COVER SHEET FOR PROPOSAL TO THE NATIONAL SCIENCE FOUNDATION

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1 COVER SHEET FOR PROPOSAL TO THE NATIONAL SCIENCE FOUNDATION PROGRAM ANNOUNCEMENT/SOLICITATION NO./CLOSING DATE/if not in response to a program announcement/solicitation enter NSF 02-2 PD /10/02 FOR CONSIDERATION BY NSF ORGANIZATION UNIT(S) DEB - Ecology (Indicate the most specific unit known, i.e. program, division, etc.) FOR NSF USE ONLY NSF PROPOSAL NUMBER DATE RECEIVED NUMBER OF COPIES DIVISION ASSIGNED FUND CODE DUNS# (Data Universal Numbering System) FILE LOCATION EMPLOYER IDENTIFICATION NUMBER (EIN) OR TAXPAYER IDENTIFICATION NUMBER (TIN) NAME OF ORGANIZATION TO WHICH AWARD SHOULD BE MADE Western Washington University AWARDEE ORGANIZATION CODE (IF KNOWN) NAME OF PERFORMING ORGANIZATION, IF DIFFERENT FROM ABOVE SHOW PREVIOUS AWARD NO. IF THIS IS A RENEWAL AN ACCOMPLISHMENT-BASED RENEWAL IS THIS PROPOSAL BEING SUBMITTED TO ANOTHER FEDERAL AGENCY? YES NO IF YES, LIST ACRONYM(S) ADDRESS OF AWARDEE ORGANIZATION, INCLUDING 9 DIGIT ZIP CODE Western Washington University 516 High Street Bellingham, WA ADDRESS OF PERFORMING ORGANIZATION, IF DIFFERENT, INCLUDING 9 DIGIT ZIP CODE PERFORMING ORGANIZATION CODE (IF KNOWN) IS AWARDEE ORGANIZATION (Check All That Apply) (See GPG II.C For Definitions) FOR-PROFIT ORGANIZATION SMALL BUSINESS MINORITY BUSINESS WOMAN-OWNED BUSINESS TITLE OF PROPOSED PROJECT REQUESTED AMOUNT PROPOSED DURATION (1-60 MONTHS) REQUESTED STARTING DATE SHOW RELATED PREPROPOSAL NO., $ 390, months 07/01/02 IF APPLICABLE CHECK APPROPRIATE BOX(ES) IF THIS PROPOSAL INCLUDES ANY OF THE ITEMS LISTED BELOW BEGINNING INVESTIGATOR (GPG I.A) HUMAN SUBJECTS (GPG II.C.11) DISCLOSURE OF LOBBYING ACTIVITIES (GPG II.C) Exemption Subsection or IRB App. Date PROPRIETARY & PRIVILEGED INFORMATION (GPG I.B, II.C.6) HISTORIC PLACES (GPG II.C.9) INTERNATIONAL COOPERATIVE ACTIVITIES: COUNTRY/COUNTRIES INVOLVED (GPG II.C.9) SMALL GRANT FOR EXPLOR. RESEARCH (SGER) (GPG II.C.11) VERTEBRATE ANIMALS (GPG II.C.11) IACUC App. Date HIGH RESOLUTION GRAPHICS/OTHER GRAPHICS WHERE EXACT COLOR REPRESENTATION IS REQUIRED FOR PROPER INTERPRETATION (GPG I.E.1) PI/PD DEPARTMENT Department of Biology PI/PD FAX NUMBER PI/PD POSTAL ADDRESS NAMES (TYPED) High Degree Yr of Degree Telephone Number Electronic Mail Address PI/PD NAME CO-PI/PD Patterns of community assembly in serpentine grasslands: the roles of initial plant composition and functional complementarity Dept. of Biology Western Washington University Bellingham, WA United States David U Hooper Ph.D hooper@biol.wwu.edu CO-PI/PD CO-PI/PD CO-PI/PD Page 1 of 2 Electronic Signature

2 Patterns of community assembly in serpentine grasslands: the roles of initial plant composition and functional complementarity Project Summary The search for patterns of community assembly has occupied the last 25 years in community ecology and remains controversial today. A commonly sought assembly rule is that species coexistence in communities will be enhanced by differences in traits related to competition for limiting resources (i.e., that niche overlap is minimized). Put another way, do species from complementary guilds or functional groups have a better chance of persisting than those with strong overlap in resource use characteristics? Evidence for the role of complementarity among plant groups in structuring communities is sporadic. The balance between rates of external stochastic processes (such as disturbance or priority effects of colonization) compared to internal community dynamics (such as competition or facilitation) may be critical in determining the degree to which internal assembly dynamics are manifested. Gaining further insight into this question is critical for understanding how communities might be structured, and for the implications of that structure for ecosystem dynamics. We propose a five-year experiment to assess the roles of initial plant composition and functional complementarity in structuring communities of serpentine annual grasslands. We seek to test several hypotheses centered on the following questions, generally grouped into two themes: 1) Does the initial composition of a community shape its subsequent development? If so, for how long? Are there different stable endpoints? 2) To what extent does complementary resource use (i.e., niche differentiation) versus dominance among plant species govern patterns of community development? Are other factors, such as seed input or climatic conditions more important in determining community composition? Our goal is not to prove or disprove the predominance of assembly rules, but rather to better understand the conditions under which they might strongly influence community composition versus be overwhelmed by other factors. We will use as our experimental system a series of California serpentine grassland communities that vary in functional group composition and richness. Functional composition has been maintained since the plots were established in In fall 1999 and 2000, seeds of six additional species (3 species each from two groups) were added to test the effects of functional characteristics and diversity on invasibility of ecosystems. Thus we now have a system with treatments that differ in initial functional composition, that have had common seed sources for a number of additional species, and that will be subject to continuing seed rain from surrounding grasslands. Our plan is to track changes in community composition over the next five years as the communities develop from their initially different starting points. The measurements we plan are fairly simple: 1) nondestructive measurements of species abundances by the point quadrat method; 2) mapping of potential seed sources from the surrounding community; and 3) measurement of weather variables (temperature, precipitation, wind speed and direction). Patterns of community composition and change will be assessed by a variety of statistical techniques including nonparametric indices of community concordance, similarity indices, cluster analysis, detrended correspondence analysis (DCA), Analysis of Variance, and null models. Monitoring of vegetation changes in these experimental communities through the next several years provides a unique opportunity to gain insight into interactions among a variety of processes that help shape ecological communities.

3 TABLE OF CONTENTS For font size and page formatting specifications, see GPG section II.C. Section Total No. of Page No.* Pages in Section (Optional)* Cover Sheet (NSF Form 1207) (Submit Page 2 with original proposal only) A Project Summary (not to exceed 1 page) 1 B Table of Contents (NSF Form 1359) 1 C D E F Project Description (plus Results from Prior NSF Support) (not to exceed 15 pages) (Exceed only if allowed by a specific program announcement/solicitation or if approved in advance by the appropriate NSF Assistant Director or designee) References Cited Biographical Sketches (Not to exceed 2 pages each) Budget (NSF Form 1030, plus up to 3 pages of budget justification) G Current and Pending Support (NSF Form 1239) H Facilities, Equipment and Other Resources (NSF Form 1363) 1 1 I Special Information/Supplementary Documentation 2 J Appendix (List below. ) (Include only if allowed by a specific program announcement/ solicitation or if approved in advance by the appropriate NSF Assistant Director or designee) Appendix Items: *Proposers may select any numbering mechanism for the proposal. The entire proposal however, must be paginated. Complete both columns only if the proposal is numbered consecutively. NSF Form 1359 (10/99)

4 Community assembly in serpentine grassland 1 RESULTS FROM PRIOR NSF SUPPORT NSF Pre-doctoral fellowship ( ) and NSF Doctoral Dissertation Improvement Grant (DEB ) $15,000. ( ). "Effects of Plant Species Diversity on Nutrient Cycling in a Restored Serpentine Grassland in California", with Dr. P.M. Vitousek (P.I.). I studied the effects of plant diversity on ecosystem processes using California serpentine grassland as an experimental system. I established experimental plots with four plant functional types: early season annual forbs (E), late seasonal annual forbs (L), perennial bunchgrasses (P), and nitrogen-fixers (N). Two or three species of each group were planted in single functional group treatments, and in 2-, 3-, and 4- way combinations of functional groups. This design allowed me to assess effects of differences in functional group richness, as well as differences in plant composition within levels of functional group richness. We were thereby able to differentiate among mechanisms (e.g., complementarity vs. the sampling effect) by which plant diversity might affect ecosystem productivity and resource dynamics. The study assessed how plant composition and richness affected productivity, soil resource dynamics, and nitrogen retention and distribution within the ecosystem. The study led to several novel findings, which were published in a number of first-authored and co-authored articles (Vitousek and Hooper 1993, Hooper et al. 1995, Chapin et al. 1997a, Chapin et al. 1997b, Hooper and Vitousek 1997, Hooper 1998, Hooper and Vitousek 1998, Chapin et al. 2000). The paper by Hooper and Vitousek (1998) won the Mercer Award for an outstanding paper in the field of ecology given by the Ecological Society of America in In addition, an undergraduate student at Stanford did her Honors Thesis in collaboration with this project (Cynthia Benton, "Timing of release of labile nutrients in decomposition of plant litter on the serpentine grasslands"). Effects of Plant Diversity and Functional Characteristics on Species Invasions (DEB ) In collaboration with Jeffrey Dukes and Julia Verville (University of Utah), I have been using the experimental plots established in the previous study to investigate how plant functional composition and diversity influence invasibility of ecosystems. We are testing the hypothesis that greater plant functional diversity leads to decreased success of invading species. The factorial design of the study also allows us to test how the functional characteristics of the invaders interact with the functional characteristics of species already in the community to determine invasion success. Invaders were seeded in known amounts into all treatments in the experimental diversity gradient (described above). We selected three species from each of two functional groups (E and L; 6 species total) to use as invaders. Each of these species was seeded into a separate subplot of each community to avoid confounding the success or failure of an invader with the establishment of other invaders. This project just finished its second field season. The data show strong effects of both functional group traits and functional group diversity on invasibility. For early season annual (E) invaders, the presence of other species from the same functional group in the original community is the strongest determinant of invasion success. For late season annual (L) invaders, both the presence of other L's in the original community, as well as the functional diversity of the original community influenced invasion success. Finally, more diverse plots had lower average success for all invaders considered together. Results of this work have been presented at several meetings (Hooper et al. 2000, Dukes et al. 2001a, b, Hooper and Dukes 2001). One manuscript is in preparation (Hooper et al., in prep) and at least two others are planned. I have also been extensively involved in efforts to synthesize and come to consensus on the state of research on questions of biodiversity and ecosystem function (Hughes and Petchey 2001). Training has included three Research

5 Community assembly in serpentine grassland 2 Experience for Undergraduate (REU) students during summer 2000 and Results from one student s undergraduate thesis based on this work will be submitted to Soil Biology and Biochemistry (Chiarelli et al., in prep). Two other undergraduate students have done independent study projects based at least in part on this research. PROJECT DESCRIPTION INTRODUCTION A fundamental question in ecology is why different species assemblages occur next to one another under what appear to be identical environmental conditions (e.g., McCune and Allen 1985, Drake 1991, Petraitis and Latham 1999). Theoretically, communities of different composition might arise from 1) differences in starting conditions, or from 2) disturbances that alter species densities sufficiently to push the community across a threshold and towards a new equilibrium state (Gilpin and Case 1976, Hughes 1989, Laycock 1991, Knowlton 1992). In what cases do different starting points lead to different stable states? If communities converge from different starting points, how long does it take them to converge, and what determines the trajectory? How do external factors (climate, disturbance) influence the outcome? Similar questions have been addressed in some models (e.g., Law and Morton 1996) and microcosm studies (e.g., Robinson and Edgemon 1988), but the identification of basic rules governing the development of complex communities remains on the frontier of ecological research (Thompson et al. 2001). The study of assembly rules for ecological communities seeks to understand the processes that select for membership in a given community from a regional pool of species (Belyea and Lancaster 1999, Keddy and Weiher 1999). Assembly rules may be studied across a range of different scales and settings, from the regional scale of variation in species composition across islands to local variation in community composition within a habitat type (Cody 1999). Our study proposes to investigate plant community composition at this smallest scale. Given the same habitat type (climate, soil type, soil depth), what factors determine plant community composition? We ask this question in the context of trying to understand patterns of species invasion as a function of initial community composition. In the proposed experiment, we focus on two primary groups of questions. First, does the initial composition of a community shape its subsequent development? If so, for how long? Do different initial starting points lead to different stable endpoints? Second, to what extent does complementary resource use (i.e., niche differentiation) among plant species influence community composition? Is plant community assembly dependent on different functional groups of species, or is dominance by one or a few species the rule? Or, are stochastic factors, such as seed input or climatic conditions, more important in determining community composition? Our goal is not to prove or disprove the generality of an assembly rule based on complementarity, but rather to better understand the conditions, if any, under which such a process might strongly influence community composition. While of theoretical interest to basic ecology, these questions are relevant to many applied issues as well. First, ecologists and land managers have a major challenge in trying to understand patterns of spread in exotic species (Luken and Thieret 1997, Mack et al. 2000). Many questions in this field are directly relevant to issues of community assembly, including: Which types of species are most likely to invade? Which types of ecosystems are likely to be invaded? How do traits of invading species interact with those in the extant community? A second applied issue is trying to understand how community composition will change in response to global environmental changes, and the resulting consequences for ecosystem processes and

6 Community assembly in serpentine grassland 3 services (Dukes and Mooney 1999, Walker et al. 1999b). Finally, improvement of restoration ecology techniques depends on prediction of the trajectory of community composition based on initial composition (Lockwood and Pimm 1999). If restoration ecologists can determine which initial community compositions will lead to a desired endpoint, this might simplify initial restoration efforts and reduce the management necessary to guide the community towards the target outcome. BACKGROUND Assembly rules? The term "assembly rules" has been controversial in ecology. In a nutshell, ecologists want to know whether particular processes govern the inclusion or exclusion of species from communities and if such processes lead to any predictability in the composition of those communities. A primary issue in the debate over assembly rules in ecology focused on the most appropriate null models with which to compare the composition of pre-existing communities (for example, Diamond 1975, Connor and Simberloff 1979, Colwell and Winkler 1984, Gotelli and Graves 1996). When selecting a null model, it can be difficult to determine which species should be included in the potential species pool and how to deal with non-overlapping or partially overlapping geographic ranges, differences in relative abundance, differences in habitat preferences, or any other factors that might influence the probability of species being found together (Fox 1999, Simberloff et al. 1999). Much of this difficulty disappears, however, when sampling occurs only on a local level under common conditions, such that all species considered share ranges and habitat preferences (Simberloff et al. 1999). Potential rules involving functional groups To test for the possibility of complementarity in community structure, traits of species, rather than species themselves, need to be the focus of investigation. One of the first assembly rules proposed (Diamond 1975), and one that is still commonly analyzed and debated, is that competition limits the similarity of species able to persist in communities. A common test for this involves whether certain species are more or less likely to be found together in natural communities than at random. However, many researchers have argued that a more general approach would look at species traits rather than taxonomic affiliations (Fox and Brown 1993, Weiher and Keddy 1998, Belyea and Lancaster 1999). Stated another way, are coexisting species more likely to be from different guilds or functional groups (presumably based on suites of traits related to resource utilization) than would be expected from random communities? Many studies - particularly with mammals and birds - have found evidence for such community structure, while others have either not found patterns or have questioned the validity of those that have (see references in Weiher et al. 1995, plus the ongoing debate: Kelt and Brown 1999, Fox 1999, Simberloff et al. 1999). Much of this debate turns on the questions of appropriate null models and test statistics outlined above. Importantly, the models of Fox, Kelt, and coworkers specifically attempt to test for complementary guild or functional group structure. However, they have not been applied to plant communities to our knowledge. The model of Wilson (1989), on the other hand, looks for constant proportionality of different guilds or functional groups across different areas of a sampled community. It asks whether certain functional groups are found in more constant proportions than would be expected at random across many samples of a community or across communities (Wilson 1999). If such a pattern holds, as it does for at least some groups in several plant communities (Wilson and Roxburgh 1994, Wilson et al. 1995, Wilson and Whittaker 1995), then an assembly rule may bring about that pattern. This approach does not test directly for complementarity, although

7 Community assembly in serpentine grassland 4 competition or complementarity interactions could be the underlying mechanism that leads to a guild proportionality pattern. A third trait-based approach looks at the distribution of various morphological or functional traits, and asks whether the distribution of those traits is broader (overdispersed) or narrower (underdispersed) than would be expected at random (Weiher and Keddy 1998). To the extent that the traits analyzed are relevant to resource acquisition or utilization, overdispersed traits would indicate that competition for a limiting resource leads to selection for complementary traits at the community level. Underdispersed traits are thought to indicate environmental constraints or filters allowing access to a given type of habitat (Weiher et al. 1995, 1999). The latter approach is consistent with classifications of plant functional groups across environmental gradients (c.f. Grime 1979, Chapin et al. 1996, Díaz et al. 1999). Because both of these processes could operate simultaneously, the logical question follows: To what extent does complementarity among plants actually influence community structure in natural plant assemblages? The role of complementarity in structuring communities is of great interest to ecological disciplines outside of community ecology, particularly to the debate on diversity effects on ecosystem function (Kaiser 2000). Some groups argue that complementarity among species or functional groups has a strong effect on ecosystem processes (Tilman et al. 1996, Tilman et al. 1997, Hector et al. 1999, Loreau and Hector 2001, Tilman et al. 2001), while others argue that the traits of dominant species are the primary biotic determinant of process patterns and rates (Huston 1997, Wardle et al. 1997a, Wardle et al. 1997b). The latter group has also criticized the experimental design of the former, saying that because ecological communities follow distinct (but unspecified) assembly rules, randomized experiments cannot provide insight into the effects of diversity on processes (Wardle 1999). If complementarity among functional groups is one such strong community assembly rule, it has immediate implications for this debate. While there is some evidence for plant community structure responding to guild or functional group complementarity, the evidence so far is mixed. Positive associations among plant species of different functional groups at individual sampling points suggests that complementary plant associations affect community composition and structure at relatively small neighborhood scales (Wilson and Roxburgh 1994). Weiher and Keddy (1995) found qualitative evidence for complementary distribution of dominant species among functional groups of different plant growth forms in experimental wetland communities. In grazed grasslands in Australia, those species with the highest rank abundances were from different functional groups and additional species from the same groups had relatively low rank abundances (Walker et al. 1999a). This pattern suggests complementarity among dominant species, and dominance patterns among species of the same functional group. Our own studies have found evidence of complementarity among plant functional groups (Hooper 1998, Dukes 2001b), as have many others in both experimental ecosystems and intercropping (e.g., Trenbath 1974, Vandermeer 1990, Haggar and Ewel 1997). However, demonstrating that complementary interactions influence primary production in manipulated communities does not necessarily demonstrate that they are a driving force in community assembly in natural systems. Studies of community development under primary succession point to simultaneous effects of facilitation, competition, and tolerance among species in structuring communities (Chapin et al. 1994). It remains to be seen how guild or functional group complementarity influences community composition and how such internal community dynamics interact with stochastic forces from outside the community. Interaction of assembly rules with stochastic forces

8 Community assembly in serpentine grassland 5 There is abundant evidence in both model (e.g. Lockwood et al. 1997) and microcosm communities (e.g. Robinson and Dickerson 1987, Robinson and Edgemon 1988, Samuels and Drake 1997) that colonization precedence can lead to alternate stable states. Weiher and Keddy (1995) focused on different environmental conditions and how they affect community structure and convergence in wetland communities. Some differences in environmental conditions still allowed for community convergence (differences in fertility), but others did not (differences in water level). There was a stochastic component of community composition in terms of relative abundances of both core species (those that were in all replicates of a given environmental treatment) and occasional species. Questions remain about the relationship between species and functional composition, however. For example, are species compositions relatively labile and subject to priority effects within the constraints of assembly guided by functional traits, or do the predominant functional traits vary as well? Other stochastic processes such as disturbance and climate variability can also have profound and persistent effects on community structure and composition. In aquatic microcosms, Fukami (2001) found that different sequences in disturbance led to different final community composition. In California serpentine grasslands, rainfall variability, timing of soil disturbance by gophers, and harvester ant activity all influence local plant species composition (Hobbs 1985). A critical question is whether species composition within functional groups might vary, even as relative proportions of functional groups stay comparatively constant. In addition, structuring of community composition by assembly rules may depend on rates of colonization relative to rates at which the community comes to equilibrium. Modeling studies suggest that if rates of colonization exceed the rate at which species are selected by assembly rules, then assembly rules may be disrupted (Belyea and Lancaster 1999). Modeling studies also suggest that simultaneous species additions will confound any structured assembly process (Belyea and Lancaster 1999, and references therein). While these reviewers express concern that testing these hypotheses in the real world will be difficult or impossible, we suggest that our experimental system is in a good position to do just that. Serpentine grassland as an experimental system California serpentine grassland is a promising system in which to address questions of community assembly. This ecosystem is characterized by small-statured, predominantly annual species with a variety of growth strategies. Several plant functional types based on phenology, growth form, and nutritional dynamics (e.g., N-fixation) have been identified by previous researchers. For example, in this Mediterranean-type climate, annual species germinate after the first significant rains in the fall. Early season annuals set seed and die by early in the dry season (~May), whereas late season annuals remain in rosette form through the wet season, then flower from June - October (Gulmon et al. 1983, Mooney et al. 1986, Chiariello 1989). Perennial bunchgrasses set seed in late May, then senesce aboveground during the dry season (Jackson and Roy 1986, Jackson and Roy 1989). Other functional groups that have been identified include perennial forbs (both mono- and dicotyledonous, including geophytes) and annual grasses (both native and exotic). In addition to differing in phenology and morphology, these groups differ in several traits relevant to resource acquisition and nutrient dynamics, including rooting depth, root:shoot ratio, size, and leaf C:N content (Gulmon et al. 1983, Hobbs 1985, Mooney et al. 1986, Chiariello 1989, Armstrong 1991). These characteristics allow easy experimental manipulations using species with well-defined suites of functional traits. In a previous experiment, we established plots differing in both functional group composition and richness (Hooper and Vitousek 1997, Hooper 1998, Hooper and Vitousek

9 Community assembly in serpentine grassland ). We chose species from four groups: 1) early season annual forbs, 2) late season annual forbs, 3) perennial bunchgrasses, and 4) nitrogen-fixers (hereafter abbreviated as E, L, P and N, respectively). In that experiment, we focused on the effects of functional group composition and richness on plant productivity and nutrient cycling (see Results of Prior NSF Support). Initial studies in these plots (Hooper 1998, Hooper and Vitousek 1998), subsequent measurements, and other projects (Dukes 2001b) have shown strong evidence for complementarity among at least some functional groups. These functional groups had different seasonal patterns of resource use, particularly of nitrogen and water. Other studies have found similar patterns in this system (Gulmon et al. 1983). If complementarity has a strong role in structuring natural serpentine communities, then we predict that species from complementary functional groups will be community dominants. Furthermore, functional group composition, richness, and complementarity can all influence the success of subsequent invasions. In a microcosm experiment, Dukes (2001c, 2001a) found that mixtures of species from complementary functional groups decreased invasion success of the aggressive exotic weed Centaurea solstitialis (yellow star-thistle). In a follow-up experiment to Hooper and Vitousek (1998), we used the original experimental gradient of functional composition and diversity to evaluate the dynamics of species invasion. We established six subplots within each plot, and introduced one invading species into each of the six subplots. This design allowed us to study which factors are most important in determining the success of an invader: the characteristics of the invading species, the characteristics of the community being invaded, or the interaction of these two factors. We have found interactions between invader and community functional groups, as well as effects of overall community diversity (See Results from Prior NSF Support). We are currently working to understand how patterns of resource availability, as influenced by the different species, influenced these results. These plots, and species from the surrounding natural serpentine grassland, are now an excellent resource to investigate patterns and mechanisms of community assembly, including the extent to which initial composition determines future composition, the importance of complementarity in structuring community composition, and the rate at which communities converge. We have variable and known initial composition, known seed sources, and some species common to all experimental treatments (the seeded invaders). This arrangement will allow us to test a number of hypotheses related to complementarity among functional groups and effects of initial composition on subsequent community trajectories (see below). In addition, substantial areas of intact serpentine grassland outside of the experimental plots are available for simultaneous monitoring of natural communities for comparison with our plots. We strongly expect that community composition in all experimental treatments, even the most initially diverse, will change through time. What is not clear, however, is how these communities might change. These plots do not currently represent natural serpentine grassland, particularly because of our strict control of functional composition. Given the patterns of weeding over the last several years, it is apparent that none of the experimental communities are "saturated" with respect to species richness. Clearly, the bare plots most closely resemble a recently disturbed site. Because of colonization of additional species, we anticipate that species and functional composition will more closely mimic unmanipulated grassland over the next several years. But we could be wrong and that would be interesting. Will the roughly complementary structure of the most diverse communities stand up to invasion by new species, or will they become dominated by particularly aggressive competitors? Will the seeded invaders dominate the bare plots indefinitely, or will there be a succession of species? Will initially bare or depauperate communities eventually resemble the more diverse complementary communities,

10 Community assembly in serpentine grassland 7 or will the original species continue to dominate? Will deterministic forces (e.g., complementarity), govern species additions to the communities, or will more random forces (proximity of seed sources, disturbance history) predominate? This experimental system is poised to address these questions. HYPOTHESES We propose to test several hypotheses related to community assembly in these experimental plots. We describe the more prominent ones here. Patterns of dominance and composition 1. Patterns of composition across treatments H1. The same species will dominate all experimental plots regardless of initial composition. (null hypothesis.) H1a. Differences in community composition among treatments prior to this experiment ( growing season) will persist through time. H1b. Differences in community composition prior to this experiment will lead to new but different community compositions. Explanation. We will be able to test these hypotheses by comparing the eventual compositions of replicate plots from across the initial gradient of diversity and community composition. If community dynamics occur quickly in this annual grassland and community assembly is relatively deterministic, then it is possible that species composition in the different treatments will converge within the time of study. This would allow us to rule out H1a and H1b. On the other hand, if the initial composition strongly governs community dynamics over the first several years and communities do not converge by the end of the funding period, we will not be able to rule out H1 - it may be that more time is required for communities to converge. However, we will be able to observe rates of community convergence among treatments of different initial composition to see which ones become most similar most quickly over the duration of the experiment. We can test for similarity in patterns of dominance by using nonparametric tests [e.g., Kendall's coefficient of concordance (Zar 1996)] and for overall similarity in community composition by using Sorenson s similarity index, cluster analysis (Magurran 1988), and ordination techniques such as detrended correspondence analysis (DCA) (e.g., Weiher and Keddy 1995, Fukami 2001) (see Statistics, below). H2. New species establishment in plots will reflect proximity to seed sources, independent of initial community composition (i.e., propagule pressure is more important than initial community functional composition). H2a. New species establishment will depend on initial composition but not proximity to seed sources. H2b. Neither initial composition nor seed source proximity matters. H2c. Both matter. Explanation. This hypothesis and H6 (below) make interesting tests of the generality of the results we are currently getting with our invasibility experiment (see Results of Prior NSF Support), as well as other such experiments (e.g., Levine 2000). We will be able to test directly for effects of propagule pressure on success of individual species (focusing on those that were not part of the original factorial design of functional groups). By mapping potential seed sources and keeping track of wind speed and direction during seed dispersal periods for the various species, we can weight distance to nearest seed source by these variables. We can then include

11 Community assembly in serpentine grassland 8 this estimate of propagule pressure as a covariate in an Analysis of Covariance of individual species abundances in response to initial functional group composition (see Statistics, below). 2. Within treatment comparisons Comparison of replicates within a treatment will give a better understanding of stochastic components of community change as well as effects of dispersal and invasion loci. Do replicates of the same initial composition diverge or stay the same? If replicates diverge initially, do they converge again later? If replicates diverge, does this depend on proximity of sources for new colonists? H3. Species composition is similar among all replicates of a given treatment. H4. Functional group composition is similar among all replicates of a given treatment. H5. Divergence among replicates depends on proximity to invasion loci for new species. Explanation. Hypotheses 3 and 4 can be tested in a couple of different ways. First, do replicates group together in a cluster analysis based on the relative abundances of either species or functional groups when compared to other treatments. Is this consistent through time? Second, do similarity indices among replicates of a treatment, based on either species or functional characteristics, show significant linear or nonlinear trends over time? Hypothesis 5 can be tested by regression of abundances of new species (not those in the original community) against estimates of propagule pressure for replicate plots within treatments. 3. Tests of focal species: the seeded invaders. H6. Abundance of seeded invaders will be equal throughout all plots (null hypothesis). Explanation. Because the six species introduced in our previous invasion study were all sown in constant, known amounts in all treatments, they provide excellent test species to follow. While we state the null hypothesis, we predict that those species we seeded in as invaders in 1999 and 2000 will have greatest dominance in experimental plots that were initially less diverse. So far, seed input of these experimental invaders has largely been restricted to what we have added ourselves. Under these conditions, initial community composition has strongly affected the success of these invaders (Fig. 1). However, population dynamics of the original invaders and colonization of new species could alter the interactions that determine final community composition. Testing these hypotheses can be done with ANOVA of invader abundances in response to the initial functional group treatments (see Statistics, below). Patterns of diversity H7. After 5 years, there will be similar species diversity in all plots, regardless of initial composition. (null hypothesis.) H7a. Final species diversity will be a function of initial composition, independent of initial diversity. H7b. Final species diversity will be correlated with initial diversity. Explanation. Hypothesis 7 and its alternates are somewhat similar to H1- H1b, but are worth testing in their own right, because the overall diversity of subordinate species may differ despite similarities in dominants, or vice versa. Testing this hypothesis can be done with ANOVA of plot species richness in response to the initial functional group composition (see Statistics, below). In addition, we can compare variation in diversity with variation in different micro-environments in the natural grassland to get a better understanding of how it fits with natural variation (see Measurements, below). Role of functional composition of structuring communities

12 Community assembly in serpentine grassland 9 We predict that if complementarity is a strong community assembly rule, then in a given location the species that are most abundant will be from different functional groups (c.f. Walker et al. 1999a). We propose to test the following hypotheses in intact grassland, as well as in the experimental plots. H8. The most abundant species will be random with respect to functional groups. (null hypothesis) H8a. The proportion of the most abundant species that belong to complementary functional groups will be greater than that expected at random. H9. The degree of functional complementarity among abundant species is independent of micro-environment in natural grasslands. (null hypothesis) Explanation. We have information about the functional characteristics and groupings of many serpentine species - both those already in the plots and those likely to invade. The simplest test for H8 involves contingency tests (Chi-square or G-test) of co-occurrence of species from similar or contrasting functional groups (Bowers and Brown 1982, Kelt and Brown 1999). This approach could also be used for tests of natural communities in different microenvironments (i.e., slope and aspect, which vary significantly in species composition; see Measurements, below). Tests in different micro-environments in natural communities will help shed light on factors that may control the degree of complementarity vs. dominance, such as soil fertility. Null models can also be used to test hypotheses 8 and 9. Douglas A. Kelt, University of California, Davis, has developed a suite of null models (Kelt et al. 1995, Kelt and Brown 1999) that are applicable to our system, and has agreed to help us with these calculations (see Statistics, below, and attached letter). Because performing the necessary measurements in the intact grassland will require substantial additional work, we request funding for two sequential masters student research assistantships to carry out the aspect of the project involving the intact grassland. METHODS Approach Our overall approach is to monitor changes in species composition over time in experimental plots that initially differ in plant functional group composition and diversity. The measurements we propose are simple non-destructive estimates of plant community composition. The original experimental treatments were actively weeded to maintain control of species composition. The current invasion experiment added seeds in controlled amounts and restricted invading individuals to separate subplots. However, we plan no further manipulations or control of species composition, and no further control of seed inputs. We will compare species and functional composition in these plots with that of the surrounding natural grassland. Experimental design Study site Hooper (1998) established experimental plots at Kirby Canyon in south San Jose, California (37 o 15 N, 121 o 45 W) near a landfill operated by Waste Management, Inc. This region experiences a Mediterranean-type climate, with cool wet winters and a dry season extending from approximately May to October. Average rainfall is ~370 mm/yr (Huenneke et al. 1990), though both the timing and amount are highly variable from year to year (Armstrong 1991, Hobbs and Mooney 1995). Species composition in the native grassland at this site depends on a variety of factors, but is strongly influenced by local topography (Huenneke 1990). A previously denuded area of grassland was used for the experimental plots. Topsoil stockpiled from the landfill was placed over serpentinitic subsoil (C horizon) to a depth of approximately 30 cm., covering an area of approximately 0.25 ha. This gave a fairly homogeneous substrate on

13 Community assembly in serpentine grassland 10 which to plant the experimental treatments (Hooper and Vitousek 1998). The area was fenced to exclude mammalian herbivores (e.g., gophers), and has so far been successful at doing so. Experimental plots For manipulations of functional group diversity in the original experimental communities, species from four distinct functional categories were used, all of which are major components of the surrounding grasslands (Gulmon et al. 1983, Hobbs and Mooney 1985, Mooney et al. 1986, Chiariello 1989, Hobbie et al. 1992). The groups are early season annuals (E), late season annuals (L), perennial bunchgrasses (P), and nitrogen-fixers (N). Each functional group was planted alone and in various combinations in experimental plots measuring 1.5 by 1.5 meters separated by m buffer strips, using two or three of the most common species from each group (Hooper 1998). Because of the small stature of serpentine vegetation, these plots are large enough for self-supporting populations of all plants: hundreds (late season annuals) or thousands (early season annuals) of individuals are encompassed in a 1 m 2 area in native serpentine grassland (McNaughton 1968, Gulmon et al. 1983). The experimental design is a randomized complete block with six replicate blocks and a total of 10 treatments in each block. Treatments include a full factorial cross of E s, L s and P s: bare plots (B), each group alone (E, L, P), all 2-way combinations (EL, EP, LP), and the 3-way combination (ELP). In addition, there were two treatments including N-fixers: a single functional group treatment (N) and a 4-way combination (ELPN). This provided a range of functional group richness from 0 (bare plots) to 4 (0-9 species). Treatments were established in Jan at total densities and biomass that approximated the natural community (~200 g/m 2 ). Species not originally seeded in have been continually weeded out to maintain the functional group treatments. Weed biomass has been less than 5% of total biomass in most treatments (except bare and N-fixer) and years. No effort has been made to control densities of individuals of the originally planted species. For the initial invasion experiment, 6 species (3 each from 2 functional groups E s and L s) were seeded into separate subplots (30 cm x 30 cm each) in each experimental plot, as described above. Equal amounts of seed were added to all treatments in Fall 1999 and again in Fall Seeds germinating outside of the invading species subplot were counted to estimate total germination success and then weeded out to avoid interactions among invaders. Seed germinating in the 2000/2001 growing season is a combination of seeds we added and seeds deposited from the previous years growth. We plan no further control of seed input or weeding. Natural grassland In addition to measurements in our experimental plots, we propose to measure species composition, diversity, and relative abundances in natural serpentine grasslands adjacent to our experimental plots. These measurements will give us a comparison of expected levels of species diversity, and year-to-year and plot-to-plot (within treatment) variability in species composition. They will also allow testing of effects of complementarity in community structure in natural communities (via null models, as described below) for comparison with results in the experimental plots. We will use 24 replicate plots (1.5 m x 1.5 m) randomly stratified across four different micro-environments (6 plots per micro-environment) to capture the range of natural variability in species composition and densities of individuals most relevant to what we anticipate in our experimental plots. The four micro-environments are swales, level hilltops, moderately sloping north aspects, and moderately sloping south aspects. Permanent plots will be established and measurements will be by the point quadrat method in years 2-5, as described below.

14 Community assembly in serpentine grassland 11 Suitability of the experimental design Previous reviewers questioned if the experimental plots were appropriate for examining the hypotheses of community assembly outlined above. We strongly feel that they are, for the following reasons: 1. Plot size. The plots are large enough relative to the size of individuals that they can support self-sustaining populations of all species. At the same time, they are small enough and close enough together that seed dispersal should differ minimally among the individual plots by the end of the experiment (see 4, below for more discussion on this issue). 2. The different initial community compositions allow us to directly test for priority effects among treatments. Previous reviewers commented critically on the fact that these plots were weeded for previous experiments but will not be weeded for this one, and that there are likely to be differences in bare area among treatments. Contrary to being a weakness in the experiment, we feel that this is an important component of the experiment. The very question we are trying to test is how community composition changes when more species are allowed to establish. The initial communities had been in place for eight years before the invasion experiment. Because we weeded constantly throughout each year, weed biomass was always a very small percentage (<5%) of total community biomass, and therefore should have had only minor effects on plant densities. Therefore, differences in the amount of bare ground among treatments are not an experimental artifact, but are a reflection of the maximum natural density of individuals in any particular species composition treatment. Difference in available space is one potential reason that different initial communities might diverge or take longer to converge. Our point framing measures have quantified the amount of bare area in all plots prior to cessation of weeding, so we can statistically test for such effects. Finally, disturbance by gopher mounds is a common generator of bare ground in serpentine grassland (Hobbs and Mooney 1991), so understanding community dynamics in such situations is directly applicable to surrounding natural systems. 3. Similar species were seeded in during the invasion study. Three species from each of two functional groups (early and late season annuals) were added to the plots as part of the invasion study. Total experimental seed rain for these six species was identical across all treatments. In the first two years of that study, however, strong differences developed in total biomass of those invaders, but again, this is not an artifact. It is a result of the species interactions in those different treatments arising from the differences in initial community composition. This serves as the beginning step of our experiment on community assembly. These initial six invaders can now serve as focal species. If the differences in invader success persist throughout the proposed study, then that provides evidence for strong priority effects. If, on the other hand, the initial differences decrease with subsequent invasion of new species from both adjacent plots and from outside the experimental area, then depending on the patterns, that could provide evidence for either random assembly or species interactions as strong factors in community assembly. 4. Because we seek to test ideas related to species interactions as forces structuring communities, we ideally would have identical seed rain for all species in this experiment so we could control for dispersal limitations. We have that case for the six initial invaders. Seed rain from species colonizing from the surrounding grassland will not necessarily be identical initially (the experimental optimum). Some original invasion loci will undoubtedly be closer to some plots than others. This is why we intend to continue mapping species composition in the vicinity of the plots. In the long run, however, dispersal limitations to colonization should be minimal because of the small plot size relative to seed dispersal distance of these species, because most