diversity is still relatively weak. For 1999). This law posits that one resource limits a population of example, plant richness only explains ~ 20% of the variation organisms at any given time, and that the limiting resource is in herbivore richness across 320 studies (Castagneyrol & Jactel that which has the highest demand to supply ratio (but see 2012). Similarly, other single factors like top-down consumers, Wilder & Eubanks 2010). For example, in the case of single plant productivity, and plant nutritional quality (often mea- nutrient limitation, the nutrient with the highest ratio of con- sured as the stoichiometry of nitrogen, N, and phosphorus, P) centration in an organism’s body compared to the concentra- clearly inﬂuence herbivore communities, but considerable vari- tion of the nutrient in the organism’s food would be assumed ation remains unexplained (e.g. Letourneau et al. 2009), even to be the single limiting nutrient in that ecosystem. Nutrient when more than one of these factors are considered together limitation of herbivores, in particular, has largely focused on

1Department of Biology, Radford University, Radford, VA 46556, USA †Present address: Department of Surgical Pathology, Northwestern University, 2Department of Biology, University of Dayton, Dayton, OH 45469, USA 251 E. Huron, Galter 7-281A, Chicago, IL 60611, USA 3Department of Biology and Biochemistry, University of Houston, Houston, *Correspondence and present address: Chelse M. Prather, Department of TX 77204, USA Biology, University of Dayton, Dayton, OH 45469, USA 4The Xerces Society, Sacramento, CA 95814, USA E-mail: [email protected]

© 2018 John Wiley & Sons Ltd/CNRS 1468 C. M. Prather et al. Letter single nutrient limitation, with first a focus on nitrogen (Matt- membranes (e.g. the sodium pump); generating electrical son 1980; White 1984), and later on phosphorus (Urabe & impulses in excitable cells, like neurons, via sodium channels; Watanabe 1992; DeMott et al. 1998; Sterner & Schulz 1998), maintaining hydrologic homeostasis; and supporting neural especially in cases when N was abundant (Elser et al. 2001; and brain development (Snell-Rood et al. 2014). Na may also Makino et al. 2002; Huberty & Denno 2006; Tao & Hunter be important for reproduction in orthopterans, as suggested 2012). This focus on N and P was logical: both have relatively by changes in nymphal Na concentrations: newly hatched high demand to supply ratios in herbivores with much higher nymphs have the highest Na which steadily decreases over N and P concentrations in herbivore body tissues than plants their development (Boswell et al. 2008). However, with a few (Sterner et al. 1992). exceptions (Beanland et al. 2003; Kaspari et al. 2008, 2010, Plant nutrients, however, have complementary functions 2014, 2017), studies showing the potential importance of whereby some basic physiological function relies on more than micronutrients in animal communities have been correlative one resource being at critical thresholds simultaneously; too or laboratory experiments, and as such, are open to criticisms little of any one of these complementary nutritional resources that they simply reflect unmeasured variables or do not reflect impacts physiological functioning, in particular the mainte- natural conditions. nance of cell and tissue homeostasis. For this reason, the We tested the overarching idea that micronutrients would applicability of Leibig’s law of the minimum to populations alter the abundance and community structure of herbivores and communities has been questioned (e.g. Danger et al. (orthopterans) using a factorial field experiment in a Texas 2008), and now multiple resource limitation is generally recog- prairie with large plots (30 9 30 m2). We chose to focus on nised to likely be common in plant communities (Harpole orthopterans because they are relatively large (for insects), et al. 2011, 2017; Fay et al. 2015). Co-limitation may occur ecologically and economically important, generalist herbivores via several different pathways (Sperfeld et al. 2016): indepen- that are relatively easy to identify to genus or species. We first dent co-limitation occurs when two different nutrients sepa- determined how well orthopteran communities at our site rately enhance populations, and these effects are further were predicted by the canonical variables described above vs. enhanced when both nutrients are abundant: simultaneous relatively understudied plant and soil micronutrients, using an limitation occurs when neither nutrient positively affects an observational approach. Based on these results, we then chose organism, but together they produce a positive effect when a three micronutrients to manipulate (Ca, K and Na) that have critical threshold of both is reached; and serial co-limitation been implicated in structuring insect (Kaspari et al. 2017) and occurs when one of the nutrients produces a moderately posi- orthopteran (Joern et al. 2012) communities in other studies. tive effect that is enhanced by the addition of some other lim- We then tested the hypotheses that micronutrients affect the iting nutrient. Many different reasons may exist for these abundance, diversity, and composition of common grassland different types of co-limitation in consumer communities. For insect herbivores (orthopterans), and that these effects depend instance, Kaspari et al. (2017) suggest serial limitation of on the availability of macronutrients (i.e. that micronutrients insects by macronutrients and Na at their Oklahoma grass- and macronutrients are co-limiting). We measured nutrient land site occurred because macronutrients limited plant bio- effects on plant biomass and chemistry, and also conducted mass whereas both N and Na altered leaf nutritional quality. feeding trials to determine if observed effects were due to Mounting evidence suggests that nutrient co-limitation may changes in resource availability or nutritional quality. Because occur not only in plant communities, but also in herbivores our coastal tallgrass prairie field site likely receives marine- (Raubenheimer & Simpson 2004; Sperfeld et al. 2012; Simp- derived inputs of Na and Ca, we expected that these two son et al. 2015; Kaspari & Powers 2016). For example, Joern nutrients alone would not have strong effects on insects as et al. (2012) found that the canonical factors associated with ambient levels may already be high. However, we hypothe- herbivore community structure (plant biomass, diversity, and sised that these nutrients might become limiting when macronutrient concentrations) explained little variance in macronutrient limitation of plant biomass was alleviated. We regional grasshopper community composition or the abun- also predicted that orthopterans that eat either a mixture of dance and diversity of different feeding guilds. When multiple grass and forb or woody species (mixed plant feeders), or plant nutrients (including the micronutrients, Na and K) were both plants and animal prey (omnivores) were less likely to be considered together with some of these canonical variables, limited by these micronutrients than those that mainly eat however, much more of the variation in herbivore community grasses (grass feeders) because forbs and animals are much structure was explained. higher in micronutrients than grasses. Recent research has shown that certain micronutrients may limit consumer communities, further explaining variation in MATERIALS AND METHODS herbivore abundance and diversity (Joern et al. 2012; Kaspari & Powers 2016). Besides macronutrients, around 23 other ele- We worked in a coastal tallgrass prairie in Texas, about 45 ments are also crucial to basic physiological functioning of all kilometers south of Houston and 20 kilometers north of the organisms (Kaspari & Powers 2016), including Ca, K and Na, Gulf Coast at the University of Houston’s Coastal Center which act as electrolytes and are crucial for multiple physio- (UHCC; 29°23026.96″ N; 95°1051.95″ W). UHCC prairies are logical processes. For example, Na, known to limit ants managed using fire (on average, every 8 years) or mowing (ev- through a hypothesised increase in metabolism (Kaspari et al. ery winter) to prevent woody encroachment and the spread of 2017), is important for many physiological processes (Chown invasive species. Around 1070 mm of rain falls throughout the & Nicolson 2004): regulating gradients of ions across cell year, and the average temperature is around 20.9 °C. This

© 2018 John Wiley & Sons Ltd/CNRS Letter Macronutrients and sodium co-limit herbivores 1469 prairie is dominated by graminoids (early summer – sedges, e.g. sufficient to cause differences in soil moisture at our site, Rhyncospora caduca Elliot, later summer – tall grasses, e.g. which has a flat topography. The large size of the plots acted Andropogon gerardii Vitman and Schizacrium scoparium to minimise artifacts caused by transient arthropods, and (Michx) Nash). Common forbs are largely in the aster (e.g. Lia- allowed us to sample plants and arthropods without markedly tris pycnostaycha Michx and Helianthus angustafolius L.) and affecting the densities of either in the plots. carrot families (e.g. Centella erecta (L. f.) Fernald and Eryn- We added macro- and micronutrient fertilisers to the plots gium yuccafolium Michx), and several woody plant species are in late winter (in early March before the growing season common (Myrica cerifera L., Baccharus halimifolia L., and began after plots were mowed) in 2016 and 2017. We chose Rubus riograndis Bailey; Siemann et al. 2007). Around 16 spe- forms of nutrients that were (1) simple, so as to not add com- cies of orthopterans (excluding crickets) occur at the site, which plex molecules or other non-target nutrients into the plot, (2) can be classified into four feeding guilds based on preliminary relatively water soluble, (3) granular instead of powder for feeding trials at our site (Prather and Pennings, unpublished): ease of spreading and more precise application, and (4) not grass feeders (e.g. Orphulella speciose Scudder), forb feeders overly cost prohibitive (Table 1). Macronutrients were each (e.g. Hesperotettix speciosus Scudder), mixed feeders (which added at 10 g m2 as is common in fertilisation experiments feed on both grasses and forbs or woody plants, e.g. Melano- (e.g. Nutrient Network: Borer et al. 2014). Micronutrients plus femurrubrum De Geer), and omnivores (which eat both were added at levels needed to create soil concentrations of plants and animal prey, e.g. Orchelimum vulgare Scudder). each that were ~ 30% higher than average ambient levels We conducted a field survey at 12 locations in prairies at ( 1 standard deviation above the average) found in the top UHCC in 2011 (detailed methodology in Appendix S1) to 10 cm of soil our site (Table 1), and all final levels were con- determine correlational relationships between orthopteran centrations that naturally occur at the site. We measured communities and canonical factors as well as micronutrients. plant biomass via vegetation clipping (0.1 9 1m2 strips; 5 per We measured orthopteran abundance and diversity, soil char- plot), and measured the nutrient content of 4 common plant acteristics, plant biomass and diversity along with soil and species on which we have witnessed abundant herbivory (2 plant nutrients at each location. We used step-wise multiple graminoids: Rhyncospora cauduca and Schizacrium scoparium; regression to determine what factor or combination of factors 2 forbs: Liatris pyncostachya and Centella erecta) on at least best predicted herbivore abundance and diversity. The result- three plant individuals per plot and nutrient concentrations in ing relationships suggested the importance of certain micronu- the soil using a pooled sample of three soil cores (n = 8 plots trients (Ca, Na and K), leading to their inclusion as per treatment). To assess the response of orthopterans to treatments in the subsequent fertilisation experiment. nutrient treatments, we measured the abundance and diversity We conducted a large fertilisation experiment in which we of grasshoppers each summer (June) in each plot using sweep manipulated macronutrients (N and P combined) and three net samples (100 sweeps per plot), and identified all orthopter- micronutrients (Ca, K and Na, each manipulated individually) ans to species (unless nymphs were too small to identify to beginning in 2015. Although it would have been interesting to species, in which case they were identified to genus). We also include N and P as separate treatments, the resulting experi- conducted feeding trials with seven orthopteran species from ment would have been twice as large and logistically infeasi- three feeding guilds to determine if feeding preferences ble, and our interest was primarily in the understudied role of matched results from our fertilisation experiment (i.e. if micronutrients. We fertilised large (30 9 30 m2) plots using a orthopterans chose leaves from treatments where orthopteran fully-crossed, factorial design: 2 macronutrient levels (ambient abundances were high over those with low abundances; meth- vs. fertilised) 9 2 Ca levels (ambient vs. fertilised) 9 2K ods in Appendix S2). levels (ambient vs. fertilised) 9 2 Na levels (ambient vs. fer- We report data from the second year of the fertilisation tilised) 9 8 replicates of each treatment combination for a experiment (2017) as effects did not fully manifest until the total of 16 treatments and 128 plots. Plots were located in second year, and so the intermediate results are of less interest. eight blocks that spanned a 2 cm gradient in elevation, with To determine the effect of macro- and micronutrients and each block containing a single replicate of each treatment. their interactions on plant biomass and orthopteran communi- Although 2 cm is not a large elevational difference, it was ties (abundance, richness, and diversity calculated as the

Table 1 Naturally occurring concentrations of macro- and micronutrients in the top 10 cm of soil at the study site were obtained from a correlational study that was 2 years before starting the fertilisation experiment, and these values were used to calculate the amounts of fertilisers used in the ﬁeld experiment

Amount of Rate of fertiliser Average Range of % above nutrient % element applied concentration concentration Form of fertiliser used average applied in annually Nutrient (mg kg1) (mg kg1) in experiment Chemical formula ambient (g m2 year1) fertiliser (g m2)

N 0.70 (NH4) 0.6–1.06 Granular MAP + granular urea NH4H2PO4 + N2H4CO 53% 10 46 192 + 36 P 2.52 0.86–5.01 Granular MAP NH4H2PO4 63% 10 32 192 Ca 2597 1530–3380 Granular calcium carbonate CaCO3 28% 46.5 41 1134 K 156 84–238 Granular potassium chloride KCl 32% 3.1 52 52

Na 296 200–409 Granular soda ash Na2CO3 33% 6.2 43 144

© 2018 John Wiley & Sons Ltd/CNRS 1470 C. M. Prather et al. Letter inverse Simpson’s index) and on individual orthopteran species plus Na treatments (Fig. 2a), and foliar P was higher in all (for those that were abundant enough throughout the treat- plants when macronutrients were added (Table S3). ments to perform statistical analyses), we used GLMs with Orthopterans collected from the fertilisation experiment rep- macronutrients, Ca, K and Na as fixed factors (each with 2 resented 15 species: 6 tettigoniids, 2 gryllids and 7 acridids levels – ambient or addition). For orthopteran total abun- (Table S4). Overall orthopteran abundance was serially co- dance and species abundance, we used Poisson log-linear mod- limited by macronutrients and Na (Figs 2c and 3a,b). Total els, and for the other variables (richness and diversity) we used numbers increased slightly with macronutrients (GLM macro linear models. To determine the effect of nutrient treatments effect: < 0.001), but Na enhanced the positive effect that on nutrient chemistry and stoichiometry, we used GLMs with macronutrients had on orthopteran abundance (Macro 9 Na: nutrient treatments (macro, Ca, K and Na) and plant species P = 0.005; Table 2, Fig. 3a) in a pattern consistent with serial (4 species) as factors for several plant chemistry variables and co-limitation, with over 1.5 times more individuals where both soil chemistry variables (log Ca, log K, log Na, log N, log P, macronutrients and Na were added than when neither was N : Na and N : P), and performed Bonferroni corrections as added (Figs 2c and 3). However, no single micronutrient, these concentrations and ratios were not independent of one including Na, or combination of micronutrients affected another (P = 0.006 for significance). We used Cohen’s d (the orthopteran abundance in the absence of macronutrients difference between the means of treatments and controls (GLM Ca, Na, and K effects & their interactions all had divided by the pooled standard deviation) to determine the P > 0.05). Effect sizes for all treatments that including both effect sizes of different treatments on orthopteran abundance, Na and macronutrients were higher than when macronutrients richness, and diversity. We examined how orthopteran com- were added alone or when macronutrients were added with munity structure changed using PCA to simplify the six most micronutrients other than Na (Fig. 3b). common orthopteran species found in our sampling into two Despite our prediction that mixed plant-feeders and omni- major axes, and visualised these differences by plotting the vores were less likely to be limited by Na, the orthopteran average and standard error of each of the 16 treatment combi- species found in the plots were all mixed feeders and omni- nation for each principle component on a scatterplot. vores that were indeed co-limited by macronutrients and Na. Within the orthopteran community, different species responded in different ways to the treatments (Appendix S3, RESULTS Table S2, Fig. S4). Of note was that the most strongly In 2011, prior to starting the fertilisation experiment, total responding species, Scudderia texensis Saussure and Pictet orthopteran abundance at the site was correlated with a com- was positively affected by macronutrients, and this effect bination of macronutrients (soil nitrate concentration) and was enhanced by the addition of Na. Males of this species micronutrients (the concentration of foliar K) (Table S1). The abundance of different feeding guilds were similarly predicted by various combinations of macronutrients (soil and foliar N and P, and foliar C : N : P) and micronutrients (Ca, Na and K), except for grass feeders, which were only related to plant species richness (Table S1). Based on these results, we chose to manipulate the three micronutrients (Ca, Na and K) that were correlated with aspects of the orthopteran community at the site in the fertilisation experiment. We did not consider crickets further, other than herbivorous tree crickets, because our sampling methods were not optimised to collect them and because our focus was on the taxa that eat significant amounts of living plant material. In the fertilisation experiment, plant biomass was enhanced with any addition of macronutrients (P < 0.001), but was not affected by micronutrients and the positive effect of macronutrients did not change when any micronutrient or combination thereof was added (all micronutrients & all interactions: P > 0.05; Fig. 1). Soil nutrient concentrations of Na, N and P each increased when their respective nutrient was applied, and there was a trend for Ca and K concentrations to be higher in the soil when each of these nutrients was added (Figs S1 and S2). Nutrient treatment effects on leaf nutrient concentrations were more complicated, and differed by nutrient or ratio examined and plant species (Figs S1 and S2). Of note was Figure 1 Macronutrients increased plant biomass in all treatments where they were added (P < 0.001), but no other nutrient or combination of that leaf Na was not higher for any plant species when Na nutrients altered plant biomass. Each bar represents the average plant was added, but was higher in both forb species when biomass (g m2) 1 SE in one micronutrient treatment (labelled on macronutrients were added (Fig. S3). Additionally, foliar x-axis) crossed with one macronutrient treatment (white bars represent N : Na ratios in all plants were lower in the macronutrient ambient macronutrients; grey bars represent added macronutrients).

Figure 2 (a) Leaves (4 plant species pooled) from plots with macronutrients and Na added differed in leaf chemistry, including lower N : Na ratios. (b) Potentially as a result of altered leaf chemistry, orthopterans preferred to feed on leaves from plots where both macronutrients and Na were added, as shown by laboratory feeding trials with individuals pooled across seven species of orthopterans and four species of plants (P = 0.01). Feeding preferences were shown as the percentage of total leaf consumption that consisted of each leaf treatment. The dashed line represented null hypothesis values (total consumption should be made up of equal amounts of all available leaves, or total consumption should be equal to 25% for all four leaf treatments in each test). (c) The abundance of orthopterans was significantly higher when both macronutrients and Na were added (P = 0.005). Asterisks indicate that the result is significantly different than the null hypothesis. were actually only found in plots with both macronutrients the macronutrients plus Na treatment or the NaK treatment and Na. fell in the upper right quadrant of the figure. In contrast, Sodium and macronutrients added together increased treatment combinations including macronutrients plus Ca fell orthopteran richness and diversity, and changed composition. in the upper left quadrant of the figure. Neither single micronutrients nor macronutrients alone affected orthopteran richness or diversity (Table 2, Fig. 3; DISCUSSION GLM for all main effects > 0.05). In fact, species richness and diversity tended to be lower with the addition of macronutri- With this first manipulation of single micronutrients and ents alone than in control plots (Fig. 3e,f; d = 0.63 and macronutrients in concert in plots of large size (30 9 30 m2), 0.56, respectively). However, the addition of macronutrients we show a grassland herbivore community limited by plus Na increased richness and diversity (Table 2, Fig. 3; macronutrients and Na. In a pattern consistent with serial co- GLM macro 9 Na interaction P = 0.007 and 0.004, respec- limitation, the increases in abundance with these co-limiting tively). As for abundance, effect sizes for richness and diver- nutrients (~ 60% more individuals) were much greater than sity for all treatment combinations including both for Na alone (which had no significant effect, but tended to macronutrients and Na were greater than or equal to effect suppress abundance), or for macronutrients alone, which sizes for macronutrients alone, or any other treatment combi- slightly increased orthopteran abundance. Here, we discuss nations. Orthopterans preferred to eat leaves from NP 9 Na the potential reasons for Na having a strong effect, propose treatments: on average, around one-third of total leaf con- mechanisms for co-limitation, and put our results in perspec- sumption consisted of leaves from the NP 9 Na treatment, tive in light of biogeographic and anthropogenically-altered whereas with no preference each leaf would make up an equal distributions of these nutrients. amount of total consumption (i.e. 25% for the 4 leaves in Anecdotally, natural historians have long known that Na is each trial; Fig. 3b, all plant and orthopteran species pooled, important to animals. Mammals and birds sometimes use P = 0.01). Orthopterans did not prefer leaves from other sodium rich salt licks or wood with little nutritional value treatments. besides high concentrations of Na (gorillas – Rothman et al. PCA reduced the six most abundant species, which repre- 2006; parrots – Lee et al. 2010). Lepidopterans often puddle sented ~ 80% of all the orthopterans, to two principle compo- at salty soil, carcasses, tears and perspiration (Arms et al. nents (PC1 and PC2; Fig. 4) that collectively explained 43% 1974; Hilgartner et al. 2007). Salty human urine has been used of the variance in the samples. PC1 represented three tettigo- as insect bait, both to catch insect pests (Martın & Pinero~ niid species (O. vulgare, 0.50; Orchelimum concinnum Scud- 2004) and in ecological studies (e.g. Gordon 2012). Sodium is der, 0.51; and Conocephalus strictus Scudder, 0.54), and one known to be crucial for the intact functioning of many physi- acridid species (Paroxya atlantica Scudder, 0.64). PC2 repre- ological processes, such as the sodium pump, cell signalling, sented two tettigoniid species (Neoconocephalus sp., 0.59; and the maintenance of hydrologic homeostasis, and neural and S. texensis, 0.67). The macronutrient and micronutrient treat- brain development (Chown & Nicolson 2004; Snell-Rood ments had different effects on orthopteran community compo- et al. 2014). sition. Orthopteran communities in the control treatment and Despite this extensive anecdotal information and the in treatments with micronutrients alone (open circles) or in known importance of Na in animal physiology, it was only the macronutrients alone treatment (filled circle without label) recently that the first terrestrial small-scale (1 m2) field stud- clustered in the middle of the figure. Communities in either ies were done experimentally manipulating Na (Kaspari et al.

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Figure 3 Orthopteran community measurements were all highest in treatments with both macronutrients and Na added: (a) orthopteran abundance (P = 0.005), (b) effect size on abundance, (c) richness (P = 0.007), (d) effect size on richness, (e) diversity (P = 0.004), and (f) effect size on diversity. Each bar (averages 1 SE) and point (Cohen’s d) represents one micronutrient treatment (labelled on x-axis) and macronutrient treatment (white bars/points represent ambient macronutrients; grey bars/points represent added macronutrients). These data suggest that this orthopteran community is serially co- limited by macronutrients and Na.

2014). These studies showed that Na played an important inconsistencies in these results might point out the unrelia- role in determining the structure of termites (Kaspari et al. bility of correlations between soil and foliar nutrient con- 2014), ants (Clay et al. 2014), and more recently, prairie centrations and the abundance of consumers that might insect communities (Kaspari et al. 2017). In the most recent arise because of unmeasured variables, and also indicate the study with prairie insect communities, above-ground insect importance of testing putative relationships with manipula- abundance increased by over 50% when macronutrients plus tive experiments. Because both the survey and manipulative Na were added (Kaspari et al. 2017), which is qualitatively experiment were conducted in field plots rather than enclo- very similar to how orthopterans responded in our larger- sures, we do not know whether positive responses of organ- scale study. isms in some treatments reflect rates of movement among Although our 2011 correlational sampling indicated that plots, with organisms moving to or remaining in preferred micronutrients were important, it did not predict orthop- plots, or reflect increased population growth or survival teran responses to particular treatments in the manipulative rates. Understanding whether these differences are a result experiment, especially the importance of Na. In the observa- of orthopteran movement or changes to reproductive rates tional study, although soil nitrate was correlated with total requires further experiments that we are currently attempt- orthopteran abundance, Na was not. Na was correlated ing. Moreover, grasshopper densities were lower at our site with the abundance of forb feeders (not collected in the fer- than at other grasslands (e.g. Batary et al. 2007; O’Neill tilisation experiment) and crickets (less than 0.01% of the et al. 2010), perhaps indicating that additional factors sampled individuals in the experiment). However, the beyond those we studied limit grasshopper densities at our

Figure 4 The effect of nutrient treatments on orthopteran community composition, represented by average principle component values ( SE) for PCA 1 and PCA 2, two principle components that collectively explained 43% of the variance. A PCA reduced the most six abundant species to two axes, labelled on the ﬁgure. Codes for grey circles indicate micronutrient treatments. Circle symbols indicate either without macronutrients added (open circles), or macronutrients added (grey circles). Micronutrient codes are shown only for the macronutrient combinations, because all treatment combinations of micronutrients without macronutrients overlapped. Macronutrients alone (the unlabelled grey point) also overlapped with controls and micronutrient only plots.

Table 2 Summary of P-values resulting from GLM models to determine the effects of experimental nutrient treatments on orthopteran numbers, species whereby ample amounts of one nutrient could cause others to = richness and diversity. Macro macronutrient treatment, consisting of N become limiting. Sodium has been hypothesised to be crucial plus P. Bolded values show statistically significant effects or interactions. in limitation cascades in herbivores (Kaspari & Powers 2016) Total number Richness Diversity (1/D) because Na concentrations that are 100–1000 times higher in herbivore bodies than plant tissue, and insect herbivores have Macro <0.001 0.13 0.30 no stable storage mechanism for Na. Ca 0.64 0.69 0.83 K 0.22 0.73 0.80 This serial co-limitation may be occurring via direct or Na 0.33 0.20 0.23 indirect mechanisms, as has been shown for other orthop- Macro 9 Ca 0.55 0.62 0.56 teran species (tettigoniids – Mormon crickets, Simpson et al. Macro 9 K 0.19 0.96 0.80 2006). We hypothesise that co-limitation is mainly occurring Macro 9 Na 0.005 0.007 0.004 via direct mechanisms, not indirect changes in plant composi- 9 Ca K 0.29 0.62 0.88 tion. Macronutrients increased the amount of food available 9 Ca Na 0.80 0.81 0.77 to these generalist herbivores, but increased orthopteran Na 9 K 0.49 0.92 0.64 Macro 9 Ca 9 K 0.57 0.62 0.93 abundance was not due solely to a direct increase in plant Macro 9 Ca 9 Na 0.55 0.40 0.36 biomass (which only responded to macronutrients), but Macro 9 K 9 Na 0.43 0.62 0.23 instead likely occurred through some change in plant palata- C 9 K 9 Na 0.98 0.59 0.93 bility. Although we need to conduct experiments with artifi- Macro 9 Ca 9 K 9 Na 0.61 0.15 0.27 cial diets to confirm this, the most obvious trend in the data is that the N : Na ratio decreased in treatments with added Na (Fig. 2c), so Na was likely more bioavailable. Addition- site. Thus, cross-site experiments are needed to corroborate ally, foliar P was higher in these leaves. An alternative our results. hypothesis is that plant composition changed; however, our Our results are consistent with a serial co-limitation path- feeding experiments where plant composition was held con- way: orthopteran abundance responded positively but weakly stant showed that a change in plant quality alone is likely to macronutrients, did not respond to sodium addition, and sufficient to explain our results (however, we are currently strongly increased when both macronutrients and sodium doing experiments that explore this potential mechanism). were added (Table 2, Figs 2c and 3). Serial limitation such as Additionally, differences in the orthopteran community were this has previously been indicated in ‘limitation cascades’ not seen until the second year of fertilisation, likely because

© 2018 John Wiley & Sons Ltd/CNRS 1474 C. M. Prather et al. Letter treatments did not induce wide-spread changes in plant stoi- 1995). Therefore, current agricultural management practices chiometry until the second year. may unintentionally be benefiting orthopteran herbivores One notable feature of the orthopteran community at this that cost ~ $1.25 billion in agricultural plant production site is that it is dominated by tettigoniids (katydids made annually in the US alone (Branson et al. 2006). up 80% of the individuals and 7 out of 15 species) that are often omnivorous, eating not only leaves, but seeds and CONCLUSIONS pollen as well as small animals. Accordingly, this increase in plant biomass likely also resulted in increases in seed or The canonical explanations for patterns in herbivore commu- flower abundance, or increases in prey availability for nity structure have largely ignored the possible role of omnivorous species, and these increases in resource avail- micronutrients. We provide further evidence for an alternative ability could have increased performance of the tettigoniid possibility: certain herbivore communities are co-limited by members of the community. macronutrients and micronutrients, especially Na. Given our The combination of macronutrients and Na altered leaf still rather limited ability to predict the structure of herbivore chemistry in ways what could affect herbivore populations communities, a better understanding of possible herbivore co- directly through altered physiological functioning, as is sug- limitation by macro- and micronutrients is necessary. This gested by our leaf chemistry and feeding trial data (Fig. 2b,c). understanding is especially important with human disruption Na has been suggested to be important for reproduction in of ambient biogeography of almost all the macro- and micro- other orthopterans (Boswell et al. 2008). Scudderia texensis, nutrients that are crucial to life. This study highlights that the species that responded mostly strongly to macronutrient altered distributions and availability of macronutrients and plus Na treatments uses nuptial gifts in its mating system. Na could potentially have large, long-term effects on insect Although females were spread across treatments, male S. tex- herbivore communities. Considering these factors in future ensis were collected only in macronutrient plus Na plots, sug- research may lead to a better understanding of herbivore com- gesting that this treatment was especially attractive to males. munity structure and have wide-ranging implications for her- In most lepidopteran species, it is males that most commonly bivore pest management and the promotion of beneficial exhibit puddling behaviour, especially those that offer nuptial herbivore groups. gifts (Sculley & Boggs 1996; Molleman et al. 2005). Male diet also affects egg nutrients: females that receive these nuptial gifts from males that have consumed Na-rich diets lay ACKNOWLEDGEMENTS eggs that are higher in sodium (Smedley & Eisner 1996). Egg This work was supported by DEB NSF grants #1457114 and nutrients may benefit larvae that have little opportunity for #1724663 to Prather. Reihart was supported by a Graduate sodium acquisition in their diets. In katydids, better nutri- Student Summer Fellowship from the University of Dayton. tional condition in males (determined by feeding them high We thank Tim Becker, Jennifer Blake-Mahmud, Denise Mon- protein diets) increases the size of nuptial gifts (Jia et al. telongo, Shania Hurst, Kiersten Angelos and many other 2000), but nothing is known about the importance of Na in undergraduates and volunteers that helped with this field nuptial gifts in katydids. If similar relationships between work, and three anonymous reviewers that greatly improved sodium and nuptial gifts exist in katydids as in lepidoptera, this manuscript. We also thank the orthoptera in a rare this suggests that future studies of Na limitation should look coastal grassland that allowed this study to occur. for differential effects in males, which may seek out Na in order to enhance the quality of nuptial gifts or other reasons. The biogeography of nutrients influences whether organ- AUTHORSHIP isms are limited by a particular nutrient. For instance, ant communities have been shown to be limited by Na inland CP and SP conceived of this project; CP, AL, JC, KG, and but not in coastal areas where soil Na concentrations are RR collected the data; CP performed statistical analyses, and high from marine-derived sources (Kaspari et al. 2008). wrote the first draft, and all authors contributed substantially Additionally, the assumed high Na inputs at our coastal tall- to revisions. grass prairie site may explain the lack of a direct effect of Na. If Na limitation of orthopterans occurs on a wider geo- REFERENCES graphic scale, these results may have important implications Arms, K., Feeny, P. & Lederhouse, R.C. (1974). Sodium: stimulus for in light of the wide-ranging anthropogenic effects on global puddling behavior by tiger swallowtail butterflies, Papilio glaucus. Na distribution. 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