Michael A. Huston

Hidden treatments in ecological experiments:re-evaluating the ecosystem function of biodiversity

Received: 16 December 1996 /Accepted: 2 March 1997

Abstract Interactions between biotic and abiotic pro-cesses complicate the design and interpretation of eco-logical experiments. Separating causality from simplecorrelation requires distinguishing among experimentaltreatments, experimental responses, and the many pro-cesses and properties that are correlated with either thetreatments or the responses, or both. When an experi-mental manipulation has multiple components, but onlyone of them is identi®ed as the experimental treatment,erroneous conclusions about cause and e�ect relation-ships are likely because the actual cause of any observedresponse may be ignored in the interpretation of theexperimental results. This unrecognized cause of anobserved response can be considered a ``hidden treat-ment.'' Three types of hidden treatments are potentialproblems in biodiversity experiments: (1) abiotic condi-tions, such as resource levels, or biotic conditions, suchas predation, which are intentionally or unintentionallyaltered in order to create di�erences in species numbersfor ``diversity'' treatments; (2) non-random selection ofspecies with particular attributes that produce treatmentdi�erences that exceed those due to ``diversity'' alone;and (3) the increased statistical probability of including aspecies with a dominant negative or positive e�ect (e.g.,dense shade, or nitrogen ®xation) in randomly selectedgroups of species of increasing number or ``diversity.'' Ineach of these cases, treatment responses that are actuallythe result of the ``hidden treatment'' may be inadver-tently attributed to variation in species diversity. Casestudies re-evaluating three di�erent types of biodiversityexperiments demonstrate that the increases found insuch ecosystem properties as productivity, nutrient usee�ciency, and stability (all of which were attributed tohigher levels of species diversity) were actually caused by

``hidden treatments'' that altered plant biomass andproductivity.

Key words Species diversity áExperiment áProductivity áStability áResources

Introduction

The ®eld of ecology is distinguished by the complexity ofthe processes and interactions that are its primary focusand also the primary excuse for ecologists' failure toe�ectively address major environmental problems. Re-peated calls for more rigorous and relevant ecology (e.g.,Suter 1981; Peters 1991; Shrader-Frechette and McCoy1994; Sarkar 1996) have led to an increasing emphasison the need for ``mechanistic models'' (Schoener 1986;Tilman 1987a) and the use of an experimental approachthat manipulates such factors as resources and predationrates (Hairston 1989; Underwood 1996). Rigorous ex-perimental tests of hypotheses are essential as ecologyincreasingly addresses issues of political, social, andeconomic importance. Experimental results typicallyhave a much greater impact than new theory, models, orobservations. However, poorly designed experiments ormisinterpretations of experimental results have the po-tential to mislead scientists and policy makers alike.

Biodiversity has recently emerged as an issue of bothscienti®c (Wilson 1988; Ehrlich and Wilson 1991; Petersand Lovejoy 1992) and political (United Nations Envi-ronment Programme 1992; World Resources Institute1992; Heywood and Watson 1995) concern primarilybecause of an increase in extinction rates caused byhuman activities (Myers 1979; Ehrlich and Ehrlich 1981;Lawton and May 1995; Pimm et al. 1995). Biodiversity isconsidered to be important for a variety of reasons(Old®eld 1989; Randall 1994; Rolston 1994), but recentattention has focused on its potential importance for theadequate functioning of the Earth's ecosystems (Schulzeand Mooney 1993). This concern about the environ-mental consequences of biodiversity loss (and thus, a

Oecologia (1997) 110:449±460 Ó Springer-Verlag 1997

Michael A. HustonEnvironmental Sciences Division, Oak Ridge National Laboratory,Oak Ridge, TN 37831-6335 USAFax: 423-574-2232; e-mail: mhu@ornl.gov

potential justi®cation for biodiversity conservation) haselevated biodiversity to near the top of the researchagenda for ecology and environmental sciences in gen-eral (Solbrig 1991; Lubchenco et al. 1991; NationalScience and Technology Council 1995a, b; President'sCouncil on Sustainable Development 1996). Strongtheoretical and experimental results are needed to pro-vide guidance for environmental policy and resourcemanagement. However, it is not always simple to ``rig-orously assess the ecosystem function of biodiversity in amanner that speaks plainly to the concerns of the publicand policy makers'' (Kareiva 1996).

Biodiversity is a quintessential ecological phenome-non because it represents the net result of a complex setof interacting ecological, evolutionary, biogeographical,and physical processes. A particular problem associatedwith biodiversity experiments is that the primaryexperimental treatment of varying the number of speciesis often correlated with variation in other biological orphysical factors that can have a stronger e�ect on theexperimental response than the putative primary treat-ment. These ``hidden treatments'' can produce strongbiological responses that may be misinterpreted asconsequences of a particular level of species diversity.

A brief history of biodiversity experimentation

Species diversity is easily manipulated, and consequentlythere is a long history of ``biodiversity'' experiments,beginning in 1843 with the Park Grass Experiments atthe Rothamsted Experimental Station in Great Britain(Lawes et al. 1882). In these experiments, various fer-tilizer and liming treatments were evaluated for theire�ect on the yield of pastures for grazing and hay pro-duction. An early result that strengthened over time wasa decrease in the number of species that occurred in theplots with the highest yield (resulting from the highestfertilizer application) (Silvertown 1980).

Many other experiments have produced similar re-sults, with the number of species of terrestrial or aquaticplants almost always decreasing in response to increasedproductivity caused by nutrient addition (see Grime1973a, b, 1979; Huston 1979, 1980, 1994; Austin andAustin 1980; Tilman 1987b, 1993). Similar patterns arefound along natural gradients of plant productivity(Keddy and MacLellan 1990; Al-Mufti et al. 1977; Ba-kker 1989; Huston 1980, 1994). The well-known phe-nomenon of eutrophication of aquatic systems resultsfrom a dramatic increase in algal productivity and bio-mass (and an equally dramatic reduction in the numberof algal species) caused by nutrient enrichment(Schindler 1974; Proulx et al. 1996). The mechanismgenerally presumed to cause this decrease in diversity isan increased intensity of competition at higher levels ofproductivity (Grime 1979; Huston 1979, 1994; Tilman1988; Keddy 1989; Reader and Best 1989), althoughseparation of the e�ects of competition from other

e�ects of the physical conditions that in¯uence produc-tivity is not always straightforward.

Thus, in most situations, an increase in plant pro-ductivity (typically associated with larger plants and/orhigher plant biomass) that results from addition of nu-trients results in a decrease in the number of plant spe-cies coexisting in a given area or volume. In somesituations where soil fertility or other factors limit plantproductivity to extremely low levels, an increase in plantproductivity from very low to moderately low levels al-lows more species to survive and reproduce, leading toan increase in plant diversity. Consequently, along theentire gradient from very low to very high productivity,the response of diversity to productivity is unimodal,with a maximum at intermediate levels (the ``hump-backed'' response, sensu Grime 1973a, b).

Current interest in the ecosystem functions of biodi-versity (e.g., Schulze and Mooney 1993) requiresexperimental approaches that are able to distinguish thee�ect of biodiversity on ecosystem processes from thee�ect of ecosystem processes on biodiversity. Anincreasing understanding of ecological processes andimproved technology encourage the hope that ``thein¯uence of biodiversity can be elegantly dissectedthrough experimental manipulations'' (Kareiva 1994).

Three types of hidden treatments

In any experiment to investigate the e�ect of diversity onecosystem properties, the primary treatment is logicallythe number of species, with di�erent treatment levelsbeing di�erent number of species. One or more ecosys-tem properties or processes would be measured to lookfor a response to the treatment, with the speci®c pro-cesses determined by the hypotheses that motivated theexperiment. Since productivity is one of the most basicand important ecosystem processes, there is considerableinterest in the e�ect of species diversity on productivity,particularly the primary productivity of plants. How-ever, the strong e�ect of productivity (as manipulated byaddition of nutrients or other resources) on speciesdiversity makes evaluating the e�ect of species diversityon productivity particularly complex.

The following three examples illustrate di�erent sit-uations in which experimental responses attributed tovariation in species diversity are much more likely to bethe result of ``hidden treatments'' that altered plantproductivity or biomass within the experimental design.Three di�erent types of hidden treatments are illus-trated, each representing a di�erent pitfall for ecologi-cal experiments. As a consequence, properties that areattributed to species diversity may have nothing to dowith the diversity levels intended to be the experimentaltreatments, but rather are caused by other factorsintentionally or unintentionally manipulated in thecourse of the experiments, that is, the ``hidden treat-ments.''

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Use of nutrient additionsto create ``diversity treatments''

A recently published long-term experiment (Tilman andDowning 1994; Tilman 1996) used a combination ofexperimental nutrient additions and natural climaticvariability to evaluate the e�ect of species diversity onthe temporal stability of primary productivity. Theoriesabout the relationship between diversity and the stabilityof community and ecosystem processes have a longhistory in theoretical ecology (Elton 1958; MacArthur1972; May 1973; DeAngelis 1975; McNaughton 1977;Pimm 1979, 1984) and direct relevance to such currentissues as biodiversity loss and environmental sustaina-bility (Schulze and Mooney 1993; Vitousek and Hooper1993; Kareiva 1994, 1996; Mo�at 1996).

In an analysis of 13 years of biomass production ofover 200 plots (4 ´ 4 m; 4 ®elds ´ 9 nutrient treat-ments ´ 6 replicates per ®eld) Tilman (1996, p. 361)concluded that ``greater plant species diversity led togreater stability of plant community biomass after aperturbation. This supports the hypothesis that diversitystabilizes community and ecosystem processes.'' In thisexperiment, the only measured response variable was themaximum annual aboveground biomass in the experi-mental plots, which in herbaceous plant communities isstrongly correlated with net primary productivity. Thestability of primary productivity in plots with di�erentnumbers of species was evaluated in response to thenatural variability in precipitation, in particular thee�ect of a severe drought in 1988.

The conclusion that diversity increased stability wasbased on the statistical signi®cance of ANOVAs andregression analyses, with the following derived statisticsused to characterize di�erent aspects of the stability ofbiomass production: (1) coe�cient of variation o/f bio-mass for each plot across all years �CV � 100� 1SD/mean) as an indication of year-to-year variation in bio-mass (i.e., the inverse of stability); (2) rate of biomasschange from two years before the drought (1986) to thedrought (1988), expressed as a yearly rate, loge (bio-mass1988/biomass1986)/2 as a measure of ``drought resis-tance''; and (3) relative biomass deviation for post-drought years [(mean pre-drought biomass ) biomass ofa particular post-drought year)/(mean pre-drought bio-mass ) drought, 1988, biomass)] as a normalized mea-sure of the rate of return toward ``equilibrium'' followinga perturbation, also called ``resilience'' (Pimm 1984). Allthree of these measures of stability were positively cor-related with species richness in the experimental plots.

The experimental treatment is presumed to be num-ber of plant species per plot, subdivided into eight cat-egories for ANOVA (Fig. 4 in Tilman 1996) or used as acontinuous variable in regression analysis. However, oncloser examination it becomes apparent that the exper-imental treatments are actually varying levels of nitrogenaddition, which caused variation in both net primaryproductivity and species richness. The primary responsevariable in this experiment is the stability of net primary

productivity, and not simply the rate of net primaryproductivity. Consequently, the di�culty of separatingthe strong e�ects of fertilization on the magnitude of theresponse variable, from the e�ects of other factors, suchas species richness, on the variability of the responsevariable, is much greater than it would have been ifspecies richness had been manipulated independently ofproductivity. The following points must be kept in mindto understand the experimental results:

1. The variation in species diversity among the plots wasprimarily a result of nitrogen addition, which produceda gradient from low biomass-high diversity plots to highbiomass-low diversity plots. Based on the published®gures, there was a strong negative correlation betweenspecies number and both the nitrogen addition rate andthe actual biomass in the treatment plots (Fig. 1A, B).Added to the changes in species diversity that resultedfrom nitrogen addition, there was also variation in di-versity between the four experimental ®elds (rangingfrom an average of 8.6 to 14.9 species /0.3 m2), as aresult of di�erences in time since abandonment andperhaps other factors (Tilman 1993). This between-®eldvariation in the number of available species may havepartially obscured the relationship between individualplot biomass and plot species number, since ®eld identitywas a signi®cant factor in the multiple regressions thatwere presented (Tables 1 and 2 in Tilman 1996). In ad-dition to causing a reduction in species richness, thefertilizer addition may also have selected for species withlower root:shoot ratios, as well as caused a shift to lowerroot:shoot ratios within species. A lower root:shoot ra-tio would make plants more susceptible to drought, andis yet another factor confounding the analysis of theseexperiments (Givnish 1994).2. The nitrogen addition treatment was constantthrough time for each plot. Each set of replicates re-ceived the same nitrogen addition (ranging from 1 to28 g/m2) each year. The rainfall was highly variablebetween years, with all plots receiving the same rainfalleach year regardless of their nitrogen level. Conse-quently, biomass production in the low-nitrogen plotswas chronically limited by nitrogen and never achievedhigh levels, even in years of abundant rainfall. Totalbiomass in these plots was low, and interannual vari-ability in biomass production was low because biomasswas limited by a resource (nitrogen) that was applied ata constant rate through time. In contrast, biomass pro-duction in the high-nitrogen plots was limited primarilyby water availability rather than nitrogen. Because pre-cipitation varied greatly from year to year, biomassproduction in the high-nitrogen plots varied greatlythrough time, reaching high levels during wet years whennet primary productivity (NPP) was unlimited by eitherwater or nitrogen and dropping to low levels during dryyears. During the extreme drought of 1988, biomassproduction in all plots was similarly low, regardless ofnitrogen treatment (Fig. 2A in Tilman 1996). The con-sequences of interactions between limiting resources are

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well known (e.g., Liebig's ``law of the minimum'') andvariation in limiting resources is the basis of many of thecurrent theories and models of species diversity (Grime1979; Huston 1979; Tilman 1982; Pastor and Post 1986;O'Neill et al. 1989; Keddy 1989; DeAngelis 1992; Hus-ton and DeAngelis 1994). A similar interactive e�ect ofnitrogen and rainfall on herbaceous biomass has beenreported for the long-term Park Grass Experiments inGreat Britain (Cashen 1947).

When the interannual variation in biomass produc-tion is considered in terms of limiting resources, itbecomes clear why species diversity was positively cor-related with the various measures of stability. The highnitrogen-high biomass plots had variable biomass pro-duction because of the high variability of their primary

limiting resource (water). These high biomass plots alsohad low diversity, presumably because of the intensecompetition associated with their higher productivity.The lower variability in biomass production of the lownutrient-low biomass (high diversity) plots is a conse-quence of the relative insensitivity of these plots tovariation in precipitation because their productivity waslimited primarily by continually low nitrogen availabil-ity. Because diversity was reduced by the same ex-perimental treatments (nitrogen addition) that shiftedresource limitation from nitrogen to water and increasedvariability in net primary production, there appeared tobe a positive relationship between species diversity andstability. However, this was a spurious correlation, nota causal relationship. The treatment levels of speciesdiversity were simply another response to the hidden

Fig. 1A±F Relationships of thestability of plant biomass pro-duction to plant diversity andplant biomass in the CedarCreek long-term diversity ex-periments. A Average numberof plant species in each nitrogentreatment after 4 years ofnutrient addition. B Averagenumber of plant species per plotin relation to average biomassfor each level of nitrogen addi-tion. Note that species diversityis reduced in direct proportionto the increase in biomasscaused by fertilization. CDecreasing drought-inducedrelative reduction of annualbiomass production at higherlevels of species richness (lowerbiomass and nitrogen addition).D Increasing drought-inducedreduction of annual biomassproduction at higher levels ofplant biomass (higher biomassand nitrogen addition). E De-creasing recovery of annualbiomass production at higherlevels of species richness (lowernitrogen addition) following1988 drought. F Increasing re-covery of annual biomass pro-duction at higher levels of plantbiomass (higher nitrogen addi-tion) during recovery fromdrought. Note that both therelative decrease in biomassproduction caused by the 1988drought (D) and the relativeincrease in biomass productionfollowing the drought (F) arepositively correlated with bio-mass (fertilization level) whichis consistent with the resourcelimitation hypothesis. However,the negative correlation of spe-cies richness with recovery fromthe drought (E) is inconsistentwith the species diversityhypothesis (see text) (based onFig. 2 from Tilman 1996)

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treatment (nitrogen addition), not the cause of theobserved biomass responses.

The relationship of resilience and resistance to theexperimental nitrogen additions con®rms that speciesdiversity per se has little direct e�ect on the stability ofbiomass production in these experiments. While theproportional reduction in biomass during the droughtyear was greater in the low diversity plots than in thehigh diversity plots (Fig. 1C), the correlation is evenstronger with pre-drought biomass (Fig. 1D). The highbiomass (low diversity) plots experienced the greatestabsolute and proportional reduction in biomass in re-sponse to the drought, as would be expected on the basisof the limiting resource interactions described above.Recovery from the drought (i.e., resilience, expressed asthe proportional increase in biomass from 1988 to 1989)is actually negatively correlated with species richness(Fig. 1E) and positively correlated with pre-droughtbiomass (Fig. 1F). This pattern is inconsistent with thehypothesized positive e�ect of diversity on stability andresilience, but ®ts perfectly with the limiting resourcehypothesis proposed above. When the e�ects of biomassare factored out by dividing the post-drought biomassrecovery by the pre-drought biomass decrease (called the``relative biomass deviation''), the signi®cance ofthe diversity-resilience correlation is greatly reduced(Tilman 1996, p. 354).

A shift between two limiting resources with con-trasting variability is the most probable explanation forthe patterns of variation in productivity in this experi-ment. While some species in these experiments appar-ently demonstrated compensatory responses thatreduced variability in productivity (Fig. 8 in Tilman1996), these e�ects were small in comparison to the ef-fects of variation in the two primary resources, nitrogenand water. Thus, the conclusion that diversity increasesecosystem stability is not supported by these experi-mental results. Species diversity was not the true treat-ment variable, and consequently the only legitimateconclusion is that, in this experiment, temporal vari-ability in primary productivity is a simple consequenceof variability in the limiting resources.

Published analyses of the long-term fertilization re-sponses at the British Park Grass Experiment indicatethat, as at Cedar Creek, plot biomass (manipulated byfertilization) has a much stronger correlation with thestability of biomass production than does species rich-ness (Dodd et al. 1994). Out of 42 years sampled be-tween 1862 and 1991, plot biomass was signi®cantlycorrelated with the coe�cient of variation (CV) of bio-mass production in 30 cases, while species richness wassigni®cantly correlated only in 3 cases. In contrast to theCedar Creek results, in the Park Grass Experiments thehigh biomass plots were most stable, re¯ected in a neg-ative correlation between plot biomass and the CV ofbiomass production. Di�erences in the treatment typesand duration between the two experiments may helpexplain this discrepancy. In the Park Grass Experimentsthe long-term e�ects of ammoni®cation and leaching by

sulfate and nitrate resulted in extreme acidi®cation ofsoils that did not receive supplemental lime (Johnstonet al. 1986). The stunted vegetation of these extremelyacidic soils is much more sensitive to variation in pre-cipitation than vegetation on the limed soils (Dodd et al.1994; Silvertown et al. 1994). Because all plots in theCedar Creek fertilization experiment receive lime toprevent acidi®cation, this phenomenon has not occurredthere.

Creating ``diversity treatments'' using non-randomselections of species

Arti®cially constructed and highly controlled experi-mental ecosystems have great potential for evaluatingthe mechanisms responsible for observed changes inecosystem function in response to variation in speciesnumber or in such physical conditions as temperature orcarbon dioxide concentration (Kareiva 1994). In theEcotron experiment (Naeem et al. 1994a, 1995), the in-vestigators carefully constructed replicate ecosystems ina total of 14 climate-controlled growth chambers (8 m3).Three levels of diversity were created using a nestedgrouping of plants and animals (including bacteria,nematodes, Collembola, earthworms, and insects) inwhich the low diversity treatment (9 species ± of which 2were plants, 4 identical replicates) was a subset of thespecies in the intermediate diversity treatment (15 spe-cies ± of which 5 were plants, 4 identical replicates),which was a subset of the species in the high diversitytreatment (31 species ± of which 16 were plants, sixidentical replicates).

These treatments were envisioned as representing``increasingly depauperate versions of the high diversitymesocosms.'' Response variables included ``communityproperties'': (1) percentage of the ground surface areacovered by plants; (2) height distribution of the volumeoccupied by plants; (3) number of individuals of snails,insects, earthworms, Collembola, and insect larvae pa-rasitized by wasps; as well as ``ecosystem process'' rates:(1) carbon dioxide ¯ux rates; (2) percentage of photo-synthetically active radiation (PAR) intercepted by thevegetation, which was used as a surrogate for produc-tivity; (3) decomposition of surface grass litter andburied birch sticks; (4) nutrient ``retention'' in soil, basedon tri-weekly chemical analyses of soil samples; and (5)water retention, based on the amount of water thatdrained out of the chambers.

The complexity and cost of this type of highly con-trolled experiment impose constraints on the number oftreatments and replicates in the experimental design.Adequate and appropriate replication is a concern inany experimental design, but a type of false replication,called pseudo-replication, was identi®ed as a particularproblem in ecological experiments (Hurlbert 1984). Thisproblem arises when multiple samples from a single ex-perimental unit (e.g., plot, chemostat) are counted asreplicates for statistical purposes. Multiple instances of

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the same treatment (e.g., plots in di�erent locations in a®eld that all receive the same amount of fertilizer) are thereplicates with regard to the experimental treatment, notmultiple samples from a single unit.

For any experiment, treatments must be carefullyde®ned (presumably in the context of speci®c hypothe-ses), before the issue of appropriate replication can beresolved. When species diversity is the experimentaltreatment, with treatment levels being di�erent numbersof species, the de®nition of replicates is particularlycritical. Species number is a simple concept that is dis-tinct from the identities or properties of the species in-volved. For example, a set of replicates of the treatment``three species'', could include three species of bacteria,three species of plants, and three species of birds. Thenear impossibility of identifying any dependent variablesthat could be used to compare the responses of di�erentlevels of this type of treatment con®rms the absurdity ofthis example.

However, the degree of taxonomic or functionalconstraint that must be imposed on comparisons of dif-ferent levels of species diversity is not a trivial issue. Forplants, one must ask whether the e�ect of species diver-sity (i.e., species number) can be distinguished from thee�ect of species identity by restricting the eligible speciesto angiosperms (ignoring gymnosperms), or to trees (ig-noring shrubs and herbs), or to shade intolerant trees(ignoring other tolerance classes), or to shade intoleranttrees of similar maximum height, etc. The importance ofthese distinctions is revealed by the possibility of suchpointless experiments as evaluating the e�ect of speciesnumber on biomass production with treatments of threespecies of trees versus six species of grass.

Once the experimental organisms have been appro-priately selected, one can ask how the speci®c treatmentscan be replicated. Even among such a constrained subsetas non-nitrogen ®xing summer annuals with a maximumheight between 25 and 50 cm, many di�erent sets ofthree species could be selected. If the treatment is to bespecies number, then the replicates of each level of spe-cies number must be sets that di�er in the identity of thecomponent species. Multiple sets of the same species arenot replicates with regard to species number, but arereplicates for an experiment investigating the propertiesof that speci®c set of species.

In the Ecotron experiments (Naeem et al. 1994a,1995), the replicates of the diversity treatments wereidentical chambers with exactly the same speciescomposition in each. Thus, there was no replication ofspecies number, the putative treatment, and the actualtreatments were simply di�erent sets of species, whichwere replicated. As a result, the e�ect of the numberof species present in each treatment cannot be distin-guished from the e�ect of the particular species chosenfor that treatment, and the results of this experimentcannot be generalized beyond the group of species thatwas used. The failure to replicate the species diversitytreatment was compounded by a strong size bias in thegroupings of species used for implementing the experi-

mental treatments. Speci®cally, the species chosen forthe low diversity treatment were plants that only grow toa small maximum size, while the intermediate and higherdiversity treatments included species of greater maxi-mum size, with the highest number of large species in thehigh diversity treatment (Table 1, Fig. 2A). All plants inthis experiment were herbaceous annuals, so the largespecies also tended to be fast-growing species.

The size bias in species selection dominates the resultsof the Ecotron experiment. All of the measurements thatwere signi®cantly correlated with species diversity aresimple properties of plant tissue. Consequently, theamount or rate of these properties inevitably increasewith increasing plant biomass, including (1) plant cover(larger plants cover more ground area than smallplants); (2) height distribution of vegetation volume(taller plants have more leaves high above the groundthan short plants); (3) insect numbers (more plant-feeding insects can be supported on large plants thanon small plants: Gilbert and Smiley 1978; Bach 1980;Cytrynowicz 1991); (4) carbon dioxide ¯ux (plants withlarge total leaf area can take up more carbon dioxidethan plants with small total leaf area: Fields et al. 1992);and (5) light interception (more light is intercepted bylarge plants with a high total leaf area than by smallplants with lower total leaf area).

The issue of size bias in this experiment has beenraised previously with regard to three of the species(Andre et al. 1994; Naeem et al. 1994b). However, theproblem is more extensive than suggested in the previousdiscussion, which presented results of glasshouse exper-iments to support the validity of the Ecotron experi-ments (Naeem et al. 1994b, 1995). The results of theglasshouse pot experiments cannot legitimately becompared to the results of the Ecotron growth chambersfor two primary reasons. First, plant species areextremely plastic in terms of size, with most speciesreaching much larger sizes under conditions of high re-source availability (e.g., large pots or fertilized soil) thanunder low resource availability (e.g., small pots or lownutrient soil). Since the glasshouse experiments wereconducted in 20-cm pots, which have much less total soilvolume than the 1 ´ 1 ´ 0.3 m soil volume of theEcotron experiments, plants in the Ecotron experimentshad much higher availability of soil resources, whichwould have allowed species with large maximum sizes(e.g., Sonchus oleraceus) to achieve sizes much closer totheir maximum in the Ecotron than in the 20-cm pots.Second, the spatial density of individual plants in thepots was 6 times higher than in the Ecotron experiment,which further invalidates any comparison of either totalproductivity or individual species responses between thepot studies and the Ecotron experiments. The pattern ofspecies performance in the pot experiments does notdisprove the presence of size bias in the main Ecotronexperiment. This size bias is con®rmed by the heightpro®les of the three treatments (Fig. 2A), which clearlydemonstrate the smaller size of the species in thelow-diversity treatment.

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Thus, none of the conclusions of the experiment(Naeem et al. 1994a, 1995; Kareiva 1994, 1996) withregard to the e�ects of loss of biodiversity on ecosystemfunction are valid. The only legitimate conclusions arethat certain ecosystem processes occur at higher rates ingroups of large plants than in groups of small plantsunder the particular conditions of this experiment.

Creating ``diversity treatments'' using randomlyselected sets of species

The constructed grassland experiments at Cedar Creek,Minnesota, United States (Tilman et al. 1996) avoidedboth the confounding e�ect of fertilization and theproblem of pseudoreplication and non-random orbiased species groupings. However, a third type ofsampling problem a�ected these experiments, illustrat-ing one of the most subtle hidden treatments in ex-perimental ecology: the increasing probability ofselecting species with a speci®c property (e.g., largemaximum height, stress tolerance, nitrogen-®xationability, high seed germination rate) in samples ofincreasing number that are randomly selected from anygroup of species. This phenomenon can be called the``selection probability e�ect.'' A related phenomenon isthe ``variance reduction e�ect,'' which is the higherprobability of similarity or overlap in composition and/or performance between samples of many species drawnfrom a given pool than between samples of few speciesdrawn from the same pool. This e�ect reduces the

variance among replicate large samples of a populationin comparision with replicate small samples. In addition,it increases the probability that large ``natural'' sampleswill be similar to large statistical samples from the samepopulation (e.g., certain types of null models).

Unlike the Ecotron experiments, the grasslandexperiments at Cedar Creek focused on the e�ect ofvariation in the number of plant species alone. In theexperimental design, 147 plots (3 ´ 3 m) were randomlyassigned to one of seven species-richness treatments thatwere planted as seeds on bare ground, and allowed togrow under ambient climate with supplemental wateringand weeding to maintain species composition. The spe-cies-richness treatments were 1, 2, 4, 6, or 8 species (20replicates of each), 12 species (23 replicates), and 24species (24 replicates). Each replicate was created byrandomly drawing (with replacement) from the totalpool of 24 species, so each replicate at a given level ofspecies diversity was potentially composed of a di�erentsubset of the available species, with the inevitable in-crease in similarity between the replicates in the higherspecies richness treatments. The plants were grown for2 years, after which time measurements were made. Al-though no ecosystem processes were actually measured,the properties that were measured are assumed to rep-resent the net result of ecosystem processes operating atdi�erent rates over the same time period: (1) percentageof ground surface covered by plants, which was used as asurrogate for productivity; and (2) soil nitrate concen-tration in the plant rooting zone (0±20 cm depth) andbelow the rooting zone (40±60 cm depth), used as asurrogate for e�ciency of nutrient uptake.

Table 1 Species used in twoexperiments on the e�ect ofbiodiversity on ecosystemprocesses. The maximum andminimum heights (cm) are fromthe species descriptions in Stace(1991) for the EcotronExperiment and from Gleasonand Cronquist (1963) for theCedar Creek Experiment. Anasterisk indicates that thetypical minumum size wasestimated as 33% of themaximum from Gleason andCronquist (1963)

Ecotron Experiment Cedar Creek Constructed Grassland Experiment

Species Typicalmaximum

Species Minimum Maximum

I. (Low diversity) Andropogon gerardii 100 300Senecio vulgaris 30 Achillea millefolium 20 100Stellaria media 50 Bouteloua gracilis 15 60

Lespedeza capitata 60 150II. (Med. diversity) + I. Rudbeckia hirta 30 100Chenopodium album 150 Agropyron smithii 40 90Spergula arvensis 40 Anemone cylindrica 30 100Cardamine hirsuta 60 Asclepias tuberosa 30 70

Aster azureus 20 150III. (High diversity) + I + II Astragalus canadensis* 50 150Aphanes arvensis 10 Buchloe dactyloides 30 100Arabidopsis thaliana 30 Coreopsis palmata 50 90Capsella bursa-pastoris 40 Elymus canadensis 100Conyza canadensis 100 Euphorbia corollata 30 100Lamium purpureum 45 Koeleria cristata 30 60Poa annua 20 Liatris aspera 40 120Sinapsis arvensis 100 Panicum virgatum* 67 200Sonchus oleraceus 150 Petalostemum purpureum 30 100Tripleurospermum inodorum 60 Poa pratensis 30 100Veronica arvensis 30 Schizachyrium scoparium 50 120Veronica persica 50 Solidago nemoralis 10 100

Sorghastrum nutans 100 200Sporobolis cryptandrus 30 100Vivia villosa* 33 100

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In these experiments, the various diversity treatmentswere appropriately replicated. However, the interactionof the ``selection probability e�ect'' with the particularset of species chosen for implementing the design con-founds analysis of the e�ects of species diversity by in-troducing a correlation between the number of plantspecies in each treatment and the maximum possibleamount of plant biomass in that treatment. The 24species used in the experiment vary greatly in size(Table 1), with the result that the probability of areplicate containing a species that can grow to a largesize increases with the number of species in the replicate.

This bias is particularly signi®cant because multi-speciesgroups of plant are typically dominated by individualsof the largest species (Grime 1979; Austin and Austin1980; Huston and Smith 1987). Consequently, most ofthe biomass in each treatment was contributed by one ora few of the dominant species in that treatment (withdominance based on height, seed production, or otherproperties that confer a competitive advantage), andre¯ects the e�ects of those few species rather than someaverage response of the total number of species origi-nally planted in the plot. This inference is supported by:(1) the authors' observation that ®ve of the species ``hadsigni®cantly greater abundance in the higher diversitytreatments than expected on the basis of their proportionin the seed mixture'' [italics added] (Tilman et al. 1996,p. 719); (2) a published photograph of the experiment(Kareiva 1996) that showed a large proportion of theheavily vegetated plots dominated by a single species(apparently the yellow-¯owered Rudbeckia hirta, a fast-growing invasive species of roadsides and disturbedprairies), while many of the other plots were mostly bareground; and (3) published data (Fig. 1A in Tilman et al.1996) that reveal that ``e�ective species richness'' (thenumber of equally abundant species needed to producethe observed value of the Shannon-Weaver index, H 0;e�ective species richness is calculated as eH

0, where

H 0 �P p i � ln p i, and p i is the proportion of the totalbiomass composed of species i ) is half or less of thenumber of species originally planted per treatment,particularly in the higher diversity treatments, where the``selection probability e�ect'' for large dominant speciesis greatest.

Because the higher diversity treatments in this ex-periment inevitably have more large species, the primaryecosystem responses of the experiment are simply theresult of higher plant biomass, rather than higher speciesdiversity. Plant cover (the percentage of the groundsurface covered by plant tissue when viewed from above)has a near linear positive correlation with increasingplant biomass up to 100% cover, above which biomasscan continue to increase with no further increase in plantcover. A simple simulation of the experimental design, inwhich the height of each replicate was determined by thetallest of the randomly selected species (Table 1) repro-duces the observed pattern of an increasing amount ofvegetation (total plant cover) with an increasing numberof species per treatment (compare Fig. 3A and B). Thepot experiments discussed previously, that were done inconjunction with the Ecotron experiments (Naeem et al.1994a, 1995), were similar in number of replicatesand treatments to the Cedar Creek experiments anddemonstrate that this ``hidden treatment'' (the selectionprobability e�ect) also applies to total biomass(Fig. 3C). Note that the maximum biomass in the single-species pots was as great or greater than the biomass ofthe high-diversity pots. This demonstrates that theamount of biomass in the high-diversity pots is set by thesize of the largest species, and is not some aggregateproperty of high diversity.

Fig. 2A, B Qualitative similarity between height distribution of plantvolume observed at three diversity levels and simple calculations basedon the sizes of plant species used in the Ecotron experiment. AObserved vertical distribution of volume occupied by plant leaves inthe Ecotron growth chambers, in 10 cm increments (fromNaeem et al.1995). Note the low maximum height of leaf distribution in the low-diversity treatment. B Calculated proportion of the total number ofspecies in each diversity level that would be present in each 10-cmheight increment, assuming that each species has leaf biomass in theupper three-quarters of the typical maximum height reported for thatspecies in a standard ¯ora (Stace 1991). Note the small maximum sizeof species used in the low-diversity treatment

456

The other signi®cant treatment response in the CedarCreek constructed grassland experiments, variation insoil nitrate, is also a consequence of plant biomass ratherthan plant species diversity. Nitrogen in the soil is ofteninversely correlated with root biomass because moreroot biomass can take up more nitrogen, thus depletingthis element in the soil (Barber 1984). Previous work atthe Cedar Creek (Tilman and Wedin 1991) has demon-strated that soil nitrate is negatively correlated with rootbiomass �r2 � 0:72�. These results were obtained usingmonocultures of ®ve grass species, clearly demonstratingthat the e�ect of root biomass on soil nitrate at CedarCreek is independent of both the number of species in-volved (i.e., monocultures reduce nitrate to levels as lowor lower than the high-diversity treatments in the presentstudy) and species identity (i.e., at the same level of rootbiomass, the e�ects of the species did not di�er signi®-cantly).

The above problems with the design of the constructedgrassland experiment would not be expected to apply tothe results obtained from the natural grassland at CedarCreek, which had relationships of plant cover and soilnitrate with species richness that were very similar tothose found in the experiment. If the experimental resultsare interpreted improperly, how can the similar resultsfrom the natural grassland be explained? The answer maybe found in the extremely poor soils of the Cedar Creeksite, which is located on an ancient sand plain.

The common feature between the experiment and thenatural grassland is the extremely low productivity andthe large amount of open ground that was not coveredby plants (Fig. 2A in Tilman 1996). In the experiment,low productivity and low plant cover (27±56% of theground surface area) were maintained by a combinationof poor soil, disturbance due to hand weeding and theresulting limitation of many of the lower diversityplots to a species composition that only includes small-statured species. In the natural grassland, the low plantcover (26±88%) is apparently caused by the poor soils(low nitrogen, low water-holding capacity), and perhapsthe e�ect of gopher disturbances (Inouye et al. 1987).Under natural conditions a gradient in soil fertilityranging from extremely low to moderately low wouldtypically be associated with a gradient of increasingplant cover and species richness. Under such conditions,species richness generally increases up to a productivitylevel at which 100% plant cover is reached, above whichrichness declines due to competitive interactions.

Published information and the details of the presentexperiment make it clear that the Cedar Creek experi-mental site and prairies are at the lower end of theproductivity gradient, and that consequently resultsfrom this site re¯ect that portion of the natural range ofplant productivity over which plant diversity typicallyincreases with increasing productivity (Grime 1973a,1979; Huston 1994). A comparison of primary produc-tivity at 13 grassland and forest ecosystem sites acrossNorth America revealed that only a single site hadproductivity lower than that at Cedar Creek, and that

Fig. 3A±C Illustration of the ``selection probability e�ect'' in randomdraws of species from a common pool. A larger number of randomlydrawn species has a higher probability of containing species with aspeci®c property (e.g., large size, stress tolerance) than a lower numberof species. Note reduction in variance (SEM) and assymptotic increasein mean with larger numbers of randomly drawn species.A Simulatedvalues for average plant height at di�erent levels of species diversity,based on randomly selecting species from the pool of 24 species usedin the Cedar Creek Experiment and assuming that the plant height ineach treatment is the height of the tallest of the randomly selectedspecies (using typical minimum heights reported in a standard ¯ora,Gleason and Cronquist 1963, see Table 1). Mean and standard errorof the mean are the averages of ten separate simulations of the fullexperimental design. B Observed vegetative cover at each level ofspecies richness in the Cedar Creek constructed grassland experiment(mean and SEM from Tilman et al. 1996). C Total biomass per potproduced in a glasshouse experiment with pots planted with di�erentnumbers of species randomly selected from the pool of species used inthe Ecotron experiment (see Table 1). Small dots indicate the variousreplicates of di�erent species combinations at each level of speciesrichness. Large o�set dots indicate mean with SEM (based on Fig. 13in Naeem et al. 1995)

457

the productivity at Cedar Creek was less than 20% ofthat recorded at the other tall-grass prairie site (Zak et al.1994). The levels of plant biomass reported from un-fertilized plots at Cedar Creek (100±200 g/m2) placethese plots at the low end of the productivity gradientreported for 13 natural herbaceous sites studied in GreatBritain (100±2000 g/m2, Al-Mufti et al. 1977). Amongthe British sites, the number of plant species increasedfrom a minimum at a biomass of 100 g/m

2

to a maxi-mum at 500 g/m2 and then declined sharply with in-creasing biomass. Past fertilization experiments at CedarCreek have demonstrated that plant species diversity atthis site also decreases with increasing plant biomass(live and dead) above a level of 400±500 g/m2 (Tilman1993, 1996), which is consistent with data from thenatural prairies at Cedar Creek suggesting that speciesrichness begins to decrease at levels of plant covergreater than 90% (Fig. 2A in Tilman et al. 1996).

In the analysis of the Cedar Creek experimental data,the e�ect of the dominant role of above and below-ground plant biomass in causing the treatment e�ects isobscured by inappropriate interpretation of multipleregression analysis (Sokal and Rohlf 1981, pp. 642±656).The published analysis shows that both species richnessand root mass are signi®cantly and positively correlatedwith total plant cover, which is itself negatively corre-lated with soil nitrate (Table 1 in Tilman et al. 1996).Failure to consider the correlation between the threeproperties (root mass, species richness, and total plantcover) that were treated as independent variables in theregression on soil nitrate (Table 2 in Tilman et al. 1996)leads to the misleading inference that the e�ect of rootmass on soil nitrate is insigni®cant, and to the erroneousconclusion that the e�ect of species richness is theexplanation for the observed results. The primaryconclusions of the paper, that the results ``supportthe diversity-productivity and diversity-sustainabilityhypotheses'' and that ``the loss of species threatensecosystem functioning and sustainability'' are not sup-ported by the data.

Thus, in the previous three ``biodiversity'' experi-ments, the true experimental treatments were actuallyvariation in plant biomass or plant productivity ratherthan variation in the number of plant species. Becauseall of the ecosystem response variables that were mea-sured or estimated are consequences of the amount ofactive plant biomass, the experiments simply demon-strate that large plants produce more biomass (and takeup more CO2 and nutrients) than small plants. Toseparate the e�ect of plant species diversity from thee�ect of plant biomass (or other species-speci®c pro-perties such as nitrogen ®xation), the experimental de-sign must include at least one treatment in addition tothe properly replicated species richness treatment. Atleast two levels of plant size (e.g., large species versussmall species, or fertilized versus unfertilized) must becrossed with the number of species in order to determinewhether the ecosystem function of many di�erent speciesof plants di�ers from that of an equivalent biomass

composed of a few species. As in any ecological experi-ment, all physical conditions that might a�ect thetreatment response (e.g., soil water potential, soil nu-trient availability, temperature, solar radiation, herbi-vory) should be carefully monitored.

Discussion

If the above experiments provide no evidence thatincreasing biodiversity improves ecosystem function,what, if any, is the relationship between biodiversity andecosystem processes? It is certainly easy to envision sit-uations in which addition of more species might increasethe net primary productivity (or other process rate) of aplant community. The addition of species that increasenutrient availability, such as nitrogen ®xers or my-corrhizal fungi, would likely increase the net primaryproductivity of an ecosystem, as would the addition ofshade tolerant plant species under an overstory of shadeintolerant species. Processes such as these underlie thephenomenon known as ``overyielding,'' (de Wit et al.1966) which is the basis for intercropping and relatedagricultural practices (Vandermeer 1989). The increasein productivity associated with intercropping results notfrom adding many di�erent species, but from adding oneor a few species selected for very speci®c properties, suchas growth form or shade tolerance (Swift and Anderson1993; Anderson 1994; for examples from naturalsystems, see Ewel and Bigelow 1996; Denslow 1996).Likewise, compensatory growth among species withdi�erent physiological optima or tolerances for stressfulconditions could reduce the temporal variability innet primary production (i.e., increase stability) asapparently happened in the Cedar Creek long-termexperiments. This type of yield compensation is ahighly desirable property in agricultural systems as well.

Nonetheless, the biological interactions associatedwith higher species diversity apparently have only amarginal e�ect on the level or stability of productivityin comparison with variation in the availability ofresources such as nitrogen, phosphorus, or water. The300% increase in biomass that resulted from nitrogenaddition in the Cedar Creek Experiments (Fig. 2Ain Tilman 1996) vastly exceeds the possibly 10±20%biomass increase between the low and high diversityplots during the drought year (Fig. 4 in Tilman 1996).

The tremendous variation in net primary productivityacross the globe, or even along a single hillslope, is not aresult of variation in species diversity, but simply theconsequence of variation in resources that results fromthe interaction of climate, geology, and soil processes(Huston 1994). The simple observation that the Earth'smost productive ecosystems generally have low plantdiversity while high plant diversity is found under muchless productive conditions demonstrates that the numberof plant species has relatively little e�ect on productivity.The number of plant species in a local area is best un-derstood as a response to the level of plant productivity

458

allowed by local conditions of soil and climate, ratherthan being the cause of that level of productivity.

The three experiments discussed above providestrong support for the conclusion that both local speciesdiversity and the rates of ecosysem processes such asproductivity are determined by the amount and vari-ability of the fundamental environmental resources thatregulate plant growth and ecosystem productivity.Although the investigators who conducted each of theexperiments came to the conclusion that higher diversityincreased productivity, stability, sustainability, andother ecosystem functions, careful evaluation of theexperiments reveals that the experimental responses ineach case actually resulted from a ``hidden treatment''that was ignored while attributing the responses to theputative treatment. In reality, the putative ``diversity''treatments were either one of many responses to thefactors that produced variation in plant productivityand/or biomass (experiment 1), or an irrelevant correlateof the particular groupings of species used as treatments(experiments 2 and 3). The potential for such ``hiddentreatments'' should be carefully evaluated in all ecolog-ical experiments that investigate complex phenomena.

Acknowledgements I thank my colleagues and friends who pro-vided helpful comments and encouragement during the variousmetamorphoses of this paper. Those who helped sharpen the logicand soften the prose include Antoinette Brenkert, Don DeAngelis,Lou Gross, Bill Lauenroth, Craig Loehle, Michel Loreau, BobO'Neill, Mac Post, and Stuart Hurlbert. This study was supportedby the Environmental Sciences Division, O�ce of Health andEnvironmental Research of the U.S. Department of Energythrough the Walker Branch Watershed Project and the GlobalCarbon Cycle Program (contract DE-AC05-96OR22464 withLockheed Martin Energy Research, Inc.). This is publicationnumber 4642 of the Oak Ridge National Laboratory Environ-mental Sciences Division.

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